NZ731011B2 - Fusion proteins, recombinant bacteria, and methods for using recombinant bacteria - Google Patents

Abstract

Fusion proteins containing a targeting sequence, an exosporium protein, or an exosporium protein fragment that targets the fusion protein to the exosporium of a Bacillus cereus family member are provided. Recombinant Bacillus cereus family members expressing such fusion proteins are also provided. Genetically inactivated Bacillus cereus family members and recombinant Bacillus cereus family members that overexpress exosporium proteins are also provided. Seeds coated with the recombinant Bacillus cereus family members and methods for using the recombinant Bacillus cereus family members (e.g., for stimulating plant growth) are also provided. Various modifiations of the recombinant Bacillus cereus family members that express the fusion proteins are further provided. Fusion proteins comprising a spore coat protein and a protein or peptide of interest, recombinant bacteria that express such fusion proteins, seeds coated with such recombinant bacteria, and methods for using such recombinant bacteria (e.g., for stimulating plant growth) are also provided. In particular, the present invention relates to a recombinant B. cereus family member that expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant B. cereus family member. The recombinant B. cereus family member comprises a mutation in an ExsY gene, CotE gene, CotY gene, ExsA gene, CotO gene, overexpresses a ExsY protein, overexpresses a BclB protein or overexpresses a YjcB protein, which results in the B. cereus family member spores having an easier to remove exosporium from the spore compared to a wild-type spore. enetically inactivated Bacillus cereus family members and recombinant Bacillus cereus family members that overexpress exosporium proteins are also provided. Seeds coated with the recombinant Bacillus cereus family members and methods for using the recombinant Bacillus cereus family members (e.g., for stimulating plant growth) are also provided. Various modifiations of the recombinant Bacillus cereus family members that express the fusion proteins are further provided. Fusion proteins comprising a spore coat protein and a protein or peptide of interest, recombinant bacteria that express such fusion proteins, seeds coated with such recombinant bacteria, and methods for using such recombinant bacteria (e.g., for stimulating plant growth) are also provided. In particular, the present invention relates to a recombinant B. cereus family member that expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant B. cereus family member. The recombinant B. cereus family member comprises a mutation in an ExsY gene, CotE gene, CotY gene, ExsA gene, CotO gene, overexpresses a ExsY protein, overexpresses a BclB protein or overexpresses a YjcB protein, which results in the B. cereus family member spores having an easier to remove exosporium from the spore compared to a wild-type spore.

Description

FUSION NS, RECOMBINANT BACTERIA, AND METHODS FOR USING RECOMBINANT IA CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to US. Provisional Application No. 62/051,885, filed September 17, 2014, the content of which is incorporated herein by reference in its entirety.

REFERENCE TO CE LISTING SUBMITTED ELECTRONICALLY The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII-formatted sequence g with a file named “3005.WO Gene Sequence Listing.txt” created on ber 10, 2015, and having a size of 488 kilobytes, and is filed concurrently with the specification. The sequence listing ned in this ASCII-formatted document is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION The present invention generally relates to fusion proteins containing a targeting sequence, an exosporium protein, or an exosporium protein nt that targets the fusion protein to the exosporium of a Bacillus cereus family member. The invention also relates to recombinant Bacillus cereus family members expressing such fusion proteins, formulations containing the recombinant Bacillus cereus family members, seeds coated with the recombinant Bacillus cereus family members, and methods for using the recombinant Bacillus cereus family members (e.g., for stimulating plant growth, protecting a plant from a en, ing stress resistance in a plant, lizing a recombinant Bacillus cereus family member spore on a plant, stimulating germination of plant seeds, and delivering nucleic acids to plants). The invention additionally relates to recombinant Bacillus cereus family members that overexpress a protease or a nuclease, wherein overexpression of the protease or se partially or completely vates spores of the Bacillus cereus family member or renders the spores more susceptible to physical or chemical inactivation. The present invention further relates to recombinant Bacillus cereus family members that overexpress exosporium proteins, seeds coated with such recombinant Bacillus cereus family members, and methods of using such recombinant Bacillus cereus family members (e.g., for stimulating plant growth, ing stress resistance in plants, and protecting plants from ens).

The invention further relates to various modifications of the inant Bacillus cereus family members that express the fusion proteins, including: (i) overexpression of tor proteins that modulate the expression of the fusion protein in the recombinant Bacillus cereus members; (ii) genetic inactivation of the recombinant Bacillus cereus family WO 44661 PCT/U82015/050807 members; and (iii) mutations or other genetic alterations of the recombinant Bacillus cereus family members that allow for the tion of exosporium fragments containing the fusion protein. The invention also relates to s methods for using the exosporium fragments.

The invention further relates to fusion proteins comprising a spore coat protein and a protein or peptide of interest, recombinant bacteria that s such fusion proteins, seeds coated with such recombinant bacteria, and methods for using such inant bacteria (e.g., for ating plant growth, protecting a plant from a pathogen, enhancing stress ance in a plant, immobilizing a recombinant ial spore on a plant, ating germination of plant seeds, and ring nucleic acids to plants).

The present invention further relates to biologically pure bacterial cultures of novel strains of bacteria.

The present invention additionally relates to plant seeds coated with an enzyme that catalyzes the production of nitric oxide or a superoxide dismutase, or with a recombinant spore-forming bacterium that overexpresses an enzyme that catalyzes the production of nitric oxide or a superoxide ase.

The invention also relates to methods for delivering cial bacteria and enzymes or vaccines to animals, and other methods of use.

BACKGROUND OF THE INVENTION Within the zone surrounding a plant’s roots is a region called the rhizosphere. In the rhizosphere, bacteria, fungi, and other organisms compete for nutrients and for binding to the root structures of the plant. Both detrimental and beneficial bacteria and fungi can occupy the rhizosphere. The bacteria, fungi, and the root system of the plant can all be influenced by the s of peptides, enzymes, and other proteins in the rhizosphere. Augmentation of soil or treatment of plants with certain of these es, enzymes, or other proteins would have beneficial effects on the overall populations of beneficial soil bacteria and fungi, create a healthier l soil environment for plant growth, improve plant growth, and provide for the protection of plants against certain bacterial and fungal pathogens. However, previous attempts to introduce peptides, enzymes, and other proteins into soil to induce such beneficial effects on plants have been ed by the low survival of enzymes, proteins, and peptides in soil.

Additionally, the prevalence of proteases naturally present in the soil leads to degradation of the proteins in the soil. The environment around the roots of a plant (the rhizosphere) is a unique mixture of bacteria, fungi, nutrients, and roots that has different qualities than that of native soil. 2015/050807 The symbiotic relationship between these organisms is unique, and could be altered for the better with inclusion of exogenous proteins. The high concentration of fungi and bacteria in the rhizosphere causes even greater degradation of proteins due to abnormally high levels of proteases and other elements detrimental to proteins in the soil. In addition, enzymes and other proteins uced into soil can dissipate away from plant roots quickly.

Thus, there exists a need in the art for a method for effectively delivering peptides, enzymes, and other proteins to plants (e.g., to plant root systems) and for extending the period of time during which such molecules remain active. Furthermore, there exists a need in the art for a method of selectively targeting such peptides, enzymes, and proteins to the rhizosphere and to plant leaves and plant roots in ular.

SUMMARY OF THE INVENTION The features of the invention are defined in the ed claims and the list of embodiments provided below in the Section ed “EMBODIMENTS.” Other s and features will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE GS FIGS. 1A and 1B show alignments of the amino acid sequence of an amino- terminal portion of Bacillus anthracis Sterne strain BclA and with the corresponding region from various exosporium proteins from Bacillus cereus family members. shows exemplary fluorescent copy results for the expression of fusion proteins containing various rium proteins linked to an mCherry reporter on the rium of a recombinant Bacillus cereus family member.

FIG 3 provides data showing to recombinant us thuringiensis BT013A spores expressing a fusion protein comprising a DNA binding n. is a transmission electron micrograph showing exosporium fragments and a Bacillus cereus family member spore from which the exosporium has been lost, generated using a recombinant Bacillus cereus family member having a knock-out mutation of its CotE gene. is a photograph of an SDS-PAGE gel showing a protein marker standard (lane 1) and proteins from exosporium fragments generated using a recombinant Bacillus cereus family member having a knock-out mutation of its CotE gene (lane 2).

W0 2016/044661 PCT/U82015/050807 provides data illustrating enzyme activity of an acid phosphatase in exosporium fragments derived from a Bacillus cereus family member having a knock-out mutation of its CotE gene. es data illustrating that Bacillus cereus family member EE349 reduces the inhibitory effects of ide on root length in lentils. provides data illustrating increased phosphatase ty in a Bacillus cereus family member modified to overexpress acid phosphatase (AcpC). provides data showing the endoglucanase activity of recombinant Bacillus thuringiensis spores expressing a CotC-endoglucanase fusion protein. provides -field and fluorescence microscopy images showing detection ofRNA on the surface of recombinant B. thuringiensis spores expressing a fusion protein comprising amino acids 20—35 of SEQ ID NO: 1 and SspC bound to either single- stranded RNA ) or double-stranded RNA (dsRNA). provides a photograph showing the s of the NA MIR3 19 on soy height and root pment, following delivery to soybean plants using recombinant B. thuringiensis spores expressing a fusion protein comprising amino acids 20—35 of SEQ ID NO: 1 and SspC bound to MIR319. provides bright-field and fluorescence microscopy images showing detection of GFP and mCherry in the gut of nematodes fed normal OPSO E. c0li bacterial food (two right-hand panels) or nematodes fed B. thuringiensis spores sing a fusion n comprising amino acids 20—35 of SEQ ID NO: 1 and either GFP or mCherry (three left-hand panels). provides a fluorescence microscopy image showing detection of endophytic bacteria isolated from inside of corn plants treated with Bacillus thuringiensis EEBOOl84expressing a fusion protein comprising amino acids 20—35 of SEQ ID NO: 1 and GFP.

Arrows denote single . provides a photograph showing fluorescence of bacterial colonies containing recombinant Bacillus cereus family members expressing a fusion protein comprising amino acids 20—35 of SEQ ID NO: 1 and GFP, isolated from inside of corn plants grown from seeds coated with the recombinant bacteria. provides a transmission electron micrographs showing: (A) intact spores ofBacillus thuringiensis BT013A nded by attached exosporium; (B) spores of CotE knockout strain ofBacillus thuringiensis BT013A, with detached exosporium; and (C) a purified exosporium fragment preparation of exosporium nts derived from a CotE knockout strain ofBacillus thuringiensis BTOl3A.

DEFINITIONS When the articles “a,” “an,” “one,” “the,” and “said” are used herein, the mean “at least one” or “one or more” unless otherwise indicated.

The terms “agriculturally acceptable carrier” and “carrier” are used hangeably herein.

The term “animal” encompasses any non-human animal as well as humans. For example, where the term “animal” is used herein, the animal can be a mammal (e.g., a human, a sheep, goat, cow, pig, deer, alpaca, bison, camel, donkey, horse, mule, llama, , dog, or cat), a bird (e.g., a chicken, turkey, duck, goose, quail, or pheasant), a fish (e.g., almon, trout, tilapia, tuna, catfish, or a carp), or a crustacean (e. g., a , prawn, lobster, crab, or crayfish).

A “biologically pure bacterial culture” refers to a culture of bacteria ning no other bacterial species in quantities sufficient to interfere with the replication of the culture or be detected by normal bacteriological techniques. Stated another way, it is a culture wherein Virtually all of the bacterial cells present are of the ed strain.

The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be onal elements other than the listed elements.

The term “bioactive peptide” refers to any peptide that exerts a biological actiVity. “Bioactive peptides” can be generated, for example, Via the cleavage of a protein, peptide, proprotein, or preproprotein by a protease or peptidase.

The term “effective amount” refers to a quantity which is sufficient to result in a statistically significant increase of growth and/or of protein yield and/or of grain yield of a plant as compared to the growth, protein yield and grain yield of the control-treated plant.

An “enzyme involved in the production or activation of a plant growth ating compound” es any enzyme that catalyzes any step in a biological synthesis y for a compound that stimulates plant growth or alters plant structure, or any enzyme that catalyzes the conversion of an inactive or less active derivative of a compound that stimulates plant growth or alters plant structure to an active or more active form of the compound. Such compounds include, for example, but are not limited to, small molecule plant es such as auxins and nins, bioactive peptides, and small plant growth stimulating molecules synthesized by ia or fungi in the rhizosphere (e.g., 2,3-butanediol).

WO 44661 PCT/U82015/050807 The term n protein” as used herein refers to a protein having a polypeptide sequence that comprises sequences d from two or more separate proteins. A fusion protein can be generated by joining together a nucleic acid molecule that encodes all or part of a first polypeptide with a nucleic acid molecule that encodes all or part of a second polypeptide to create a nucleic acid sequence which, when expressed, yields a single polypeptide having functional properties derived from each of the original proteins.

The term “germination rate” as used herein refers to the number of seeds that germinate during a particular time period. For e, a germination rate of 85% indicates that 85 out of 100 seeds germinate during a given time period.

The term ivate” or “inactivation” as used herein in reference to the inactivation of spores of a recombinant Bacillus cereus family member or a recombinant spore- forming ium means that the spores are unable to germinate, or that the spores can germinate, but are damaged such that germination does not result in a living bacterium. The terms “partially inactivate” or “partial inactivation” mean that a percentage of the spores are vated, but that some spores retain the ability to germinate and return to a live, replicating state. The term “genetic inactivation” refers to vation of spores a recombinant Bacillus cereus family member or recombinant spore-forming bacterium by a on of the spore’s DNA that results in complete or partial inactivation of the spore. The terms “physical inactivation” and cal inactivation refer to inactivation of spores using any physical or chemical means, e.g., by heat treatment, gamma irradiation, x-ray irradiation, UV-A irradiation, UV-B irradiation, or treatment with a solvent such as gluteraldehyde, formaldehyde, hydrogen peroxide, acetic acid, bleach, chloroform, or phenol, or any combination thereof.

The terms “immobilizing a recombinant Bacillus cereus family member spore on a plant” and “immobilizing a spore of a recombinant spore-forming bacterium on a plant” refers to the binding of a recombinant Bacillus cereus family member spore or a spore of a inant spore-forming bacterium to plant, e. g., to a root of a plant or to an aerial portion of a plant such as a leaf, stem, flower, or fruit, such that the spore is maintained at the plant’s root structure or aerial portion instead of dissipating into the plant growth medium or into the environment surrounding the aerial portions of the plant.

The term “inoculant” as bed in this invention is defined in several l, or State regulations as (1) “soil or plant inoculants shall include any carrier or culture of a specific micro-organism or mixture of micro-organisms represented to improve the soil or the growth, quality, or yield of plants, and shall also include any seed or fertilizer represented to be PCT/U82015/050807 inoculated with such a culture” (New York State lO-A Consolidated Law); (2) “substances other than fertilizers, manufactured, sold or represented for use in the improvement of the physical condition of the soil or to aid plant growth or crop yields” (Canada izers Act); (3) “a formulation ning pure or predetermined mixtures of living bacteria, fungi or virus particles for the treatment of seed, seedlings or other plant propagation material for the purpose of enhancing the growth capabilities or disease resistance or otherwise altering the properties of the al plants or crop” (Ad hoc European Working Group, 1997) or (4) “meaning any chemical or biological substance of e of substances or device distributed in this state to be applied to soil, plants or seeds for soil corrective purposes; or which is intended to improve germination, growth, quality, yield, product quality, reproduction, flavor, or other desirable characteristics of plants or which is intended to produce any al, biochemical, biological or physical change in soil” (Section 14513 of the California Food and Agriculture Code).

A “modulator protein” es any protein that, when overexpressed in a Bacillus cereus family member expressing any of the fusion proteins described herein, modulates expression of the fusion protein, such that the expression of the fusion n is increased or decreased as compared to expression of the fusion protein in a Bacillus cereus family member that does not press the modulator protein.

A “plant growth medium” es any material that is capable of supporting the growth of a plant.

A “plant immune system enhancer protein or e” as used herein includes any protein or peptide that has a beneficial effect on the immune system of a plant.

The term “plant growth stimulating protein or peptide” as used herein includes any protein or peptide that increases plant growth in a plant exposed to the protein or peptide.

The term “probiotic” as used herein refers to microorganisms (e.g., bacteria) that e health benefits when consumed by or administered to an animal.

The terms “promoting plant ” and “stimulating plant growth” are used interchangeably herein, and refer to the ability to enhance or increase at least one of the plant's height, weight, leaf size, root size, or stem size, to increase n yield from the plant or to increase grain yield of the plant.

A “protein or peptide that protects a plant from a pathogen” as used herein includes any protein or peptide that makes a plant exposed to the protein or peptide less susceptible to infection with a pathogen.

PCT/U82015/050807 A “protein or e that enhances stress resistance in a plant” as used herein includes any protein or peptide that makes a plant exposed to the protein or peptide more resistant to stress.

The term “plant g protein or peptide” refers to any e or protein capable of specifically or non-specifically binding to any part of a plant (e.g., roots or aerial portions of a plant such as leaves foliage, stems, flowers, or fruits) or to plant .

The term “pyrethrinase” refers to any enzyme that degrades a pyrethrin or a pyrethroid.

The term “rhizosphere” is used interchangeably with “root zone” to denote that segment of the soil that surrounds the roots of a plant and is ced by them.

The term “targeting sequence” as used herein refers to a polypeptide sequence that, when present as part of a longer polypeptide or a protein, results in the localization of the longer polypeptide or the protein to a specific subcellular location. The targeting sequences bed herein result in localization of proteins to the exosporium of a Bacillus cereus family DESCRIPTION OF THE INVENTION 1. Fusion Proteins for Expression in Bacillus Cereus Family Members and Recombinant Bacillus Cereus Family Members Expressing such Fusion Proteins The t ion relates to fusion proteins comprising a targeting sequence, an rium protein, or an exosporium protein fragment targets the fusion protein to the exosporium of a Bacillus cereus family member and at least one protein or e of interest.

When expressed in Bacillus cereus family member bacteria, these fusion proteins are targeted to the exosporium layer of the spore and are physically oriented such that the protein or peptide of interest is displayed on the outside of the spore.

This Bacillus exosporium display (BEMD) system can be used to deliver peptides, enzymes, and other proteins to plants (e. g., to plant foliage, fruits, flowers, stems, or roots) or to a plant growth medium such as soil. Peptides, enzymes, and ns delivered to the soil or another plant growth medium in this manner persist and t actiVity in the soil for extended periods of time. Introduction of recombinant Bacillus cereus family member bacteria expressing the fusion proteins described herein into soil or the rhizosphere of a plant leads to a beneficial enhancement of plant growth in many different soil conditions. The use of the BEMD to create these enzymes allows them to continue to exert their beneficial results to the plant and the rhizosphere over the first months of a plants life.

A. Targeting Sequences, rium Proteins, and Exosgorium Protein Fragments [or Tar etin Proteins or Pe tia’es 0 Interest to the Exos orium o a Bacillus cereus amil member For ease of reference, descriptions of the amino acid sequences for the ing sequences, exosporium proteins, and exosporium protein fragments that can be used for targeting of proteins or peptides of interest to the exosporium of a Bacillus cereus family members, are provided in Table 1 together With their SEQ ID NOs.

Table 1. Peptide and protein sequences used for ing of proteins or peptides of interest to the exos n orium of Bacillus cereus famil members Protein, nrotein fra ment, or tarI I etin_ sen SEQ ID NO.

AA 1—41 of BclA (B. anthracis Sterne) * Full len h BclA B. anthracis Sterne * AA 1—33 of BetA/BAS3290 B. anthracis Sterne Full len_th AS3290 B. anthracis Sterne Met + AA 2—43 of BAS4623 B. cis Sterne Full length BAS4623(B. anthracis Sterne) AA 1—34 of BclB B. anthracis Sterne Full len h BclB B. anthracis Sterne AA 1—30 of BAS1882 B. anthracis Sterne Full lenth BAS1882 B. anthracis Sterne n—n O AA 1—39 of _ene 2280 B. weihenstenhensis KBAB4 Full len_th KBAB4 _ene 2280 B. weihenstenhensis KBAB4 AA 1—39 of ene 3572 B. weihenstenhensis KBAB4 Full Len_th KBAB4 _ene 3572 B. stenhensis KBAB4 AA 1—49 of Exos norium Leader Pe tide B. cereus VD200 Full Lenth Exos norium Leader Pe tide B. cereus VD200 AA 1—33 of EXOSnorium Leader Pe tide B. cereus VD166 Full Lenth Exos norium Leader Pe tide B. cereus VD166 AA 1—39 of h nothetical nrotein IKG 04663 B. cereus VD200 H nothetical nrotein IKG 04663, nartial B. cereus VD200 NH O\O in—nAA 1—39 ofYVTN B-nnroeller nrotein B. weihenstenhensis KBAB4 Full len_th YVTN B-nnroeller nrotein B. weihenstenhensis KBAB4 AA 1—30 of hypothetical n bcerkbab4_2363 NNDJN B. weihenstenhensis KBAB4 Full length hypothetical protein bcerkbab4_2363 NJ; B. weihenstenhensis KBAB4 AA 1—30 of etical protein bcerkbab4_2l3l B. stenhensis KBAB4 Full length hypothetical protein bcerkbab4_2l3l B. stenhensis KBAB4 AA 1—36 of triple helix repeat containing collagen B. weihenstenhensis KBAB4 Full length triple helix repeat-containing collagen KBAB4 B. weihenstenhensis KBAB4 AA 1—39 ofh nothetical nrotein bm c00001 21660 B. m coia’es 2048 Protein, orotein fra_ment, or tar__etin sen SEQ ID NO.

Full len_th h oothetical rootein brn c00001 21660 B. m coides 2048 AA 1—30 of h oothetical rootein brn c0001 22540 B. m coides 2048 Full len_th h oothetical rootein brn c0001 22540 B. m coides 2048 AA 1—21 ofh oothetical rootein brn c0001 21510 B. m coides 2048 Full len_th h oothetical rootein brn c0001 21510 B. m coides 2048 AA 1—22 of en triole helix reoeat rootein B. thurin l'ensz's 35646 en triole helix reoeat orotein B. thurz'n l'ensz's 35646 AA 1—35 of h ical rootein WP 69652 B. cereus Full len_th h oothetical rootein WP 69652 B. cereus AA 1—41 of ex05oorium leader WP016117717 B. cereus Full len_th exos ooriurn leader WP016117717 B. cereus AA 1—49 of ex05oorium ooetide WP002105192 B. cereus Full len_th ex05oorium ooetide 05192 B. cereus AA 1—38 of h oothetical rootein WP87353 B. cereus Full len_th h oothetical rootein WP87353 B. cereus Ui-h-h-h-h-h-h-h OOOONOUI-bb.) AA 1—39 of exOSooriurn oeotide 02112369 B. cereus Full len_th exOSoorium oeotide 02112369 B. cereus AA 1—39 of exOSooriurn rootein WP016099770 B. cereus Kink/1K1} WNn—n Full len_th exOSoorium rootein 99770 B. cereus AA 1—36 of h oothetical orotein YP006612525 B. thurin l'ensz's kink/1 Ui-h Full len_th h oothetical orotein YP006612525 B. thurin iensz's £11 0 AA 1—136 of h oothetical orotein TIGR03720 B. m coides Full len_th h oothetical orotein 720 B. m coides AA 1—36 of collagen triple helix repeat domain n B. cereus ATCC 10987 Full length en triple helix repeat domain n B. cereus ATCC 10987 AA 1—39 of colla_en-like rootein B. cereus E33L Full len_th en-like rootein B. cereus E33L AA 1—41 of triple helix repeat-containing collagen B. weihensteohanensis KBAB4 Full length triple helix repeat-containing collagen B. weihensteohanensz’s KBAB4 AA 1—30 of hypothetical protein BALH_2230 B. thurz’n; iensz’s str. Al Hakam Full length hypothetical protein BALH_2230 B. thurz’n; iensz’s str. Al Hakam AA 1—44 of collaen triole helix reoeat B. cereus Full len; h n triole helix reoeat B. cereus AA 1—30 of h oothetical rootein BCZK1835 B. cereus E33L Full len_th h oothetical orotein BCZK1835 B. cereus E33L AA 1—48 of triple helix repeat-containing collagen B. weihensteohanensz’s KBAB4 SEQ ID NO.

Full length triple helix repeat-containing collagen B. weihensteIhanensz’s KBAB4 AA 1—39 of h Iothetical Irotein BC4725 B. cereus ATCC 14579 Full len_th h Iothetical Irotein BC4725 B. cereus ATCC 14579 AA 1—44 of h Iothetical Irotein BCZK4476 B. cereus E33L Full len_th h Iothetical Irotein BCZK4476 B. cereus E33L AA 1—40 of triple helix repeat-containing collagen B. anthracis str. ‘Arnes or’ Full length triple helix repeat-containing en B. anthracis str. ‘Arnes Ancestor’ AA 1—34 of BclA Irotein B. nz'ensz‘s serovar konkukian str. 97-27 Irotein B. thurinz'ensz’s serovar konkukian str. 97-27 AA 1—34 of conserved h Iothetical Irotein B. cereus ATCC 10987 Full len_th conserved h ical Irotein B. cereus ATCC 10987 AA 1—99 of exosmrium leader IIetide Iartial seIuence B. cereus Exosmrium leader IIetide Iartial seIuence B. cereus AA 1—136 of hypothetical protein ER45_27600, partial sequence B. weihensteIhanensis H Iothetical Irotein ER45 27600, Iartial seIuence B. weihensteIhanensz's 94 AA 1—196 of BclA B. anthracis Sterne Vlet -- AA 20—35 of BclA B. anthracis Sterne S('DH, |- AA 12—27 of BetA/BAS3290 B. anthracis Sterne OO \]O\ iiCDCDI—rI—r||- AA 18—33 of ,ene 2280 B. weihensteIhensis KBAB4 - AA 18—33 of ,ene 3572 B. weihensteIhensis KBAB4 \O\O \OOO wéwéwéég|g|g||- AA 12—27 of Exos Iorium Leader Pe Itide B. cereus VD166 - AA 18—33 ofYVTN B-propeller protein eihensteIhensz's KBAB4 - AA 9—24 of hypothetical protein bcerkbab4_2363 eihensteIhensz's KBAB4 - AA 9—24 of etical protein ab4_2131 . eihensteIhensz's KBAB4 Vlet -- AA 9—24 of hypothetical protein bmyc0001_22540 B. m coides 2048 Vlet -- AA 9—24 of BAS1882 B. anthracis Sterne - AA 20—35 of exosmriurn leader 17717 B. cereus - AA 9—24 of hypothetical protein BALH_2230 B. thurz’n; s str. Al Hakam Full len_th InhA B. m coides Full len_th BAS1141 Est B. anthracis Sterne Full len_th BAS1144 BXIB/ExsFA B. anthracis Sterne Full lenth BAS1145 CotY B. anthracis Sterne Full lenth BAS1140 B. anthracis Sterne Full len_th ExsFB B. anthracis H9401 n, rotein fra_ment, or tar__etin se SEQ ID NO.

Full len_th InhAl B. thurin iensis HD74 Full len_ h Est B. cereus ATCC 10876 Full len_ h Est B. cereus Full len_ h Y'cA B. anthracis Ames Full len_ h Y'cB B. cis Full len h Bch B. anthracis Sterne Full length acid phosphatase Bacillus thuriniensis serovar konkukian str. 97-27 Full len_th InhA2 B. thurin iensis HD74 Full len_th InhA3 B. m c0ia’es AA = amino acids * B. cis Sterne strain BclA has 100% sequence identity with B. thuringiensis BclA. Thus, SEQ ID NOs: 1, 2, and 95 also represent amino acids 1—41 of B. thuringiensis BclA, full length B. thuringiensis BclA, and amino acids 1—196 of B. thuringiensis BclA, respectively. Likewise, SEQ ID NO: 96 also represents a methionine e plus amino acids 20—35 of B. thuringiensis BclA.

** B. mycoia’es hypothetical protein TIGR03720 has 100% sequence identity with B. mycoia’es hypothetical protein WP003189234. Thus, SEQ ID NOs: 57 and 58 also represent amino acids 1—136 of B. mycoia’es hypothetical protein WP003189234 and full length B. mycoia’es hypothetical protein WP003 , respectively.

Bacillus is a genus of rod-shaped bacteria. The Bacillus cereus family of bacteria includes any Bacillus species that is capable of producing an exosporium. Thus, the us cereus family of bacteria es the species Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoia’es, Bacillus pseudomycoia’es, Bacillus samanii, Bacillus gaemokensis, Bacillus weihenstephensis, and Bacillus t0y0iensis. Under stressful environmental conditions, us cereus family bacteria undergo sporulation and form oval endospores that can stay dormant for extended periods of time. The outermost layer of the endospores is known as the exosporium and comprises a basal layer surrounded by an external nap of ike projections. nts on the hair-like nap are predominantly formed by the collagen-like glycoprotein BclA, while the basal layer is comprised of a number of different proteins. Another en-related protein, BclB, is also present in the rium and exposed on endospores of Bacillus cereus family members. BclA, the major constituent of the surface nap, has been shown to be attached to the exosporium with its amino-terminus (N-terminus) positioned at the basal layer and its carboxy-terminus (C-terminus) extending d from the spore.

It was previously discovered that certain sequences from the N-terminal regions of BclA and BclB could be used to target a peptide or protein to the rium of a Bacillus cereus endospore (see US. Patent Application Publication Nos. 2010/0233124 and 2011/0281316, and Thompson et al., Targeting 0fthe BclA and BclB proteins to the Bacillus anthracis spore surface, lar Microbiology 70(2):42l—34 (2008)). It was also found that the AS3290 protein ofBacillus anthracis zed to the exosporium.

In particular, amino acids 20—35 of BclA from Bacillus anthracis Sterne strain have been found to be sufficient for targeting to the exosporium. A sequence alignment of amino acids 1—41 of BclA (SEQ ID NO: 1) with the corresponding N—terminal regions of several other us cereus family exosporium proteins and us cereus family proteins having related sequences is shown in Figures 1A and 1B. As can be seen from s 1A and 1B, there is a region of high-homology among all of the proteins in the region corresponding to amino acids —41 of BclA. However, in these sequences, the amino acids corresponding to amino acids 36— 41 of BclA contain secondary structure and are not necessary for fusion protein localization to the exosporium. The ved targeting sequence region of BclA (amino acids 20—35 of SEQ ID NO: 1) is shown in bold in Figures 1A and 1B and corresponds to the minimal targeting sequence needed for zation to the exosporium. A more highly conserved region spanning amino acids 25—35 of BclA within the targeting sequence is underlined in the sequences in Figures 1A and 1B, and is the recognition sequence for ExsFA/BxpB/ExsFB and homologs, which direct and assemble the described proteins on the surface of the exosporium. The amino acid sequences of SEQ ID NOs. 3, 5, and 7 in Figure 1A are amino acids 1—33 of Bacillus anthracis Sterne strain BetA/BAS3290, a methionine followed by amino acids 2—43 ofBacillus anthracis Sterne strain 3, and amino acids 1—34 ofBacillus anthracis Sterne strain BclB, respectively. (For BAS4623, it was found that replacing the valine present at position 1 in the native protein with a methionine resulted in better expression.) As can be seen from Figure 1A, each of these sequences contains a conserved region corresponding to amino acids 20—35 of BclA (SEQ ID NO: 1 ; shown in bold), and a more highly ved region corresponding to amino acids 20—35 of BclA (underlined).

Additional proteins from Bacillus cereus family members also n the conserved targeting region. In particular, in s 1A and 1B, SEQ ID NO: 9 is amino acids 1—30 of Bacillus anthracis Sterne strain BASl882, SEQ ID NO: ll is amino acids 1—39 of the Bacillus weihenstephensis KBAB4 2280 gene t, SEQ ID NO: 13 is amino acids 1—39 of the Bacillus weihenstephensis KBAB4 3572 gene product, SEQ ID NO: 15 is amino acids 1—49 ofBacillus cereus VD200 exosporium leader peptide, SEQ ID NO: 17 is amino acids 1—33 of Bacillus cereus VDl66 exosporium leader peptide, SEQ ID NO: 19 is amino acids 1—39 of Bacillus cereus VD200 hypothetical protein IKG_04663, SEQ ID NO: 21 is amino acids 1—39 ofBacillus weihenstephensis KBAB4 YVTN B-propeller protein, SEQ ID NO: 23 is amino acids 2015/050807 1—30 of Bacillus weihenstephensis KBAB4 hypothetical protein bcerkbab4_2363, SEQ ID NO: is amino acids 1—30 of Bacillus weihenstephensis KBAB4 hypothetical protein bcerkbab4_2l3l, SEQ ID NO: 27 is amino acids 1—36 of Bacillus weihenstephensis KBAB4 triple helix repeat containing collagen, SEQ ID NO: 29 is amino acids 1—39 ofBacillus mycoia’es 2048 hypothetical protein brnyc00001_21660, SEQ ID NO: 31 is amino acids 1—30 of Bacillus mycoia’es 2048 hypothetical protein bmyc0001_22540, SEQ ID NO: 33 is amino acids 1—21 of Bacillus mycoia’es 2048 hypothetical n brnyc0001_21510, SEQ ID NO: 35 is amino acids 1—22 ofBacillus thuringiensis 35646 collagen triple helix repeat protein, SEQ ID NO: 43 is amino acids 1—35 ofBacillus cereus etical protein WP_69652, SEQ ID NO: 45 is amino acids 1—41 ofBacillus cereus exosporiurn leader WP016117717, SEQ ID NO: 47 is amino acids 1—49 ofBacillus cereus exosporiurn e WP002105192, SEQ ID NO: 49 is amino acids 1—38 ofBacillus cereus hypothetical protein WP87353, SEQ ID NO: 51 is amino acids 1—39 ofBacillus cereus exosporiurn peptide 02112369, SEQ ID NO: 53 is amino acids 1— 39 of Bacillus cereus exosporiurn protein WP016099770, SEQ ID NO: 55 is amino acids 1—36 llus thuringiensis hypothetical protein YP006612525, SEQ ID NO: 57 is amino acids 1— 136 of Bacillus mycoia’es hypothetical protein TIGR03720, SEQ ID NO: 59 is amino acids 1—36 ofB. cereus ATCC 10987 collagen triple helix repeat domain protein, SEQ ID NO: 61 is amino acids 1—39 of B. cereus E33L collagen-like protein, SEQ ID NO: 63 is amino acids 1—41 of B. weihenstephanensis KBAB4 triple helix repeat-containing collagen, SEQ ID NO: 65 is amino acids 1—30 of B. thuringiensis str. Al Hakam hypothetical protein BALH_2230, SEQ ID NO: 67 is amino acids 1—33 of B. cereus ATCC 14579 triple helix repeat-containing collagen, SEQ ID NO: 69 is amino acids 1—44 ofB. cereus collagen triple helix repeat, SEQ ID NO: 71 is amino acids 1—38 of B. cereus ATCC 14579 triple helix -containing collagen, SEQ ID NO: 73 is amino acids 1—30 ofB. cereus E33L hypothetical n 35, SEQ ID NO: 75 is amino acids 1—48 of B. stephanensis KBAB4 triple helix repeat-containing collagen, SEQ ID NO: 77 is amino acids 1—30 ofB. cereus ATCC 14579 triple helix repeat-containing collagen, SEQ ID NO: 79 is amino acids 1—39 of B. cereus ATCC 14579 hypothetical protein BC4725, SEQ ID NO: 81 is amino acids 1—44 of B. cereus E33L hypothetical protein BCZK4476, SEQ ID NO: 83 is amino acids 1—40 of B. anthracis str. ‘Ames Ancestor’ triple helix repeat- containing collagen, SEQ ID NO: 85 is amino acids 1—34 of B. thuringiensis serovar ian str. 97-27 BclA n, SEQ ID NO: 87 is amino acids 1—34 of B. cereus ATCC 10987 conserved hypothetical protein, SEQ ID NO: 89 is amino acids 1—34 of B. cereus ATCC 14579 triple helix repeat-containing collagen, SEQ ID NO: 91 is amino acids 1—99 of B. cereus exosporium leader peptide partial sequence, and SEQ ID NO: 93 is amino acids 1—136 of B. weihenstephanensz’s hypothetical protein ER45_27600. As shown in Figures 1A and 1B, each of the N—terminal regions of these proteins contains a region that is conserved With amino acids 20— of BclA (SEQ ID NO: I), and a more highly conserved region corresponding to amino acids —35 of BclA.

Any portion of BclA Which includes amino acids 20—35 can be used as to target a fusion n to the exosporium. In addition, full-length exosporium proteins or exosporium protein fragments can be used for targeting the fusion proteins to the rium. Thus, fulllength BclA or a fragment of BclA that es amino acids 20—35 can be used for targeting to the exosporium. For e, full length BclA (SEQ ID NO: 2) or a midsized nt of BclA that lacks the carboxy-terminus such as SEQ ID NO: 95 (amino acids 1—196 of BclA) can be used to target the fusion proteins to the exosporium. Midsized nts such as the fragment of SEQ ID NO: 95 have less secondary structure than full length BclA and has been found to be suitable for use as a targeting sequence. The targeting sequence can also comprise much shorter portions of BclA Which include amino acids 20—35, such as SEQ ID NO: 1 (amino acids 1—41 of BclA), amino acids 1—35 of SEQ ID NO: 1, amino acids 20—35 of SEQ ID NO: 1, or SEQ ID NO: 96 (a methionine residue linked to amino acids 20—35 of BclA). Even shorter nts of BclA Which include only some of amino acids 20—35 also exhibit the ability to target fusion proteins to the rium. For example, the targeting ce can comprise amino acids 22— 31 of SEQ ID NO: 1, amino acids 22—33 of SEQ ID NO: 1, or amino acids 20—3l of SEQ ID NO: 1.

Alternatively, any portion of AS3290, BAS4623, BclB, BASl882, the KBAB4 2280 gene product, the KBAB4 3572 gene product, B. cereus VD200 exosporium leader peptide, B. cereus VDl66 exosporium leader peptide, B. cereus VD200 hypothetical protein 663, B. stephensz’s KBAB4 YVTN B-propeller protein, B. weihenstephensz’s KBAB4 hypothetical protein bcerkbab4_2363, B. weihenstephensz’s KBAB4 hypothetical protein bcerkbab4_2l3 l , B. weihenstephensz’s KBAB4 triple helix repeat containing collagen, B. mycoz'des 2048 hypothetical protein bmycoOOOl_2l660, B. mycoz'des 2048 hypothetical protein bmchOOl_22540, B. mycoz'des 2048 hypothetical protein bmchOOl_2l5 10, B. thurz'ngz'ensz’s 35646 collagen triple helix repeat protein, B. cereus hypothetical protein WP_69652, B. cereus exosporium leader WPO l 61 177 l 7, B. cereus exosporium peptide WP002lO5 192, B. cereus hypothetical protein WP87353, B. cereus exosporium peptide 021 12369, B. cereus exosporium protein WPOl6099770, B. thurz'ngz'ensz’s hypothetical protein YP006612525, B. mycoz'des hypothetical protein 720, B. cereus ATCC 10987 collagen triple helix repeat domain protein, B. cereus E33L en-like protein, B. weihenstephanensz’s KBAB4 triple helix repeat-containing collagen, B. thuringz'ensz’s str. Al Hakam hypothetical protein BALH_2230, B. cereus ATCC 14579 triple helix -containing collagen, B. cereus collagen triple helix repeat, B. cereus ATCC 14579 triple helix repeatcontaining collagen, B. cereus E33L hypothetical protein BCZK1835, B. weihenstephanensis KBAB4 triple helix repeat-containing collagen, B. cereus ATCC 14579 triple helix repeat- containing collagen, B. cereus ATCC 14579 hypothetical protein BC4725, B. cereus E33L hypothetical protein BCZK4476, B. anthracis str. ‘Arnes Ancestor’ triple helix repeat-containing collagen, B. thurz'ngz'ensz’s serovar konkukian str. 97-27 BclA protein, B. cereus ATCC 10987 conserved hypothetical protein, B. cereus ATCC 14579 triple helix repeat-containing collagen, B. cereus exosporiurn leader peptide partial sequence, or B. weihenstephanensz‘s hypothetical protein ER45_27600 which includes the amino acids corresponding to amino acids 20—35 of BclA can serve as the ing sequence.

As can be seen from Figure 1A, amino acids 12—27 of BetA/BAS3290, amino acids 23—38 of 3, amino acids 13—28 of BclB, amino acids 9—24 of BASl882, amino acids 18—33 of KBAB4 2280 gene product, amino acids 18—33 of KBAB4 3572 gene product, amino acids 28—43 ofB. cereus VD200 exosporiurn leader peptide, amino acids 12—27 of B. cereus VD166 exosporiurn leader peptide, amino acids 18—33 of B. cereus VD200 hypothetical protein 663, amino acids 18—33 B. weihenstephensz’s KBAB4 YVTN B-propeller protein, amino acids 9—24 ofB. weihenstephensz’s KBAB4 hypothetical protein bcerkbab4_23 63, amino acids 9—24 of B. weihenstephensz’s KBAB4 hypothetical protein bcerkbab4_2131, amino acids —30 of B. weihenstephensz’s KBAB4 triple helix repeat containing collagen, amino acids 18— 33 of B. mycoz'des 2048 hypothetical protein brnycoOOOl_21660, amino acids 9—24 of B. mycoz'des 2048 hypothetical protein brnyc0001_22540, amino acids 1—15 of B. des 2048 hypothetical protein brnyc0001_21510, amino acids 1—16 ofB. thurz'ngz'ensz’s 35646 en triple helix repeat protein, amino acids 14—29 of B. cereus hypothetical protein 52, amino acids 20—35 ofB. cereus exosporiurn leader WP016117717, amino acids 28—43 of B. cereus exosporiurn peptide WP002105192, amino acids 17—32 of B. cereus hypothetical protein WP87353, amino acids 18—33 ofB. cereus exosporiurn peptide 02112369, amino acids 18—33 of B. cereus exosporiurn protein WP016099770, amino acids 15—30 of B. thurz'ngz‘ensz‘s hypothetical protein YP006612525, and amino acids 0 of B. mycoz'des hypothetical protein 720 correspond to amino acids 20—35 of BclA. As can be seen from Figure 1B, 2015/050807 amino acids 15—30 ofB. cereus ATCC 10987 collagen triple helix repeat domain protein, amino acids 18—33 of B. cereus E33L collagen-like protein, amino acids 20—35 of B. stephanensz’s KBAB4 triple helix -containing collagen, amino acids 9—24 of B. thurz'ngz'ensz’s str. Al Hakam hypothetical protein BALH_2230, amino acids 12—27 of B. cereus ATCC 14579 triple helix repeat-containing collagen, amino acids 23—38 of B. cereus collagen triple helix repeat, amino acids 17—32 of B. cereus ATCC 14579 triple helix repeat-containing collagen, amino acids 9—24 of B. cereus E33L hypothetical protein BCZK1835, amino acids 27— 42 of B. weihenstephanensz’s KBAB4 triple helix repeat-containing collagen, amino acids 9—24 ofB. cereus ATCC 14579 triple helix repeat-containing collagen, amino acids 18—33 of B. cereus ATCC 14579 hypothetical protein BC4725, amino acids 23—38 ofB. cereus E33L hypothetical protein BCZK4476, amino acids 19—34 B. anthracis str. ‘Ames Ancestor’ triple helix repeat-containing collagen, amino acids 13—28 of B. thurz'ngz'ensz’s serovar konkukian str. 97-27 BclA n, amino acids 13—28 of B. cereus ATCC 10987 conserved hypothetical protein, amino acids 13—28 of B. cereus ATCC 14579 triple helix repeat-containing collagen, amino acids 78—93 ofB. cereus exosporium leader peptide partial sequence, and amino acids 115—130 of B. weihenstephanensz‘s hypothetical protein ER45_27600 correspond to amino acids —35 of BclA. Thus, any portion of these proteins that includes the above-listed corresponding amino acids can serve as the targeting sequence.

Furthermore, any amino acid ce comprising amino acids 20—35 of BclA, or any of the above-listed corresponding amino acids can serve as the targeting sequence.

Thus, the targeting sequence can comprise amino acids 1—35 of SEQ ID NO: 1, amino acids 20—35 of SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 96, amino acids 22—31 of SEQ ID NO: 1, amino acids 22—33 of SEQ ID NO: 1, or amino acids 20—31 of SEQ ID NO: 1.

Alternatively, the targeting sequence consists of amino acids 1—35 of SEQ ID NO: 1, amino acids 20—35 of SEQ ID NO: 1, SEQ ID NO: 1, or SEQ ID NO: 96. Alternatively, the ing sequence can consist of amino acids 22—31 of SEQ ID NO: 1, amino acids 22—33 of SEQ ID NO: 1, or amino acids 20—31 of SEQ ID NO: 1. Alternatively, the exosporium protein can comprise full length BclA (SEQ ID NO: 2), or the exosporium n fragment can comprise a ed fragment of BclA that lacks the carboxy-terminus, such as SEQ ID NO: 59 (amino acids 1—196 of BclA). Alternatively, the exosporium protein fragment can consist of SEQ ID NO: 59. 2015/050807 The ing sequence can se amino acids 2—35 of SEQ ID NO: 1 ; amino acids 5—35 of SEQ ID NO: 1; amino acids 8—35 of SEQ ID NO: 1; amino acids 10—35 of SEQ ID NO: 1; or amino acids 15—35 of SEQ ID NO: 1.

The targeting sequence can also se amino acids 1—27 of SEQ ID NO: 3, amino acids 12—27 of SEQ ID NO: 3, or SEQ ID NO: 3, or the exosporium protein can comprise full length BetA/BAS3290 (SEQ ID NO: 4). It has also been found that a methionine residue linked to amino acids 12—27 of BetA/BAS3290 can be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 97. The targeting sequence can also comprise amino acids 14—23 of SEQ ID NO: 3, amino acids 14—25 of SEQ ID NO: 3, or amino acids 12— 23 of SEQ ID NO: 3.

The targeting sequence can comprise amino acids 2—27 of SEQ ID NO: 3; amino acids 5—27 of SEQ ID NO: 3; amino acids 8—27 of SEQ ID NO: 3; or amino acids 10—27 of SEQ ID NO: 3.

The targeting sequence can also comprise amino acids 1—38 of SEQ ID NO: 5, amino acids 23—38 of SEQ ID NO: 5, or SEQ ID NO: 5, or the exosporium protein can comprise full length BAS4623 (SEQ ID NO: 6).

The targeting sequence can comprise amino acids 2—38 of SEQ ID NO: 5; amino acids 5—38 of SEQ ID NO: 5; amino acids 8—38 of SEQ ID NO: 5; amino acids 10—38 of SEQ ID NO: 5; amino acids 15—38 of SEQ ID NO: 5; or amino acids 20—38 of SEQ ID NO: 5.

Alternatively, the targeting sequence can comprise amino acids 1—28 of SEQ ID NO: 7, amino acids 13—28 of SEQ ID NO: 7, or SEQ ID NO: 7, or the exosporium protein can comprise full length BclB (SEQ ID NO:8).

The targeting sequence can comprise amino acids 2—28 of SEQ ID NO: 7; amino acids 5—28 of SEQ ID NO: 7; amino acids 8—28 of SEQ ID NO: 7; or amino acids 10—28 of SEQ ID NO: 7.

The targeting ce can also comprise amino acids 1—24 of SEQ ID NO: 9, amino acids 9—24 of SEQ ID NO: 9, or SEQ ID NO: 9, or the exosporium protein can comprise full length BASl882 (SEQ ID NO: 10). A methionine residue linked to amino acids 9—24 of 2 can also be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 105.

The targeting sequence can comprise amino acids 2—24 of SEQ ID NO: 9; amino acids 5—24 of SEQ ID NO: 9; or amino acids 8—24 of SEQ ID NO: 9.

The targeting sequence can also comprise amino acids 1—33 of SEQ ID NO:11, amino acids 18—33 of SEQ ID NO: 11, or SEQ ID NO: 11, or the exosporium protein can comprise the full length B. weihenstephensz’s KBAB4 2280 gene product (SEQ ID NO: 12). A nine e linked to amino acids 18—33 of the B. weihenstephensz’s KBAB4 2280 gene product can also be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 98.

The targeting sequence can comprise amino acids 2—33 of SEQ ID NO: 11; amino acids 5—33 of SEQ ID NO: 11; amino acids 8—33 of SEQ ID NO: 11; amino acids 10—33 of SEQ ID NO: 11; or amino acids 15—33 of SEQ ID NO: 11.

The targeting sequence can also comprise amino acids 1—33 of SEQ ID NO: 13, amino acids 18—33 of SEQ ID NO: 13, or SEQ ID NO: 13, or the exosporium protein can comprise the full length B. weihenstephensz’s KBAB4 3572 gene product (SEQ ID NO: 14). A methionine residue linked to amino acids 18—33 of the B. weihenstephensis KBAB4 3572 gene t can also be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 99.

The targeting sequence can se amino acids 2—33 of SEQ ID NO: 13; amino acids 5—33 of SEQ ID NO: 13; amino acids 8—33 of SEQ ID NO: 13; amino acids 10—33 of SEQ ID NO: 13; or amino acids 15—33 of SEQ ID NO: 13; Alternatively, the targeting sequence can comprise amino acids 1—43 of SEQ ID NO: 15, amino acids 28—43 of SEQ ID NO: 15, or SEQ ID NO:15, or the exosporium protein can comprise full length B. cereus VD200 exosporium leader e (SEQ ID NO: 16).

The targeting sequence can comprise amino acids 2—43 of SEQ ID NO: 15; amino acids 5—43 of SEQ ID NO: 15 ; amino acids 8—43 of SEQ ID NO: 15 ; amino acids 10—43 of SEQ ID NO: 15; amino acids 15—43 of SEQ ID NO: 15 ; amino acids 20—43 of SEQ ID NO: ; or amino acids 25—43 of SEQ ID NO: 15.

The targeting sequence can also comprise amino acids 1—27 of SEQ ID NO: 17, amino acids 12—27 of SEQ ID NO: 17, or SEQ ID NO:17, or the exosporium n can comprise full-length B. cereus VD166 exosporium leader peptide (SEQ ID NO:18). A methionine residue linked to amino acids 12—27 of the B. cereus VD166 exosporium leader peptide can also be used as a targeting sequence. Thus, the ing sequence can comprise SEQ ID NO: 100.

WO 44661 The ing sequence can comprise amino acids 2—27 of SEQ ID NO: 17; amino acids 5—27 of SEQ ID NO: 17; amino acids 8—27 of SEQ ID NO: 17; or amino acids 10— 27 of SEQ ID NO: 17.

The targeting sequence can also comprise amino acids 1—33 of SEQ ID NO: 19, amino acids 18—33 of SEQ ID NO: 19, or SEQ ID NO:19, or the exosporium protein can comprise full length B. cereus VD200 hypothetical protein IKG_04663 (SEQ ID NO:20).

The ing sequence can comprise amino acids 2—33 of SEQ ID NO: 19; amino acids 5—33 of SEQ ID NO: 19; amino acids 8—33 of SEQ ID NO: 19; amino acids 10—33 of SEQ ID NO: 19; or amino acids 15—33 of SEQ ID NO: 19.

Alternatively, the targeting sequence comprises amino acids 1—33 of SEQ ID NO: 21, amino acids 18—33 of SEQ ID NO: 21, or SEQ ID NO:21, or the exosporium protein can comprise full length B. weihenstephensz’s KBAB4 YVTN B-propeller protein (SEQ ID NO:22). A methionine residue linked to amino acids 18—33 of the B. weihenstephensz’s KBAB4 YVTN B-propeller n can also be used as a targeting sequence. Thus, the targeting sequence can se SEQ ID NO: 101.

The targeting sequence can comprise amino acids 2—33 of SEQ ID NO: 21 ; amino acids 5—33 of SEQ ID NO: 21; amino acids 8—33 of SEQ ID NO: 21; amino acids 10—33 of SEQ ID NO: 21; or amino acids 15—33 of SEQ ID NO: 21.

The targeting sequence can also comprise amino acids 1—24 of SEQ ID NO: 23, amino acids 9—24 of SEQ ID NO: 23, or SEQ ID NO:23, or the rium protein can comprise full length B. weihenstephensz’s KBAB4 hypothetical protein bcerkbab4_2363 (SEQ ID NO:24). A methionine residue linked to amino acids 9—24 of B. weihenstephensz’s KBAB4 hypothetical protein bcerkbab4_2363 can also be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 102.

The targeting sequence can comprise amino acids 2—24 of SEQ ID NO:23; amino acids 5—24 of SEQ ID NO: 23; or amino acids 8—24 of SEQ ID NO: 23.

The targeting sequence comprise amino acids 1—24 of SEQ ID NO: 25, amino acids 9—24 of SEQ ID NO: 25, or SEQ ID NO:25, or the exosporium protein can comprise full length B. weihenstephensz’s KBAB4 hypothetical protein bcerkbab4_2131 (SEQ ID NO:26). A methionine residue linked to amino acids 9—24 of B. weihenstephensz’s KBAB4 hypothetical protein bcerkbab4_2131 can also be used as a ing sequence. Thus, the targeting sequence can comprise SEQ ID NO: 103.

The targeting sequence can comprise amino acids 2—24 of SEQ ID NO: 25; amino acids 5—24 of SEQ ID NO: 25; or amino acids 8—24 of SEQ ID NO: 25.

Alternatively, the targeting sequence comprises amino acids 1—30 of SEQ ID NO: 27, amino acids 15—30 of SEQ ID NO: 27, or SEQ ID NO:27, or the exosporium protein can comprise full length B. weihenstephensz’s KBAB4 triple helix repeat containing collagen (SEQ ID NO:28).

The targeting sequence can comprise amino acids 2—30 of SEQ ID NO: 27; amino acids 5—30 of SEQ ID NO: 27; amino acids 8—30 of SEQ ID NO: 27; or amino acids 10— of SEQ ID NO: 27.

The targeting sequence can also comprise amino acids 1—33 of SEQ ID NO: 29, amino acids 18—33 of SEQ ID NO: 29, or SEQ ID NO:29, or the exosporium protein can comprise full length B. mycoz'des 2048 hypothetical protein bmycoOOOl_21660 (SEQ ID NO:30).

The targeting sequence can se amino acids 2—33 of SEQ ID NO: 29; amino acids 5—33 of SEQ ID NO: 29; amino acids 8—33 of SEQ ID NO: 29; amino acids 10—33 of SEQ ID NO: 29; or amino acids 15—33 of SEQ ID NO: 29.

The targeting sequence can also comprise amino acids 1—24 of SEQ ID NO: 31, amino acids 9—24 of SEQ ID NO: 31, or SEQ ID NO:31, or the exosporium protein can comprise full length B. mycoz'des 2048 hypothetical protein bmyc0001_22540 (SEQ ID NO:32).

A methionine e linked to amino acids 9—24 of B. mycoides 2048 hypothetical protein bmyc0001_22540 can also be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 104.

The targeting sequence can comprise amino acids 2—24 of SEQ ID NO: 31 ; amino acids 5—24 of SEQ ID NO: 31; or amino acids 8—24 of SEQ ID NO: 31.

Alternatively, the ing sequence comprises amino acids 1—15 of SEQ ID NO: 33, SEQ ID NO:33, or the exosporium protein comprises full length B. mycoz'des 2048 hypothetical protein bmyc0001_21510 (SEQ ID .

The targeting sequence can also comprise amino acids 1—16 of SEQ ID NO: 35, SEQ ID NO:35, or the exosporium protein can comprise full length B. thuringz‘ensz‘s 35646 collagen triple helix repeat n (SEQ ID NO:36).

The targeting ce can se amino acids 1—29 of SEQ ID NO:43, amino acids 14—29 of SEQ ID NO: 43, or SEQ ID NO: 43, or the exosporium protein can comprise full length B. cereus hypothetical n WP_69652 (SEQ ID NO: 44).

The targeting sequence can comprise amino acids 2—29 of SEQ ID NO: 43; amino acids 5—29 of SEQ ID NO: 43; amino acids 8—29 of SEQ ID NO: 43; or amino acids 10— 29 of SEQ ID NO: 43.

Alternatively, the targeting sequence can comprise amino acids 1—35 of SEQ ID NO: 45, amino acids 20—35 of SEQ ID NO: 45, or SEQ ID NO: 45, or the exosporium protein can comprise full length B. cereus exosporium leader 17717 (SEQ ID NO: 46). A nine residue linked to amino acids 20—35 of B. cereus rium leader WP016117717 can also be used as a targeting ce. Thus, the targeting sequence can comprise SEQ ID NO: 106.

The targeting sequence can comprise amino acids 2—35 of SEQ ID NO: 45; amino acids 5—35 of SEQ ID NO: 45 ; amino acids 8—35 of SEQ ID NO: 45 ; amino acids 10—35 of SEQ ID NO: 45; or amino acids 15—35 of SEQ ID NO: 45.

The targeting sequence can se amino acids 1—43 of SEQ ID NO: 47, amino acids 28—43 of SEQ ID NO: 47, or SEQ ID NO: 47, or the exosporium protein can comprise full length B. cereus exosporium peptide WP002105192 (SEQ ID NO: 48).

The targeting sequence can comprise amino acids 2—43 of SEQ ID NO: 47; amino acids 5—43 of SEQ ID NO: 47; amino acids 8—43 of SEQ ID NO: 47; amino acids 10—43 of SEQ ID NO: 47; amino acids 15—43 of SEQ ID NO: 47; amino acids 20—43 of SEQ ID NO: 47; or amino acids 25—43 of SEQ ID NO: 47.

The targeting sequence can comprise amino acids 1—32 of SEQ ID NO: 49, amino acids 17—32 of SEQ ID NO: 49, or SEQ ID NO: 49, or the exosporium protein can comprise full length B. cereus hypothetical protein 3 (SEQ ID NO: 50).

The targeting sequence can comprise amino acids 2—32 of SEQ ID NO: 49; amino acids 5—32 of SEQ ID NO: 49; amino acids 8—32 of SEQ ID NO: 49; amino acids 10—32 of SEQ ID NO: 49; or amino acids 15—32 of SEQ ID NO: 49.

Alternatively, the targeting sequence can comprise amino acids 1—33 of SEQ ID NO: 51, amino acids 18—33 of SEQ ID NO: 51, or SEQ ID NO: 51, or the rium protein can comprise full length B. cereus exosporium peptide 02112369 (SEQ ID NO: 52).

The targeting sequence can comprise amino acids 2—33 of SEQ ID NO: 51 ; amino acids 5—33 of SEQ ID NO: 51; amino acids 8—33 of SEQ ID NO: 51; amino acids 10—33 of SEQ ID NO: 51; or amino acids 15—33 of SEQ ID NO: 51; ] The targeting ce can comprise amino acids 1—33 of SEQ ID NO: 53, amino acids 18—33 of SEQ ID NO: 53, or SEQ ID NO: 53, or the exosporium protein can comprise full length B. cereus exosporium protein WP016099770 (SEQ ID NO: 54).

The targeting sequence can comprise amino acids 2—33 of SEQ ID NO: 53; amino acids 5—33 of SEQ ID NO: 53; amino acids 8—33 of SEQ ID NO: 53; amino acids 10—33 of SEQ ID NO: 53; or amino acids 15—33 of SEQ ID NO: 53.

Alternatively, the targeting sequence can comprise acids 1—30 of SEQ ID NO: 55, amino acids 15—30 of SEQ ID NO: 55, or SEQ ID NO: 55, or the exosporium protein can comprise full length B. thurz’ngz’ensz’s hypothetical protein YP006612525 (SEQ ID NO: 56).

The targeting sequence can comprise amino acids 2—30 of SEQ ID NO: 55; amino acids 5—30 of SEQ ID NO: 55; amino acids 8—30 of SEQ ID NO: 55; or amino acids 10— of SEQ ID NO: 55.

The targeting sequence can also comprise amino acids l—l30 of SEQ ID NO: 57, amino acids 115—130 of SEQ ID NO: 57, or SEQ ID NO: 57, or the exosporium protein can comprise full length B. mycoz'des hypothetical protein TIGR03720 (SEQ ID NO: 58).

The targeting sequence can comprise amino acids 2—130 of SEQ ID NO: 57; amino acids 5—130 of SEQ ID NO: 57; amino acids 10—130 of SEQ ID NO: 57; amino acids 20— 130 of SEQ ID NO: 57; amino acids 30—130 of SEQ ID NO: 57; amino acids 40—130 of SEQ ID NO: 57; amino acids 50—130 of SEQ ID NO: 57; amino acids 60—130 of SEQ ID NO: 57; amino acids 70—130 of SEQ ID NO: 57; amino acids 80—130 of SEQ ID NO: 57; amino acids 90—130 of SEQ ID NO: 57; amino acids 100—130 of SEQ ID NO: 57; or amino acids 110—130 of SEQ ID NO: 57.

The targeting sequence can se amino acids 1—30 of SEQ ID NO: 59; or SEQ ID NO: 59; or the rium protein can comprise full length B. cereus ATCC 10987 collagen triple helix repeat domain protein (SEQ ID NO: 60).

] The targeting sequence can comprise amino acids 2—30 of SEQ ID NO: 59; amino acids 4—30 of SEQ ID NO: 59; or amino acids 6—30 of SEQ ID NO: 59.

] The targeting sequence can comprise amino acids 1—33 of SEQ ID NO: 61; amino acids 18—33 of SEQ ID NO: 61; or SEQ ID NO: 61; or the exosporium protein can comprise full length B. cereus E33L collagen-like protein (SEQ ID NO: 62).

The targeting ce can comprise amino acids 2—33 of SEQ ID NO: 61; amino acids 5—33 of SEQ ID NO: 61; amino acids 10—33 of SEQ ID NO: 61; or amino acids 15— 33 of SEQ ID NO: 61.

The targeting sequence can comprise amino acids l—35 of SEQ ID NO: 63 ; or SEQ ID NO: 63; or the exosporium protein can comprise full length B. weihenstephanensz‘s KBAB4 triple helix repeat-containing collagen (SEQ ID NO: 64).

The targeting sequence can comprise amino acids 2—35 of SEQ ID NO: 63; amino acids 5—35 of SEQ ID NO: 63; amino acids 8—35 of SEQ ID NO: 63 ; amino acids lO—35 of SEQ ID NO: 63; or amino acids 15—35 of SEQ ID NO: 63.

The targeting sequence can se amino acids l—24 of SEQ ID NO: 65; acids 9—24 of SEQ ID NO: 65 ; SEQ ID NO: 65 ; or SEQ ID NO: 107; or the exosporium protein can comprise full length B. thurz’ngz’ensz’s str. Al Hakam hypothetical protein BALH_2230 (SEQ ID NO: 66).

The ing sequence can se amino acids 2—24 of SEQ ID NO: 65; or amino acids 5—24 of SEQ ID NO: 65.

The targeting ce can comprise acids l—27 of SEQ ID NO: 67; amino acids 12—27 of SEQ ID NO: 67; or SEQ ID NO: 67; or the exosporium n can comprise full length B. cereus ATCC 14579 triple helix repeat-containing collagen (SEQ ID NO: 68).

The targeting sequence can comprise amino acids 2—27 of SEQ ID NO: 67; amino acids 5—27 of SEQ ID NO: 67; or amino acids lO—27 of SEQ ID NO: 67.

The targeting sequence can comprise amino acids l—38 of SEQ ID NO: 69; amino acids 23—38 of SEQ ID NO: 69; or SEQ ID NO: 69; or the exosporium protein can comprise full length B. cereus collagen triple helix repeat (SEQ ID NO: 70).

The targeting sequence can comprise amino acids 2—38 of SEQ ID NO: 69; amino acids 5—38 of SEQ ID NO: 69; amino acids lO—38 of SEQ ID NO: 69; or amino acids 15— 38 of SEQ ID NO: 69.

The exosporium protein can comprise full length B. cereus ATCC 14579 triple helix repeat-containing collagen (SEQ ID NO: 72).

] The targeting sequence can comprise SEQ ID NO: 73, or the exosporium n can comprise full length B. cereus E33L hypothetical n BCZKl835 (SEQ ID NO: 74).

The targeting sequence can comprise amino acids l—42 of SEQ ID NO: 75; amino acids 27—42 of SEQ ID NO: 75; or SEQ ID NO: 75; or the exosporium protein can comprise full length B. weihenstephanensz’s KBAB4 triple helix repeat-containing collagen (SEQ ID NO: 76).

The targeting sequence can comprise amino acids 2—42 of SEQ ID NO: 75; amino acids 5—42 of SEQ ID NO: 75 ; amino acids 10—42 of SEQ ID NO: 75 ; amino acids 15—42 of SEQ ID NO: 75; amino acids 20—42 of SEQ ID NO: 75 ; or amino acids 25—42 of SEQ ID NO: 75.

The targeting sequence can comprise amino acids 1—24 of SEQ ID NO: 77; amino acids 9—24 of SEQ ID NO: 77; or SEQ ID NO: 77; or the exosporium protein can comprise full length B. cereus ATCC 14579 triple helix repeat-containing collagen (SEQ ID NO: 78).

The targeting sequence can comprise amino acids 2—24 of SEQ ID NO: 77; or amino acids 5—24 of SEQ ID NO: 77; The exosporium protein can comprise full length B. cereus ATCC 14579 hypothetical protein BC4725 (SEQ ID NO: 80).

The targeting sequence can comprise amino acids 1—38 of SEQ ID NO: 81 ; amino acids 23—38 of SEQ ID NO: 81 ; or SEQ ID NO: 81 ; or the exosporium n can comprise full length B. cereus E33L hypothetical protein BCZK4476 (SEQ ID NO: 82).

The targeting sequence can comprise amino acids 2—38 of SEQ ID NO: 81 ; acids 5—38 of SEQ ID NO: 81; amino acids 10—38 of SEQ ID NO: 81; amino acids 15—38 of SEQ ID NO: 81; or amino acids 20—38 of SEQ ID NO: 81.

The targeting ce can comprise amino acids 1—34 of SEQ ID NO: 83; or SEQ ID NO: 83; or the exosporium protein can comprise full length B. anthracis str. ‘Ames Ancestor’ triple helix -containing collagen (SEQ ID NO: 84).

The exosporium protein can comprise full length B. ngz’ensz’s serovar konkukian str. 97-27 BclA protein (SEQ ID NO: 86).

The targeting sequence can comprise amino acids 1—28 of SEQ ID NO: 87; amino acids 13—28 of SEQ ID NO: 87; or SEQ ID NO: 87; or the rium protein can comprise full length B. cereus ATCC 10987 conserved hypothetical protein (SEQ ID NO: 88).

The targeting sequence can comprise amino acids 2—28 of SEQ ID NO: 87; amino acids 5—28 of SEQ ID NO: 87; or amino acids 10—28 of SEQ ID NO: 87.

The targeting sequence can se amino acids 1—28 of SEQ ID NO: 89; or SEQ ID NO: 89; or the exosporium n can comprise full length B. cereus ATCC 14579 triple helix repeat-containing collagen (SEQ ID NO: 90).

The targeting ce can comprise amino acids 2—28 of SEQ ID NO: 89; amino acids 5—28 of SEQ ID NO: 89; or amino acids 10—28 of SEQ ID NO: 89 The targeting sequence can comprise amino acids 1—93 of SEQ ID NO: 91; or SEQ ID NO: 91 ; or the exosporium protein can comprise B. cereus exosporium leader peptide partial ce (SEQ ID NO: 92).

The targeting sequence can comprise amino acids 2—93 of SEQ ID NO: 91 ; amino acids 10—93 of SEQ ID NO: 91; amino acids 20—93 of SEQ ID NO: 91; amino acids 30— 93 of SEQ ID NO: 91; amino acids 40—93 of SEQ ID NO: 91; amino acids 50—93 of SEQ ID NO: 91; or amino acids 60—93 of SEQ ID NO: 91.

The targeting sequence can comprise amino acids 1—130 of SEQ ID NO: 93 ; or SEQ ID NO: 93; or the exosporium protein can comprise B. weihenstephanensis) hypothetical protein ER45_27600, partial sequence (SEQ ID NO: 94).

The targeting sequence can se amino acids 2—130 of SEQ ID NO: 93; amino acids 10—130 of SEQ ID NO: 93; amino acids 20—130 of SEQ ID NO: 93 ; or amino acids —130 of SEQ ID NO: 93.

Furthermore, as rated in the Examples provided hereinbelow, it has been found that sequences shorter than amino acids 20—35 of BclA can be used to target a fusion protein to the rium of a recombinant Bacillus cereus family member. In particular, amino acids 20—33 of BclA, amino acids 20—31 of BclA, amino acids 21—33 of BclA, or amino acids 23—31 of BclA can be used to target a fusion n to the exosporium of a recombinant Bacillus cereus family member. Thus, the targeting sequence can consist of amino acids 20—33 of SEQ ID NO: 1, amino acids 20—31 of SEQ ID NO: 1, amino acids 21—33 of SEQ ID NO: 1, or amino acids 23—31 of SEQ ID NO: 1. The corresponding s of any of the SEQ ID NOs. shown in Figures 1A and 1B can also be used to target a fusion protein to the exosporium of a recombinant Bacillus cereus family member. By “corresponding regions,” it is meant that when the sequences are aligned with SEQ ID NO: 1, as shown in Figure 1A and 1B, the regions of the other amino acid ces that align with the amino acids of SEQ ID NO: are the “corresponding s” of those sequences. Thus, for example, amino acids 12—25 of SEQ ID NO: 3, amino acids 23—36 of SEQ ID NO: 5, amino acids 13—26 of SEQ ID NO: 7, etc. can be used to target a fusion protein to the exosporium of a recombinant Bacillus cereus family member, since these regions align with amino acids 20—33 of SEQ ID NO: 1 as shown in Figure Even shorter regions within amino acids 20—35 of BclA can also be used for targeting a fusion protein to the exosporium of a recombinant Bacillus cereus family member. In particular, any amino acid sequence that includes amino aicds 25—30 of SEQ ID NO: 1 or the corresponding amino acids from any of the sequences shown in Figures 1A and 1B can be used.

A skilled person will recognize that starting with amino acids 25—30 of SEQ ID NO: 1 or the corresponding region of any of the sequences shown in s 1A and 1B, additional amino acids can be added to the amino-terminus, the carboxy terminus, or both the amino- and carboxy termini to create a targeting sequence that will be effective for targeting a fusion n to the exosporium of a recombinant Bacillus cereus family member.

] In addition, it can readily be seen from the sequence alignment in Figures 1A and 1B that while amino acids 20—35 of BclA are conserved, and amino acids 25—35 are more conserved, some degree of variation can occur in this region without affecting the ability of the targeting sequence to target a protein to the exosporium. Figures 1A and 1B list the percent identity of each of corresponding amino acids of each sequence to amino acids 20—35 of BclA (“20—35% Identity”) and to amino acids 25—35 of BclA 5 % Identity”). Thus, for example, as compared to amino acids 20—35 of BclA, the corresponding amino acids of BetA/BAS3290 are about 81.3% identical, the corresponding amino acids of BAS4623 are about 50.0% identical, the corresponding amino acids of BclB are about 43.8% identical, the ponding amino acids of BAS1882 are about 62.5% identical, the ponding amino acids of the KBAB4 2280 gene product are about 81.3% identical, and the corresponding amino acids of the KBAB4 3572 gene product are about 81.3% cal. The sequence identities over this region for the remaining ces are listed in Figures 1A and 1B.

With respect to amino acids 25—35 of BclA, the corresponding amino acids of BetA/BAS3290 are about 90.9% identical, the corresponding amino acids of BAS4623 are about 72.7% identical, the corresponding amino acids of BclB are about 54.5% identical, the corresponding amino acids of BAS1882 are about 72.7% identical, the corresponding amino acids of the KBAB4 2280 gene product are about 90.9% identical, and the corresponding amino acids of the KBAB4 3572 gene product are about 81.8% identical. The sequence identities over this region for the remaining ces are listed in Figures 1A and 1B.

Thus, the targeting sequence can comprise an amino acid sequence having at least about 43% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 54%. Alternatively, the ing sequence consists of an amino acid sequence consisting of 16 amino acids and having at least about 43% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 54%.

The targeting sequence can also comprise an amino acid sequence having at least about 50% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 63%. Alternatively the targeting sequence consists of an amino acid sequence consisting of 16 amino acids and having at least about 50% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 63%.

The targeting sequence can also se an amino acid sequence having at least about 50% identity with amino acids 20—35 of SEQ ID NO: 1, n the identity with amino acids 25—35 is at least about 72%. Alternatively, the targeting sequence consists of an amino acid sequence consisting of 16 amino acids and having at least about 50% identity with amino acids 20—35 of SEQ ID NO: 1, n the identity with amino acids 25—35 is at least about 72%.

The targeting ce can also se an amino acid sequence having at least about 56% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 63%. Alternatively, the ing sequence consists of an amino acid sequence consisting of 16 amino acids and having at least about 56% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 63%.

Alternatively, the targeting sequence can comprise an amino sequence having at least about 62% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 72%. The targeting sequence can also consist of an amino acid sequence consisting of 16 amino acids and having at least about 62% ty with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 of SEQ ID NO:1 is at least about 72%.

The targeting ce can comprise an amino acid sequence having at least 68% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids —35 is at least about 81%. Alternatively, the targeting sequence consists of an amino acid sequence consisting of 16 amino acids and having at least 68% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 81%.

The targeting sequence can also comprises an amino sequence having at least about 75% identity with amino acids 20—35 of SEQ ID NO: 1, n the identity with amino acids 25—35 is at least about 72%. Alternatively, the targeting sequence consists of an amino acid sequence consisting of 16 amino acids and having at least about 75% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 of SEQ ID NO:1 is at least about 72%.

The targeting sequence can also comprise an amino ce having at least about 75% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 81%. Alternatively, the targeting sequence consists of an amino acid sequence ting of 16 amino acids and having at least about 75% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 of SEQ ID NO:1 is at least about 81%.

The targeting sequence can also comprise an amino acid sequence having at least about 81% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 81%. Alternatively, the targeting sequence consists of an amino acid ce consisting of 16 amino acids and having at least about 81% identity with amino acids 20—35 of SEQ ID NO:1, wherein the identity with amino acids 25—35 is at least about 8 1 %.

The targeting sequence can comprise an amino acid sequence having at least about 81% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 90%. Alternatively, the ing sequence consists of an amino acid sequence ting of 16 amino acids and having at least about 81% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 90%.

] The skilled person will recognize that variants of the above sequences can also be used as targeting sequences, so long as the targeting sequence comprises amino acids —35 of BclA, the ponding amino acids of BetA/BAS3290, BAS4263, BclB, BAS1882, the KBAB4 2280 gene product, or the KBAB 3572 gene product, or a sequence comprising any of the above noted ce identities to amino acids 20—35 and 25—35 of BclA is t.

Certain Bacillus cereus family exosporium proteins which lack regions having homology to amino acids 25—35 of BclA can also be used to target a peptide or protein to the exosporium of a Bacillus cereus family member. In particular, the fusion proteins can comprise an exosporium protein comprising SEQ ID NO: 108 (B. mycoz'des InhA), an exosporium protein comprising SEQ ID NO: 109 (B. anthracis Sterne BAS1141 (Est)), an exosporium protein comprising SEQ ID NO: 110 (B. cis Sterne BAS1144 (BxpB/ExsFA)), an exosporium protein comprising SEQ ID NO: 111 (B. anthracis Sterne BAS1145 (CotY)), an exosporium protein sing SEQ ID NO: 112 (B. anthracis Sterne 0), an exosporium protein comprising SEQ ID NO: 113 (B. cis H9401 ExsFB), an exosporium protein comprising SEQ ID NO: 114 (B. thuringiensis HD74 InhAl), an exosporium protein comprising SEQ ID NO: 115 (B. cereus ATCC 10876 Est), an rium protein comprising SEQ ID NO: 116 (B. cereus Est), an exosporium protein comprising SEQ ID NO: 117 (B. anthracis Ames chA), an exosporium protein comprising SEQ ID NO: 118 (B. anthracis chB), an exosporium protein comprising SEQ ID NO: 119 (B. anthracis Sterne Bch), an exosporium protein comprising SEQ ID NO: 120 (Bacillus thuringiensis serovar konkukian str. 97-27 acid phosphatase), an exosporium protein comprising SEQ ID NO: 121 (B. thuringiensis HD74 InhA2), or an exosporium protein comprising SEQ ID NO: 122 (B. mycoia’es . Inclusion of an exosporium protein comprising any of SEQ ID NOs: 108—122 in the fusion proteins described herein will result in ing to the exosporium of a B. cereus family member.

Moreover, exosporium ns having a high degree of sequence identity with any of the full-length exosporium proteins or the exosporium protein fragments bed above can also be used to target a peptide or protein to the exosporium of a Bacillus cereus family member. Thus, the fusion n can comprise an exosporium n or exosporium protein fragment comprising an amino acid sequence having at least 85% identity with any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, and 122. Alternatively, the fusion protein can comprise an rium n having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84,86,88,90,92,94,95,108,109,110,111,112,113,114,115,116,117,118,119,120, 121, and 122.

During sporulation of a recombinant us cereus family member expressing any of the fusion proteins described herein, the targeting motif, exosporium protein, or exosporium protein fragment is recognized by the spore exosporium assembly machinery and ed to the exosporium, resulting in display of the protein or peptide of interest portion of the fusion protein on the outside of the spore.

As illustrated further by the Examples provided hereinbelow, the use of different targeting sequences allows for control of the expression level of the fusion protein on the surface of the Bacillus cereus family member spore. Use of certain of the targeting sequences described herein Will result in a higher level of expression of the fusion protein, Whereas use of others of the targeting sequences Will result in lower levels of expression of the fusion n on the surface of the spore.

In any of the fusion proteins bed herein, the targeting sequence, exosporium protein, or exosporium protein fragment can comprise the amino acid sequence GXT at its carboxy terminus, wherein X is any amino acid.

In any of the fusion proteins described herein, the targeting sequence, exosporium n, or exosporium protein fragment, can comprise an alanine residue at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1.

In any of the fusion proteins described herein, the targeting sequence, exosporium protein, or exosporium protein nt can further comprise a methionine, serine, or threonine residue at the amino acid position immediately preceding the first amino acid of the targeting ce, exosporium protein, or exosporium protein fragment or at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1.

B. Fusion ns [or Expression in Recombinant Bacillus cereus Family Members The present invention relates to fusion proteins comprising at least one protein or peptide of interest and a targeting sequence or exosporium protein. When the protein or peptide of interest is any protein or peptide of interest, the fusion protein can comprise: (l) a targeting ce sing amino acids 1—30 of SEQ ID NO: 59; (2) a targeting sequence comprising SEQ ID NO: 59; (3) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 60; (4) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 59; (5) a targeting sequence comprising amino acids 4—30 of SEQ ID NO: 59; (6) a targeting sequence sing amino acids 6—30 of SEQ ID NO: 59; (7) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 61 ; (8) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 61 ; (9) a targeting sequence comprising SEQ ID NO: 61 ; (10) an exosporium n comprising an amino acid sequence having at least 85% sequence ty With SEQ ID NO: 62; (ll) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 61; (12) a ing sequence comprising amino acids 5—33 of SEQ ID NO: 61; (13) a ing sequence comprising amino acids 10—33 of SEQ ID NO: 61 ; (14) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 61 ; (15) a ing sequence comprising amino acids 1—35 of SEQ ID NO: 63; (16) a targeting sequence comprising SEQ ID NO: 63; (17) an exosporium protein comprising an amino acid sequence having at least 85% WO 44661 identity with SEQ ID NO: 64; (18) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 63 ; (19) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 63 ; (20) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 63; (21) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 63; (22) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 63 ; (23) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 65; (24) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 65 ; (25) a targeting sequence comprising SEQ ID NO: 65; (26) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 66; (27) a targeting sequence comprising SEQ ID NO: 107; (28) a ing sequence comprising amino acids 2—24 of SEQ ID NO: 65; (29) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 65; (30) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 67; (31) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 67; (32) a targeting sequence comprising SEQ ID NO: 67; (33) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 68; (34) an targeting sequence comprising amino acids 2—27 of SEQ ID NO: 67; (35) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 67; (36) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 67; (37) a targeting sequence comprising amino acids 1—38 of SEQ ID NO: 69; (38) a targeting sequence sing amino acids 23—38 of SEQ ID NO: 69; (39) a targeting sequence comprising SEQ ID NO: 69; (40) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 70; (41) a ing sequence comprising amino acids 2—38 of SEQ ID NO: 69; (42) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 69; (43) a ing sequence comprising amino acids 10—38 of SEQ ID NO: 69; (44) a targeting ce comprising amino acids 15—38 of SEQ ID NO: 69; (45) an exosporium protein comprising SEQ ID NO: 72; (46) a targeting ce comprising SEQ ID NO: 73; (47) an exosporium protein comprising an amino acid sequence haVing at least 95% identity With SEQ ID NO: 74; (48) a targeting sequence comprising amino acids 1—42 of SEQ ID NO: 75; (49) a targeting ce comprising amino acids 27—42 of SEQ ID NO: 75; (50) a targeting ce comprising SEQ ID NO: 75; (51) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 76; (52) a targeting sequence comprising amino acids 2—42 of SEQ ID NO: 75 ; (53) a targeting sequence comprising amino acids 5—42 of SEQ ID NO: 75 ; (54) a targeting sequence comprising amino acids 10—42 of SEQ ID NO: 75; (55) a targeting sequence comprising amino acids 15—42 of SEQ ID NO: 75; (56) a targeting ce comprising amino acids 20—42 of SEQ ID NO: 75; (57) a targeting sequence comprising amino acids 25—42 of WO 44661 SEQ ID NO: 75; (58) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 77; (59) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 77; (60) a targeting sequence comprising SEQ ID NO: 77; (61) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 78; (62) a targeting sequence sing amino acids 2—24 of SEQ ID NO: 77; (63) a targeting ce comprising amino acids 5—24 of SEQ ID NO: 77; (64) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 80; (65) a ing sequence comprising amino acids 1—38 of SEQ ID NO: 81; (66) a ing sequence comprising amino acids 23—38 of SEQ ID NO: 81; (67) a targeting sequence comprising SEQ ID NO: 81; (68) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 82; (69) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 81; (70) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 81; (71) a targeting sequence sing amino acids 10—38 of SEQ ID NO: 81; (72) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 81; (73) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 81; (74) a targeting sequence comprising amino acids 1—34 of SEQ ID NO: 83; (75) a targeting sequence comprising SEQ ID NO: 83; (76) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 84; (77) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 86; (78) a targeting sequence comprising amino acids 1—28 of SEQ ID NO: 87; (79) a targeting sequence comprising amino acids 13—28 of SEQ ID NO: 87; (80) a targeting sequence comprising SEQ ID NO: 87; (81) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 88; (82) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 87; (83) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 87; (84) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 87; (85) a targeting sequence sing amino acids 1—28 of SEQ ID NO: 89; (86) a targeting sequence comprising SEQ ID NO: 89; (87) an exosporium protein comprising an amino acid ce having at least 85% identity With SEQ ID NO: 90; (88) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 89; (89) a targeting ce comprising amino acids 5—28 of SEQ ID NO: 89; (90) a ing sequence comprising amino acids 10—28 of SEQ ID NO: 89; (91) a targeting sequence comprising amino acids 1—93 of SEQ ID NO: 91; (92) a targeting sequence comprising SEQ ID NO: 91 ; (93) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 92; (94) a targeting sequence comprising amino acids 2—93 of SEQ ID NO: 91; (95) a targeting sequence comprising amino acids 10—93 of SEQ ID NO: 91; (96) a ing sequence comprising amino acids 20—93 of SEQ ID NO: 91; (97) a targeting sequence comprising amino acids 30—93 of SEQ ID NO: 91; (98) a ing sequence comprising amino acids 40—93 of SEQ ID NO: 91; (99) a targeting sequence comprising amino acids 50—93 of SEQ ID NO: 91; (100) a targeting sequence sing amino acids 60—93 of SEQ ID NO: 91; (101) a targeting sequence comprising amino acids 1—130 of SEQ ID NO: 93; (102) a targeting sequence sing SEQ ID NO: 93; (103) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 94; (104) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 93; (105) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 93 ; (106) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 93 ; (107) a targeting sequence comprising amino acids 30—130 of SEQ ID NO: 93 ; or (108) an exosporium protein comprising an amino acid sequence haVing at least 85% sequence identity with SEQ ID NO: 122.

For example, When the protein or peptide of interest is any protein or peptide of interest, the fusion protein can comprise: (1) a targeting sequence comprising amino acids 2— of SEQ ID NO: 59; (2) a targeting sequence comprising amino acids 4—30 of SEQ ID NO: 59; (3) a targeting sequence comprising amino acids 6—30 of SEQ ID NO: 59; (4) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 61 ; (5) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 61 ; (6) a ing sequence comprising amino acids 10—33 of SEQ ID NO: 61; (7) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 61; (8) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 63; (9) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 63; (10) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 63 ; (11) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 63 ; (12) a ing sequence comprising amino acids 15—35 of SEQ ID NO: 63 ; (13) a targeting ce comprising amino acids 2—24 of SEQ ID NO: 65; (14) a targeting sequence sing amino acids 5—24 of SEQ ID NO: 65; (15) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 67; (16) a targeting ce comprising amino acids 5—27 of SEQ ID NO: 67; (17) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 67; (18) a ing sequence comprising amino acids 2—38 of SEQ ID NO: 69; (19) a targeting sequence sing amino acids 5—38 of SEQ ID NO: 69; (20) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 69; (21) a targeting ce comprising amino acids 15—38 of SEQ ID NO: 69; (22) a targeting sequence comprising amino acids 2—42 of SEQ ID NO: 75; (23) a targeting sequence comprising amino acids 5—42 of SEQ ID NO: 75; (24) a targeting sequence comprising amino acids 10—42 of SEQ ID NO: 75; (25) a targeting sequence 2015/050807 sing amino acids 15—42 of SEQ ID NO: 75; (26) a targeting sequence comprising amino acids 20—42 of SEQ ID NO: 75; (27) a targeting sequence comprising amino acids 25—42 of SEQ ID NO: 75; (28) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 77; (29) a targeting sequence sing amino acids 5—24 of SEQ ID NO: 77; (30) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 81 ; (31) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 81 ; (32) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 81; (33) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 81 ; (34) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 81 ; (35) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 87; (36) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 87; (37) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 87; (38) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 89; (39) a targeting sequence sing amino acids 5—28 of SEQ ID NO: 89; (40) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 89; (41) a targeting sequence comprising amino acids 2—93 of SEQ ID NO: 91; (42) a targeting ce comprising amino acids 10—93 of SEQ ID NO: 91; (43) a targeting sequence comprising amino acids 20—93 of SEQ ID NO: 91; (44) a ing sequence comprising amino acids 30—93 of SEQ ID NO: 91; (45) a ing sequence comprising amino acids 40—93 of SEQ ID NO: 91; (46) a targeting sequence sing amino acids 50—93 of SEQ ID NO: 91; (47) a targeting sequence comprising amino acids 60—93 of SEQ ID NO: 91; (48) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 93; (49) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 93 ; (50) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 93; or (51) a targeting sequence sing amino acids 30—130 of SEQ ID NO: Alternatively, When the n or peptide of interest is any protein or peptide of interest, the fusion protein can comprise: (1) a targeting sequence consisting of amino acids —33 of SEQ ID NO: 1; (2) a targeting ce consisting of amino acids 21—33 of SEQ ID NO: 1; (3) a targeting sequence consisting of amino acids 23—31 of SEQ ID NO: 1; (4) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 96; (5) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 96; (6) a targeting sequence consisting of amino acids 12—25 of SEQ ID NO: 3; (7) a targeting sequence consisting of amino acids 13—25 of SEQ ID NO: 3; (8) a targeting sequence consisting of amino acids 15—23 of SEQ ID NO: 3; (9) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 97; (10) a targeting sequence ting of amino acids 1—13 of SEQ ID NO: 98; (11) a targeting sequence consisting of amino acids 23—36 of SEQ ID NO: 5; (12) a targeting sequence consisting of amino acids 23—34 of SEQ ID NO: 5; (13) a targeting sequence consisting of amino acids 24—36 of SEQ ID NO: 5; (14) a targeting sequence consisting of amino acids 26—34 of SEQ ID NO: 5; (15) a targeting sequence consisting of amino acids 13—26 of SEQ ID NO: 7; (16) a targeting sequence consisting of amino acids 13—24 of SEQ ID NO: 7; (17) a targeting sequence consisting of amino acids 14—26 of SEQ ID NO: 7; (18) a targeting ce consisting of amino acids 16—24 of SEQ ID NO: 7; (19) a targeting sequence ting of amino acids 9—22 of SEQ ID NO: 9; (20) a targeting sequence consisting of amino acids 9—20 of SEQ ID NO: 9; (21) a targeting sequence consisting of amino acids 10—22 of SEQ ID NO: 9; (22) a targeting sequence consisting of amino acids 12—20 of SEQ ID NO: 9; (23) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 105; (24) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 105; (25) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 11; (26) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 11; (27) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 11; (28) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 98; (29) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 98 ; (30) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 13; (31) a targeting sequence consisting of amino acids 18— 29 of SEQ ID NO: 13; (32) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 13 ; (33) a targeting sequence ting of amino acids 21—29 of SEQ ID NO: 13 ; (34) a ing sequence ting of amino acids 1—15 of SEQ ID NO: 99; (35) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 99; (36) a ing sequence consisting of amino acids 28—41 of SEQ ID NO: 15; (37) a targeting sequence consisting of amino acids 28— 39 of SEQ ID NO: 15; (38) a targeting sequence consisting of amino acids 29—41 of SEQ ID NO: 15; (39) a targeting sequence consisting of amino acids 31—39 of SEQ ID NO: 15; (40) a targeting sequence consisting of amino acids 12—25 of SEQ ID NO: 17; (41) a targeting ce consisting of amino acids 13—25 of SEQ ID NO: 17; (42) a ing sequence consisting of amino acids 1—15 of SEQ ID NO: 100; (43) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 19; (44) a targeting ce consisting of amino acids 18— 29 of SEQ ID NO: 19; (45) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 19; (46) a targeting sequence consisting of amino acids 21—29 of SEQ ID NO: 19; (47) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 21; (48) a targeting sequence ting of amino acids 18—29 of SEQ ID NO: 21; (49) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 21; (50) a ing sequence consisting of amino acids 21—29 of SEQ ID NO: 21; (51) a targeting ce consisting of amino acids 1—15 of SEQ ID NO: 101; (52) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 101; (53) a targeting sequence consisting of amino acids 9—22 of SEQ ID NO: 23 ; (54) a targeting sequence consisting of amino acids 9—20 of SEQ ID NO: 23; (55) a ing sequence consisting of amino acids 10—22 of SEQ ID NO: 23; (56) a targeting sequence consisting of amino acids 12—20 of SEQ ID NO: 23; (57) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 102; (58) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 102; (59) a targeting ce consisting of amino acids 9—22 of SEQ ID NO: 25; (60) a targeting sequence consisting of amino acids 9—20 of SEQ ID NO: 25; (61) a targeting sequence consisting of amino acids 10—22 of SEQ ID NO: 25; (62) a targeting sequence consisting of amino acids 12—20 of SEQ ID NO: 25; (63) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 103; (64) a ing sequence consisting of amino acids 1—13 of SEQ ID NO: 103; (65) a targeting sequence consisting of amino acids 15—28 of SEQ ID NO: 27; (66) a targeting sequence consisting of amino acids 15—26 of SEQ ID NO: 27; (67) a targeting sequence consisting of amino acids 16—28 of SEQ ID NO: 27; (68) a targeting sequence consisting of amino acids 18—26 of SEQ ID NO: 27; (69) a ing sequence consisting of amino acids 1—15 of SEQ ID NO: 104; (70) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 104; (71) a targeting ce consisting of amino acids 1—13 of SEQ ID NO: 33; (72) a targeting sequence ting of amino acids 1—11 of SEQ ID NO: 33; (73) a targeting sequence consisting of amino acids 3—11 of SEQ ID NO: 33; (74) a targeting sequence consisting of amino acids 1—14 of SEQ ID NO: 35; (75) a targeting sequence consisting of amino acids 1—12 of SEQ ID NO: 35; (76) a targeting sequence consisting of amino acids 2—14 of SEQ ID NO: 35; (77) a targeting sequence consisting of amino acids 14—27 of SEQ ID NO: 43; (78) a targeting sequence consisting of amino acids 14—25 of SEQ ID NO: 43 ; (79) a targeting sequence consisting of amino acids 15—27 of SEQ ID NO: 43; (80) a targeting sequence consisting of amino acids 20—33 of SEQ ID NO: 45; (81) a targeting ce consisting of amino acids 20—31 of SEQ ID NO: 45; (82) a targeting sequence consisting of amino acids 21—33 of SEQ ID NO: 45; (83) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 106; (84) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 106; (85) a targeting sequence consisting of amino acids 28—41 of SEQ ID NO: 47; (86) a targeting sequence consisting of amino acids 28—39 of SEQ ID NO: 47; (87) a targeting ce consisting of amino acids 18—31 of SEQ ID NO: 53; (88) a targeting ce consisting of amino acids 18—29 of SEQ ID NO: 53; (89) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 53; (90) a targeting sequence comprising amino acids 18—31 of SEQ ID NO: 61; (91) a targeting sequence sing amino acids 18—29 of SEQ ID NO: 61; (92) a targeting sequence comprising amino acids 19—31 of SEQ ID NO: 61; (93) a ing sequence comprising amino acids 9—22 of SEQ ID NO: 65; (94) a ing sequence comprising amino acids 9—20 of SEQ ID NO: 65; (95) a targeting ce comprising amino acids 10—22 of SEQ ID NO: 65; (96) a targeting sequence comprising amino acids 1—15 of SEQ ID NO: 107; (97) a targeting sequence comprising amino acids 1—13 of SEQ ID NO: 107; (98) a targeting sequence comprising amino acids 12—25 of SEQ ID NO: 67; (99) a targeting sequence comprising amino acids 12—23 of SEQ ID NO: 67; (100) a targeting sequence comprising amino acids 13—25 of SEQ ID NO: 67; (101) a targeting sequence comprising amino acids 15—23 of SEQ ID NO: 67; (102) a targeting sequence comprising amino acids 23—36 of SEQ ID NO: 69; (103) a ing sequence comprising amino acids 23—34 of SEQ ID NO: 69; (104) a targeting sequence comprising amino acids 24—36 of SEQ ID NO: 69; (105) a targeting sequence comprising amino acids 26—34 of SEQ ID NO: 69; (106) a targeting ce comprising amino acids 27—40 of SEQ ID NO: 75; (107) a targeting sequence comprising amino acids 27—38 of SEQ ID NO: 75; (108) a targeting sequence comprising amino acids 9—22 of SEQ ID NO: 77; (109) a targeting sequence comprising amino acids 9—20 of SEQ ID NO: 77; (110) a targeting sequence comprising amino acids 10—22 of SEQ ID NO: 77; (111) a targeting sequence comprising amino acids 12—20 of SEQ ID NO: 77; (112) a targeting sequence comprising amino acids 23—36 of SEQ ID NO: 81; (113) a targeting sequence comprising amino acids 23—34 of SEQ ID NO: 81; (114) a targeting sequence sing amino acids 24—36 of SEQ ID NO: 81; (115) a targeting sequence comprising amino acids 26—34 of SEQ ID NO: 81; (116) a targeting ce comprising amino acids 13—26 of SEQ ID NO: 87; (117) a targeting sequence comprising amino acids 13—24 of SEQ ID NO: 87; or (118) a targeting sequence comprising amino acids 14—26 of SEQ ID NO: 87. The ing sequence can also t of any of these sequences.

The present invention relates to fusion proteins comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment. The protein or peptide of interest can be an enzyme that catalyzes the production of nitric oxide or a c acid binding protein or peptide. When the protein or peptide of interest comprises an enzyme that zes the production of nitric oxide or a nucleic acid binding protein or peptide, the targeting sequence, exosporium protein, or exosporium protein nt can be any targeting sequence, exosporium protein, or rium protein fragment that targets the fusion protein to the exopsorium of a recombinant Bacillus cereus family . For example, the targeting sequence exosporium protein or rium protein fragment can be any of the targeting sequences, exosporium ns, or exosporium protein fragments listed above in aphs [00166]—[00168] for use with any protein or peptide of interest or: (1) a targeting sequence comprising an amino acid sequence haVing at least about 43% identity With amino acids 20—35 of SEQ ID NO: 1, wherein the identity With amino acids —35 is at least about 54%; (2) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 1; (3) a targeting sequence comprising amino acids 20—35 of SEQ ID NO: 1; (4) a targeting sequence comprising SEQ ID NO: 1; (5) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 2; (6) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 1; (7) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 1; (8) a targeting sequence sing amino acids 8—35 of SEQ ID NO: 1; (9) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 1; (10) a ing sequence comprising amino acids 15—35 of SEQ ID NO: 1; (11) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 3; (12) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 3; (13) a targeting sequence sing SEQ ID NO: 3; (14) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 4; (15) a targeting ce comprising amino acids 2—27 of SEQ ID NO: 3; (16) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 3; (17) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 3; (18) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 3; (19) a targeting sequence comprising amino acids 1—38 of SEQ ID NO: 5; (20) a targeting sequence comprising amino acids 23—38 of SEQ ID NO: 5; (21) a targeting sequence comprising SEQ ID NO: 5; (22) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 6; (23) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 5; (24) a ing sequence comprising amino acids 5—38 of SEQ ID NO: 5; (25) a targeting sequence comprising amino acids 8—38 of SEQ ID NO: 5; (26) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 5; (27) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 5; (28) a ing sequence comprising amino acids 20—38 of SEQ ID NO: 5; (29) a targeting ce comprising amino acids 1—28 of SEQ ID NO: 7; (30) a targeting sequence comprising amino acids 13—28 of SEQ ID NO: 7; (31) a targeting sequence comprising SEQ ID NO: 7; (32) an exosporium protein sing an amino acid sequence having at least 85% identity With SEQ ID NO: 8; (33) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 7; (34) a ing sequence comprising amino acids 5—28 of SEQ ID NO: 7; (35) a ing sequence comprising amino acids 8—28 of SEQ ID NO: 7; (36) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 7; (37) a ing sequence comprising amino acids 1—24 of SEQ ID NO: 9; (38) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 9; (39) a targeting ce comprising SEQ ID NO: 9; (40) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 10; (41) a targeting ce comprising amino acids 2—24 of SEQ ID NO: 9; (42) a targeting sequence sing amino acids 5—24 of SEQ ID NO: 9; (43) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 9; (44) a targeting sequence comprising amino acids l—33 of SEQ ID NO: I l ; (45) a targeting ce comprising amino acids 18—33 of SEQ ID NO: ll; (46) a targeting sequence comprising SEQ ID NO: 11; (47) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 12; (48) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 11; (49) a targeting ce comprising amino acids 5—33 of SEQ ID NO: 11 ; (50) a ing sequence comprising amino acids 8—33 of SEQ ID NO: 11 ; (5 l) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 11; (52) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 11; (53) a targeting sequence sing amino acids l—33 of SEQ ID NO: 13; (54) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 13; (55) a targeting sequence comprising SEQ ID NO:l3; (56) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 14; (57) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 13; (58) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13; (59) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 13; (60) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 13; (61) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 13; (62) a targeting sequence comprising amino acids l—43 of SEQ ID NO: 15; (63) a targeting sequence sing amino acids 28—43 of SEQ ID NO: 15; (64) a targeting sequence comprising SEQ ID NO:15; (65) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:l6; (66) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 15; (67) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 15; (68) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 15; (69) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 15; (70) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 15; (71) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 15; (72) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 15; (73) a targeting sequence comprising amino acids l—27 of SEQ ID NO: 17; (74) a targeting sequence WO 44661 comprising amino acids 12—27 of SEQ ID NO: 17; (75) a targeting sequence comprising SEQ ID NO:17; (76) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:18; (77) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 17; (78) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 17; (79) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 17; (80) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 17; (81) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 19; (82) a ing sequence comprising amino acids 18—33 of SEQ ID NO: 19; (83) a targeting sequence comprising SEQ ID NO:19; (84) an exosporium protein comprising an amino acid ce having at least 85% identity With SEQ ID NO:20; (85) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 19; (86) a targeting ce comprising amino acids 5—33 of SEQ ID NO: 19; (87) a ing ce comprising amino acids 8—33 of SEQ ID NO: 19; (88) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 19; (89) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 19; (90) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 21 ; (91) a ing sequence comprising amino acids 18—33 of SEQ ID NO: 21; (92) a targeting sequence sing SEQ ID NO:21; (93) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:22; (94) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 21; (95) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 21; (96) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 21 ; (97) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 21 ; (98) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 21; (99) a targeting sequence sing amino acids 1—24 of SEQ ID NO: 23; (100) a targeting ce comprising amino acids 9—24 of SEQ ID NO: 23; (101) a targeting sequence comprising SEQ ID NO:23; (102) an exosporium protein comprising an amino acid ce having at least 85% identity With SEQ ID NO:24; (103) a targeting sequence comprising amino acids 2—24 of SEQ ID NO:23; (104) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 23; (105) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 23; (106) a targeting ce comprising amino acids 1—24 of SEQ ID NO: 25; (107) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 25 ; (108) a targeting sequence comprising SEQ ID NO:25; (109) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:26; (110) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 25; (111) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 25; (112) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 25; (113) a targeting sequence comprising amino acids 1—30 of SEQ WO 44661 ID NO: 27; (114) a targeting sequence comprising amino acids 15—30 of SEQ ID NO: 27; (115) a targeting sequence comprising SEQ ID NO:27; (116) an exosporium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO:28; (117) a targeting ce comprising amino acids 2—30 of SEQ ID NO: 27; (118) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 27; (119) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 27; (120) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 27; (121) a targeting ce comprising amino acids 1—33 of SEQ ID NO: 29; (122) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 29; (123) a targeting sequence comprising SEQ ID NO:29; (124) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:30; (125) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 29; (126) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 29; (127) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 29; (128) a ing sequence comprising amino acids 10—33 of SEQ ID NO: 29; (129) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 29; (130) a targeting ce sing amino acids 1—24 of SEQ ID NO: 31 ; (131) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 31 ; (132) a targeting sequence comprising SEQ ID NO:31; (133) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:32; (134) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 31; (135) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31; (136) a targeting ce comprising amino acids 8—24 of SEQ ID NO: 31 ; (137) a targeting sequence comprising amino acids 1—15 of SEQ ID NO: 33; (138) a targeting sequence comprising SEQ ID NO:33; (139) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:34; (140) a targeting sequence comprising amino acids 1—16 of SEQ ID NO: 35; (141) a targeting sequence comprising SEQ ID NO:35; (142) an rium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO:36; (143) a targeting sequence comprising amino acids 1—29 of SEQ ID NO:43; (144) a targeting sequence comprising amino acids 14—29 of SEQ ID NO: 43; (145) a targeting sequence comprising SEQ ID NO: 43; (146) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 44; (147) a targeting ce comprising amino acids 2—29 of SEQ ID NO: 43 ; (148) a targeting sequence comprising amino acids 5—29 of SEQ ID NO: 43 ; (149) a targeting sequence sing amino acids 8—29 of SEQ ID NO: 43; (150) a targeting sequence comprising amino acids 10—29 of SEQ ID NO: 43; (151) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 45; (152) a ing sequence comprising amino acids 20—35 of SEQ ID NO: 45 ; (153) a targeting sequence comprising SEQ ID NO: 45 ; (154) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 46; (155) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 45; (156) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 45; (157) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 45; (158) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45; (159) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 45 ; (160) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 47; (161) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 47; (162) a targeting sequence comprising SEQ ID NO: 47; (163) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 48; (164) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 47; (165) a ing sequence comprising amino acids 5—43 of SEQ ID NO: 47; (166) a targeting sequence sing amino acids 8—43 of SEQ ID NO: 47; (167) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 47; (168) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 47; (169) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (170) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 47; (171) a targeting sequence comprising amino acids 1—32 of SEQ ID NO: 49; (172) a targeting sequence comprising amino acids 17—32 of SEQ ID NO: 49; (173) a ing sequence comprising SEQ ID NO: 49; (174) an exosporium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO: 50; (175) a targeting sequence comprising amino acids 2—32 of SEQ ID NO: 49; (176) a targeting sequence comprising amino acids 5—32 of SEQ ID NO: 49; (177) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (178) a targeting sequence sing amino acids 10—32 of SEQ ID NO: 49; (179) a ing sequence comprising amino acids 15—32 of SEQ ID NO: 49; (180) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 51; (181) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 51; (182) a targeting sequence comprising SEQ ID NO: 51; (183) an rium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 52; (184) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 51; (185) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 51; (186) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 51; (187) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 51; (188) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 51; (189) a ing sequence comprising amino acids 1—33 of SEQ ID NO: 53; (190) a ing sequence comprising amino acids 18—33 of SEQ ID NO: 53; (191) a targeting sequence comprising SEQ ID NO: 53; (192) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 54; (193) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 53; (194) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53; (195) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53; (196) a ing sequence sing amino acids 10—33 of SEQ ID NO: 53; (197) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 53; (198) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 55; (199) a targeting sequence comprising amino acids 15—30 of SEQ ID NO: 55 ; (200) a targeting sequence comprising SEQ ID NO: 55 ; (201) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 56; (202) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 55; (203) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 55; (204) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 55; (205) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 55; (206) a targeting ce comprising amino acids 1—130 of SEQ ID NO: 57; (207) a targeting sequence comprising amino acids 115—130 of SEQ ID NO: 57; (208) a targeting sequence sing SEQ ID NO: 57; (209) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 58; (210) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 57; (211) a ing sequence comprising amino acids 5—130 of SEQ ID NO: 57; (212) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (213) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 57; (214) a targeting sequence comprising amino acids 30— 130 of SEQ ID NO: 57; (215) a targeting sequence comprising amino acids 40—130 of SEQ ID NO: 57; (216) a targeting sequence comprising amino acids 50—130 of SEQ ID NO: 57; (217) a targeting sequence comprising amino acids 60—130 of SEQ ID NO: 57; (218) a targeting sequence comprising amino acids 70—130 of SEQ ID NO: 57; (219) a targeting ce comprising amino acids 80—130 of SEQ ID NO: 57; (220) a targeting sequence sing amino acids 90—130 of SEQ ID NO: 57; (221) a targeting sequence comprising amino acids 100—130 of SEQ ID NO: 57; (222) a targeting ce comprising amino acids 0 of SEQ ID NO: 57; (223) an exosporium protein fragment comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 95; (224) a targeting sequence comprising SEQ ID NO: 96; (225) a targeting sequence comprising SEQ ID NO: 97; (226) a targeting sequence comprising SEQ ID NO: 98; (227) a ing sequence comprising SEQ ID NO: 99; (228) a targeting sequence comprising SEQ ID NO: 100; (229) a targeting sequence comprising SEQ ID NO: 101; (230) a targeting sequence comprising SEQ ID NO: 102; (231) a targeting sequence comprising SEQ ID NO: 103; (232) a targeting sequence comprising SEQ ID NO: 104; (233) a ing sequence comprising SEQ ID NO: 105; (234) a targeting sequence comprising SEQ ID NO: 106; (235) an exosporium protein comprising an amino acid sequence having at least 85% ty with SEQ ID NO: 108; (236) an exosporium protein sing an amino acid sequence having at least 85% identity With SEQ ID NO: 109; (237) an exosporium protein sing an amino acid sequence haVing at least 85% ty With SEQ ID NO: 110; (238) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 111; (239) an rium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO: 112; (240) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 113; (241) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 114; (242) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 115; (243) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 116; (244) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 117; (245) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 118; (246) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 119; (247) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 120; (248) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 121 ; (249) a targeting ce comprising amino acids 22—31 of SEQ ID NO: 1 ; (250) a targeting sequence comprising amino acids 22—33 of SEQ ID NO: 1 ; (251) a targeting sequence comprising amino acids 20—31 of SEQ ID NO: 1 ; (252) a targeting sequence comprising amino acids 14—23 of SEQ ID NO: 3; (253) a targeting sequence comprising amino acids 14—25 of SEQ ID NO: 3; or (254) a targeting sequence comprising amino acids 12—23 of SEQ ID NO: 3.

For example, When the protein or peptide of interest ses an enzyme that catalyzes the production of nitric oxide or a nucleic acid binding protein or peptide, the ing sequence, exosporium protein, or exosporium protein fragment can be: (1) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 1 ; (2) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 1; (3) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 1; (4) a ing sequence comprising amino acids 10—35 of SEQ ID NO: 1; (5) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 1 ; (6) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 3; (7) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 3; (8) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 3; (9) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 3; (10) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 5; (11) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 5; (12) a targeting sequence comprising amino acids 8—38 of SEQ ID NO: 5; (13) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 5; (14) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 5; (15) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 5; (16) a targeting sequence sing amino acids 2—28 of SEQ ID NO: 7; (17) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 7; (18) a targeting sequence comprising amino acids 8—28 of SEQ ID NO: 7; (19) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 7; (20) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 9; (21) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 9; (22) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 9; (23) a targeting ce sing amino acids 2—33 of SEQ ID NO: 11; (24) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 11; (25) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 11; (26) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 11; (27) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 11; (28) a targeting ce comprising amino acids 2—33 of SEQ ID NO: 13; (29) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13; (30) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 13; (31) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 13; (32) a ing sequence sing amino acids 15—33 of SEQ ID NO: 13; (33) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 15; (34) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 15; (35) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 15; (36) a ing sequence comprising amino acids 10—43 of SEQ ID NO: 15; (37) a ing sequence comprising amino acids 15—43 of SEQ ID NO: 15; (38) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 15; (39) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 15; (40) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 17; (41) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 17; (42) a ing sequence comprising amino acids 8—27 of SEQ ID NO: 17; (43) a targeting ce comprising amino acids 10—27 of SEQ ID NO: 17; (44) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 19; (45) a targeting sequence sing amino acids 5—33 of SEQ ID NO: 19; (46) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 19; (47) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 19; (48) a targeting ce comprising amino acids 15—33 of SEQ ID NO: 19; (49) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 21; (50) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 21; (51) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 21; (52) a targeting ce comprising amino acids 10—33 of SEQ ID NO: 21; (53) a targeting ce comprising amino acids 15—33 of SEQ ID NO: 21; (54) a targeting ce comprising amino acids 2—24 of SEQ ID NO:23; (55) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 23; (56) a targeting ce comprising amino acids 8—24 of SEQ ID NO: 23; (57) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 25; (58) a targeting ce comprising amino acids 5—24 of SEQ ID NO: 25; (59) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 25; (60) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 27; (61) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 27; (62) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 27; (63) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 27; (64) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 29; (65) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 29; (66) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 29; (67) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 29; (68) a ing ce comprising amino acids 15—33 of SEQ ID NO: 29; (69) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 31; (70) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31; (71) a ing sequence comprising amino acids 8—24 of SEQ ID NO: 31 ; (72) a targeting sequence comprising amino acids 2—29 of SEQ ID NO: 43; (73) a targeting sequence comprising amino acids 5—29 of SEQ ID NO: 43; (74) a targeting sequence comprising amino acids 8—29 of SEQ ID NO: 43; (75) a targeting sequence comprising amino acids 10—29 of SEQ ID NO: 43; (76) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 45; (77) a targeting ce comprising amino acids 5—35 of SEQ ID NO: 45; (78) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 45 ; (79) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45 ; (80) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 45 ; (81) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 47; (82) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 47; (83) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 47; (84) a targeting sequence sing amino acids 10—43 of SEQ ID NO: 47; (85) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 47; (86) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (87) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 47; (88) a targeting sequence comprising amino acids 2—32 of SEQ ID NO: 49; (89) a ing sequence comprising amino acids 5—32 of SEQ 2015/050807 ID NO: 49; (90) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (91) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (92) a targeting sequence comprising amino acids 15—32 of SEQ ID NO: 49; (93) a ing sequence comprising amino acids 2—33 of SEQ ID NO: 51; (94) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 51; (95) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 51; (96) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 51; (97) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 51; (98) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 53; (99) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53; (100) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53; (101) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 53; (102) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 53; (103) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 55; (104) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 55; (105) a targeting sequence sing amino acids 8—30 of SEQ ID NO: 55; (106) a ing ce comprising amino acids 10—30 of SEQ ID NO: 55; (107) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 57; (108) a targeting sequence comprising amino acids 5—130 of SEQ ID NO: 57; (109) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (110) a ing sequence comprising amino acids 20—130 of SEQ ID NO: 57; (111) a targeting sequence comprising amino acids 30— 130 of SEQ ID NO: 57; (112) a ing ce comprising amino acids 40—130 of SEQ ID NO: 57; (113) a targeting ce comprising amino acids 50—130 of SEQ ID NO: 57; (114) a targeting sequence sing amino acids 60—130 of SEQ ID NO: 57; (115) a targeting sequence comprising amino acids 70—130 of SEQ ID NO: 57; (116) a targeting sequence comprising amino acids 80—130 of SEQ ID NO: 57; (117) a targeting sequence comprising amino acids 90—130 of SEQ ID NO: 57; (118) a targeting sequence sing amino acids 100—130 of SEQ ID NO: 57; or (119) a targeting sequence comprising amino acids 0 of SEQ ID NO: 57.

A fusion protein is provided which comprises an antigen or a remediation enzyme and a targeting sequence or exosporium protein. The targeting sequence or exosporium protein can comprise any of the targeting sequences or exosporium proteins listed above in paragraphs [00166]—[00168] for use With any protein or peptide of interest or: (1) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 1; (2) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 1; (3) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 1; (4) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 1; (5) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 1; (6) a targeting sequence comprising amino acids 22—31 of SEQ ID NO: 1; (7) a targeting sequence comprising amino acids 22—33 of SEQ ID NO: 1; (8) a targeting sequence sing amino acids 20—31 of SEQ ID NO: 1; (9) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 3; (10) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 3; (11) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 3; (12) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 3; (13) a targeting ce comprising amino acids 14—23 of SEQ ID NO: 3; (14) a targeting sequence sing amino acids 14—25 of SEQ ID NO: 3; (15) a ing sequence comprising amino acids 12—23 of SEQ ID NO: 3; (16) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 5; (17) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 5; (18) a targeting sequence comprising amino acids 8—38 of SEQ ID NO: 5; (19) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 5; (20) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 5; (21) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 5; (22) a ing sequence comprising amino acids 2—28 of SEQ ID NO: 7; (23) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 7; (24) a targeting sequence comprising amino acids 8—28 of SEQ ID NO: 7; (25) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 7; (26) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 9; (27) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 9; (28) a targeting ce comprising SEQ ID NO: 9; (29) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 10; (30) a targeting sequence sing amino acids 2—24 of SEQ ID NO: 9; (31) a targeting ce comprising amino acids 5—24 of SEQ ID NO: 9; (32) a targeting sequence sing amino acids 8—24 of SEQ ID NO: 9; (33) a targeting sequence comprising amino acids 1—33 of SEQ ID NO:11; (34) a targeting sequence sing amino acids 18—33 of SEQ ID NO: 11 ; (35) a targeting sequence comprising SEQ ID NO: 11 ; (36) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 12; (37) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 11 ; (38) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 11 ; (39) a targeting sequence sing amino acids 8—33 of SEQ ID NO: 11 ; (40) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 11; (41) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 11; (42) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 13; (43) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 13; (44) a targeting sequence comprising SEQ ID NO:13; (45) an exosporium n comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 14; (46) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 13 ; (47) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13 ; (48) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 13; (49) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 13; (50) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 13 ; (51) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 15; (52) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 15; (53) a targeting sequence comprising SEQ ID NO:15; (54) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 16; (55) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 15; (56) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 15 ; (57) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 15 ; (58) a ing sequence comprising amino acids 10—43 of SEQ ID NO: 15 ; (59) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 15; (60) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 15; (61) a targeting ce comprising amino acids 25—43 of SEQ ID NO: 15; (62) a ing sequence comprising amino acids 1—27 of SEQ ID NO: 17; (63) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 17; (64) a targeting sequence comprising SEQ ID NO:17; (65) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:18; (66) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 17; (67) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 17; (68) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 17; (69) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 17; (70) a targeting ce comprising amino acids 1—33 of SEQ ID NO: 19; (71) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 19; (72) a targeting sequence comprising SEQ ID NO:19; (73) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO:20; (74) a targeting ce comprising amino acids 2—33 of SEQ ID NO: 19; (75) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 19; (76) a targeting sequence sing amino acids 8—33 of SEQ ID NO: 19; (77) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 19; (78) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 19; (79) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 21; (80) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 21; (81) a targeting sequence comprising SEQ ID NO:21; (82) an exosporium n comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO:22; (83) a ing sequence comprising amino acids 2—33 of SEQ ID NO: 21 ; (84) a targeting sequence sing amino acids 5—33 of SEQ ID NO: 21; (85) a targeting sequence comprising amino acids 8—33 of SEQ WO 44661 ID NO: 21; (86) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 21; (87) a targeting sequence sing amino acids 15—33 of SEQ ID NO: 21; (88) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 23; (89) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 23 ; (90) a ing sequence comprising SEQ ID NO:23; (91) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:24; (92) a targeting sequence comprising amino acids 2—24 of SEQ ID NO:23; (93) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 23; (94) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 23; (95) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 25; (96) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 25; (97) a ing sequence comprising SEQ ID NO:25; (98) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:26; (99) a targeting ce comprising amino acids 2—24 of SEQ ID NO: 25; (100) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 25; (101) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 25; (102) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 27; (103) a targeting sequence comprising amino acids 15—30 of SEQ ID NO: 27; (104) a targeting sequence comprising SEQ ID NO:27; (105) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:28; (106) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 27; (107) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 27; (108) a targeting ce comprising amino acids 8—30 of SEQ ID NO: 27; (109) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 27; (110) a ing sequence comprising amino acids 1—33 of SEQ ID NO: 29; (111) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 29; (112) a ing sequence comprising SEQ ID NO:29; (113) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:30; (114) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 29; (115) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 29; (116) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 29; (117) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 29; (118) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 29; (119) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 31 ; (120) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 31 ; (121) a targeting sequence comprising SEQ ID NO:31; (122) an exosporium protein sing an amino acid ce haVing at least 85% identity With SEQ ID NO:32; (123) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 31; (124) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31; (125) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 31; (126) a targeting sequence sing amino acids 1—15 of SEQ ID NO: 33; (127) a targeting sequence comprising SEQ ID NO:33; (128) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:34; (129) a targeting sequence comprising amino acids 1—16 of SEQ ID NO: 35; (130) a targeting ce comprising SEQ ID NO:35; (131) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO:36; (132) a targeting sequence comprising amino acids 1—29 of SEQ ID NO:43; (133) a targeting sequence comprising amino acids 14—29 of SEQ ID NO: 43; (134) a targeting sequence sing SEQ ID NO: 43; (135) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 44; (136) a targeting sequence comprising amino acids 2—29 of SEQ ID NO: 43 ; (137) a targeting sequence comprising amino acids 5—29 of SEQ ID NO: 43 ; (138) a ing sequence comprising amino acids 8—29 of SEQ ID NO: 43; (139) a targeting sequence comprising amino acids 10—29 of SEQ ID NO: 43; (140) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 45; (141) a targeting sequence comprising amino acids 20—35 of SEQ ID NO: 45 ; (142) a targeting ce comprising SEQ ID NO: 45 ; (143) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 46; (144) a targeting sequence sing amino acids 2—35 of SEQ ID NO: 45 ; (145) a targeting ce comprising amino acids 5—35 of SEQ ID NO: 45 ; (146) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 45; (147) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45; (148) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 45 ; (149) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 47; (150) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 47; (151) a ing sequence comprising SEQ ID NO: 47; (152) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 48; (153) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 47; (154) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 47; (155) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 47; (156) a ing sequence comprising amino acids 10—43 of SEQ ID NO: 47; (157) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 47; (158) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (159) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 47; (160) a targeting sequence comprising amino acids 1—32 of SEQ ID NO: 49; (161) a targeting sequence comprising amino acids 17—32 of SEQ ID NO: 49; (162) a ing ce comprising SEQ ID NO: 49; (163) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 50; (164) a targeting ce comprising amino acids 2—32 of SEQ ID NO: 49; (165) a targeting sequence comprising amino acids 5—32 of SEQ ID NO: 49; (166) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (167) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (168) a targeting sequence sing amino acids 15—32 of SEQ ID NO: 49; (169) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 51 ; (170) a targeting ce sing amino acids 18—33 of SEQ ID NO: 51 ; (171) a targeting sequence comprising SEQ ID NO: 51 ; (172) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 52; (173) a ing sequence comprising amino acids 2—33 of SEQ ID NO: 51 ; (174) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 51 ; (175) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 51; (176) a ing sequence comprising amino acids 10—33 of SEQ ID NO: 51; (177) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 51; (178) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 53 ; (179) a ing sequence comprising amino acids 18—33 of SEQ ID NO: 53; (180) a targeting sequence comprising SEQ ID NO: 53; (181) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 54; (182) a targeting ce comprising amino acids 2—33 of SEQ ID NO: 53; (183) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53 ; (184) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53 ; (185) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 53; (186) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 53; (187) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 55; (188) a targeting sequence comprising amino acids 15—30 of SEQ ID NO: 55 ; (189) a targeting sequence comprising SEQ ID NO: 55 ; (190) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 56; (191) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 55; (192) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 55; (193) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 55; (194) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 55; (195) a targeting sequence comprising amino acids 1—130 of SEQ ID NO: 57; (196) a targeting sequence comprising amino acids 115—130 of SEQ ID NO: 57; (197) a targeting sequence comprising SEQ ID NO: 57; (198) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 58; (199) a ing sequence comprising amino acids 2—130 of SEQ ID NO: 57; (200) a targeting ce comprising amino acids 5—130 of SEQ ID NO: 57; (201) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (202) a ing sequence comprising amino acids 20—130 of SEQ ID NO: 57; (203) a targeting ce comprising amino acids 30— 130 of SEQ ID NO: 57; (204) a targeting sequence comprising amino acids 40—130 of SEQ ID NO: 57; (205) a targeting sequence comprising amino acids 50—130 of SEQ ID NO: 57; (206) a ing sequence comprising amino acids 60—130 of SEQ ID NO: 57; (207) a targeting sequence comprising amino acids 70—130 of SEQ ID NO: 57; (208) a targeting sequence comprising amino acids 80—130 of SEQ ID NO: 57; (209) a ing sequence comprising amino acids 90—130 of SEQ ID NO: 57; (210) a targeting sequence comprising amino acids 100—130 of SEQ ID NO: 57; (211) a ing sequence comprising amino acids 110—130 of SEQ ID NO: 57; (212) a targeting sequence comprising SEQ ID NO: 97; (213) a ing sequence comprising SEQ ID NO: 98; (214) a targeting sequence comprising SEQ ID NO: 99; (215) a targeting sequence comprising SEQ ID NO: 100; (216) a ing sequence comprising SEQ ID NO: 101; (217) a targeting sequence comprising SEQ ID NO: 102; (218) a targeting sequence comprising SEQ ID NO: 103; (219) a targeting sequence sing SEQ ID NO: 104; (220) a targeting sequence comprising SEQ ID NO: 105; (221) a targeting sequence comprising SEQ ID NO: 106; (222) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 108; (223) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 109; (224) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 110; (225) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 111; (226) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 112; (227) an exosporium n comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 113; (228) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 114; (229) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 115; (230) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 116; (231) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 117; (232) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 118; (233) an exosporium n comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 119; (234) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 120; or (235) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 121.

A fusion protein is provided which comprises an enzyme le for breaking an emulsion or gel in a hydraulic fracturing fluid or an antibacterial protein or peptide and a targeting sequence, exosporium protein, or exosporium protein fragment. The ing sequence, rium protein, or exosporium protein fragment can se any of the targeting sequences or exosporium proteins listed above in paragraphs ]—[00168] for use With any protein or peptide of interest or: (1) a targeting sequence comprising an amino acid sequence haVing at least about 43% identity With amino acids 20—35 of SEQ ID NO: 1, wherein the identity With amino acids 25—35 is at least about 54%; (2) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 1; (3) a targeting ce comprising amino acids 20—35 of SEQ ID NO: 1 ; (4) a targeting sequence comprising SEQ ID NO: 1 ; (5) an rium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 2; (6) a ing sequence comprising amino acids 2—35 of SEQ ID NO: 1 ; (7) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 1 ; (8) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 1; (9) a ing sequence comprising amino acids 10—35 of SEQ ID NO: l; (10) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 1; (ll) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 3; (12) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 3; (13) a targeting sequence comprising SEQ ID NO: 3; (14) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 4; (15) a targeting ce comprising amino acids 2—27 of SEQ ID NO: 3; (16) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 3; (17) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 3; (18) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 3; (19) a targeting sequence comprising amino acids 1—38 of SEQ ID NO: 5; (20) a targeting sequence comprising amino acids 23—38 of SEQ ID NO: 5; (21) a targeting sequence comprising SEQ ID NO: 5; (22) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 6; (23) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 5; (24) a ing sequence comprising amino acids 5—38 of SEQ ID NO: 5; (25) a targeting sequence comprising amino acids 8—38 of SEQ ID NO: 5; (26) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 5; (27) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 5; (28) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 5; (29) a targeting sequence comprising amino acids 1—28 of SEQ ID NO: 7; (30) a targeting sequence comprising amino acids 13—28 of SEQ ID NO: 7; (31) a targeting sequence comprising SEQ ID NO: 7; (32) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 8; (33) a targeting ce comprising amino acids 2—28 of SEQ ID NO: 7; (34) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 7; (35) a targeting sequence comprising amino acids 8—28 of SEQ ID NO: 7; (36) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 7; (37) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 9; (38) a ing sequence comprising amino acids 9—24 of SEQ ID NO: 9; (39) a targeting sequence sing SEQ ID NO: 9; (40) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 10; (41) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 9; (42) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 9; (43) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 9; (44) a targeting sequence comprising amino acids 1—33 of SEQ ID NO:11; (45) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 11; (46) a targeting sequence comprising SEQ ID NO: 11; (47) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 12; (48) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 11; (49) a targeting ce comprising amino acids 5—33 of SEQ ID NO: 11; (50) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 11; (51) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 11; (52) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 11; (53) a targeting ce comprising amino acids 1—33 of SEQ ID NO: 13; (54) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 13; (55) a targeting sequence comprising SEQ ID NO:13; (56) an exosporium n comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 14; (57) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 13; (58) a targeting sequence sing amino acids 5—33 of SEQ ID NO: 13; (59) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 13 ; (60) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 13 ; (61) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 13 ; (62) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 15; (63) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 15; (64) a targeting sequence sing SEQ ID NO:15; (65) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 16; (66) a targeting ce sing amino acids 2—43 of SEQ ID NO: 15 ; (67) a targeting ce comprising amino acids 5—43 of SEQ ID NO: 15 ; (68) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 15; (69) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 15; (70) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 15 ; (71) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 15; (72) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 15; (73) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 17; (74) a targeting ce comprising amino acids 12—27 of SEQ ID NO: 17; (75) a targeting ce comprising SEQ ID NO:17; (76) an exosporium protein comprising an amino acid ce having at least 85% identity With SEQ ID NO:18; (77) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 17; (78) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 17; (79) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 17; (80) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 17; (81) a targeting ce comprising amino acids 1—33 of SEQ ID NO: 19; (82) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 19; (83) a targeting sequence comprising SEQ ID NO:19; (84) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 20; (85) a ing sequence comprising amino acids 2—33 of SEQ ID NO: 19; (86) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 19; (87) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 19; (88) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 19; (89) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 19; (90) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 21; (91) a ing sequence comprising amino acids 18—33 of SEQ ID NO: 21 ; (92) a targeting ce sing SEQ ID NO:21; (93) an exosporium n comprising an amino acid sequence having at least 85% ty With SEQ ID NO:22; (94) a targeting ce comprising amino acids 2—33 of SEQ ID NO: 21 ; (95) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 21; (96) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 21; (97) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 21; (98) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 21 ; (99) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 23; (100) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 23 ; (101) a targeting sequence comprising SEQ ID NO:23; (102) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 24; (103) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 23 ; (104) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 23; (105) a targeting sequence sing amino acids 8—24 of SEQ ID NO: 23; (106) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 25; (107) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 25; (108) a targeting sequence comprising SEQ ID NO:25; (109) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:26; (110) a targeting sequence sing amino acids 2—24 of SEQ ID NO: 25; (111) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 25; (112) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 25; (113) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 27; (114) a targeting sequence comprising amino acids 15—30 of SEQ ID NO: 27; (115) a targeting sequence comprising SEQ ID NO:27; (116) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:28; (117) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 27; (118) a ing sequence comprising amino acids 5—30 of SEQ ID NO: 27; (119) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 27; (120) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 27; (121) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 29; (122) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 29; (123) a targeting sequence comprising SEQ ID NO:29; (124) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:30; (125) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 29; (126) a ing sequence comprising amino acids 5—33 of SEQ ID NO: 29; (127) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 29; (128) a targeting ce comprising amino acids 10—33 of SEQ ID NO: 29; (129) a targeting ce comprising amino acids 15—33 of SEQ ID NO: 29; (130) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 31 ; (131) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 31 ; (132) a targeting sequence comprising SEQ ID NO:31; (133) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:32; (134) a targeting ce comprising amino acids 2—24 of SEQ ID NO: 31; (135) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31; (136) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 31 ; (137) a targeting sequence comprising amino acids 1—15 of SEQ ID NO: 33; (138) a targeting sequence sing SEQ ID NO:33; (139) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:34; (140) a targeting sequence comprising amino acids 1—16 of SEQ ID NO: 35; (141) a targeting ce comprising SEQ ID NO:35; (142) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:36; (143) a targeting sequence sing amino acids 1—29 of SEQ ID NO:43; (144) a ing sequence sing amino acids 14—29 of SEQ ID NO: 43; (145) a targeting ce comprising SEQ ID NO: 43; (146) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 44; (147) a targeting sequence comprising amino acids 2—29 of SEQ ID NO: 43 ; (148) a targeting sequence sing amino acids 5—29 of SEQ ID NO: 43 ; (149) a targeting sequence comprising amino acids 8—29 of SEQ ID NO: 43; (150) a targeting sequence comprising amino acids 10—29 of SEQ ID NO: 43; (151) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 45; (152) a targeting sequence comprising amino acids 20—35 of SEQ ID NO: 45 ; (153) a targeting sequence comprising SEQ ID NO: 45 ; (154) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 46; (155) a targeting ce comprising amino acids 2—35 of SEQ ID NO: 45; (156) a ing sequence comprising amino acids 5—35 of SEQ ID NO: 45; (157) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 45; (158) a targeting ce comprising amino acids 10—35 of SEQ ID NO: 45; (159) a ing sequence comprising amino acids 15—35 of SEQ ID NO: 45 ; (160) a targeting ce comprising amino acids 1—43 of SEQ ID NO: 47; (161) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 47; (162) a targeting sequence comprising SEQ ID NO: 47; (163) an rium protein comprising an amino acid ce having at least 85% identity With SEQ ID NO: 48; (164) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 47; (165) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 47; (166) a ing sequence comprising amino acids 8—43 of SEQ ID NO: 47; (167) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 47; (168) a targeting sequence sing amino acids 15—43 of SEQ ID NO: 47; (169) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (170) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 47; (171) a ing sequence comprising amino acids 1—32 of SEQ ID NO: 49; (172) a targeting sequence comprising amino acids 17—32 of SEQ ID NO: 49; (173) a targeting sequence comprising SEQ ID NO: 49; (174) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 50; (175) a targeting sequence comprising amino acids 2—32 of SEQ ID NO: 49; (176) a targeting sequence comprising amino acids 5—32 of SEQ ID NO: 49; (177) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (178) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (179) a targeting sequence comprising amino acids 15—32 of SEQ ID NO: 49; (180) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 51; (181) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 51; (182) a targeting sequence comprising SEQ ID NO: 51; (183) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 52; (184) a targeting sequence sing amino acids 2—33 of SEQ ID NO: 51; (185) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 51; (186) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 51; (187) a ing sequence comprising amino acids 10—33 of SEQ ID NO: 51; (188) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 51; (189) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 53; (190) a targeting sequence sing amino acids 18—33 of SEQ ID NO: 53; (191) a targeting ce comprising SEQ ID NO: 53; (192) an exosporium protein sing an amino acid sequence having at least 85% identity With SEQ ID NO: 54; (193) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 53; (194) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53; (195) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53; (196) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 53; (197) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 53; (198) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 55; (199) a targeting sequence comprising amino acids 15—30 of SEQ ID NO: 55 ; (200) a targeting sequence comprising SEQ ID NO: 55 ; (201) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 56; (202) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 55; (203) a targeting sequence sing amino acids 5—30 of SEQ ID NO: 55; (204) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 55; (205) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 55; (206) a ing sequence comprising amino acids 1—130 of SEQ ID NO: 57; (207) a targeting ce comprising amino acids 115—130 of SEQ ID NO: 57; (208) a targeting sequence comprising SEQ ID NO: 57; (209) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 58; (210) a ing sequence comprising amino acids 2—130 of SEQ ID NO: 57; (211) a targeting sequence comprising amino acids 5—130 of SEQ ID NO: 57; (212) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (213) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 57; (214) a targeting sequence comprising amino acids 30— 130 of SEQ ID NO: 57; (215) a targeting ce comprising amino acids 40—130 of SEQ ID NO: 57; (216) a targeting sequence comprising amino acids 50—130 of SEQ ID NO: 57; (217) a targeting sequence comprising amino acids 60—130 of SEQ ID NO: 57; (218) a targeting sequence comprising amino acids 70—130 of SEQ ID NO: 57; (219) a targeting sequence comprising amino acids 80—130 of SEQ ID NO: 57; (220) a targeting sequence comprising amino acids 90—130 of SEQ ID NO: 57; (221) a targeting ce comprising amino acids 100—130 of SEQ ID NO: 57; (222) a targeting sequence comprising amino acids 110—130 of SEQ ID NO: 57; (223) an exosporium protein fragment comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 95; (224) a targeting ce comprising SEQ ID NO: 96; (225) a targeting sequence comprising SEQ ID NO: 97; (226) a targeting sequence comprising SEQ ID NO: 98; (227) a targeting sequence comprising SEQ ID NO: 99; (228) a ing sequence comprising SEQ ID NO: 100; (229) a targeting sequence comprising SEQ ID NO: 101; (230) a targeting sequence comprising SEQ ID NO: 102; (231) a targeting sequence comprising SEQ ID NO: 103; (232) a targeting sequence comprising SEQ ID NO: 104; (233) a targeting sequence comprising SEQ ID NO: 105; (234) a ing ce comprising SEQ ID NO: 106; (235) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (236) an exosporium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO: 109; (237) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 110; (238) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 111; (239) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 112; (240) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 113; (241) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 114; (242) an exosporium n comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 115; (243) an exosporium n comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 116; (244) an exosporium n comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 117; (245) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 118; (246) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 119; (247) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 120; (248) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 121 ; (249) a targeting sequence comprising amino acids 22—31 of SEQ ID NO: 1; (250) a targeting sequence comprising amino acids 22—33 of SEQ ID NO: 1; (251) a targeting sequence comprising amino acids 20—31 of SEQ ID NO: 1; (252) a targeting sequence sing amino acids 14—23 of SEQ ID NO: 3; (253) a targeting sequence comprising amino acids 14—25 of SEQ ID NO: 3; or (254) a targeting sequence comprising amino acids 12—23 of SEQ ID NO: 3.

When the protein or peptide of interest ses an antigen, a remediation enzyme, an enzyme suitable for ng an emulsion or gel in a hydraulic fracturing fluid or an antibacterial protein or peptide, preferably, the targeting sequence or exosporium protein comprises any of the targeting sequences or exosporium proteins listed above in paragraph for use With any n or peptide of interest or: (1) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 1; (2) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 1; (3) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 1; (4) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 1; (5) a targeting sequence sing amino acids 15—35 of SEQ ID NO: 1; (6) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 3; (7) a targeting ce comprising amino acids 5—27 of SEQ ID NO: 3; (8) a targeting ce sing amino acids 8—27 of SEQ ID NO: 3; (9) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 3; (10) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 5; (11) a targeting ce sing amino acids 5—38 of SEQ ID NO: 5; (12) a targeting sequence comprising amino acids 8—38 of SEQ ID NO: 5; (13) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 5; (14) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 5; (15) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 5; (16) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 7; (17) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 7; (18) a targeting sequence comprising amino acids 8—28 of SEQ ID NO: 7; (19) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 7; (20) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 9; (21) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 9; (22) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 9; (23) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 11; (24) a targeting ce comprising amino acids 5—33 of SEQ ID NO: 11; (25) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 11; (26) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 11; (27) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 11; (28) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 13; (29) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13; (30) a targeting sequence sing amino acids 8—33 of SEQ ID NO: 13; (31) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 13; (32) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 13; (33) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 15; (34) a ing sequence comprising amino acids 5—43 of SEQ ID NO: 15; (35) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 15; (36) a ing sequence comprising amino acids 10—43 of SEQ ID NO: 15; (37) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 15; (38) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 15; (39) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 15; (40) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 17; (41) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 17; (42) a ing sequence comprising amino acids 8—27 of SEQ ID NO: 17; (43) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 17; (44) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 19; (45) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 19; (46) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 19; (47) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 19; (48) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 19; (49) a targeting ce comprising amino acids 2—33 of SEQ ID NO: 21; (50) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 21; (51) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 21 ; (52) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 21 ; (53) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 21 ; (54) a targeting ce sing amino acids 2—24 of SEQ ID NO:23; (55) a targeting sequence sing amino acids 5—24 of SEQ ID NO: 23 ; (56) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 23; (57) a targeting ce comprising amino acids 2—24 of SEQ ID NO: 25; (58) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 25; (59) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 25; (60) a targeting ce comprising amino acids 2—30 of SEQ ID NO: 27; (61) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 27; (62) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 27; (63) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 27; (64) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 29; (65) a targeting sequence sing amino acids 5—33 of SEQ ID NO: 29; (66) a targeting ce comprising amino acids 8—33 of SEQ ID NO: 29; (67) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 29; (68) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 29; (69) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 31; (70) a targeting sequence sing amino acids 5—24 of SEQ ID NO: 31; (71) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 31 ; (72) a targeting sequence comprising amino acids 2—29 of SEQ ID NO: 43; (73) a targeting sequence comprising amino acids 5—29 of SEQ ID NO: 43; (74) a ing sequence comprising amino acids 8—29 of SEQ ID NO: 43; (75) a targeting sequence comprising amino acids 10—29 of SEQ ID NO: 43; (76) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 45; (77) a targeting ce comprising amino acids 5—35 of SEQ ID NO: 45; (78) a targeting sequence sing amino acids 8—35 of SEQ ID NO: 45 ; (79) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45 ; (80) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 45 ; (81) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 47; (82) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 47; (83) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 47; (84) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 47; (85) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 47; (86) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (87) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 47; (88) a targeting sequence comprising amino acids 2—32 of SEQ ID NO: 49; (89) a targeting sequence comprising amino acids 5—32 of SEQ ID NO: 49; (90) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (91) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (92) a targeting sequence comprising amino acids 15—32 of SEQ ID NO: 49; (93) a ing sequence comprising amino acids 2—33 of SEQ ID NO: 51; (94) a targeting ce comprising amino acids 5—33 of SEQ ID NO: 51; (95) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 51; (96) a ing sequence comprising amino acids 10—33 of SEQ ID NO: 51; (97) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 51; (98) a targeting ce sing amino acids 2—33 of SEQ ID NO: 53; (99) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53; (100) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53; (101) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 53; (102) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 53; (103) a targeting ce comprising amino acids 2—30 of SEQ ID NO: 55; (104) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 55; (105) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 55; (106) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 55; (107) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 57; (108) a targeting sequence comprising amino acids 5—130 of SEQ ID NO: 57; (109) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (110) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 57; (111) a targeting ce comprising amino acids 30— 130 of SEQ ID NO: 57; (112) a targeting sequence comprising amino acids 40—130 of SEQ ID NO: 57; (113) a targeting ce comprising amino acids 50—130 of SEQ ID NO: 57; (114) a targeting sequence comprising amino acids 60—130 of SEQ ID NO: 57; (115) a targeting sequence comprising amino acids 70—130 of SEQ ID NO: 57; (116) a targeting sequence comprising amino acids 80—130 of SEQ ID NO: 57; (117) a targeting sequence comprising amino acids 90—130 of SEQ ID NO: 57; (118) a targeting sequence comprising amino acids 100—130 of SEQ ID NO: 57; (119) a targeting ce comprising amino acids 110—130 of SEQ ID NO: 57.

When the protein or peptide of interest comprises an antigen, a ation enzyme, an enzyme suitable for breaking an emulsion or gel in a hydraulic fracturing fluid or an antibacterial protein or peptide, more preferably, the targeting sequence or exosporium protein comprises any of the targeting sequences or exosporium proteins listed above in paragraph for use With any protein or peptide of interest or: (1) a targeting ce comprising amino acids 2—24 of SEQ ID NO: 9; (2) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 9; (3) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 9; (4) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 11; (5) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 11; (6) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 11; (7) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 11; (8) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 11; (9) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 13; (10) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13; (11) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 13; (12) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 13 ; (13) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 13 ; (14) a targeting sequence sing amino acids 2—43 of SEQ ID NO: 15; (15) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 15; (16) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 15 ; (17) a ing sequence comprising amino acids 10—43 of SEQ ID NO: 15 ; (18) a targeting ce comprising amino acids 15—43 of SEQ ID NO: 15 ; (19) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 15; (20) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 15; (21) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 17; (22) a ing sequence comprising amino acids 5—27 of SEQ ID NO: 17; (23) a targeting ce comprising amino acids 8—27 of SEQ ID NO: 17; (24) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 17; (25) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 19; (26) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 19; (27) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 19; (28) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 19; (29) a targeting sequence sing amino acids 15—33 of SEQ ID NO: 19; (30) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 21 ; (31) a ing sequence comprising amino acids 5—33 of SEQ ID NO: 21; (32) a targeting sequence sing amino acids 8—33 of SEQ ID NO: 21; (33) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 21; (34) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 21 ; (35) a targeting sequence comprising amino acids 2—24 of SEQ ID NO:23; (36) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 23; (37) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 23; (38) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 25; (39) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 25; (40) a ing sequence sing amino acids 8—24 of SEQ ID NO: 25; (41) a targeting sequence comprising amino 2015/050807 acids 2—30 of SEQ ID NO: 27; (42) a ing sequence comprising amino acids 5—30 of SEQ ID NO: 27; (43) a ing sequence sing amino acids 8—30 of SEQ ID NO: 27; (44) a targeting sequence sing amino acids 10—30 of SEQ ID NO: 27; (45) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 29; (46) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 29; (47) a targeting ce sing amino acids 8—33 of SEQ ID NO: 29; (48) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 29; (49) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 29; (50) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 31 ; (51) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31 ; (52) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 31; (53) a targeting sequence comprising amino acids 2—29 of SEQ ID NO: 43; (54) a targeting sequence comprising amino acids 5—29 of SEQ ID NO: 43; (55) a targeting sequence comprising amino acids 8—29 of SEQ ID NO: 43; (56) a targeting sequence comprising amino acids 10—29 of SEQ ID NO: 43; (57) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 45 ; (58) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 45 ; (59) a ing sequence comprising amino acids 8—35 of SEQ ID NO: 45; (60) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45; (61) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 45; (62) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 47; (63) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 47; (64) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 47; (65) a targeting sequence sing amino acids 10—43 of SEQ ID NO: 47; (66) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 47; (67) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (68) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 47; (69) a targeting sequence comprising amino acids 2—32 of SEQ ID NO: 49; (70) a targeting sequence sing amino acids 5—32 of SEQ ID NO: 49; (71) a ing ce comprising amino acids 8—32 of SEQ ID NO: 49; (72) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (73) a targeting sequence comprising amino acids 15—32 of SEQ ID NO: 49; (74) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 51; (75) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 51 ; (76) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 51 ; (77) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 51 ; (78) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 51 ; (79) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 53; (80) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53 ; (81) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53; (82) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 53; (83) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 53; (84) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 55; (85) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 55; (86) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 55; (87) a targeting sequence sing amino acids 10—30 of SEQ ID NO: 55; (88) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 57; (89) a ing sequence comprising amino acids 5—130 of SEQ ID NO: 57; (90) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (91) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 57; (92) a targeting sequence comprising amino acids 30—130 of SEQ ID NO: 57; (93) a targeting sequence comprising amino acids 40—130 of SEQ ID NO: 57; (94) a targeting sequence sing amino acids 50—130 of SEQ ID NO: 57; (95) a targeting sequence comprising amino acids 60—130 of SEQ ID NO: 57; (96) a targeting sequence comprising amino acids 70—130 of SEQ ID NO: 57; (97) a targeting sequence comprising amino acids 80—130 of SEQ ID NO: 57; (98) a targeting ce sing amino acids 90—130 of SEQ ID NO: 57; (99) a ing sequence comprising amino acids 100—130 of SEQ ID NO: 57; (100) a targeting sequence comprising amino acids 110—130 of SEQ ID NO: 57.

When the protein or e of st comprises an antigen, a remediation enzyme, an enzyme suitable for breaking an emulsion or gel in a hydraulic fracturing fluid or an antibacterial n or peptide, even more ably, the targeting sequence or exosporium protein comprises: (1) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 9; (2) a ing sequence comprising amino acids 8—24 of SEQ ID NO: 9; (3) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 11; (4) a targeting sequence sing amino acids 8—33 of SEQ ID NO: 11; (5) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 11; (6) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 11; (7) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13; (8) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 13; (9) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 13; (10) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 13; (11) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 15 ; (12) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 15; (13) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 15; (14) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 15; (15) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 15; (16) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 15 ; (17) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 17; (18) a targeting ce comprising amino acids 8—27 of SEQ ID NO: 17; (19) a targeting ce comprising amino acids 10—27 of SEQ ID NO: 17; (20) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 19; (21) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 19; (22) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 19; (23) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 19; (24) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 21 ; (25) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 21 ; (26) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 21; (27) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 21; (28) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 23; (29) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 23; (30) a targeting sequence sing amino acids 5—24 of SEQ ID NO: 25; (31) a ing sequence comprising amino acids 8—24 of SEQ ID NO: 25; (32) a targeting ce comprising amino acids 5—30 of SEQ ID NO: 27; (33) a targeting sequence sing amino acids 8—30 of SEQ ID NO: 27; (34) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 27; (35) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 29; (36) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 29; (37) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 29; (38) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 29; (39) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31; (40) a ing sequence comprising amino acids 8—24 of SEQ ID NO: 31 ; (41) a targeting sequence comprising amino acids 5—29 of SEQ ID NO: 43; (42) a targeting sequence comprising amino acids 8—29 of SEQ ID NO: 43; (43) a targeting sequence comprising amino acids 10—29 of SEQ ID NO: 43; (44) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 45; (45) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 45 ; (46) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45; (47) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 45; (48) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 47; (49) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 47; (50) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 47; (51) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 47; (52) a targeting ce comprising amino acids 20—43 of SEQ ID NO: 47; (53) a ing sequence comprising amino acids 25—43 of SEQ ID NO: 47; (54) a targeting sequence comprising amino acids 5—32 of SEQ ID NO: 49; (55) a targeting sequence sing amino acids 8—32 of SEQ ID NO: 49; (56) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (57) a targeting sequence comprising amino acids 15—32 of SEQ ID NO: 49; (58) a targeting sequence comprising amino WO 44661 acids 5—33 of SEQ ID NO: 51; (59) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 51; (60) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 51; (61) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 51; (62) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53; (63) a targeting ce comprising amino acids 8—33 of SEQ ID NO: 53 ; (64) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 53; (65) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 53 ; (66) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 55; (67) a targeting sequence sing amino acids 8—30 of SEQ ID NO: 55; (68) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 55 ; (69) a targeting sequence comprising amino acids 5—130 of SEQ ID NO: 57; (70) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (71) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 57; (72) a ing sequence comprising amino acids 30—130 of SEQ ID NO: 57; (73) a targeting sequence comprising amino acids 40—130 of SEQ ID NO: 57; (74) a targeting sequence comprising amino acids 50—130 of SEQ ID NO: 57; (75) a targeting sequence comprising amino acids 60—130 of SEQ ID NO: 57; (76) a targeting sequence comprising amino acids 70—130 of SEQ ID NO: 57; (77) a targeting sequence sing amino acids 80—130 of SEQ ID NO: 57; (78) a targeting ce comprising amino acids 90—130 of SEQ ID NO: 57; (79) a targeting sequence comprising amino acids 100—130 of SEQ ID NO: 57; (80) a targeting ce comprising amino acids 110—130 of SEQ ID NO: 57; (81) a targeting sequence comprising amino acids 4— of SEQ ID NO: 59; (82) a targeting sequence comprising amino acids 6—30 of SEQ ID NO: 59; (83) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 61; (84) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 61 ; (85) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 61 ; (86) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 63; (87) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 63 ; (88) a targeting ce comprising amino acids 10—35 of SEQ ID NO: 63 ; (89) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 63; (90) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 65; (91) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 67; (92) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 67; (93) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 69; (94) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 69; (95) a targeting sequence sing amino acids 15—38 of SEQ ID NO: 69; (96) a ing sequence comprising amino acids 5—42 of SEQ ID NO: 75; (97) a targeting sequence comprising amino acids 10—42 of SEQ ID NO: 75; (98) a targeting ce comprising amino acids 15—42 of SEQ ID NO: 75; (99) a targeting sequence comprising amino acids 20—42 of SEQ ID NO: 75; (100) a targeting sequence comprising amino acids 25—42 of SEQ ID NO: 75; (101) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 77; (102) a ing sequence comprising amino acids 5—38 of SEQ ID NO: 81 ; (103) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 81; (104) a targeting sequence comprising amino acids l5—38 of SEQ ID NO: 81; (105) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 81 ; (106) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 87; (107) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 87; (108) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 89; (109) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 89; (110) a targeting ce comprising amino acids 10—93 of SEQ ID NO: 91; (111) a targeting sequence comprising amino acids 20—93 of SEQ ID NO: 91 ; (112) a targeting sequence comprising amino acids 30—93 of SEQ ID NO: 91 ; (113) a ing ce comprising amino acids 40—93 of SEQ ID NO: 91 ; (114) a targeting sequence comprising amino acids 50—93 of SEQ ID NO: 91; (115) a targeting sequence sing amino acids 60—93 of SEQ ID NO: 91; (116) a targeting sequence sing amino acids 10—130 of SEQ ID NO: 93 ; (117) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 93; or (118) a targeting sequence comprising amino acids 30—130 of SEQ ID NO: 93.

The protein or peptide of st of the fusion protein described above can comprise an n.

The protein or peptide of interest of the fusion protein described above can comprise a remediation enzyme.

The protein or peptide of interest of the fusion protein described above can comprise an enzyme suitable for breaking an emulsion or gel in a lic fracturing fluid.

The protein or peptide of st of the fusion protein described above can comprise an antibacterial protein or peptide.

C. Recombinant Bacillus cereus family members that exgress fusion groteins The present invention r relates to recombinant Bacillus cereus family s that express a fusion protein. The fusion protein can be any of the fusion proteins described above in Section I.B.

II. Modulation of Fusion Protein Expression in Recombinant Bacillus cereus Family Members that Express a Fusion Protein by Co-Overexpression of Modulator Proteins Recombinant Bacillus cereus family s that express the fusion proteins described herein display the protein or peptide of interest portion of the fusion protein on the outside of their spores. It has been found that overexpression of certain exosporium proteins (referred to herein as “modulator proteins”) in a recombinant us cereus family member that also ses a fusion protein allows for modulation (i.e., increasing or decreasing) the expression level of the fusion protein, thereby increasing or decreasing the amount of the protein or e of interest that is displayed on the outside of the spore. The ability to the control the amount of the protein or peptide of interest that is displayed on the outside of the spore is beneficial, since in some cases, it will be desirable to increase the amount of the protein or peptide of interest that is displayed. For example, where the protein of interest is an enzyme that degrades a plant nutrient source, it may be desirable to se the amount of the enzyme displayed on the spore, such that greater tic activity and greater stimulation of plant growth can be achieved upon introducing the spores into a plant growth medium or application of the spores to a plant or plant seed or an area surrounding a plant or a plant seed. In other ces, it will be desirable to decrease the amount of the protein or peptide of interest that is displayed. For example, where the protein or peptide of st comprises a plant immune system enhancer protein or peptide, it may be desirable to decrease the amount of the n or peptide displayed on the spore, since excess stimulation of a plant’s immune system can lead to undesirable s.

As is described further hereinbelow, the recombinant us cereus family members that s a tor protein can be used in any of the various fields and methods described herein, and for any of the uses described herein. For example, the recombinant Bacillus cereus family members that express a modulator protein can be used in methods for stimulating plant growth; methods for protecting a plant from a pathogen; methods for ing stress resistance in plants; methods for immobilizing recombinant Bacillus cereus family member spores on ; methods for stimulating germination of a plant seed; methods for delivering nucleic acids to a plant; methods for delivering nucleic acids to animals, insects, worms (e.g., nematodes), fungi, or protozoans; methods for delivering s to a plant; methods for ng a property of a plant; methods for delivering proteins or peptides to an animal; vaccines and methods of producing an immunogenic response in a subject; methods for reducing contaminants in an environment; s for phytoremediation of contaiminated soil; methods of treating a hydraulic fracturing fluid to break an on or gel within the fluid; methods of disinfecting a surface; and for uses such as , oil, or fat treatment or degumming; r hide processing; biofuel, biodiesel, or anol formation; sugar processing or sion; starch treatment; paper or linen processing; animal or fungal uct treatment or amino acid recovery; targeted digestion of facility ; feed or food additives; dietary supplements; animal nutrition; industrial cleaning; grain processing; cosmetic cturing; odor control; food or beverage processing; brewing enhancement or additives; detergent additives; or textile or yarn sing.

For many applications of proteins (e.g., enzymes), there is a biological response curve wherein an optimal tration of a protein or enzyme leads to the desired , and an excess of the n or too small of an amount of the protein leads to rable or diminished effects. One example of this biological curve is the demonstration that a biological drug, such as the protein drug insulin for diabetes treatment, requires an optimum dose in order to reduce blood sugar levels in diabetic patients. Too little insulin leads to an insufficient response and maintenance of undesired elevated blood sugar levels and potential hyperkalemia.

Too great of a dose of insulin leads to low blood sugar levels and potential hypokalemia and related morbidity.

Similar biological response curves exist for many of the proteins and peptides of interest comprised within the fusion proteins described herein. Thus, for the various fields of use and methods for the recombinant Bacillus cereus family members described herein, it may be desirable to modulate the expression level of the protein or peptide of interest on the exosporium. By increasing or decreasing the expression levels of the protein or peptide of st on the exosporium of the recombinant Bacillus cereus family member, expression levels can be optimized to maintain the overall expression level of the protein or peptide of interest at the most effective concentration.

For e, it would be desirable to modulate expression levels of the fusion protein in cases where the protein or peptide of interest comprises a protein or peptide involved in direct signaling in plants, such as the flagellin peptide flg22, and the recombinant Bacillus cereus family member expressing the fusion protein is to be applied to a plant to e a beneficial effect to the plant. Such modulation would be beneficial to avoid a signaling response that is great enough that it would lead to detrimental responses to the plant (e.g., too great of a response to flg22 can result in necrosis), or a signaling response that is low enough that it would yield a poor or cient response to the peptide. 2015/050807 A biological response curve would also be relevant for recombinant Bacillus cereus family members sing a fusion protein wherein the protein or e of interest comprises an antigen. In such cases, it would be desirable to modulate the expression level of the fusion protein comprising the antigen to e an optimal range for generating a proper immune response in an animal. Too large of a dose could lead to injection site edema and unwanted inflammation, s too small of a dose could lead to insufficient vaccination or immune response.

Modulation of the expression level of a fusion protein on the exosporium of a recombinant Bacillus cereus family member also provides benefits, for example, when the recombinant Bacillus cereus family member is used for breaking an emulsion or gel in a hydraulic ring fluid. Polysaccharide gels are frequently used in the hydraulic fracturing processing gels. These gels e breaking. When the gel on is ready to break, the operator will desire that the break, which is an enzymatic reaction, happen at a particular optimized rate. Breaking the gel too quickly can lead to undesired side effects such as pooling of undigested gel fragments. On the other hand, breaking the gel too slowly leads to long wait times and increased expense. Using the techniques described hereinbelow, the enzyme levels on the exosporium of a recombinant Bacillus cereus family member expressing a fusion protein comprising an enzyme suitable for breaking an emulsion or gel in a hydraulic ring fluid can be ted to ensure that an zed level of enzyme is present for breaking gels, g to preferred results when used in the field.

A recombinant Bacillus cereus family member is provided that expresses: (i) a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant us cereus family member; and (ii) a modulator n, n the sion of the modulator protein is increased as compared to expression of the modulator protein in a wild-type Bacillus cereus family member under the same conditions. The modulator protein, when co-expressed with the fusion protein in the recombinant Bacillus cereus family member, results in increased or decreased expression of the fusion protein as compared to the expression level of the fusion protein in a recombinant Bacillus cereus family member that does not express the modulator protein at an increased level under the same conditions as compared to the expression of the modulator protein in a wild-type Bacillus cereus family member.

The modulator protein can se an 13st protein, an ExsFA/BxpB protein, a CotY protein, a CotO protein, an ExsFB protein, an InhAl protein, an InhA2 n, an 13st protein, an 13st protein, a YjCA protein, a YjCB n, a Bch protein, an AcpC protein, an InhA3 protein, an alanine racemase 1, an alanine racemase 2, a BclA protein, a BclB protein, a Bpr protein, a BclE protein, a BetA/BAS3290 protein, a CotE protein, an 13st protein, an ExsK protein, an 13st protein, a YabG protein, a Tgl protein, a SODAl protein, a SODAZ protein, a variant of any thereof, or a combination of any thereof.

For example, the modulator protein, When co-expressed in the recombinant Bacillus cereus family member With the fusion protein, results in increased expression of the fusion protein as ed to the expression level of the fusion protein in a recombinant Bacillus cereus family member that does not express the modulator n at an increased level under the same conditions as compared to the expression of the modulator protein in a wild-type Bacillus cereus family member. Where the tor protein, When co-expressed in the recombinant Bacillus cereus family member With the fusion n, results in such increased expression of the fusion protein, the modulator protein can comprise a BclB protein, a CotE protein, a BxpB protein, a CotO protein, a BclA protein, a variant of any thereof, or a combination of any thereof ] Alternatively, the modulator protein, When co-expressed in the recombinant Bacillus cereus family member With the fusion protein, s in decreased expression of the fusion n as compared to the expression level of the fusion protein in a recombinant Bacillus cereus family member that does not express the modulator protein at an increased level under the same conditions as compared to the expression of the modulator n in a Wild-type Bacillus cereus family member. Where the modulator protein, When co-expressed in the recombinant Bacillus cereus family member With the fusion protein, s in such decreased expression of the fusion protein, the modulator protein can comprise a Bch protein, an Ach protein, a YjcB n, a variant of any thereof, or a ation of any thereof.

For example, the modulator protein can comprise a CotO protein, a BclB protein, an ExsFA/BxpB protein, a YjcB protein, a variant of any thereof, or a combination of any f For ease of reference, descriptions of the tor proteins and their SEQ ID NOs. are listed in Table 2 below.

Table 2. Amino Acid Se uences for Modulator ns Modulator Protein SEQ ID NO.

Est, Bacillus ; iensis 123 ExsFA/BXoB, Bacillus ; iensis 124 CotY, Bacillus cereus 125 CotO, Bacillus anthracis 126 ExsFB, Variant l, us cereus 127 ExsFB, Variant 2, Bacillus cereus 128 InhAl, Bacillus cereus 129 InhA3, Bacillus m c0ia’es 130 Est, Bacillus cereus ATCC 10876 131 Est, Bacillus cereus 132 Y'CA, Bacillus cereus 133 Y'cB, Variant l, Bacillus cereus 134 Y'cB, Variant 2, Bacillus cereus 135 Bch, us anthracis 136 ACoC, Bacillus cereus 137 InhA2, Bacillus cereus 138 Alanine racemase 1, Bacillus cereus 139 Alanine racemase 2, us cereus 140 BclA, variant 1, Bacillus anthracis Sterne 141 BclA, variant 2, Bacillus anthracis 142 BclB, variant 1, Bacillus anthracis Sterne 143 BclB, variant 2, Bacillus anthracis Sterne 144 BX oA, Bacillus anthracis 145 BAS4623/BclE, variant 1, Bacillus anthracis Sterne 146 BAS4623/BclE, variant 2, Bacillus anthracis Sterne 147 BetA/BAS3290, Bacillus anthracis 148 CotE, Bacillus cereus _or0u 149 Est, Bacillus cereus 150 ExsK, us cereus AH187 151 Est, Bacillus cereus 152 YabG, us cereus 153 Tl, us cereus _or0u 154 SODAl, Bacillus cereus 155 SODA2, Bacillus thurin iensis 156 Many of the modulator proteins have homologs, paralogs, or genetic rearrangements. Thus, many proteins that have at least 70% gy to any of the modulator sequences listed above in Table 2 will retain the ability to act as modulator proteins when overexpressed in a recombinant Bacillus cereus family member that also expresses any of the fusion proteins described herein. In addition, many of the modulator proteins (e.g., BclA, BclB, and BclE) have internal repeat regions that can differ significantly between strains. Additions or reductions in the number of repeats in the internal repeat region would affect overall sequence homology, but so long as the gy of amino- and y-terminal regions of the protein retain at least 75% sequence identity to any of the amino acid sequences of the modulator proteins listed in the table above, such homologs would be expected to retain the ability to act as modulator proteins.

Thus, for e, the modulator protein can comprise an amino acid sequence having at least 70% sequence identity With any of SEQ ID NOs: 123—156.

The modulator protein can comprise an amino acid sequence having at least 75% ce identity With any of SEQ ID NOs: 123—156.

The modulator protein can comprise an amino acid sequence having at least 85% sequence identity With any of SEQ ID NOs: 123—156.

The modulator protein can comprise an amino acid sequence having at least 90% sequence ty With any of SEQ ID NOs: 123—156.

The modulator protein can comprise an amino acid sequence having at least 95% sequence identity With any of SEQ ID NOs: 123—156.

The modulator protein can se an amino acid sequence having at least 98% sequence identity With any of SEQ ID NOs: 123—156.

The modulator n can comprise an amino acid sequence having at least 99% sequence identity With any of SEQ ID NOs: 123—156.

The modulator protein can comprise an amino acid sequence having 100% sequence identity With any of SEQ ID NOs: 6.

For e, the modulator protein can comprise SEQ ID NO: 124, 126, 134, 135, 143, or 144.

The inant Bacillus cereus family s that express a tor protein can comprise a vector encoding the modulator protein. For example, the vector can comprise a multicopy plasmid. Multicopy plasmids allow for high expression levels of the modulator protein. 111. Promoters for Expression of Fusion Proteins and/0r Modulator Proteins in Recombinant Bacillus cereus Family Members When the fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that s the fusion protein to the exosporium of a Bacillus cereus family member, the DNA encoding the fusion protein is suitably under the control of a sporulation promoter Which Will cause expression of the fusion protein on the exosporium of a B. cereus family member endospore (e.g., a native bclA promoter from a B. cereus family member).

Thus, any of the fusion proteins described above in Section l.B can be expressed in the recombinant Bacillus cereus family member under the control of a sporulation promoter that is native to the targeting sequence, rium protein, or exosporium protein nt of the fusion protein, or a portion of such a promoter.

Similarly, any of the modulator proteins described above in Section II can be expressed under the control of its native promoter or a portion thereof Any of the fusion proteins or modulator proteins can be expressed under the control of a high-expression sporulation promoter.

] The high-expression ation promoter comprises a sigma-K sporulation- specific polymerase promoter sequence.

For ease of reference, exemplary nucleotide sequences for promoters that can be used to express any of the fusion proteins or any of the modulator proteins in a recombinant Bacillus cereus family member are provided in Table 3 below, together with their SEQ ID NOs.

Table 3 also provides exemplary l promoter sequences for many of the promoters. In Table 3, sigma-K sporulation-specific polymerase promoter sequences in the ers are indicated by bold and underlined text. Several of the sequences have multiple sigma K sequences that overlap with one another. The overlaps are indicated by double underlining in the table. The promoter ces are immediately upstream of the start codon for each of the indicated genes. In other words, in the sequences shown in Table 3 below, the last tide of the promoter sequence ately precedes the first nucleotide of the start codon for the coding region of the gene encoding the indicated protein.

Table 3. Promoter Sequences for Expression of Fusion Proteins and Modulator Proteins in Recombinant Bacillus cereus famil members er Promoter Sequence (SEQ ID NO) EXSY promoter TTTCTTAATCCTTTACCCTTTACTTTTGTAAAAGTTGATACACTT (B. CereuS F83 7/76) GGCTCTGTAATTTCTAATTCATCAATAAATGGTCTTCG CAAAAAGCCTGTAATTTTATCATAAACAATTAAACGAGTGAGC (SEQ ID NO: 157) CTAAAAGCAGCTAACGCGAAAATAAAAAATAAAAGCCAGCTT GTAAACAGCATAATTCCACCTTCCCTTATCCTCTTTCGCCTATT AAGGTCTTGAGATTGTGACCAAATCTCCTCAACTCCA ATATCTTATTAATGTAAATACAAACAAGAAGATAAGGA Est minimal promoter ACCAAATCTCCTCAACTCCAATATCTTATTAATGTAAATACAA (B. cereus F837/76) AGATAAGGA SEQ ID NO: 15 8 EXSFA/BX oB o romoter ACCACCTACCGACGATCCAATCTGTACATTCCTAGCTGTACCA Promoter Promoter Sequence (SEQ ID NO) (B. anthracis Sterne) AATGCAAGATTAATATCGACTAACACTTGTCTTACTGTTGATTT (SEQ ID NO: 159) AAGTTGCTTCTGTGCGATTCAATGCTTGCGTGATGTTACGATTT AAAACTAAATAATGAGCTAAGCATGGATTGGGTGGCAGAATT ATCTGCCACCCAATCCATGCTTAACGAGTATTATTATGTAAATT AATTGGGAACTTGTCTAGAACATAGAACCTGTCCTTTT CATTAACTGAAAGTAGAAACAGATAAAGGAGTGAAAAAC ExsFA/BxpB minimal AACCTGTCCTTTTCATTAACTGAAAGTAGAAACAGAT promoter (B. cis AAAGGAGTGAAAAAC Sterne) SEQ ID NO: 160 CotY/CotZ promoter (B. AAGAACGCCGACTACTTTATGTCGCAATTACACGGGC anthracis Sterne) GAAAGAAGAACTTTACATTTCCTCTCCGCAATTTTTTAGAGGA AAAAAATTAGATATATCTCGTTTTTTATACACTGTGCGAAAAG (SEQ ID NO: 161) ATTTACCTGAAAAGACATCCACTAAATAAGGATGTCTTTTTTTA TATTGTATTATGTACATCCCTACTATATAAATTCCCTGCTTTTAT CGTAAGAATTAACGTAATATCAACCATATCCCGTTCATATTGT AGTAGTGTATGTCAGAACTCACGAGAAGGAGTGAACATA CotY/CotZ minimal TCAACCATATCCCGTTCATATTGTAGTAGTGTATGTCAGAACT CACGAGAAGGAGTGAACATA promoter (B. anthracis Sterne) SEQ ID NO: 162 CotO promoter (B. cereus) TAACTCAATCTTAAGAGAAATTGAGGAGCGCGCACCACTTCGT (SEQ ID NO: 163) CGTACAACAACGCAAGAAGAAGTTGGGGATACAGCAGTATTCT TATTCAGTGATTTAGCACGCGGCGTAACAGGAGAAAACATTCA CGTTGATTCAGGGTATCATATCTTAGGATAAATATAATATTAA TTTTAAAGGACAATCTCTACATGTTGAGATTGTCCTTTTTATTT GTTCTTAGAAAGAACGATTTTTAACGAAAGTTCTTACCACGTTA TGAATATAAGTATAATAGTACACGATTTATTCAGCTACGT CotO minimal promoter (B. ATTCAGGGTATCATATCTTAGGATAAATATAATATTA cereus) ATTTTAAAGGACAATCTCTACATGTTGAGATTGTCCTTTTTATT (SEQ ID NO: 164) TGTTCTTAGAAAGAACGATTTTTAACGAAAGTTCTTACCACGTT ATGAATATAAGTATAATAGTACACGATTTATTCAGCTACGT EXSFB promoter (B. cereus CATAAAAATCTACTTTTCTTGTCAAAGAGTATGCTTATATGCGT F837/76) GCTCTTTTTATTTGGTTTTCTTTCATTTCTAAATAACATTTTCAA CTCTATTCATACTATTCTTTCAACTTTAGGTTACAAACTATTTCT (SEQ ID NO: 165) GTAGTGTTTCTTTTGTACTATAGGCAGTTAGTTTTATC CATAACAGTACACCTCTGCACTATTCACTATAAATTTTCATATA ETATTGTGCTTGTCCAAAACATGTGGTTATTACTCACGCGAT GAAAGAAAGGAGTGAAAAT EXSFB minimal promoter (B. ACTATTCACTATAAATTTTCATATATTATATTGTGCTTGTCCAA AACATGTGGTTATTACTCACGCGATCTAAATGAAAGAAAGGAG cereus F837/76) TGAAAAT SEQ ID NO: 166 InhAl promoter (B. AATACATGATAATGAAATCCGATTTTGTGTTTTATATAGTGAAT ngiensis serovar TATCAAATATTGTGTAGATGAAACAAAGATAAAATCCCCATTA kurstakz' str. HD-l) AACTCCCTCTATGGAAATTATAAATTGTTCGATAAAAACTTTCA (SEQ ID NO: 167) ATATTTTCAGAAAACATTGTTGAATTGTGATATATTCGTATGCT AACTATGAAATTTTTACAAATATATTAAAAACATTACATAATA EACTAAATATTGAAAAAATATTGAATTTTTAATAAAATTTAA TTTGTAATACATATTATTTATTAGGGGAGGAAATAAGGG Promoter Promoter Sequence (SEQ ID NO) InhAl minimal promoter (B. AAAATTTAATTTGTAATACATATTATTTATTAGGGGAGGAAAT thurz'ngiensis r kurstakz' str. HD-1) (SEQ ID NO: 168) InhA2 promoter (B. mycaides AATTGTGCATATTGTCTTTTAAATTTTCTATCTAAGTTATTTAAT strain 219298) ATATAATAAATAACTCTTTTTTGTGAGTTTTTTTGATACGAGGT (SEQ ID NO: 169) AAATAATCAGTACAGGGTCTGACCAGAGGACTGGAGGGCATG ATTCTATAAGGGAATATTTACTATTCCATGATTATAGAACTATG TCTTTTTTATTGTATATAGAAGGGGGGATAGGTCTATATTATA GAACTTATATATATTGTGCATTCCATATTATCAATTATCTAAAT TTTAAGTCTTGTTACAATTAATAAGGGAGGAAATAGTA InhA2 minimal promoter (B. ACTTATATATATTGTGCATTCCATATTATCAATTATCTAAATTT mycoides strain 219298) TAAGTCTTGTTACAATTAATAAGGGAGGAAATAGTA SEQ ID NO: 170 EXSJ promoter (B. AATGACGTTTTCAAGTTTGATTATCATTCATGTTTCCTATTTTAA thuringiensis serovar GAGAAACATATAACTCAACTACTTTTTTCAATGGCATCTTTTA kurstaki) TAGTACTTAGAATAGGAAAACACTCAACTATAAGAAAAGTAA (SEQ ID NO: 171) GGAGGAAATAA EXSJ minimal er (B. ACTACTTTTTTCAATGGCATCTTTTATAGTACTTAGAATAGGA thuringiensis r AAACACTCAACTATAAGAAAAGTAAGGAGGAAATAA kurstaki) SEQ ID NO: 172 EXSH promoter (B. cereus TAATGCTTAGTTTTTATACTCAAGTTAAAATGTGCTTT F837/76) TGGACCTAAGAGATAAACGTGGAAAAATAAAATAAACTCTTA AGTTTAGGTGTTTAATCTAAGCAGTCAATTATTAAAAACATAT (SEQ ID NO: 173) AATTAATATGTGAGTCATGAACATAATTAAATAATGTTTTCAA GTTTAATTATCGTTCATGTTTCCTATTTTAAGCAGAACAAATAA CTCAATTACTTTTTTCGATTGGATCTTTTTTAACTCTTATAATAG GAAAACACTCAACTATAAAAATAAGTAAGGAGGAAATAA EXSH minimal promoter (B. AATATGTGAGTCATGAACATAATTAAATAATGTTTTCAAGTTT AATTATCGTTCATGTTTCCTATTTTAAGCAGAACAAATAACTCA cereus F837/76) ATTACTTTTTTCGATTGGATCTTTTTTAACTCTTATAATAGGAA (SEQ ID NO: 174) AACACTCAACTATAAAAATAAGTAAGGAGGAAATAA YjCA promoter ATAAAAGGGCGTGTATTTGCTACTGATGCAGTATTGT (B. thuringiensis serovar GTGCGCCTAAAAATGGAATTTCACAACCAGATCCACATGTTGT kurstaki str. HD73) TGTAGAACAATCTTGTAATTCATTGATGAATTTTACAACGTCAA (SEQ ID NO: 175) CTACACAATGAGAAGAGCCATGGTGTTTATTTTCGTTACAACTC GTCACTCCTTATCTTCTTGTTTGTATTTACATTAATAA GATATTGGAGTTGAGGAGATTTGGTCACAATCTCAAGACCTTT AATAGGCGAAAGAGGATAAGGGAAGGTGGAATT YjCA minimal promoter TCTTGTTTGTATTTACATTAATAAGATATTGGAGTTGAGGAGAT (B. thuringiensis serovar TTGGTCACAATCTCAAGACCTTTTTTTTAAATAGGCGAAAGAG kurstaki str. HD73) GATAAGGGAAGGTGGAATT (SEQ ID NO: 176) Promoter Promoter Sequence (SEQ ID NO) chB promoter ATCAACTTTTACAAAAGTAAAGGGTAAAGGATTAAGAAAGTG (B. murmgl‘ensis serovar GATTGGCGAATTATTAAGCTGTTATTGGTGTACAGGTGTATGG kurstaki str. HD73) GCTTTTTTATTAGTTTTATATAATTGGATTCCGATCGTT (SEQ ID NO: 177) GCAGAGCCGTTACTTGCATTATTAGCTATTGCAGGAGCAGCAG CAATCATTGAAACGATTACAGGATATTTTATGGGAGAATAAT flTTTTCATAATACGAGAAAAAGCGGAGTTTAAAAGAATGAG GGAACGGAAATAAAGAGTTGTTCATATAGTAAATAGACAGAA chB minimal promoter ACGGAAATAAAGAGTTGTTCATATAGTAAATAGACAGAA (B. thuringiensis serovar kurstaki str. HD73) SEQ ID NO: 178 BCIC promoter TGAAGTATCTAGAGCTAATTTACGCAAAGGAATCTCAGGACAA (B. anthracis Sterne) CACTTTCGCAACACCTATATTTTAAATTTAATAAAAAAAGAGA (SEQ ID NO: 179) AGTCAGAAATTATAAAGCTAGCTGGGTTCAAATCAAA AATTTCACTAAAACGATATTATCAATACGCAGAAAATGGAAAA AACGCCTTATCATAAGGCGTTTTTTCCATTTTTTCTTCAAACAA ACGATTTTACTATGACCATTTAACTAATTTTTGCATCTACTATG ATGAGTTTCATTCACATTCTCATTAGAAAGGAGAGATTTA BCIC minimal promoter ACCATTTAACTAATTTTTGCATCTACTATGATGAGTTTCATTCA (B. cis Sterne) CATTCTCATTAGAAAGGAGAGATTTA SEQ ID No: 180 AcpC promoter (B. cereus GACTATGTTTATTCAGGATAAAATATAGCACTACACTCTCTCCT F837/76) CTTATTATGTAGCATCTCTCTAATCCATCATTTGTTTCATTTAGT (SEQ ID NO: 18 1) TAAAATTGTAAATAAAATCACATGATTTGTCAATTATAATTGTC ATTTCGACAATTAAACTTGTCAAAATAATTCTCATCATTTTTTC TCATCTTTCTAATATAGGACATACTACTATATATACAAAAGAC AATATGCAAATGTTCATACAAAAAATATTATTTTTCGATATAT AATATTAACTGATTTTCTAACATCAAGGAGGGTACAT AcpC l promoter (B. AGACAATATGCAAATGTTCATACAAAAAATATTATTTTTCGAI cereus F837/76) ATATAATATTAACTGATTTTCTAACATCAAGGAGGGTACAT SEQ ID NO: 182 InhA3 promoter (B. ATAGTGAGTAATATGGTAATCCATAGATTAAATAGTATAGAA mun-”gig”sis semvar AATATTTAATTCTTATTTTTATTAAAAAAGCATGAATCCCAGAT kurstaki str. HD73) TTACTGGGTTTTGATTGTAACTAAGAACATATAAAAGTTCACT (SEQ ID NO: 183) TATAGGAGAGTCTGTTTGTTTTTATATCTTATGTATTT CACCCTGCATAAAAAAATATTTCTCAACATTTTATTTGTTGAAA AATATTGAATATTCGTATTATAACGAATATTATGTTGTTATCGG CAAAAAACGATAATTTGCAGACACTGGGGAGGAAATACA InhA3 minimal er (B. TCTTATGTATTTCACCCTGCATAAAAAAATATTTCTCAACATTT thurz‘ngz‘ensz‘s semvar TATTTGTTGAAAAATATTGAATATTCGTATTATAACGAATATTA kurstaki str. HD73) TGTTGTTATCGGCAAAAAACGATAATTTGCAGACACTGGGGAG SEQ ID NO: 184 GAAATACA Alanine racemase l promoter CTTCGTCAGCAATAAGTGTGAGCGGAGAATTGGTTGATCTTGG (B. cereus F837/76) CTTTACAATTGGAGCATTGACGAAAGACTCTTTAACGTGGTCG (SEQ ID NO: 185) CATAACGGAGTAGAATATATGCTCGTGTCTAAAGGTTTAGAGC AGCTATTAATGGTTGCTCGTTCAGTTACAGAGAAGCA AGTGAAGTAAACTTCTTAGACGTGGTGATATATGTGCACCACG TCTTTTCTTAGTTTGAAGGGTGGATTTCATAAAAGAAGCATAT AAAAGAATAAGCTTCGCATATCGTGTATAAGGAAGTGTATTT Promoter Promoter Sequence (SEQ ID NO) e racemase 1 l ATAAAAGAATAAGCTTCGCATATCGTGTATAAGGAAGTGTAT promoter (B. cereus F837/76) TT SEQ ID NO: 186 Alanine racemase 2 promoter CATTTCAAATAATGAACGCTTCGATTGAATCGGAGCTATTTTCA (B. thurz‘ngz‘ensz‘s semvar AATCAATTTCAGTATATTGATCCAGCATTTGAATAGAAGTATC kurstaki str. HD73) AACAGCAACTTTAAGTTGATGCAATGCAGATTGTACAAACATT (SEQ ID N02 187) GTAATTCTCCTCTTCTCCGTATATAATAGTTTCTTGAGGGTATT ATATCATGCTCAAAATTCCGAAAATTCTAGTAGTTTGACTAGQ ATATTGAAAAGTATTATATTGTAAAAGGTCATATGAAACGTG AAATAGAATGGAATGCAATTATTGAGTTAGGAGTTAGACCA Alanine racemase 2 minimal TWAAAGGTWCGTGAAATAGAATGGAA promoter (B. thurz‘ngz‘ensz‘s TGCAATTATTGAGTTAGGAGTTAGACCA serovar kurstaki str. HD73) SEQ ID NO: 188 1301A promoter (B. cereus ATCGATGGAACCTGTATCAACCACTATAATTTCATCCACAATTT F837/76) TTTCAACTGAGTCTAAACAACGGGCTATTGTCTTCTCCTCATCT CGAACAATCATACATAAACTAATTGTAATTCCTTGCTTGTTCA (SEQ ID NO- 189)' ACATAATCACCCTCTTCCAAATCAATCATATGTTATACATATA flAACTTTCCATTTTTTTAAATTGTTCAAGTAGTTTAAGATTT CTTTTCAATAATTCAAATGTCCGTGTCATTTTCTTTCGGTTTTGQ ATCTACTATATAATGAACGCTTTATGGAGGTGAATTT 1301A minimal promoter (B. AATCAATCATATGTTATACATATACTAAACTTTCCATTTTTTT AAATTGTTCAAGTAGTTTAAGATTTCTTTTCAATAATTCAAATG cereus F837/76) TCCGTGTCATTTTCTTTCGGTTTTGCATCTACTATATAATGAAC (SEQ ID NO- 190)' GCTTTATGGAGGTGAATTT 1301B promoter (B. GACCTGTAAGTCTGTAGGGAAGAATAATTTCAAGAGCCAGTGA thuringiensis serovar TAATAGATTTTTTTGTTTTTTCATTCTTATCTTGAATATAAATCA CCTCATCTTTTAATTAGAACGTAACCAATTTAGTATTTTGAAA ian str. 97-27) TAGAGCTATCATTTTATAATATGAATACTACTAGTTATAGAAA (SEQ 113 N03 191) CGGCAAAAAGTTTAATATATGTAAAAATCATTTGGATATGAAA GCCATAGATTTTTTCGAAATGATAAATGTTTTATTTT GTTAATTAGGAAACAAAAATGTGGAATGAGGGGGATTTAA 130113 minimal promoter (B. ATATGAAAAAAGTAGCCATAGATTTTTTCGAAATGATAAATGT thuringiensis serovar TTGTTAATTAGGAAACAAAAATGTGGAATGAGGGGGA TTTAA konkukian str. 97-27) SEQ ID NO: 192 13pr promoter (B. cis TTTTCATCTGCTACATCGTGAAGTAATGCTGCCATTTCAATTAT ATTTCCTCCTTCTTGCTCGGATAAAGAAATCGCCAGTT str. Sterne) TATGTACACGCTCAATATGATACCAATCATGCCCACTGGCATC (SEQ ID NO- 193)- TTTTTCTAAAATATGTTTTACAAAAGTAATTGTTTTTTCTATCTT TTCTTGTTTTGTCATTTTATCTTCACCCAGTTACTTATTGTAACA CGCCCGCATTTTTTCATCAQATATTTTCTTGTCCGCCCATACA flGGTGGTAGGCATCATCATGAAGGAGGAATAGAT 13pr minimal promoter (B. ACATATTTTCTTGTCCGCCCATACACTAGGTGGTAGGCATCAT anthracis str. Sterne) GGAGGAATAGAT SEQ ID NO: 194 Ben; promoter (3 anthracis GGTGACGACAACATATACAAGAGGCACTCCTGCTGGTACTGTA ASteme) ACAGGAACAAATATGGGGCAAAGTGTAAATACATCGGGTATA GCTGTCCCGAATACAGATAATATGGATTCAACGGCG (SEQ ID NO‘ 195) GGACTCCCTTAAGAAATTAGGGGAGTCTTTATTTGGAAAAAGA GCTTATGTTACATAAAAACAGGAGTAATTGTTTTAAAAGTAGT ATTGGTGACGTTGTTAGAAAATACAATTTAAGTAGAAGGTGCG TTTTTATATGAAATATATTTTATAGCTGTACTTTACCTTTCAAG Promoter er Sequence (SEQ ID NO) BclE minimal promoter (B. ACAAGCTGTCCCGAATACAGATAATATGGATTCAACGGCGGG ACTCCCTTAAGAAATTAGGGGAGTCTTTATTTGGAAAAAGAGC anthracis ASterne) TTATGTTACATAAAAACAGGAGTAATTGTTTTAAAAGTAGTAT (SEQ ID NO: 196) TGGTGACGTTGTTAGAAAATACAATTTAAGTAGAAGGTGCGTT TTTATATGAAATATATTTTATAGCTGTACTTTACCTTTCAAG BetA promoter ATTTATTTCATTCAATTTTTCCTATTTAGTACCTACCGCACTCAC (B. anthracis Sterne) AAAAAGCACCTCTCATTAATTTATATTATAGTCATTGAAATCTA ATTTAATGAAATCATCATACTATATGTTTTATAAGAAGTAAAG (SEQ ID NO: 197) TACTTAATTAATACATATCTATACACTTCAATATCAC AGCATGCAGTTGAATTATATCCAACTTTCATTTCAAATTAAATA AGTGCCTCCGCTATTGTGAATGTCATTTACTCTCCCTACTAw TTAATAATTATGACAAGCAATCATAGGAGGTTACTAC BetA minimal promoter TAAGAAGTAAAGGTACCATACTTAATTAATACATATCTATACA (B. anthracis Sterne) CTTCAATATCACAGCATGCAGTTGAATTATATCCAACTTTCATT TCAAATTAAATAAGTGCCTCCGCTATTGTGAATGTCATTTACTC (SEQ ID NO: 198) CTACATTTAATAATTATGACAAGCAATCATAGGAGGT TACTAC CotE promoter (B. cereus AGTTGTACAAGAATTTAAATCTTCACAAACATATGTAAATGAC AH820) TTACTACAGCTAGTTGCAAGTACGATTTCTAACAACGTAACAG ATGAAATATTAATTTCAACTAATGGCGATGTATTGAAGGGTGA (SEQ ID NO: 199) AACGGGCGCAGCGGTAGAAAGTAAAAAAGGAAATTGTGGTTG TTAAAGAGATGTCGAAATGACATCTCTTTTTTTAGTGGATTAAA CGTAAGTTCTTCTCAAAAAAAGAATGACACATTCCGCTATTGT CACGCATATGATTAAGTGAATAGTGATTGAGGAGGGTTACGA CotE minimal promoter (B. ACATTCCGCTATTGTCACGCATATGATTAAGTGAATAGTGATT GAGGAGGGTTACGA cereus AH820) SEQ ID NO: 200 EXSA promoter (B. cereus AACGTTATTAGCGTAGACAAACAAGTAACGGCAGAAGCAGTTC strain ATCC 10876) TTGCATTAAATCGTATGTTAGAGCGTGTGTAAAGCAACGGTAT (SEQ ID NO: 201) TCCCGTTGCTTTTTTTCATACATATAATCATAACGAGAACGAA ATGGGCATACATTGTTTTGAAGAAATCATTGTGGTTCTTTATG CTTATTCCACTTCGAATGATATTGAAAATCGAAGAAGTGATAA AAGTAAAAAGAAGTTAATGTTATTTAGAAAGAGTTACTTCATG AGATTTGTTACTTATAGATAAGTTATACAGGAGGGGGAAAAT EXSA minimal promoter (B. TCATGAGATTTGTTACTTATAGATAAGTTATACAGGAGGGGGA cereus strain ATCC 10876) AAAT SEQ ID NO: 202 ExsK promoter (B. AAGCCGCGGTCAATGCTGTATATGCAAATAAGATTGCAGCTTT thuringiensis serovar AGAAGAGCGTGATAGCTTCATTGCTGAAAAACGAGA ian str. 97-27) AGAGTATAAGAAAGATATTGATATTTACCATTTAGCATCAGAG (SEQ ID NO: 203) ATGGTCATTGATGGTATTGTTCATCCAAACAATTTAAGAGAAG AGTTAAAAGGACGATTCGAAATGTATATGAGTAAATATCAAGT ATTTACGGATCGTAAACATCCTGTTTATCCAGTTTAAAAGCCC TATTTAGGGCTTTCTTGCTCAAAAAGTTAAGGAGGGGAAAACA ExsK minimal er (B. TCAAGTATTTACGGATCGTAAACATCCTGTTTATCCAGTTTAA thuringiensis serovar AAGCCCTATTTAGGGCTTTCTTGCTCAAAAAGTTAAGGAGGGG konkukian str. 97-27) AAAACA (SEQ ID NO: 204) Promoter er Sequence (SEQ ID NO) EXSB promoter (B. cereus AGGATTTCAGTGGGACGCCTCCTCTCTTCTTACATTAAATTAAT F837/76) CATACTATAAAATGAAAGAAATGAAATGAAAAATAGCGGAAA (SEQ ID NO: 205) AATCAGAAATTTTTTCTGGTAGTATACAAIATGTTACAATAAG CTTTGTCAATGAAAGAAGGAATTCCGTGCAATGCACGGGAGAG GTTCGCGAACTCCCTCTATAAAAAACTATGGAAACAACAATAT CTTTAGGTATTGTTTTGTTTTTTTATTGTGACAGTTCAAGAACG CTTCTTATTCGTAGTAGAGAAGGAGAATGAGTGAA EXSB minimal promoter (B. ACTATGGAAACAACAATATCTTTAGGTATTGTTTTGTTTTTTTA cereus F837/76) TTGTGACAGTTCAAGAACGTTCTTTCTTCTTATTCGTAGTAGAG SEQ ID NO: 206 AAGGAGAATGAGTGAA YabG promoter (B. cereus TTTTGCACAACGCCGTAAAACTTTAATGAATAATTTATCAfl AH820) AATTTAAATGGTTTCCCGAAAGATAAAGAGCTGTTGGATCGAA (SEQ ID NO: 207) TTTTAACAGAAGTAGGAATTGATCCAAAACGAAGAGGCGAAA CGCTATCTATCGAAGAGTTTGCGACATTAAGTAATGCATTAGTT CTTCATAAGTTATCATAAGAATACAAAAGGGACAGTTCAATTT GAACTGTCCCTTTTGTCACCTTTCTCCTCCTAAATTCATACTTT AAAAACAGGTAAGATGGCCTAACGAGTTTGGAGGTAGGAGA YabG l promoter (B. TCTCCTCCTAAATTCATACTTTAAAAACAGGTAAGATGGCCTA cereus AH820) ACGAGTTTGGAGGTAGGAGA SEQ ID NO: 208 Tgl promoter (B. GGAAACAGAAGTCATCCCATTTGAAAATGCAGCAGGTCGTATT thuringiensis serovar ATAGCTGATTTCGTTATGGTTTATCCGCCAGGGATTCCAATCTT konkukian str. 97-27) TACTCCGGGGGAAATTATTACACAAGACAACTTAGAGTATATT (SEQ ID NO: 209) CGTAAAAACTTAGAAGCAGGTTTACCTGTACAAGGTCCTGAAG ATATGACATTACAAACATTACGCGTGATCAAAGAGTACAAGCC TATCAGTTGATAGGCTTTTTTTCACCCTTTTTCCCTTTTCTCATA CGATATTATGTAATGTAACGTATAGGTGGGGATACTACT Tgl minimal promoter (B. ACCCTTTTTCCCTTTTCTCATACGATATTATGTAATGTAACGTA thuringiensis r TAGGTGGGGATACTACT konkukian str. 97-27) SEQ ID NO: 210 Superoxide dismutase ATTGTGGACCCTTAGCTCAGCTGGTTAGAGCAGACGGCTCATA (SODAl) er (B. ACCGTCCGGTCGTAGGTTCGAGTCCTACAGGGTCCATATCCATT TCACATGTTTATTATGTCGGCAGGAAGCTTCCTTGTAGAAGGG cereus F837/76) AGCTTTTTTTATGAAATATATGAGCATTTTAATTGAAATGAAGT (SEQ ID NO: 21 l) GGGAATTTTGCTACTTTAATGATAGCAAGACAATGTGATTTATT TGTTTGCACCCTATGGCAATTAGGGTAGAATGAAGTTGTATGT CACTTAAGTGGCAATACATAAACTGGGAGGAATATAACA xide dismutase ACTTAAGTGGCAATACATAAACTGGGAGGAATATAACA (SODAl) minimal promoter (B. cereus F837/76) SEQ ID NO: 212 Superoxide dismutase ACAGAAAATTCTGATGTTTTTTCAAATCCTATAATAAG (SODA2) promoter (B. TCCGTATGATGCCTTTATATTTTCCGGAAGATAAAAC AGAATATATTATTCCAGGGATTGTTTGTGTTCTATTTATCATCG cereus AH820) GTGCGATTGCTACGTGGCGTATGTTCATTCGTGTATCAAAACG (SEQ ID NO: 213) AGAAGCAGAGCGATTACAGAAAGTTGAAGAAAAGCTGTTAGC TGAAAAGAAACAGTAACTCATTTTTGTATGTTTCCCTCTATGCT CGGACAATCTAAGGGCAGAATGTATTTTGGAGGGAATGAA Promoter Promoter ce (SEQ ID NO) Superoxide dismutase TCCGGAAGATAAAACAGAATATATTATTCCAGGGATTGTTTGT (SODA2) minimal promoter GTTCTATTTATCATCGGTGCGATTGCTACGTGGCGTATGTTCAT TCGTGTATCAAAACGAGAAGCAGAGCGATTACAGAAAGTTGA (B. cereus AH820) AGAAAAGCTGTTAGCTGAAAAGAAACAGTAACTCATTTTTGTA (SEQ ID NO: 214) TGTTTCCCTCTATGCTCGGACAATCTAAGGGCAGAATGTATTTT GGAGGGAATGAA BclA er TAATCACCCTCTTCCAAATCAATCATATGTTATACATATACTA (B. anthracis Sterne) AACTTTCCATTTTTTTAAATTGTTCAAGTAGTTTAAGATTTCTT (SEQ ID NO: 215) TTCAATAATTCAAATGTCCGTGTCATTTTCTTTCGGTTTTGCAT CTACTATATAATGAACGCTTTATGGAGGTGAATTT BAS1882 er (B. AATTACATAACAAGAACTACATTAGGGAGCAAGCAGTCTAGCG anthracis Sterne) AACTGCTTTTTTATTAAATAACTATTTTATTAAATTTC ATATATACAATCGCTTGTCCATTTCATTTGGCTCTACCCACGQ (SEQ ID NO: 216) TTTACTATTAGTAATATGAATTTTTCAGAGGTGGATTTTATT Gene 3572 promoter CTATGATTTAAGATACACAATAGCAAAAGAGAAACATATTAT (B. weihenstephensis ATAACGATAAATGAAACTTATGTATATGTATGGTAACTGTATA TATTACTACAATACAGTATACTCATAGGAGGTAGGT KBAB 4) SEQ ID NO: 217 YVTN B-propeller protein GGTAGGTAGATTTGAAATATGATGAAGAAAAGGAATAACTAA AAGGAGTCGATATCCGACTCCTTTTAGTTATAAATAATGTGGA promoter ATTAGAGTATAATTTTATATAGGTATATTGTATTAGATGAACGC (B. weihenstephensis TTTATCCTTTAATTGTGATTAATGATGGATTGTAAGAGAAGGG KBAB 4) GCTTACAGTCCTTTTTTTATGGTGTTCTATAAGCCTTTTTAAAA (SEQ ID NO: 218) GGGGTACCACCCCACACCCAAAAACAGGGGGGGTTATAACTA CATATTGGATGTTTTGTAACGTACAAGAATCGGTATTAATTACC CTGTAAATAAGTTATGTGTATATAAGGTAACTTTATATATTCT CCTACAATAAAATAAAGGAGGTAATAAA Cry1A er AACCCTTAATGCATTGGTTAAACATTGTAAAGTCTAAAGCATG (B. thuringiensis HD-73) GATAATGGGCGAGAAGTAAGTAGATTGTTAACACCCTGGGTCA AAAATTGATATTTAGTAAAATTAGTTGCACTTTGTGCATTTTTT (SEQ ID NO: 219) CATAAGATGAGTCATATGTTTTAAATTGTAGTAATGAAAAAC AGTATTATAT; QATAATGAATTGGTATCTTAATAAAAGAGATGG AGGTAACTTA Est promoter TAATTCCACCTTCCCTTATCCTCTTTCGCCTATTTAAAAAAAGG (B. thuringiensis serovar TCTTGAGATTGTGACCAAATCTCCTCAACTCCAATATCTTATTA ATACAAACAAGAAGATAAGGA konkukian str. 97-27) SEQ ID NO: 220 CotY promoter AGGATGTCTTTTTTTATATTGTATTATGTACATCCCTACTATATA (B. thuringiensis A1 Hakarn) AATTCCCTGCTTTTATCGTAAGAATTAACGTAATATCAACCATA TCCCGTTCATATTGTAGTAGTGTATGTCAGAACTCACGAGAAG (SEQ ID NO: 221) ACATAA YjCA promoter TTAATGTCACTCCTTATCTTCTTGTTTGTATTTACATTAATAAG (B. thuringiensis serovar flTTGGAGTTGAGGAGATTTGGTCACAATCTCAAGACCTTTTT TAGGCGAAAGAGGATAAGGGAAGGTGGAATT kurstaki str. HD73) SEQ ID NO: 222 YjCB promoter TTTCATAATACGAGAAAAAGCGGAGTTTAAAAGAATG (B. thuringiensis serovar AGGGAACGGAAATAAAGAGTTGTTCATATAGTAAATAGACAG kurstaki str. HD73) SEQ ID NO: 223 ExsFA/BxpB promoter AAACTAAATAATGAGCTAAGCATGGATTGGGTGGCAGAATTAT (B. thuringiensis A1 Hakarn) CTGCCACCCAATCCATGCTTAACGAGTATTATTATGTAAATTT CTTAAAATTGGGAACTTGTCTAGAACATAGAACCTGTCCTTTTQ (SEQ ID NO: 224) ATTAACTGAAAGTAGAAACAGATAAAGGAGTGAAAAAC Promoter Promoter Sequence (SEQ ID NO) Rhamnose promoter ATTCACTACAACGGGGATGAGTTTGATGCGGATACATATGAG (B mun-”gig”sis A1 Hakam) AAGTACCGGAAAGTGTTTGTAGAACATTACAAAGATATATTAT SEQ ID NO- 225 CTCCATCATAAAGGAGAGATGCAAAG CotO promoter (B. anthracis CGCGCACCACTTCGTCGTACAACAACGCAAGAAGAAGTTGGGG Sterne) ATACAGCAGTATTCTTATTCAGTGATTTAGCACGCGGCGTAAC AGGAGAAAACATTCACGTTGATTCAGGGTATCATATCTTAGGA (SEQ ID NO- 226)- TAAATATAATATTAATTTTAAAGGACAATCTCTACATGTTGAG CTTTTTATTTGTTCTTAGAAAGAACGATTTTTAACGAA AGTTCTTACCACGTTATGAATATAAGTATAATAGTACACGATTT CTACGTA Sigma K promoter TATATCATATGTAAAATTAGTTCTTATTCCCACATATCATATAG (B. cis Sterne) AATCGCCATATTATACATGCAGAAAACTAAGTATGGTATTATT CTTAAATTGTTTAGCACCTTCTAATATTACAGATAGAATCCGTC (SEQ ID NO' 227)- ATTTTCAACAGTGAACATGGATTTCTTCTGAACACAACTCTTTT TCTTTCCTTATTTCCAAAAAGAAAAGCAGCCCATTTTAAAATAC GGCTGCTTGTAATGTACATTA InhAl promoter TATCACATAACTCTTTATTTTTAATATTTCGACATAAAGTGAAA (B. thm-ngZ-enSl-S A1 Hakam) CTTTAATCAGTGGGGGCTTTGTTCATCCCCCCACTGATTATTAA TTGAACCAAGGGATAAAAAGATAGAGGGTCTGACCAGAAAAC (SEQ ID NO- 228)' TGGAGGGCATGATTCTATAACAAAAAGCTTAATGTTTATAGAA TTATGTCTTTTTATATAGGGAGGGTAGTAAACAGAGATTTGGA CAAAAATGCACCGATTTATCTGAATTTTAAGTTTTATAAAGGG GAGAAATG BclA cluster g1ycosy1 ATTTTTTACTTAGCAGTAAAACTGATATCAGTTTTACTGCTTTTT transferase Operon 1 CATTTTTAAATTCAATCATTAAATCTTCCTTTTCTACATAGTQ TAATGTTGTATGACATTCCGTAGGAGGCACTTATA (B thurmgiensis r konkukian str. 97-27) SEQ ID NO: 229 BclA cluster glycosyl ACATAAATTCACCTCCATAAAGCGTTCATTATATAGTAGATGC transferase Operon 2 AAAACCGAAAGAAAATGACACGGACATTTGAATTATTGAAAA GAAATCTTAAACTACTTGAACAATTTAAAAAAATGGAAAGTTT (B' thufmglenm WOW” TGTATAACATATGATTGATTTGGAAGAGGGTGATTA kurstaki str. HD73) SEQ ID NO: 230 Glycosyl transferase TTCTATTTTCCAACATAACATGCTACGATTAAATGGTTTTTTGC AAATGCCTTCTTGGGAAGAAGGATTAGAGCGTTTTTTTATAGA promoter AACCAAAAGTCATTAACAATTTTAAGTTAATGACTTTTTTGTTT (B' thuringiensis A1 Hakam) GCCTTTAAGAGGTTTTATGTTACTATAATTATAGTATCAGGTAC (SEQ 113 N03 231) TAATAACAAGTATAAGTATTTCTGGGAGGATATATCA The sigma-K sporulation-specific polymerase promoter sequences in the er sequences shown in Table 3 result in high expression levels of the fusion protein or modulator protein during late sporulation. The sus sequence for the sigma-K sporulation- specific rase promoter sequence is CATANNNTN; however, this sequence can se up to two mutations and still be functional. The K sporulation-specific polymerase promoter sequence is generally found upstream of the ribosome binding site (RBS).

Promoters having a high degree of sequence identity to any of the sequences shown above in Table 3 can also be used to express the fusion proteins or the modulator proteins.

For example, the fusion protein or modulator protein can be sed under the control of a promoter sing a nucleic acid sequence having at least 80% identity With a nucleic acid ce of any one of SEQ ID NOs: 157—23 1.

The fusion protein or tor protein can be expressed under the control of a promoter sing a nucleic acid ce haVing at least 90% identity With a nucleic acid sequence of any one of SEQ ID NOs: 157—23 1.

The fusion protein or modulator protein can be expressed under the control of a promoter comprising a nucleic acid sequence haVing at least 95% identity With a nucleic acid sequence of any one of SEQ ID NOs: 157—23 1.

The fusion protein or modulator protein can be expressed under the control of a promoter comprising a nucleic acid sequence haVing at least 98%identity With a nucleic acid sequence of any one of SEQ ID NOs: 157—23 1.

The fusion n or modulator protein can be expressed under the control of a promoter sing a nucleic acid sequence haVing at least 99% identity With a nucleic acid sequence of any one of SEQ ID NOs: 157—23 1.

The fusion n or modulator protein can be expressed under the control of a promoter comprising a nucleic acid sequence haVing 100% identity With a nucleic acid sequence of any one of SEQ ID NOs: 157—23 1.

For example, the modulator protein or fusion protein can be expressed under the control ofa BclA promoter (e.g., SEQ ID NO: 189, 190, 215, 229 or 230), a CotY er (e.g., SEQ ID NO: 161, 162 or 221), an Est promoter (e.g., SEQ ID NO: 157, 158 or 220), or a rhamnose promoter (e.g., SEQ ID NO: 225). For example, the fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence haVing at least 80%identity With a nucleic acid sequence of any one of SEQ ID NOs: 157, 158, 161, 162, 189, 190, 215, 220, 221, 225, 229, or 230.

The fusion protein can be expressed under the control of a promoter comprising a c acid sequence haVing at least 85% identity With a nucleic acid sequence of any one of SEQ ID NOs: 157, 158, 161, 162, 189, 190, 215, 220, 221, 225, 229, or 230.

WO 44661 The fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having at least 90% identity with a nucleic acid ce of any one of SEQ ID NOs: 157, 158, 161, 162, 189, 190, 215, 220, 221, 225, 229, or 230.

The fusion protein can be expressed under the control of a promoter comprising a c acid sequence having at least 95% identity with a nucleic acid sequence of any one of SEQ ID NOs: 157, 158, 161, 162, 189, 190, 215, 220, 221, 225, 229, or 230.

The fusion protein can be expressed under the l of a promoter comprising a nucleic acid sequence having at least 98% identity with a nucleic acid ce of any one of SEQ ID NOs: 157, 158, 161, 162, 189, 190, 215, 220, 221, 225, 229, or 230.

The fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having at least 99% identity with a nucleic acid sequence of any one of SEQ ID NOs: 157, 158, 161, 162, 189, 190, 215, 220, 221, 225, 229, or 230.

The fusion protein can be expressed under the control of a promoter comprising a c acid sequence having 100% identity with a nucleic acid sequence of any one of SEQ ID NOs: 157, 158, 161, 162, 189, 190, 215, 220, 221, 225, 229, or 230.

The fusion protein or modulator protein can be expressed under the control of a promoter comprising a sigma-K sporulation specific polymerase promoter sequence, wherein the sigma-K sporulation-specific polymerase er ce or sequences have 100% identity with the corresponding nucleotides of any of SEQ ID NOs: 157—23 1.

The fusion proteins can be expressed under the control of a promoter that is native to the targeting sequence, exosporium protein, or exosporium protein fragment of the fusion protein. Thus, for example, where the targeting sequence is derived from BclA, the fusion protein can be expressed under the control of a native BclA promoter (e. g., SEQ ID NO: 189, 190,215,229 or 230).

The modulator proteins can be expressed under the control of their native promoters. Thus, for example, where the modulator protein comprises CotO, the CotO can be expressed under the control of a native CotO promoter (e. g., SEQ ID NO: 163 or 226). Native promoter sequences for each of the modulator proteins are provided above in Table 3.

Table 3 also provides exemplary minimal promoter sequences for each modulator protein. The modulator proteins and fusion proteins can be expressed under any of these minimal promoter sequences. For example, the tor protein can be expressed under a minimal er that comprises a portion of the native er sequence. For instance, where the modulator protein comprises CotO, the CotO can be expressed under the minimal CotO promoter (SEQ ID NO: 164).

Alternatively, the modulator proteins can be expressed under the control of any promoter sing a sigma-K sporulation-specif1c polymerase promoter sequence, regardless of whether the promoter is the native promoter for the modulator protein. As can be seen from Table 3, each of the native ers and the minimal promoters for the tor proteins contains at least one sigma-K sporulation-specif1c polymerase promoter sequence.

Thus, for example, where the modulator protein is BxpB, the BxpB can be expressed under the l ofa BclA promoter (e.g., SEQ ID NO: 189, 190, 215, 229 or 230) or any ofthe other ers listed in Table 3.

Furthermore, the modulator protein or the fusion protein can be expressed under a portion of any of the promoters listed above in Table 3, so long as the portion of the er includes a sigma-K sporulation-specif1c polymerase promoter sequence. For example, the modulator protein can be expressed under a promoter region that comprises the first 25, 50, 100, 150, 200, 250, or 300 nucleotides upstream of the start codon, so long as that region comprises a sigma-K sporulation-specif1c polymerase promoter ce.

IV. Mutations and Other Genetic Alterations to Recombinant Bacillus cereus Family Members that Allow for Collection of Free Exosporium As is described further hereinbelow, the inant Bacillus cereus family members that express fusion proteins comprising a protein or peptide of interest and a targeting sequence, an rium n, or an exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member can be used to r proteins or peptides of interest to plants, seeds, a plant growth medium, or an area surrounding a seed or a plant (e. g., via soil drench, foliar application, or as a seed treatment). In addition, the recombinant Bacillus cereus family members can be used to r nucleic acid molecules to animals, insects, worms (e. g., nematodes), fungi, and protozoans; to deliver ns or peptides to an animal; in vaccines and for producing an immunogenic response; for remediation; for treating a hydraulic fracturing fluid to break an emulsion or gel within the fluid; for disinfection; and for various other uses described hereinbelow. However, in some cases, the presence of the living microorganisms may not be desirable, and d, it would be ble to separate the living spore from the fusion proteins in the rium on the outside surface of the spore. For example, in some applications it will be desirable to increase enzyme activity without WO 44661 concern for spore integrity. In such situations, the exosporium fragments may be preferred over living rganisms having the enzyme on their rium.

] In addition, for some uses, it may be desirable to reduce the density of the product. In such instances, it would be desirable to separate the dense spore from the exosporium (containing the fusion proteins). In the field of vaccines, it may be desirable to separate the spore from the exosporium (containing fusion proteins that comprise an antigen) in order to remove ial antigens present on the spore itself from the vaccine preparation.

Furthermore, under some circumstances the presence of live spores would lead to potential for bacterial growth in a t, which would be rable for some applications (e.g., animal feed supplementation and leather hide processing).

Mutations or other genetic alterations (e. g., overexpression of a protein) can be introduced into the recombinant Bacillus cereus family members that allow free exosporium to be separated from spores of the recombinant Bacillus cereus family member. This tion process yields exosporium fragments that contain the fusion proteins but that are substantially free of the spores themselves. By “substantially free of spores” it is meant that once the free exosporium is separated from the spores, a preparation is obtained that contains less than 5% by volume of spores, preferably less than 3% by volume of spores, even more ably less than 1% by volume of spores, and most preferably contains no spores or if spores are present, they are undetectable. These exosporium fragments can be used in place of the recombinant us cereus family members themselves and can be used to deliver proteins or es of interest to plants, seeds, a plant growth medium, or an area surrounding a seed or a plant, or for any of the other purposes described herein.

Thus, a recombinant Bacillus cereus family member is provided that ses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member. The recombinant us cereus family member comprises a mutation or expresses a protein, wherein the expression of the protein is increased as compared to the expression of the protein in a wild-type Bacillus cereus family member under the same conditions. The mutation or the increased expression of the protein results in Bacillus cereus spores having an exosporium that is easier to remove from the spore as compared to the rium of a wild-type spore.

A further recombinant Bacillus cereus family member is provided that expresses a fusion protein comprising at least one protein or e of interest and a targeting sequence, exosporium protein, or exosporium n fragment that targets the fusion protein to the exosporium of the inant Bacillus cereus family member. The recombinant Bacillus cereus family member: (i) comprises a mutation in a CotE gene; (ii) expresses an 13st n, wherein the expression of the 13st protein is increased as compared to the expression of the 13st protein in a wild-type Bacillus cereus family member under the same conditions, and wherein the 13st protein comprises a carboxy-terminal tag comprising a ar protein; (iii) expresses a BclB protein, wherein the expression of the BclB protein is increased as compared to the expression of the BclB protein in a wild-type Bacillus cereus family member under the same conditions; (iV) expresses a chB protein, wherein the expression of the chB protein is increased as compared to the expression of the YjcB n in a wild-type Bacillus cereus family member under the same conditions;(V) comprises a mutation in an 13st Vi) comprises a on in a CotY gene;(Vii) comprises a mutation in an 13st gene; or (Viii) comprises a on in a CotO gene.

The recombinant Bacillus cereus family member can comprise a mutation in the CotE gene, such as a knock-out of the CotE gene or a nt negative form of the CotE gene. The mutation in the CotE gene can partially or completely inhibit the ability of CotE to attach the exosporium to the spore.

The recombinant Bacillus cereus family member can express an 13st protein. The 13st protein comprises a carboxy-terminal tag comprising a globular protein (e. g., a green fluorescent protein (GFP) or a variant f), and the expression of the 13st protein is increased as compared to the expression of the 13st protein in a wild-type Bacillus cereus family member under the same conditions. The globular protein can have a lar weight of between 25 kDa and 100 kDa. Expression of the 13st n comprising the carboxy-terminal tag comprising a globular n can also inhibit binding of the 13st protein to its targets in the exosporium.

The inant Bacillus cereus family member can express a BclB protein, which may result in the formation of a fragile rium. The expression of the BclB protein can be increased as compared to the expression of the BclB protein in a wild-type Bacillus cereus family member under the same conditions.

] The recombinant Bacillus cereus family member can express a chB protein, which may cause the exosporium to form in pieces rather than in a complete structure. The expression of the YjcB protein can be increased as compared to the expression of the YjcB protein in a wild-type Bacillus cereus family member under the same conditions.

The inant Bacillus cereus family member can comprise a mutation an 13st gene, such as a knock-out of the 13st gene. The mutation in the 13st gene can partially or completely t the ability ofEst to complete the formation of the exosporium or attach the exosporium to the spore.

The recombinant Bacillus cereus family member can comprise a on a CotY gene, such as a knock-out of the CotY gene. The mutation in the CotY gene can result in the formation of a fragile exosporium.

The recombinant Bacillus cereus family member can comprise a mutation an 13st gene, such as a knock-out of the 13st gene. The mutation in the 13st gene can result in the formation of a e exosporium.

The recombinant Bacillus cereus family member can comprise a mutation a CotO gene, such as a knock-out of the CotO gene or a dominant negative form of the CotO gene. The mutation in the CotO gene can cause the exosporium to form in strips.

Exosporium fragments can be prepared from any of these recombinant Bacillus cereus family members and used for various purposes as described further hereinbelow.

The exosporium fragments comprise the fusion proteins. Upon purification of the exosporium fragments that contain the fusion proteins from the spores, a cell-free protein preparation is obtained in which the fusion proteins are stabilized and supported through covalent bonds to the exosporium fragments.

Due to the strong covalent bonds between the fusion proteins and the exosporium nets, the fusion proteins become resistant to heat. The heat resistance of the fusion proteins bound to the exosproium fragments allows them to be used for applications that require esistant proteins or enzymes (e.g., in feed ves).

V. Inactivation of Spores of Bacillus Genus Bacteria, ing Spores of Recombinant Bacillus cereus Family Members Spores of bacteria of the genus Bacillus can be genetically vated.

Genetic inactivation of the spores can be ageous, for example e it allows for delivery of spores to a plant or a plant growth medium while eliminating any detrimental effects that the live bacteria might have on a plant. In addition, use of inactivated spores can provide many of the same s (e.g., prevention of bacterial growth in a product) as discussed above in n IV with respect to the use of exosporium fragments.

A. Genetic inactivation by overexgression 01a grotease or a nuclease A recombinant bacterium of the genus Bacillus that expresses a protease or a nuclease is provided. The expression of the protease or nuclease is increased as compared to the expression of the protease or the nuclease in a Wild-type bacterium of the genus Bacillus under the same conditions. The increased expression of the protease or the nuclease partially or completely vates spores of the recombinant bacterium of the genus Bacillus or renders spores of the recombinant bacterium of the genus Bacillus more susceptible to physical or chemical inactivation.

The recombinant bacterium of the genus Bacillus is preferably a recombinant Bacillus cereus family member.

The recombinant Bacillus cereus family member can also express a fusion protein sing at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member.

The recombinant bacterium of the genus Bacillus can express both a protease and a nuclease, n the expression of the protease is increased as compared to the expression of the protease in a Wild-type bacterium of the genus Bacillus under the same ions and the expression of the nuclease is increased as compared to the sion of the nuclease in a Wild-type ium of the genus Bacillus under the same conditions.

] The se of the recombinant bacterium can comprise a ecific protease.

The protease of the recombinant bacterium can comprise a serine protease, a threonine se, a cysteine protease, an aspartate protease, a glutamic acid protease, an alkaline protease, a subtilisin, a histidine protease, or a oprotease.

The protease of the recombinant bacterium can comprise a ation spore protease, such as a Bacillus subtilis germination spore protease, a us mycoia’es germination spore protease, or a Bacillus thuringiensis germination spore protease.

The germination spore protease can comprise an active form of the germination spore protease. This protease is naturally inactive in the spore. Upon ation, the protease becomes active due to cleavage of the se into a proprotein active form. Thus, the recombinant bacterium can comprise an active protease rather than the naturally inactive form. The active protease can digest the protective SASP proteins in the spore prior to germination.

The nuclease of the recombinant bacterium can se an endonuclease or an exonuclease. The nuclease can comprise a non-specific endonuclease, such as Bacillus subtilis endonuclease 1. For example, the germination spore protease and endonuclease lcan have the amino acid sequences listed below in Table 4.

Table 4. Amino acid sequences of a germination spore protease and endonuclease 1 Protein SEQ ID NO.

Endonuclease l, B. subtilis 168 GPR Protease, B. subtilis 168 233 GPR Protease, B. cereus 234 A protease or a nuclease having a high degree of amino acid identity to the sequences listed above in Table 4 can also be used.

] Thus, for example, the germination spore protease can comprise an amino acid sequence haVing at least 85% identity with SEQ ID NO: 233 or 234.

The germination spore protease can comprise an amino acid sequence haVing at least 90% identity with SEQ ID NO: 233 or 234.

The ation spore protease can comprise an amino acid sequence haVing at least 95% identity with SEQ ID NO: 233 or 234.

] The germination spore protease can comprise an amino acid sequence haVing at least 98% identity with SEQ ID NO: 233 or 234.

The ation spore protease can se an amino acid ce haVing at least 99% identity with SEQ ID NO: 233 or 234.

The germination spore protease can comprise an amino acid sequence haVing 100% identity with SEQ ID NO: 233 or 234.

Similarly, the non-specific endonuclease can comprise an amino acid haVing at least 85% identity with SEQ ID NO: 232.

The ecific endonuclease can comprise an amino acid haVing at least 90% identity with SEQ ID NO: 232.

The non-specific endonuclease can comprise an amino acid haVing at least 95% identity with SEQ ID NO: 232.

The non-specific endonuclease can comprise an amino acid haVing at least 98% ty with SEQ ID NO: 232.

The non-specific endonuclease can comprise an amino acid haVing at least 99% identity with SEQ ID NO: 232.

The non-specific clease can comprise an amino acid having 100% identity with SEQ ID NO: 232.

The se or nuclease can be expressed under the l of a promoter comprising a sigma G promoter sequence. For example, the promoter can have one of the sequences shown in Table 5 below. The consensus ce for binding of the sigma G transcription factor is CATNNTA, where N is any nucleotide. The sigma G promoter sequences in the promoters in Table 5 are indicated by bold and underlined text.

Table 5. Promoter Seuences havin_ sima G se uences Nucleic Acid Sequence GPR Protease, B. GTAACTAAAGCTTCTACAGTTTTAACAGCTGAACGCATGTCAGACTT ZiS 1 68 GATAGAAGCGTTATGTGCACGACGCTCTTCGCTAAGTTTAGCGCGTT TGATAGCAGATTTAATGTTTGCCATACTTTTCACCTCCCTGGTGCGA (SEQ ID NO‘ 23 5)' TCGAGTGACTCGATACTTACATAGAACAAGTGATATTCTATCAAACG GAGAAGAGAATTGCAATAGCGAGATCAATGAAATTTCATGTAAAGG AAAGAATGACCTTATATATTTTTGGGGAATCTAACTATATTTACTAT GAATTGCGGAGGAGATACG GPR Protease GCAATAGCGAGATCAATGAAATTTCATGTAAAGGAAAGAATGACCT minimal promoter, TATATATTTTTGGGGAATCTAACTATATTTACTATGAATTGCGGAGG AGATACG B. subtilis 168 SEQ ID NO: 236 GPR protease, B, TTTCACCTCCTAAGATACAACCTGTAGCACAGTGTCTTAAGGTTAAA sum-[is I68 TCTTCTTCACAATAGAACAAATTGTATTCTATCAAACACACCTTTAG ATTGCAATATAAATGTAAAGTATTTTTCATTGAAGGTTCTCTTTTTAG (SEQ ID NO' 237)- CATGATTTATTCAGCAAATGGCAACAATATAGGTACTTAATGTGAA GGAGGCCCCTGT GPR protease GAAGGTTCTCTTTTTAGCATGATTTATTCAGCAAATGGCAACAATAT minimal promoter, AGGTACTTAATGTGAAGGAGGCCCCTGT B. subtilis 168 SEQ ID NO: 238 SASPQ, B. subtilis GCTTTGTTGATTTCGAGCCGTATATTCAAGAAGCGGTAGATAACATT 168 GAGACAATGACCCTTTATAGCGAACAAGAAGCTAACGATAAATTCG CTGAACTCTTTTAAATCAATTTTCAGCTCCTGTATACAATTACCAAAG (SEQ ID NO- 239)- TTTTTCTGAATGAAGCCATGTGTTTTGACACATTCTATACTCACAAG AGACAC SASPu minimal GAATGAAGCCATGTGTTTTGACACATTCTATACTCACAAGGAGGTG AGACAC promoter, B. is SEQ ID NO: 240 SASPB, B. subtilis AAACGGCTAAGCTTTTTTTATTTCTCAAGATTTACCACACAATTCTCC 1 68 GCATGATTTTCCGGCCATTTTAACATAATACGTAGTAACAAGCCGGC AAAGCATTGGGTTACGCCGAGGCGGCAGTGACACCCGAGAAGGGTT (SEQ ID NO- 241)' CACAGATTGGTGCAACTCCAGTTAACCCAACCATACTAAATAAAAA TTTACAC SASPB minimal GATTGGTGCAACTCCAGTTAACCCAACCATACTAAATAAAAAGGAG ATTTTACAC promoter, B. subtilis (SEQ ID NO: 242) Nucleic Acid Sequence SASPY, B. subtilis TTCGCTTCTCCCACTTAATCTGATTTACATTCCAAGGAATCCAATGAT 1 68 TTATATGGAGATCTGAAACATAATCAATTTTCATTTTGTCTCCACCTT TCTTAATGAAAAATTTATTTCTTTGGCGTGTATAAATTAAAATAATCT (SEQ ID NO: 243) CTCCATAATATGATTCAAACAAGCTTGTTTTCATTACACTTTAGGAG ATGAATAAG SAspy minimal ATTAAAATAATCTCTCCATAATATGATTCAAACAAGCTTGT TTTCATTACACTTTAGGAGATGAATAAG promoter, B. subtilis SEQ ID NO: 244 sA5135, B. sum-[is TACAGTCCTCTCCATTTTGACATTCCATATTCAGGCAACCGCACATA 1 68 AAATGACAGCAGACATTCTATAGTCTGCGCCACCCCGGCTCAGAGG CCGGGGTTTTATTTTTCTCCACAACAATTGCCAGCATAAATAAACCC (SEQ ID NO- 245)- CGTATATTTCAAACTAAATACGCGTTAAGAATTTCTTTATCGAAAAA GGAGATGAAAAAG sA5135 minimal GCAACCGCACATAAAATGACAGCAGACATTCTATAGTCTGCGCCAC CCCGGCTCAGAGGCCGGGGTTTTATTTTTCTCCACAACAATTGCCAG promoter, B. ”brill-S CATAAATAAACCCCGTATATTTCAAACTAAATACGCGTTAAGAATTT 1 68 CTTTATCGAAAAAGGAGATGAAAAAG SEQ ID NO: 246 Expression of a nuclease or protease under a sigma G er allows for site-specific sion of the nuclease or protease in the forespore, where the enzyme’s activity is directed towards the forespore and, the region where the bacterial target DNA is located.

Extensive cleavage of the forespore DNA is lethal to the bacterial spore when it begins to germinate.

For example, as illustrated further in the es provided hereinbelow, overexpression of germination spore protease (GPR) in its active form in the forespore of a Bacillus cereus family member during sporulation results in proteolytic cleavage of proteins in the forespore and inactivation of the spore and/or renders the spore more sensitive to inactivation by ultraviolet or gamma irradiation. Similarly, overexpression of a non-specific endonuclease in the ore during ation destroys the DNA in the spore, leading to a high number of inactivated spore particle. These methods for inactivating Bacillus cereus family member spores can be used separately or in conjunction with each other and/or with other spore inactivation methods.

Expression of genes in Bacillus spores is y ted by expression of c sporulation sigma factors that direct the RNA rase to the genes that need to be expressed during each stage of sporulation. Late expression of genes in the forespore, where bacterial DNA and essential proteins are packaged, is regulated by the sigma factor sigma G.

During late sporulation, the bacterial DNA is packaged with protective proteins called small acid soluble proteins ). These SASP proteins include SASPa, SASPB, and SASPy, among WO 44661 others. The SASP proteins protect the bacterial DNA from UV irradiation and other assaults.

Upon germination, the proprotein germination spore protease is activated and digests these SASP proteins.

By expressing a GPR under the control of a sigma G promoter, the GPR is expressed in the forespore and the protective SASP proteins are degraded as sporulation commences, leaving the bacterial DNA more susceptible to degradation. rly, expression of a non-specific nuclease under the control of a sigma G promoter leads to digestion of the host DNA. Since the spore is unable to repair the large scale damage to its DNA, this ultimately leads to g of the spore. Overexpression of a GPR and a non-specific endonuclease can be used together to both e the protective SASP proteins and the host DNA.

The se or the nuclease can be expressed under the control of any promoter comprising a sigma G promoter sequence.

] Thus, the protease or nuclease can be expressed under the control of any of the promoters listed in Table 5 above. In addition, the protease or nuclease can be sed under the control of a promoter having a high degree of sequence identity with any of the promoter sequences listed above in Table 5.

For example, the promoter can comprise a nucleic acid sequence having at least 95% ty with a nucleic acid sequence of any of SEQ ID NOs: 23 5—246.

The promoter can comprise a nucleic acid sequence having at least 98% identity with a nucleic acid ce of any of SEQ ID NOs: 23 5—246.

The er can comprise a nucleic acid sequence having at least 99% identity with a nucleic acid sequence of any of SEQ ID NOs: 23 5—246.

The promoter can comprise a nucleic acid sequence having 100% identity with a nucleic acid sequence of any of SEQ ID NOs: 23 5—246.

In any of the recombinant bacteria of the genus Bacillus that s a protease or a nuclease, spores of the inant bacterium of the genus Bacillus can be more susceptible to inactivation, for example, by ultraviolet irradiation, gamma irradiation, or by treatment with bleach, en peroxide, chloroform, phenol, or acetic acid, as compared to the same spores that do not expresses the protease or the nuclease at an increased level as compared to expression of the protease or the nuclease in a wild-type bacterium of the genus us, treated under the same conditions. 2015/050807 B. Genetic inactivation by mutation of a gene encoding a germination recthor, a sQore core lytic l a small acid-soluble sQore grotein l or a some coat grotein Spores of any of the recombinant Bacillus cereus family member spores that express a fusion protein comprising a targeting sequence, an exosporium protein, or an rium protein fragment that targets the fusion protein to the exosporium of the recombinant us cereus family member can also be genetically inactivated or rendered more susceptible to physical or chemical inactivation by modification of the Bacillus cereus family member to comprise a mutation.

Such mutations include knock-out or other inactivating mutations in one or more genes encoding a germination receptor. The germination or genes include, for example, GerA, GerB, GerK, GerH, GerI, GerG, GerL, GerQ, GerR, GerS, GerN, GerU, or GerX.

Such mutations also include out or other inactivating mutations in spore cortex lytic enzymes. For example, the spore cortex lytic enzymes SleB and CWJ can be mutated to inactivate spores. Such mutations prevent outgrowth of the spore upon ation and effectively inactivate the spores.

Such mutations r include knock-out or other inactivating mutations of SASP genes (e. g., SASPu, SASPB, or SASPy). Such mutations eliminate the UV protection of the spores and render them more susceptible to inactivation by ultraviolet irradiation and other methods.

Such methods also include making knock-out or other inactivating mutations in genes encoding spore coat or cortex proteins (e. g., CotA, CotB, or CotC). Such mutations render the spores more tible to inactivation by physical or chemical methods such as exposure to ultraviolet irradiation, gamma irradiation, or treatment With solvents such as , hydrogen peroxide, chloroform, phenol, or acetic acid.

Thus, the present invention relates to a recombinant us cereus family member that expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or rium protein nt that targets the fusion protein to the exosporium of the inant Bacillus cereus family member. The recombinant Bacillus cereus family member comprises a mutation that partially or completely inactivates spores of the recombinant Bacillus cereus family member or renders spores of the recombinant Bacillus cereus family more susceptible to physical or chemical inactivation as compared to the same spores that do not comprise the mutation. The mutation comprises a mutation in a gene encoding a germination receptor, a on in a gene encoding a spore cortex lytic enzyme, a mutation in a gene encoding a small acid-soluble spore n (SASP), or a mutation in a gene encoding a spore coat or cortex n.

The present ion further relates to a recombinant Bacillus cereus family member that expresses a fusion protein as described in Section 1 above. The recombinant Bacillus cereus family member comprises a mutation that partially or completely inactivates spores of the recombinant Bacillus cereus family member or renders spores of the recombinant Bacillus cereus family more susceptible to physical or chemical inactivation as compared to the same spores that do not comprise the mutation.

Any of the recombinant Bacillus cereus family s described above in Section V.A that express a protease or a se can also comprise a mutation that partially or completely inactivates spores of the recombinant Bacillus cereus family member or renders spores of the recombinant Bacillus cereus family more susceptible to physical or chemical inactivation as compared to the same spores that do not comprise the on. For e, the mutation can comprise a on in a gene encoding a germination receptor, a mutation in a gene encoding a spore cortex lytic enzyme, a mutation in a gene encoding a small acid-soluble spore protein (SASP), or a mutation in a gene encoding a spore coat or cortex protein.

For e, the mutation can comprise a mutation in a gene encoding a germination receptor, such as a knock-out mutation of the gene encoding the ation receptor. The germination receptor can comprise GerA, GerB, GerK, GerH, GerI, GerG, GerL, GerQ, GerR, GerS, GerN, GerU, or GerX.

For example, the mutation can comprise a mutation in a gene encoding a spore cortex lytic enzyme, such as a knock-out mutation of the gene encoding the spore cortex lytic enzyme. The spore cortex lytic enzyme can comprise SleB or CWlJ.

For example, the on can comprise a mutation in a gene encoding a SASP, such as a mutation in a SspA gene, a mutation in a SspB gene, a mutation in a SspC gene, a mutation in a SspD gene, a mutation in a SspE gene, a on in a SspF gene, a mutation in a SspG gene, a on in a SspH gene, a mutation in a SspI gene, a mutation in a SspJ gene, a mutation in a SspK gene, a mutation in a SspL gene, a mutation in a SspM gene, a mutation in a SspN gene, a mutation in a SspO gene, a mutation in a SspP gene, or a combination thereof. The SASP can comprise SASPu, SASPB, or SASPy. The spores of the recombinant Bacillus cereus family member may be more susceptible to inactivation by ultraviolet irradiation or gamma irradiation as compared to the same spores that do not comprise the mutation in the gene encoding the SASP.

For example, the mutation can comprise a mutation in a gene encoding a spore coat or cortex protein, such as a knock-out mutation of the gene encoding the spore coat or cortex protein. The spore coat or cortex n can comprise CotA, CotB, or CotC. The spores of the recombinant Bacillus cereus family member may be more susceptible to inactivation by ultraviolet irradiation, gamma irradiation or by treatment with bleach, hydrogen peroxide, chloroform, phenol, or acetic acid, as compared to the same spores that do not comprise the mutation in the spore coat or cortex protein, treated under the same conditions.

VI. Recombinant Bacillus cereus Family Members that Overexpress rium Enzymes that Have Beneficial Effects on Plants 0r Delay Germination of Bacillus cereus Family Member Spores ] Recombinant Bacillus cereus family members that overexpress various exosporium ns to provide beneficial effects on plants or delay spore germination are also provided.

A recombinant Bacillus cereus family member that expresses an exosporium protein is provided, wherein the expression of the exosporium protein is increased as compared to the expression of the rium protein in a wild-type Bacillus cereus family member under the same conditions. The exosporium protein can se an exosporium enzyme, wherein the rium enzyme comprises an enzyme involved in nutrient solubilization, an inosine-uridine hydrolase, a protease, an enzyme that catalyzes the degradation of a free radical, an arginase, or an alanine racemase. Alternatively, the exosporium protein can comprise a BclA protein, a BclB n, a CotE protein a CotO protein, an Est n, an ExsFA/BxpB n, a CotY protein, an ExsFB protein, an Est protein, an Est protein, a chA protein, a chB protein, a Bch n, a Bpr protein, a BclE protein, a BetA/BAS3290 protein, an Est protein, an ExsK protein, an Est protein, a YabG protein, or a Tgl n.

The exosporium protein is preferably not part of a fusion protein.

Exemplary amino acid sequences for AcpC, InhAl, InhA2, InhA3, SODAl, and SODAZ are provided above in Tables 1 and 2. Exemplary sequences for alanine racemase l, alanine racemase 2, se, IunHl and IunH2 are ed by the SEQ ID NOs. referenced in Table 6 below.

Table 6. Exemplary amino acid ces for exosporium enzymes SEQ ID NO- Alanine Racemase 1, B. anthracis ASteme 247 Alanine Racemase 2, Bacillus cereus F837/78 248 e, Bacillus tlzurinriensis nondicheriensis 48.4.1 249 IunHl, B. cereus Str. CI 250 IunH2, Bacillus thurin iensis 251 Overexpression of inosine-uridine ases and alanine ses hinders the y of spores to germinate and thereby maintains the spores in a dormant stage and increases the stability of the spores.

The SODA enzymes and arginase degrade free radicals. Spores that overexpress these enzymes have increased resistance to stress caused by free radicals.

Where the rium n comprises an exosporium enzyme, and the exosporium enzyme ses an enzyme involved in nutrient solubilization, the enzyme involved in nutrient solubilization can comprise an enzyme involved in phosphate solubilization, such as an acid phosphatase (e. g., AcpC). The acid phosphatase can comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 137.

] The acid phosphatase can comprise an amino acid sequence having at least 95% identity with SEQ ID NO: 137.

The acid phosphatase can comprise an amino acid sequence having at least 98% identity with SEQ ID NO: 137.

The acid phosphatase can comprise an amino acid ce having at least 99% identity with SEQ ID NO: 137.

The acid phosphatase can comprise an amino acid sequence having 100% identity with SEQ ID NO: 137.

Where the exosporium protein comprises an exosporium enzyme, and the exosporium enzyme comprises an inosine-uridine hydrolase, the inosine-uridine hydrolase can comprise IunHl or IunH2. The inosine-uridine hydrolase can comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 250 or 251.

The inosine-uridine hydrolase can comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 250 or 251.

The inosine-uridine hydrolase can se an amino acid sequence having at least 95% identity with SEQ ID NO: 250 or 251.

The inosine-uridine hydrolase can comprise an amino acid sequence having at least 98% identity with SEQ ID NO: 250 or 251.

The inosine-uridine hydrolase can comprise an amino acid sequence having at least 99% identity with SEQ ID NO: 250 or 251.

The inosine-uridine hydrolase can comprise an amino acid ce having 100% identity with SEQ ID NO: 250 or 251.

Where the exosporium protein comprises an exosporium enzyme, and the exosporium enzyme comprises a protease, the protease can be a metalloprotease (e.g., InhAl, InhA2, or . The metalloprotease can comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

] The metalloprotease can comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

The metalloprotease can comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

The metalloprotease can comprise an amino acid sequence having at least 95% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

The metalloprotease can comprise an amino acid sequence having at least 98% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

The metalloprotease can comprise an amino acid sequence having at least 99% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

The metalloprotease can se an amino acid ce having 100% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

Where the exosporium protein comprises an exosporium enzyme, and the exosporium enzyme comprises an enzyme that zes the ation of a free radical, the enzyme that catalyzes the degradation of a free radical can comprise a superoxide dismutase (e.g., xide ase 1 (SODAl) or superoxide dismutase 2 (SODA2)). The superoxide dismutase can comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase can comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase can comprise an amino acid sequence having at least 95% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase can comprise an amino acid sequence having at least 98% identity with SEQ ID NO: 155 or 156.

The superoxide ase can comprise an amino acid sequence having at least 99% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase can comprise an amino acid sequence having 100% identity with SEQ ID NO: 155 or 156.

Where the exosporium protein comprises an exosporium , and the exosporium enzyme comprises an arginase, the arginase can comprise a us thuringiensis arginase. The se can comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 249.

The arginase can comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 249.

The arginase can comprise an amino acid ce having at least 95% identity with SEQ ID NO: 249.

The arginase can comprise an amino acid sequence having at least 98% ty with SEQ ID NO: 249.

The arginase can se an amino acid sequence having at least 99% identity with SEQ ID NO: 249.

The arginase can comprise an amino acid sequence having 100% ty with SEQ ID NO: 249.

Where the exosporium protein comprises an exosporium enzyme, and the exosporium enzyme comprises an alanine racemase, the alanine racemase can comprise alanine racemase 1 (ALR1) or alanine racemase 2 (ALR2). The alanine racemase can comprise an amino acid sequence having at least 85% ty with SEQ ID NO: 247 or 248.

The alanine racemase can comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 247 or 248.

The alanine racemase can comprise an amino acid sequence having at least 95% identity with SEQ ID NO: 247 or 248.

The alanine racemase can se an amino acid sequence having at least 98% identity with SEQ ID NO: 247 or 248.

The alanine se can comprise an amino acid sequence having at least 99% identity with SEQ ID NO: 247 or 248. 2015/050807 The alanine se can comprise an amino acid sequence having 100% identity with SEQ ID NO: 247 or 248.

The exosporium protein can comprise a BclA protein, a BclB protein, a CotE protein a CotO protein, an Est protein, an ExsFA/BxpB protein, a CotY protein, an ExsFB protein, an Est protein, an Est protein, a YjcA protein, a Yj cB protein, a Bch protein, a Bpr protein, a BclE n, a BetA/BAS3290 protein, an Est protein, an ExsK protein, an Est protein, a YabG protein, or a Tgl protein. The exosporium protein preferably comprises a BclA n, a BclB protein, a CotE protein, or a CotO protein. Exemplary amino acid sequences for these exosporium proteins can be found in Table 2 above.

The exosporium protein can comprise a BclA n. The BclA protein can comprise an amino acid sequence having at least 85% identity With SEQ ID NO: 141 or 142.

The BclA protein can comprise an amino acid sequence having at least 90% identity With SEQ ID NO: 141 or 142.

The BclA protein can comprise an amino acid sequence having at least 95% identity With SEQ ID NO: 141 or 142.

The BclA protein can comprise an amino acid sequence having at least 98% identity With SEQ ID NO: 141 or 142.

] The BclA protein can comprise an amino acid ce having at least 99% identity With SEQ ID NO: 141 or 142.

The BclA protein can comprise an amino acid sequence having 100% identity with SEQ ID NO: 141 or 142.

The exosporium n can comprise a BclB protein. The BclB protein can comprise an amino acid sequence having at least 85% identity With SEQ ID NO: 143 or 144.

The BclB protein can comprise an amino acid sequence having at least 90% identity With SEQ ID NO: 143 or 144.

The BclB protein can comprise an amino acid sequence having at least 95% identity With SEQ ID NO: 143 or 144.

The BclB protein can comprise an amino acid sequence having at least 98% identity With SEQ ID NO: 143 or 144.

The BclB protein can se an amino acid sequence having at least 99% identity With SEQ ID NO: 143 or 144.

The BclB protein can comprise an amino acid ce having 100% identity With SEQ ID NO: 143 or 144.

The exosporium protein can comprise a CotE protein. The CotE protein can comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 149.

The CotE protein can comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 149.

The CotE n can comprise an amino acid sequence having at least 95% identity with SEQ ID NO: 149.

The CotE n can se an amino acid sequence having at least 98% identity with SEQ ID NO: 149.

The CotE protein can comprise an amino acid sequence having at least 99% identity with SEQ ID NO: 149.

The CotE protein can comprise an amino acid sequence having 100% identity with SEQ ID NO: 149.

The exosporium protein can comprise a CotO protein. The CotO protein can comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 126.

The CotO protein can comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 126.

The CotO protein can se an amino acid sequence having at least 95% identity with SEQ ID NO: 126.

The CotO protein can comprise an amino acid sequence having at least 98% identity with SEQ ID NO: 126.

The CotO protein can comprise an amino acid sequence having at least 99% identity with SEQ ID NO: 126.

The CotO protein can comprise an amino acid sequence having at least 100% identity with SEQ ID NO: 126.

The exosporium n can comprise an Est protein. The Est protein can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 123.

The exosporium n can comprise an ExsFA/BxpB protein. The ExsFA/BxpB protein can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% ty with SEQ ID NO: 124.

The exosporium protein can comprise a CotY n. The CotY protein can se an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 125.

The exosporium protein can comprise an ExsFB protein. The ExsFB protein can comprise an amino acid ce having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 127 or 128.

The exosporium protein can comprise an Est protein. The Ex] protein can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity With SEQ ID NO: 131.

The exosporium protein can comprise an Est protein. The Est protein can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity With SEQ ID NO: 132.

The exosporium protein can comprise a YjcA protein. The YjcA protein can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity With SEQ ID NO: 133.

The exosporium protein can comprise a chB protein. The chB n can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% ty With SEQ ID NO: 134 or 135.

The rium protein can comprise a Bch protein. The Bch protein can comprise an amino acid ce having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% With SEQ ID NO: 136.

The exosporium n can comprise a Bpr protein. The Bpr n can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% With SEQ ID NO: 145.

The exosporium protein can comprise a BclE protein. The BclE protein can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity With SEQ ID NO: 146 or 147.

The exosporium protein can comprise a BetA/BAS3290 protein. The BetA/BAS3290 protein can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% ty With SEQ ID NO: 148.

] The exosporium protein can comprise an Est protein. The Est protein can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity With SEQ ID NO: 150.

The exosporium protein can comprise an ExsK protein. The ExsK n can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity With SEQ ID NO: 151.

The rium protein can comprise an Est protein. The Est protein can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity With SEQ ID NO: 152.

The exosporium protein can comprise a YabG protein. The YabG protein can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity With SEQ ID NO: 153.

The exosporium protein can comprise a Tgl protein. The le n can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity With SEQ ID NO: 156.

The recombinant Bacillus cereus family member can also express a fusion protein comprising at least one protein or peptide of interest and a targeting ce, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member.

VII. Expression of Fusion Proteins in Endophytic Bacillus cereus Family Members: in Bacillus cereus Family Members Capable of Degrading Herbicides or Pesticides, or in tic Bacillus cereus Family Members ] Any of the fusion proteins comprising a protein or peptide of interest and a targeting sequence, an rium protein, or an exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member, can be expressed an endophytic Bacillus cereus family member, a strain of ia that is capable of degrading an ide or a ide, or a probiotic strain of bacteria.

The expression of the fusion proteins in an endophytic strain of bacteria provides the ability to deliver the protein or peptide of interest into the plant itself. The endophytic strains can be delivered to plants using various s, e.g., the ytic s can be delivered via seed treatment, treatment of the plant growth medium (e.g., soil), irrigation, application to the plant itelf (e.g., foliar application to the aerial portions of a plant). Once inside the plant, the ia multiply and colonize the internal tissues of the plant.

As is explained further hereinbelow, probiotic strains of bacteria that s of the fusion proteins, and in particular strains that are both probiotic and endophytic that express the fusion proteins, are useful in methods for delivering the proteins or peptides of interest (e.g., enzymes) to animals.

] While any of the fusion proteins comprising a protein or peptide of interest and a targeting ce, an exosporium protein, or an exosporium protein fragment that targets 2015/050807 the fusion protein to the exosporium of a recombinant Bacillus cereus family member can be expressed in Bacillus cereus family member strain that is e of ing an herbicide or a pesticide, as explained further hereinbelow, these s are particularly useful in methods for amination of an enVironment contaminated With an herbicide and/or a pesticide.

The present invention therefore relates to a recombinant Bacillus cereus family member that expresses a fusion protein comprising at least one protein or peptide of interest and a targeting ce, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant us cereus family member, Wherein the recombinant Bacillus cereus family member comprises an endophytic strain of bacteria, a strain of ia that is capable of degrading an herbicide or a pesticide, or a probiotic strain of bacteria.

The endophytic strain of bacteria can comprise Bacillus cereus family member EE349, us cereus family member EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, or Bacillus thuringiensis EE319, Bacillus thuringiensis EE-BOOl84, Bacillus cereus family member EE-B00377, Bacillus pseudomycoia’es EE-B00366, or Bacillus mycoia’es EE-B00363.

For example, the endophytic strain of bacteria can comprise Bacillus cereus family member EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, or Bacillus thuringiensis EE3 l9, Bacillus giensis EE-B00184, Bacillus cereus family member EE- B00377, Bacillus pseudomycoia’es EE-B00366, or Bacillus ’es EE-B00363.

The strain of bacteria that is capable of degrading an herbicide or a ide can comprise Bacillus cereus family member EE349, Bacillus cereus family member EE- B00377, Bacillus pseudomycoia’es EE-B00366, or Bacillus mycoia’es EE-B00363.

The probiotic strain of bacteria can se Bacillus cereus family member EE349, Bacillus cereus family member EE439, Bacillus thuringiensis EE417, or Bacillus cereus EE444.

The present invention r s to a recombinant Bacillus cereus family member that ses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member, wherein the recombinant Bacillus cereus family member comprises an endophytic strain of bacteria, and the fusion protein comprises any of the fusion proteins described in Section 1 above.

VIII. Targeting Seguences, Exosporium Proteins, and Exosporium Protein Fragments for Use in: 1a) Recombinant us cereus Family Members that Express a Fusion Protein and Co-Overexpress a Modulator Protein; 1b) Recombinant Bacillus cereus Family s that Comprise a Mutation or Other Genetic Alteration that Allows for Collection of Free Exosporium; 1c) Recombinant Bacillus cereus Family Members that Overexpress a se or a Nuclease; 1d) Recombinant Bacillus cereus Family Members that Express a Fusion n and Overexpress an Exosporium Protein that Has Beneficial Effects on Plants; or gel or endophytic recombinant Bacillus cereus Family Members that Express Fusion ns Any of the targeting sequences, exosporium proteins, or exosporium proteins described in this section can be in any of the fusion ns in: (a) any of the recombinant Bacillus cereus family members that express a fusion protein and overexpress a modulator protein, described in Section II above; (b) any of the recombinant Bacillus cereus family members that express a fusion n and se a mutation or other genetic alteration that allows for collection of free exosporium, described in Section IV above; (c) any of the inant Bacillus cereus family members that expresses a fusion protein and overexpress a se or a nuclease, described above in Section VA; (d) any of the recombinant us cereus family members that express a fusion protein and overexpress an exosporium protein that has beneficial effects on plants, described in Section VI above; and (e) any of the endophytic recombinant Bacillus cereus family members that express a fusion protein, described in Section VII above.

In any of the recombinant Bacillus cereus members (a) through (e), the targeting ce, exosporium protein, or rium protein fragment can comprise: (l) a targeting ce comprising an amino acid sequence having at least about 43% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 54%; (2) a targeting sequence sing amino acids l—35 of SEQ ID NO: 1; (3) a targeting sequence comprising amino acids 20—35 of SEQ ID NO: 1 ; (4) a targeting ce comprising SEQ ID NO: I ; (5) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 2; (6) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 1 ; (7) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 1 ; (8) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 1 ; (9) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 1 ; (10) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: I ; (ll) a targeting sequence comprising amino acids l—27 of SEQ ID NO: 3; (12) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 3; (13) a targeting ce comprising SEQ ID NO: 3; (14) an rium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 4; (15) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 3; (16) a targeting ce comprising amino acids 5—27 of SEQ ID NO: 3; (17) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 3; (18) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 3; (19) a targeting sequence comprising amino acids 1—38 of SEQ ID NO: 5; (20) a ing sequence comprising amino acids 23—38 of SEQ ID NO: 5; (21) a targeting sequence comprising SEQ ID NO: 5; (22) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 6; (23) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 5; (24) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 5; (25) a targeting sequence comprising amino acids 8—38 of SEQ ID NO: 5; (26) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 5; (27) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 5; (28) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 5; (29) a targeting sequence comprising amino acids 1—28 of SEQ ID NO: 7; (30) a targeting sequence comprising amino acids 13—28 of SEQ ID NO: 7; (31) a targeting sequence comprising SEQ ID NO: 7; (32) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 8; (33) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 7; (34) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 7; (35) a targeting ce comprising amino acids 8—28 of SEQ ID NO: 7; (36) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 7; (37) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 9; (38) a ing sequence comprising amino acids 9—24 of SEQ ID NO: 9; (39) a ing ce comprising SEQ ID NO: 9; (40) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 10; (41) a targeting sequence sing amino acids 2—24 of SEQ ID NO: 9; (42) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 9; (43) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 9; (44) a targeting sequence comprising amino acids 1—33 of SEQ ID NO:11; (45) a targeting ce comprising amino acids 18—33 of SEQ ID NO: 11; (46) a targeting sequence comprising SEQ ID NO: 11; (47) an rium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 12; (48) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 11; (49) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 11; (50) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 11; (51) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 11; (52) a targeting ce comprising amino acids 15—33 of 1 10 SEQ ID NO: 11; (53) a targeting sequence sing amino acids 1—33 of SEQ ID NO: 13 ; (54) a ing sequence comprising amino acids 18—33 of SEQ ID NO: 13; (55) a ing sequence comprising SEQ ID NO:13; (56) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 14; (57) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 13; (58) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13 ; (59) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 13 ; (60) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 13; (61) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 13; (62) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 15; (63) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 15; (64) a targeting sequence sing SEQ ID NO:15; (65) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:16; (66) a ing sequence comprising amino acids 2—43 of SEQ ID NO: 15 ; (67) a targeting ce comprising amino acids 5—43 of SEQ ID NO: 15; (68) a ing sequence comprising amino acids 8—43 of SEQ ID NO: 15; (69) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 15 ; (70) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 15; (71) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 15; (72) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 15; (73) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 17; (74) a ing sequence comprising amino acids 12—27 of SEQ ID NO: 17; (75) a targeting sequence comprising SEQ ID NO:17; (76) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:18; (77) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 17; (78) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 17; (79) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 17; (80) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 17; (81) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 19; (82) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 19; (83) a targeting sequence comprising SEQ ID NO:19; (84) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO:20; (85) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 19; (86) a targeting sequence sing amino acids 5—33 of SEQ ID NO: 19; (87) a ing sequence comprising amino acids 8—33 of SEQ ID NO: 19; (88) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 19; (89) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 19; (90) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 21 ; (91) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 21; (92) a targeting sequence comprising SEQ 1 1 1 ID NO:21; (93) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:22; (94) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 21; (95) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 21; (96) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 21; (97) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 21; (98) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 21; (99) a targeting ce comprising amino acids 1—24 of SEQ ID NO: 23; (100) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 23; (101) a targeting sequence comprising SEQ ID NO:23; (102) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:24; (103) a targeting sequence comprising amino acids 2—24 of SEQ ID NO:23; (104) a targeting sequence sing amino acids 5—24 of SEQ ID NO: 23; (105) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 23; (106) a ing sequence sing amino acids 1—24 of SEQ ID NO: 25; (107) a targeting ce comprising amino acids 9—24 of SEQ ID NO: 25 ; (108) a targeting sequence comprising SEQ ID NO:25; (109) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:26; (110) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 25; (111) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 25; (112) a targeting sequence sing amino acids 8—24 of SEQ ID NO: 25; (113) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 27; (114) a ing sequence comprising amino acids 15—30 of SEQ ID NO: 27; (115) a targeting sequence comprising SEQ ID NO:27; (116) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:28; (117) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 27; (118) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 27; (119) a targeting ce comprising amino acids 8—30 of SEQ ID NO: 27; (120) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 27; (121) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 29; (122) a targeting ce comprising amino acids 18—33 of SEQ ID NO: 29; (123) a targeting sequence comprising SEQ ID NO:29; (124) an exosporium n sing an amino acid sequence haVing at least 85% identity With SEQ ID NO:30; (125) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 29; (126) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 29; (127) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 29; (128) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 29; (129) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 29; (130) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 31; (131) a targeting sequence 1 12 comprising amino acids 9—24 of SEQ ID NO: 31 ; (132) a targeting sequence comprising SEQ ID NO:31; (133) an exosporium protein comprising an amino acid ce having at least 85% ty With SEQ ID NO:32; (134) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 31; (135) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31; (136) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 31 ; (137) a targeting sequence comprising amino acids 1—15 of SEQ ID NO: 33; (138) a targeting ce sing SEQ ID NO:33; (139) an rium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:34; (140) a targeting ce comprising amino acids 1—16 of SEQ ID NO: 35; (141) a targeting sequence comprising SEQ ID NO:35; (142) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:36; (143) a targeting sequence comprising amino acids 1—29 of SEQ ID NO:43; (144) a targeting sequence comprising amino acids 14—29 of SEQ ID NO: 43; (145) a targeting sequence comprising SEQ ID NO: 43; (146) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 44; (147) a targeting sequence comprising amino acids 2—29 of SEQ ID NO: 43 ; (148) a targeting sequence comprising amino acids 5—29 of SEQ ID NO: 43 ; (149) a targeting ce comprising amino acids 8—29 of SEQ ID NO: 43; (150) a targeting sequence comprising amino acids 10—29 of SEQ ID NO: 43; (151) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 45; (152) a ing sequence comprising amino acids 20—35 of SEQ ID NO: 45 ; (153) a targeting sequence comprising SEQ ID NO: 45 ; (154) an rium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 46; (155) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 45; (156) a targeting sequence sing amino acids 5—35 of SEQ ID NO: 45; (157) a targeting ce comprising amino acids 8—35 of SEQ ID NO: 45; (158) a targeting sequence sing amino acids 10—35 of SEQ ID NO: 45; (159) a targeting sequence sing amino acids 15—35 of SEQ ID NO: 45 ; (160) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 47; (161) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 47; (162) a targeting sequence comprising SEQ ID NO: 47; (163) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 48; (164) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 47; (165) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 47; (166) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 47; (167) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 47; (168) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 47; (169) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (170) a targeting 1 13 sequence comprising amino acids 25—43 of SEQ ID NO: 47; (171) a targeting sequence comprising amino acids 1—32 of SEQ ID NO: 49; (172) a targeting sequence comprising amino acids 17—32 of SEQ ID NO: 49; (173) a ing sequence comprising SEQ ID NO: 49; (174) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 50; (175) a ing ce sing amino acids 2—32 of SEQ ID NO: 49; (176) a targeting ce comprising amino acids 5—32 of SEQ ID NO: 49; (177) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (178) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (179) a targeting ce comprising amino acids 15—32 of SEQ ID NO: 49; (180) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 51; (181) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 51; (182) a targeting sequence comprising SEQ ID NO: 51; (183) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 52; (184) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 51; (185) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 51; (186) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 51; (187) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 51; (188) a targeting ce comprising amino acids 15—33 of SEQ ID NO: 51; (189) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 53; (190) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 53; (191) a targeting sequence comprising SEQ ID NO: 53; (192) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 54; (193) a ing sequence comprising amino acids 2—33 of SEQ ID NO: 53; (194) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53 ; (195) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53 ; (196) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 53 ; (197) a targeting ce sing amino acids 15—33 of SEQ ID NO: 53; (198) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 55; (199) a targeting sequence comprising amino acids 15—30 of SEQ ID NO: 55 ; (200) a targeting sequence comprising SEQ ID NO: 55 ; (201) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 56; (202) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 55; (203) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 55; (204) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 55; (205) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 55; (206) a targeting sequence comprising amino acids 1—130 of SEQ ID NO: 57; (207) a targeting sequence comprising amino acids 115—130 of SEQ ID NO: 57; (208) a ing sequence comprising SEQ ID NO: 57; (209) an exosporium 1 14 protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 58; (210) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 57; (211) a targeting sequence comprising amino acids 5—130 of SEQ ID NO: 57; (212) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (213) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 57; (214) a targeting sequence comprising amino acids 30— 130 of SEQ ID NO: 57; (215) a targeting sequence comprising amino acids 40—130 of SEQ ID NO: 57; (216) a targeting sequence comprising amino acids 50—130 of SEQ ID NO: 57; (217) a targeting sequence comprising amino acids 60—130 of SEQ ID NO: 57; (218) a targeting sequence comprising amino acids 70—130 of SEQ ID NO: 57; (219) a targeting ce comprising amino acids 80—130 of SEQ ID NO: 57; (220) a targeting sequence comprising amino acids 90—130 of SEQ ID NO: 57; (221) a targeting ce comprising amino acids 100—130 of SEQ ID NO: 57; (222) a ing sequence comprising amino acids 110—130 of SEQ ID NO: 57; (223) an rium protein fragment comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 95; (224) a targeting sequence comprising SEQ ID NO: 96; (225) a targeting sequence comprising SEQ ID NO: 97; (226) a ing sequence comprising SEQ ID NO: 98; (227) a targeting ce comprising SEQ ID NO: 99; (228) a ing sequence comprising SEQ ID NO: 100; (229) a targeting sequence comprising SEQ ID NO: 101; (230) a targeting sequence comprising SEQ ID NO: 102; (231) a targeting sequence comprising SEQ ID NO: 103; (232) a targeting sequence comprising SEQ ID NO: 104; (233) a targeting ce comprising SEQ ID NO: 105; (234) a targeting sequence sing SEQ ID NO: 106; (235) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (236) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 109; (237) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 110; (238) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 111; (239) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 112; (240) an exosporium n comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 113; (241) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 114; (242) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 115; (243) an exosporium n comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 116; (244) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 117; (245) an exosporium protein comprising an amino acid 1 15 sequence having at least 85% identity with SEQ ID NO: 118; (246) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 119; (247) an exosporium protein sing an amino acid sequence having at least 85% identity With SEQ ID NO: 120; (248) an rium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 121 ; (249) a targeting sequence comprising amino acids 22—31 of SEQ ID NO: 1; (250) a targeting sequence comprising amino acids 22—33 of SEQ ID NO: 1; (251) a targeting sequence comprising amino acids 20—31 of SEQ ID NO: 1; (252) a targeting sequence comprising amino acids 14—23 of SEQ ID NO: 3; (253) a targeting sequence sing amino acids 14—25 of SEQ ID NO: 3; (254) a targeting ce comprising amino acids 12—23 of SEQ ID NO: 3; (255) a targeting ce comprising amino acids 1—30 of SEQ ID NO: 59; (256) a targeting sequence comprising SEQ ID NO: 59; (257) an exosporium protein comprising an amino acid ce having at least 85% identity With SEQ ID NO: 60; (258) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 59; (259) a targeting sequence comprising amino acids 4—30 of SEQ ID NO: 59; (260) a ing sequence comprising amino acids 6—30 of SEQ ID NO: 59; (261) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 61; (262) a ing sequence comprising amino acids 18—33 of SEQ ID NO: 61; (263) a targeting ce sing SEQ ID NO: 61 ; (264) an exosporium protein comprising an amino acid sequence having at least 85% sequence identity With SEQ ID NO: 62; (265) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 61 ; (266) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 61 ; (267) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 61; (268) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 61; (269) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 63; (270) a targeting sequence comprising SEQ ID NO: 63; (271) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 64; (272) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 63; (273) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 63; (274) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 63 ; (275) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 63 ; (276) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 63 ; (277) a targeting sequence sing amino acids 1—24 of SEQ ID NO: 65; (278) a targeting ce comprising amino acids 9—24 of SEQ ID NO: 65; (279) a targeting sequence comprising SEQ ID NO: 65; (280) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 66; (281) a targeting ce comprising SEQ ID NO: 107; (282) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 65; (283) 1 16 a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 65; (284) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 67; (285) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 67; (286) a targeting sequence comprising SEQ ID NO: 67; (287) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 68; (288) an targeting sequence comprising amino acids 2—27 of SEQ ID NO: 67; (289) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 67; (290) a targeting ce comprising amino acids 10—27 of SEQ ID NO: 67; (291) a targeting sequence comprising amino acids 1—38 of SEQ ID NO: 69; (292) a targeting sequence comprising amino acids 23—38 of SEQ ID NO: 69; (293) a targeting sequence comprising SEQ ID NO: 69; (294) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 70; (295) a targeting ce comprising amino acids 2—38 of SEQ ID NO: 69; (296) a ing ce comprising amino acids 5—38 of SEQ ID NO: 69; (297) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 69; (298) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 69; (299) an exosporium protein comprising SEQ ID NO: 72; (300) a targeting sequence comprising SEQ ID NO: 73; (301) an exosporium protein comprising an amino acid sequence haVing at least 95% identity With SEQ ID NO: 74; (302) a targeting ce comprising amino acids 1—42 of SEQ ID NO: 75; (303) a targeting sequence comprising amino acids 27—42 of SEQ ID NO: 75; (304) a targeting sequence comprising SEQ ID NO: 75; (305) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 76; (306) a targeting sequence comprising amino acids 2—42 of SEQ ID NO: 75; (307) a ing ce comprising amino acids 5—42 of SEQ ID NO: 75; (308) a targeting sequence comprising amino acids 10—42 of SEQ ID NO: 75; (309) a targeting sequence comprising amino acids 15—42 of SEQ ID NO: 75; (310) a targeting sequence comprising amino acids 20—42 of SEQ ID NO: 75; (311) a targeting sequence sing amino acids 25—42 of SEQ ID NO: 75; (312) a targeting sequence sing amino acids 1—24 of SEQ ID NO: 77; (313) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 77; (314) a targeting sequence comprising SEQ ID NO: 77; (315) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 78; (316) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 77; (317) a ing ce comprising amino acids 5—24 of SEQ ID NO: 77; (318) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 80; (319) a targeting sequence comprising amino acids 1—38 of SEQ ID NO: 81; (320) a targeting sequence comprising amino acids 23—38 of SEQ ID NO: 81; (321) a targeting sequence 2015/050807 1 17 comprising SEQ ID NO: 81; (322) an rium protein sing an amino acid sequence having at least 85% identity with SEQ ID NO: 82; (323) a targeting sequence sing amino acids 2—38 of SEQ ID NO: 81; (324) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 81; (325) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 81; (326) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 81; (327) a targeting ce comprising amino acids 20—38 of SEQ ID NO: 81; (328) a targeting sequence comprising amino acids 1—34 of SEQ ID NO: 83; (329) a targeting sequence comprising SEQ ID NO: 83; (330) an exosporium protein sing an amino acid ce having at least 85% identity With SEQ ID NO: 84; (331) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 86; (332) a targeting sequence sing amino acids 1—28 of SEQ ID NO: 87; (333) a targeting sequence comprising amino acids 13—28 of SEQ ID NO: 87; (334) a targeting sequence comprising SEQ ID NO: 87; (335) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 88; (336) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 87; (337) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 87; (338) a targeting ce comprising amino acids 10—28 of SEQ ID NO: 87; (339) a targeting sequence comprising amino acids 1—28 of SEQ ID NO: 89; (340) a targeting ce comprising SEQ ID NO: 89; (341) an exosporium n comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 90; (342) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 89; (343) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 89; (344) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 89; (345) a targeting sequence comprising amino acids 1—93 of SEQ ID NO: 91 ; (346) a targeting sequence comprising SEQ ID NO: 91 ; (347) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 92; (348) a targeting sequence comprising amino acids 2—93 of SEQ ID NO: 91 ; (349) a targeting sequence comprising amino acids 10—93 of SEQ ID NO: 91 ; (350) a targeting sequence comprising amino acids 20—93 of SEQ ID NO: 91 ; (351) a targeting sequence comprising amino acids 30—93 of SEQ ID NO: 91 ; (352) a targeting sequence comprising amino acids 40—93 of SEQ ID NO: 91 ; (353) a targeting sequence comprising amino acids 50—93 of SEQ ID NO: 91 ; (354) a targeting ce comprising amino acids 60—93 of SEQ ID NO: 91; (355) a targeting sequence comprising amino acids 1—130 of SEQ ID NO: 93; (356) a targeting sequence comprising SEQ ID NO: 93; (357) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 94; (358) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 93; (359) a targeting 1 18 sequence comprising amino acids 10—130 of SEQ ID NO: 93; (360) a ing sequence comprising amino acids 20—130 of SEQ ID NO: 93; (361) a targeting sequence comprising amino acids 30—130 of SEQ ID NO: 93; (362) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 122; (363) a targeting sequence consisting of amino acids 20—33 of SEQ ID NO: 1; (364) a targeting sequence consisting of amino acids 21—33 of SEQ ID NO: 1; (365) a targeting sequence consisting of amino acids 23— 31 of SEQ ID NO: 1; (366) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 96; (367) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 96; (368) a targeting sequence consisting of amino acids 12—25 of SEQ ID NO: 3; (369) a targeting ce consisting of amino acids 13—25 of SEQ ID NO: 3; (370) a targeting sequence consisting of amino acids 15—23 of SEQ ID NO: 3; (371) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 97; (372) a ing sequence ting of amino acids 1—13 of SEQ ID NO: 98; (373) a targeting sequence consisting of amino acids 23—36 of SEQ ID NO: ; (374) a targeting sequence consisting of amino acids 23—34 of SEQ ID NO: 5; (375) a targeting sequence consisting of amino acids 24—36 of SEQ ID NO: 5; (376) a targeting sequence consisting of amino acids 26—34 of SEQ ID NO: 5; (377) a targeting sequence consisting of amino acids 13—26 of SEQ ID NO: 7; (378) a ing ce consisting of amino acids 13—24 of SEQ ID NO: 7; (379) a targeting ce consisting of amino acids 14— 26 of SEQ ID NO: 7; (380) a targeting sequence consisting of amino acids 16—24 of SEQ ID NO: 7; (381) a targeting sequence consisting of amino acids 9—22 of SEQ ID NO: 9; (382) a targeting sequence consisting of amino acids 9—20 of SEQ ID NO: 9; (383) a targeting sequence consisting of amino acids 10—22 of SEQ ID NO: 9; (384) a ing sequence consisting of amino acids 12—20 of SEQ ID NO: 9; (385) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 105; (386) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 105; (387) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 11; (388) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 11; (389) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 11; (390) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 98 ; (391) a targeting ce consisting of amino acids 1—13 of SEQ ID NO: 98 ; (392) a ing sequence ting of amino acids 18— 31 of SEQ ID NO: 13; (393) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 13 ; (394) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 13 ; (395) a targeting sequence consisting of amino acids 21—29 of SEQ ID NO: 13; (396) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 99; (397) a targeting sequence 1 19 consisting of amino acids 1—13 of SEQ ID NO: 99; (398) a targeting sequence consisting of amino acids 28—41 of SEQ ID NO: 15; (399) a targeting sequence consisting of amino acids 28— 39 of SEQ ID NO: 15; (400) a targeting sequence consisting of amino acids 29—41 of SEQ ID NO: 15; (401) a targeting sequence consisting of amino acids 31—39 of SEQ ID NO: 15; (402) a targeting sequence ting of amino acids 12—25 of SEQ ID NO: 17; (403) a targeting sequence ting of amino acids 13—25 of SEQ ID NO: 17; (404) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 100; (405) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 19; (406) a targeting sequence consisting of amino acids 18— 29 of SEQ ID NO: 19; (407) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 19; (408) a targeting sequence consisting of amino acids 21—29 of SEQ ID NO: 19; (409) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 21; (410) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 21; (411) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 21; (412) a targeting ce consisting of amino acids 21—29 of SEQ ID NO: 21; (413) a targeting sequence ting of amino acids 1— of SEQ ID NO: 101; (414) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 101 ; (415) a targeting sequence consisting of amino acids 9—22 of SEQ ID NO: 23; (416) a targeting sequence consisting of amino acids 9—20 of SEQ ID NO: 23; (417) a targeting ce consisting of amino acids 10—22 of SEQ ID NO: 23; (418) a targeting sequence consisting of amino acids 12—20 of SEQ ID NO: 23; (419) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 102; (420) a targeting sequence consisting of amino acids 1— 13 of SEQ ID NO: 102; (421) a targeting sequence consisting of amino acids 9—22 of SEQ ID NO: 25; (422) a ing sequence consisting of amino acids 9—20 of SEQ ID NO: 25 ; (423) a targeting sequence consisting of amino acids 10—22 of SEQ ID NO: 25; (424) a targeting sequence consisting of amino acids 12—20 of SEQ ID NO: 25; (425) a targeting ce consisting of amino acids 1—15 of SEQ ID NO: 103; (426) a ing sequence consisting of amino acids 1—13 of SEQ ID NO: 103; (427) a targeting sequence consisting of amino acids 15— 28 of SEQ ID NO: 27; (428) a targeting sequence consisting of amino acids 15—26 of SEQ ID NO: 27; (429) a targeting ce consisting of amino acids 16—28 of SEQ ID NO: 27; (430) a ing sequence consisting of amino acids 18—26 of SEQ ID NO: 27; (431) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 104; (432) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 104; (433) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 33; (434) a targeting sequence consisting of amino acids 1—11 of SEQ ID NO: 33 ; (435) a targeting sequence consisting of amino acids 3—11 of SEQ ID NO: 33; (436) a targeting sequence consisting of amino acids 1—14 of SEQ ID NO: 35; (437) a targeting ce consisting of amino acids 1—12 of SEQ ID NO: 35; (438) a targeting sequence ting of amino acids 2—14 of SEQ ID NO: 35; (439) a targeting sequence consisting of amino acids 14—27 of SEQ ID NO: 43; (440) a targeting sequence consisting of amino acids 14—25 of SEQ ID NO: 43; (441) a targeting sequence consisting of amino acids 15— 27 of SEQ ID NO: 43; (442) a targeting sequence ting of amino acids 20—33 of SEQ ID NO: 45; (443) a ing sequence consisting of amino acids 20—31 of SEQ ID NO: 45 ; (444) a targeting sequence consisting of amino acids 21—33 of SEQ ID NO: 45; (445) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 106; (446) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 106; (447) a targeting sequence consisting of amino acids 28—41 of SEQ ID NO: 47; (448) a targeting sequence consisting of amino acids 28— 39 of SEQ ID NO: 47; (449) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 53; (450) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 53 ; (451) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 53; (452) a ing sequence comprising amino acids 18—31 of SEQ ID NO: 61; (453) a targeting sequence comprising amino acids 18—29 of SEQ ID NO: 61; (454) a targeting sequence comprising amino acids 19—31 of SEQ ID NO: 61; (455) a targeting ce comprising amino acids 9—22 of SEQ ID NO: 65; (456) a targeting sequence comprising amino acids 9—20 of SEQ ID NO: 65; (457) a ing sequence comprising amino acids 10—22 of SEQ ID NO: 65; (458) a targeting sequence comprising amino acids 1—15 of SEQ ID NO: 107; (459) a targeting sequence comprising amino acids 1—13 of SEQ ID NO: 107; (460) a targeting sequence comprising amino acids 12—25 of SEQ ID NO: 67; (461) a ing sequence comprising amino acids 12—23 of SEQ ID NO: 67; (462) a targeting sequence comprising amino acids 13—25 of SEQ ID NO: 67; (463) a targeting sequence comprising amino acids 15—23 of SEQ ID NO: 67; (464) a targeting sequence comprising amino acids 23—36 of SEQ ID NO: 69; (465) a targeting sequence comprising amino acids 23—34 of SEQ ID NO: 69; (466) a targeting sequence comprising amino acids 24—36 of SEQ ID NO: 69; (467) a targeting sequence sing amino acids 26—34 of SEQ ID NO: 69; (468) a targeting sequence sing amino acids 27—40 of SEQ ID NO: 75; (469) a ing sequence comprising amino acids 27—38 of SEQ ID NO: 75; (470) a targeting sequence comprising amino acids 9—22 of SEQ ID NO: 77; (471) a targeting sequence comprising amino acids 9—20 of SEQ ID NO: 77; (472) a targeting sequence comprising amino acids 10—22 of SEQ ID NO: 77; (473) a targeting sequence comprising amino acids 12—20 of SEQ ID NO: 77; (474) a targeting sequence comprising amino acids 23—36 of SEQ ID NO: 81; (475) a targeting ce comprising amino acids 23—34 of SEQ ID NO: 81; (476) a targeting sequence comprising amino acids 24—36 of SEQ ID NO: 81; (477) a targeting sequence comprising amino acids 26—34 of SEQ ID NO: 81; (478) a targeting sequence comprising amino acids 13—26 of SEQ ID NO: 87; (479) a targeting sequence comprising amino acids 13—24 of SEQ ID NO: 87; or (480) a targeting sequence comprising amino acids 14—26 of SEQ ID NO: For example, the targeting sequence can comprise an amino acid sequence haVing at least about 50% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 63%.

For example, the targeting sequence can comprise an amino acid sequence haVing at least about 56% ty with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 63%.

For example, the targeting ce can se an amino acid sequence haVing at least about 50% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the ty with amino acids 25—35 is at least about 72%.

For example, the targeting sequence can comprise an amino acid ce haVing at least about 62% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 72%.

For example, the targeting sequence can comprise an amino acid sequence haVing at least about 75% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the ty with amino acids 25—35 is at least about 72%.

For example, the targeting sequence can comprise an amino acid sequence haVing at least about 68% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 81%.

For example, the targeting sequence can comprise an amino acid sequence haVing at least about 75% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 81%.

For example, the targeting sequence can comprise an amino acid sequence haVing at least about 81% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 81%.

For example, the ing sequence can se an amino acid sequence haVing at least about 81% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 90%.

WO 44661 For example, the targeting sequence can consist of: (a) an amino acid sequence ting of 16 amino acids and having at least about 43% identity with amino acids —35 of SEQ ID NO: 1, wherein the identity With amino acids 25—35 is at least about 54%; (b) amino acids 1—35 of SEQ ID NO: 1; (c) amino acids 20—35 of SEQ ID NO: 1; (d) SEQ ID NO: 1; (e) SEQ ID NO: 96; or (f) SEQ ID NO: 120.

The targeting sequence can consist of the amino acid sequence as described in these examples.

The fusion protein can comprise an exosporium protein or an exosporium protein fragment comprising an amino acid ce haVing at least 90% identity With SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113,114,115,116,117,118,119,120,121,and122.

The fusion protein can se an exosporium protein or an exosporium protein fragment comprising an amino acid ce haVing at least 95% identity With SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113,114,115,116,117,118,119,120,121,and122.

The fusion protein can comprise an exosporium protein or an exosporium n fragment comprising an amino acid sequence haVing at least 98% identity With SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113,114,115,116,117,118,119,120,121,and122.

The fusion protein can comprise an exosporium protein or an exosporium protein fragment comprising an amino acid sequence haVing at least 99% identity With SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113,114,115,116,117,118,119,120,121,and122.

The fusion protein can comprise an exosporium protein or an exosporium protein fragment comprising an amino acid sequence haVing 100% identity With SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113, 114,115,116,117,118,119,120,121,and122.

The fusion protein can comprise an exosporium protein comprising an amino acid sequence having at least 90% identity With SEQ ID NO: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 122.

The fusion protein can comprise an exosporium protein comprising an amino acid sequence haVing at least 95% identity With SEQ ID NO: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 122.

The fusion protein can comprise an exosporium protein comprising an amino acid ce haVing at least 98% identity With SEQ ID NO: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 122.

The fusion protein can comprise an exosporium protein comprising an amino acid ce haVing at least 99% identity With SEQ ID NO: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 122.

The fusion protein can comprise an exosporium protein comprising an amino acid sequence haVing 100% identity With SEQ ID NO: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 122.

The targeting ce, exosporium protein, or exosporium protein fragment of the fusion protein can comprise the amino acid sequence GXT at its carboxy terminus, wherein X is any amino acid.

The targeting sequence, exosporium protein, or exosporium n fragment can se an e residue at the on of the ing sequence that corresponds to amino acid 20 of SEQ ID NO: 1.

The targeting ce, exosporium protein, or exosporium protein fragment can further comprise a methionine, serine, or threonine residue at the amino acid position immediately preceding the first amino acid of the targeting sequence, exosporium protein, or exosporium protein fragment or at the position of the targeting sequence that ponds to amino acid 20 of SEQ ID NO: 1.

IX. Fusion Proteins Comprising a Protein or Peptide of Interest and a Spore Coat Protein, Recombinant Coat Forming Bacteria, and Seeds Coated with Recombinant Spore-Coat Forming Bacteria A. Spore coat proteins that can be usea1 to target a fusion protein sing the spore coat protein to a surface of a spore of a recombinant spore-Zorming bacterium A number of spore coat proteins can be used to y proteins or peptides of interest on a surface of a spore of a recombinant spore-forming bacterium. Such bacteria include any spore-forming bacteria, and in particular include spore-forming bacteria of the genuses Bacillus, Lysinibacillus, Virginibacillus, Clostria’ia, and Paenibacillus. Spore-forming bacteria of the genus Bacillus include Bacillus cereus family members as well as other Bacillus s that are not Bacillus cereus family members (e. g., Bacillus species bacteria that lack an rium). These spore coat proteins include CotB, CotC, CgeA, CotB/H, CotG, spore coat protein X, and CotY. For ease of reference, the descriptions of the amino acid sequences for exemplary spore coat proteins that can be used for targeting of proteins or peptides of interest to a spore surface of a recombinant spore-forming bacterium are ed in Table 7 below, together with their SEQ ID NOs.

Table 7. Spore coat protein sequences used for targeting of proteins and peptides of st to a spore surface of a recombinant spore-forming bacterium B. Fusion proteins comprising a protein or peptide of interest and a spore coat protein The present invention also s to fusion ns comprising at least one protein or e of interest and a spore coat protein, wherein the spore coat protein comprises a CotB/H protein, a spore protein X n, or a CotY protein, wherein the CotY n comprises an amino acid sequence haVing at least 80% identity with SEQ ID NO: 258 or 259.

For example, the spore coat protein can comprise a CotB/H protein.

For example, the spore coat protein can comprise a spore protein X protein.

For example, the spore coat protein can comprise a CotY n, wherein the CotY protein comprises an amino acid sequence haVing at least 80% ty with SEQ ID NO: 258 or 259.

The spore coat protein can comprises an amino acid sequence haVing at least 85% identity with SEQ ID NO: 255, 257, 258, or 259.

The spore coat protein can comprises an amino acid sequence haVing at least 90% identity with SEQ ID NO: 255, 257, 258, or 259.

The spore coat protein can ses an amino acid sequence having at least 95% identity with SEQ ID NO: 255, 257, 258, or 259.

The spore coat protein can comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 255, 257,258, or 259.

The spore coat protein can comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 255, 257,258, or 259.

The spore coat protein can comprises an amino acid sequence having at least 100% identity with SEQ ID NO: 255, 257, 258, or 259.

C. Recombinant sgore-coat forming bacteria that exgress fusion groteins and giant seeds coated with recombinant sgore-coat forming bacteria Recombinant spore-forming bacteria that expresses any of the fusion proteins described in Section IX.B are ed. The recombinant spore-forming bacteria can comprise an endophytic strain of bacteria, a plant growth-promoting strain of bacteria, or a strain of ia that is both ytic and plant -promoting.

The present invention further relates to a recombinant spore-forming bacterium that expresses a fusion protein comprising at least one n or peptide of interest and a spore coat protein that s the fusion protein to the surface of a spore of the bacterium, wherein the spore coat protein comprises a CotB protein, a CotC protein, a CgeA protein, a CotB/H protein, a CotG protein, a spore coat n X protein, or a CotY protein; and wherein the recombinant spore-forming bacterium comprises an endophytic strain of bacteria, a plant growth-promoting strain of bacteria, or a strain of bacteria that is both endophytic and plant -promoting.

Expression of the fusion protein in an endophytic strain of bacteria allows for delivery of the protein or peptide of interest internally to a plant. The endophytic strains can be delivered to plants using various methods, e.g., the endophytic strains can be red Via seed treatment, treatment of the plant growth medium (e.g., soil), irrigation, application to the plant itelf (e. g., foliar application to the aerial portions of a plant). Once inside the plant, the ia multiply and colonize the internal tissues of the plant.

The present ion also relates to plant seeds coated with a recombinant spore-forming bacterium, n the inant spore-forming ium expresses a fusion protein comprising at least one protein or peptide of interest and a spore coat protein that targets the fusion protein to the surface of a spore of the bacterium, wherein the spore coat protein comprises a cotB protein, a CotC protein, a CgeA protein, a CotB/H protein, a Cot G n, a spore protein X protein, or a cotY protein.

The recombinant spore-coat forming ium can comprise a bacterium of the genus Bacillus or Lysinibacillus.

] The present invention further relates to a recombinant ium of the genus Bacillus, wherein the recombinant bacterium comprises a recombinant spore-forming bacterium and wherein the recombinant forming bacterium expresses a fusion protein comprising at least one protein or peptide of interest and a spore coat protein that targets the fusion protein to the surface of a spore of the bacterium, wherein the spore coat protein comprises a CotB protein, a CotC protein, a CgeA protein, a CotB/H protein, a Cot G protein, a spore coat protein X protein, or a CotY protein. The recombinant spore-coat forming bacterium ses a protease or a nuclease, wherein the expression of the se or nuclease is increased as compared to the expression of the protease or the nuclease in a wild-type bacterium of the genus Bacillus under the same conditions, and wherein the increased sion of the protease or the nuclease partially or tely inactivates spores of the recombinant bacterium of the genus Bacillus or renders spores of the recombinant bacterium of the genus Bacillus more susceptible to physical or al inactivation. The protease or nuclease can be any of the proteases or nucleases described above in Section V.A, and can be expressed under the control of any of the promoters described above in Section VA. The invention further relates to plant seeds coated with such spore-forming bacteria. The recombinant ium can se an endophytic strain of bacteria, a plant growth-promoting strain of bacteria, or a strain of bacteria that is both endophytic and plant growth-promoting.

In any of the plant seeds described in this Section, the recombinant spore- forming bacterium can comprise an endophytic strain of bacteria, a plant growth-promoting strain of bacteria, or a strain of ia that is both ytic and plant -promoting.

In any of the recombinant spore-forming bacteria or seeds, the endophytic strain of bacteria, the plant growth-promoting strain of bacteria, or the strain of bacteria that is both ytic and plant growth-promoting can comprise Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillusfusiformis EE442, or Lysinibacillus sphaericus EE443, Bacillus pumilus EE-B00143, Bacillus subtilis EEl48, Bacillus subtilis EE2l8, or Bacillus megaterium EE28l. For example, the endophytic strain of bacteria can comprise Bacillus subtilis EE405 or Bacillus megaterium EE385.

Alternatively, the endophytic strain, the plant growth-promoting strain of bacteria, or the strain of bacteria that is both endophytic and plant growth-promoting of bacteria can comprise Bacillus cereus family member EE349, Bacillus cereus family member EE439, Bacillus thuringiensis EE4l7, Bacillus cereus EE444, or Bacillus giensis EE319, Bacillus thuringiensis EE-B00184, us cereus family member EE-B003 77, Bacillus pseudomycoia’es EE-B00366, Bacillus mycoia’es EE-B00363, Bacillus mycoia’es BT155, Bacillus mycoia’es EE118, Bacillus mycoia’es EEl4l, Bacillus mycoia’es BT46-3, Bacillus cereus family member EE128, Bacillus thuringiensis BT013A, or Bacillus cereus family member EE349.

In any of the inant spore-forming bacteria or seeds, the spore coat protein can comprise an amino acid sequence having at least 85% identity With any of SEQ ID NOs: 252—259.

] The spore coat protein can comprise an amino acid ce having at least 90% identity With any of SEQ ID NOs: 252—259.

The spore coat protein can comprise an amino acid sequence having at least 95% identity With any of SEQ ID NOs: 9.

The spore coat protein can comprise an amino acid sequence having at least 98% identity With any of SEQ ID NOs: 252—259.

The spore coat protein can comprise an amino acid sequence having at least 99% identity With any of SEQ ID NOs: 252—259.

The spore coat protein can comprise an amino acid ce having 100% identity With any of SEQ ID NOs: 252—259.

A recombinant spore-forming bacterium that expresses a fusion protein comprising at least one protein or e of interest and a protein that targets the fusion protein to the surface of a spore of the bacterium is also provided. The recombinant spore-forming bacterium is not a recombinant us cereus family member. The protein that targets the fusion n to the surface of a spore of the bacterium comprises amino acids 20—35 of SEQ ID NO: 1, SEQ ID NO: 96, or an amino acid sequence having at least 85% identity With SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO: 114, SEQ ID NO: 120, or SEQ ID NO: 121.

] The n that targets the fusion protein of the surface of a spore of the bacterium can comprise an amino acid sequence having at least 90% identity With SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO: 114, SEQ ID NO: 120, or SEQ ID NO:121.

The protein that targets the fusion protein of the surface of a spore of the bacterium can comprise an amino acid sequence having at least 95% identity With SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO: 114, SEQ ID NO: 120, or SEQ ID NO: 121.

The protein that targets the fusion protein of the surface of a spore of the ium can se an amino acid sequence having at least 98% identity With SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO: 114, SEQ ID NO: 120, or SEQ ID NO:121.

The protein that targets the fusion protein of the surface of a spore of the bacterium can comprise an amino acid ce having at least 99% identity With SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO: 114, SEQ ID NO: 120, or SEQ ID NO:121.

The protein that targets the fusion protein of the surface of a spore of the bacterium can comprise an amino acid sequence having at least 100% identity With SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO: 114, SEQ ID NO: 120, or SEQ ID NO:121.

For example, the protein that targets the fusion protein to a surface of a spore of the bacterium can comprise amino acids 20—35 of SEQ ID NO: 1, SEQ ID NO: 96, SEQ ID NO: 108, SEQ ID NO: 120, or SEQ ID NO:121.

The recombinant-spore forming bacterium comprises an endophytic strain of bacteria, a plant growth-promoting strain of bacteria, or a strain of ia that is both endophytic and plant growth-promoting. For example, the endophytic strain of bacteria, the plant growth-promoting strain of bacteria, or the strain of bacteria that is both endophytic and plant growth-promoting ses us rium EE385, Bacillus sp. EE3 87, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillusfusiformis EE442, Lysinibacillus sphaericus EE443, Bacillus s 143, Bacillus subtilis EE148, Bacillus is EE218, or Bacillus megaterium EE281. The endophytic strain of bacteria preferably comprises Bacillus sp. EE387.

X. Methods for Making the Fusion Proteins Any of the fusion proteins described herein can be made using standard cloning and molecular biology methods known in the art. For example, a gene encoding a protein or e of interest (e.g., a gene encoding a plant growth stimulating protein or peptide) can be ed by rase chain reaction (PCR) and ligated to DNA coding for any of the above-described targeting sequences, exosporium proteins, exosporium protein fragments, or spore coat proteins, to form a DNA molecule that encodes the fusion protein. The DNA molecule encoding the fusion protein can be cloned into any suitable vector, for example a WO 44661 d vector. The vector suitably comprises a multiple cloning site into which the DNA molecule encoding the fusion protein can be easily inserted. The vector also suitably contains a selectable marker, such as an antibiotic resistance gene, such that bacteria transformed, transfected, or mated with the vector can be readily identified and isolated. Where the vector is a plasmid, the plasmid suitably also comprises an origin of replication. Alternatively, DNA coding for the fusion protein can be integrated into the chromosomal DNA of the B. cereus family member or spore-forming bacterium host.

XI. Tags, Markers, and Linkers that Can be Included in the Fusion Proteins Any of the fusion ns described herein can also comprise additional polypeptide sequences that are not part of the targeting sequence, exosporium protein, exosporium protein fragment, or the plant growth ating protein or peptide, the protein or peptide that protects a plant from a pathogen, the protein or peptide that enhances stress resistance in a plant, or the plant binding protein or peptide. For example, the fusion protein can e tags or markers to facilitate purification or visualization of the fusion n (e.g., a polyhistidine tag or a cent protein such as GFP or YFP) or visualization of recombinant us cereus family member spores expressing the fusion protein. sion of fusion proteins on the exosporium of a us cereus family member or on a surface of a spore of a spore-forming bacterium using the targeting sequences, exosporium proteins, exosporium protein nts, and spore coat proteins described herein is enhanced due to a lack of secondary structure in the amino-termini of these sequences, which allows for native folding of the fused proteins and retention of activity. Proper folding can be further enhanced by the inclusion of a short amino acid linker between the targeting sequence, exosporium protein, exosporium protein fragment, spore coat protein, and the protein or peptide of interest.

Thus, any of the fusion ns described herein can comprise an amino acid linker between the targeting sequence, the exosporium protein, the exosporium protein nt, or the spore coat protein and the protein or peptide of interest.

The linker can comprise a polyalanine linker or a ycine linker. A linker comprising a mixture of both alanine and e residues can also be used.

For example, in a fusion protein where the targeting ce comprises SEQ ID NO: 1, a fusion protein can have one of the ing structures: No linker: SEQ ID NO: 1 — POI Alanine Linker: SEQ ID NO: l—An—POI Glycine : SEQ ID NO: l—Gn—POI Mixed Alanine and Glycine Linker: SEQ ID NO: 1 — (A/G)Il — POI where An, G11, and (A/G)Il are any number of alanines, any number of glycines, or any number of a mixture of alanines and glycines, respectively. For example, 11 can be 1 to 25, and is preferably 6 to 10. Where the linker comprises a mixture of alanine and glycine residues, any ation of glycine and alanine residues can be used. In the above structures, “POI” represents the n or peptide of interest.

Alternatively or in addition, the linker can comprise a protease recognition site. Inclusion of a protease recognition site allows for targeted removal, upon exposure to a protease that recognizes the protease recognition site, of the protein or peptide of interest.

XII. Proteins and Peptides of Interest The protein or peptide of interest can comprise any protein or peptide.

The n or peptide of interest in the fusion proteins bed herein can se, for e: (a) a plant growth stimulating protein or peptide; (b) a protein or peptide that protects a plant from a pathogen; (c) a protein or peptide that enhances stress resistance of a plant; (d) a plant binding protein or peptide; (e) an enzyme that catalyzes the production of nitric oxide; (f) a nucleic acid binding protein or peptide; or (g) a plant signaling molecule or a protein or peptide that alters the composition of a plant; (h) an antigen; (i) a remediation enzyme; (j ) an enzyme suitable for breaking an emulsion or gel in a hydraulic fracturing fluid; or (k) an antibacterial n or peptide.

A. Plant growth stimulating ns or gegtides The protein or peptide of interest can comprise a plant growth stimulating protein or peptide.

The plant growth stimulating protein or peptide can comprise a peptide hormone, a non-hormone e, an enzyme involved in the production or activation of a plant growth ating compound, or an enzyme that degrades or es a bacterial, fungal, or plant nutrient source. 13 1 For example, the plant growth stimulating n or peptide can comprise a peptide hormone.

The peptide hormone can comprise a phytosulfokine (e.g., phytosulfokine-u), a 3 (CLV3), systemin, ZmlGF, or a SClVSPl 1.

The plant growth stimulating protein or peptide can comprise a non-hormone peptide.

The non-hormone peptide can comprise a RKN 16D10, Hg-Syy46, an eNOD40 peptide, melittin, mastoparan, Mas7, RHPP, S, or kunitz trypsin inhibitor (KTI).

The plant growth stimulating protein or peptide can comprise an enzyme involved in the production or activation of a plant growth stimulating compound. The enzyme involved in the production or activation of a plant growth stimulating compound can be any enzyme that catalyzes any step in a biological synthesis pathway for a compound that stimulates plant growth or alters plant structure, or any enzyme that catalyzes the conversion of an inactive or less active tive of a compound that ates plant growth or alters plant structure into an active or more active form of the compound.

The plant growth stimulating compound can comprise a compound produced by bacteria or fungi in the rhizosphere, e.g., 2,3-butanediol.

Alternatively, the plant growth ating compound can comprise a plant growth hormone.

The plant growth hormone can comprise a nin or a cytokinin derivative, ethylene, an auxin or an auxin derivative, a gibberellic acid or a gibberellic acid derivative, abscisic acid or an abscisic acid derivative, or a jasmonic acid or a jasmonic acid derivative.

] Where the plant growth stimulating compound comprises a cytokinin or a cytokinin derivative, the cytokinin or the cytokinin tive can se kinetin, cis-zeatin, zeatin, 6-benzylaminopurine, dihydroxyzeatin, N6-(D2-isopentenyl) adenine, ribosylzeatin, N6-(D2—isopentenyl) ine, 2-methylthio-cis-ribosylzeatin, cis-ribosylzeatin, trans-ribosylzeatin, 2-methylthio-trans-ribosylzeatin, lzeatinmonosphosphate, N6- methylaminopurine, N6-dimethylaminopurine, 2’-deoxyzeatin riboside, 4-hydroxymethyl- transbutenylaminopurine, ortho-topolin, meta-topolin, benzyladenine, ortho-methyltopolin, meta-methyltopolin, or a combination thereof.

Where the plant growth ating compound comprises an auxin or an auxin derivative, the auxin or the auxin derivative can comprise an active auxin, an inactive auxin, a conjugated auxin, a naturally occurring auxin, or a synthetic auxin, or a combination thereof. For example, the auxin or auxin derivative can comprise indoleacetic acid, indole pyruvic acid, indoleacetaldoxime, indoleacetamide, indoleacetonitrile, indoleethanol, pyruvate, indoleacetaldoxime, indolebutyric acid, a phenylacetic acid, 4- indoleacetic acid, a glucose-conjugated auxin, or a combination thereof.

The enzyme involved in the production or activation of a plant growth stimulating compound can comprise an acetoin reductase, an indoleacetamide hydrolase, a tryptophan monooxygenase, an acetolactate synthetase, an (x-acetolactate decarboxylase, a pyruvate decarboxylase, a yl reductase, a diol dehydrogenase, an aminotransferase (e.g., tryptophan aminotransferase), a tryptophan decarboxylase, an amine oxidase, an indole pyruvate decarboxylase, an acetaldehyde dehydrogenase, a tryptophan side chain oxidase, a nitrile hydrolase, a nitrilase, a peptidase, a protease, an adenosine phosphate isopentenyltransferase, a phosphatase, an adenosine , an adenine phosphoribosyltransferase, CYP735A, a nucleotide phosphohydrolase, an adenosine nucleosidase, a zeatin cis-trans isomerase, a zeatin O-glucosyltransferase, a B-glucosidase, a cis- hydroxylase, a CK cis-hydroxylase, a CK N—glucosyltransferase, a 2,5-ribonucleotide phosphohydrolase, an adenosine sidase, a purine nucleoside phosphorylase, a zeatin reductase, a hydroxylamine reductase, a lutarate dioxygenase, a ellic 2B/3B hydrolase, a gibberellin 3-oxidase, a gibberellin 20-oxidase, a chitosanase, a chitinase, a B-l,3- glucanase, a glucanase, a B-l,6-glucanase, an aminocyclopropane-l-carboxylic acid deaminase, or an enzyme involved in producing a nod factor (e.g., nodA, nodB, or nodI).

] Where the enzyme comprises a protease or peptidase, the protease or peptidase can be a protease or peptidase that cleaves proteins, peptides, proproteins, or preproproteins to create a bioactive peptide. The ive peptide can be any peptide that exerts a biological activity.

Examples of bioactive peptides include RKN 16D10 and RHPP.

The protease or peptidase that cleaves proteins, peptides, proproteins, or preproproteins to create a ive peptide can comprise subtilisin, an acid protease, an alkaline protease, a proteinase, an endopeptidase, an exopeptidase, thermolysin, papain, pepsin, trypsin, pronase, a carboxylase, a serine se, a glutamic protease, an aspartate protease, a cysteine protease, a threonine protease, or a metalloprotease.

The protease or peptidase can cleave proteins in a n-rich meal (e.g., soybean meal or yeast extract).

Where the enzyme comprises a chitosanase, the chitosanase can comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 3 13.

The chitosanase can comprise an amino acid ce having at least 90% identity with SEQ ID NO: 3 13.

] The chitosanase can comprise an amino acid sequence having at least 95% identity with SEQ ID NO: 3 13.

The chitosanase can comprise an amino acid sequence having at least 98% identity with SEQ ID NO: 3 13.

The chitosanase can se an amino acid sequence having at least 99% identity with SEQ ID NO: 3 13.

The chitosanase can comprise an amino acid sequence having at least 100% ty with SEQ ID NO: 3 13.

For example, the fusion protein can comprise amino acids 20—35 of BclA (amino acids 20—35 of SEQ ID NO: 1) as the targeting ce and an amino acid sequence comprising SEQ ID NO: 3 13 as the enzyme that is specific for a ar component of a bacterium or fungus. The fusion protein can further comprise a linker (e.g., a polyalanine linker) between the ing sequence and the enzyme.

The plant growth stimulating protein or peptide can comprise an enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source.

The enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source can comprise a cellulase, a lipase, a lignin oxidase, a protease, a glycoside hydrolase, a phosphatase, a nitrogenase, a nuclease, an amidase, a nitrate reductase, a nitrite reductase, an amylase, an ammonia oxidase, a ligninase, a glucosidase, a phospholipase, a phytase, a pectinase, a glucanase, a ase, a urease, a xylanase, or a siderophore.

When introduced into a plant growth medium or applied to a plant, seed, or an area surrounding a plant or a plant seed, fusion proteins comprising enzymes that e or modify a bacterial, fungal, or plant nutrient source can aid in the processing of nutrients in the ty of the plant and result in enhanced uptake of nutrients by the plant or by beneficial bacteria or fungi in the vicinity of the plant.

The enzyme that degrades or modifies a bacterial, , or plant nutrient source can comprise a ase.

The cellulase can comprise an llulase (e. g., an ucanase such as a Bacillus subtilis endoglucanase, a Bacillus thuringiensis endoglucanase, a Bacillus cereus endoglucanase, or a Bacillus clausii endoglucanase), an exocellulase (e.g., a Trichoa’erma reesei exocellulase), or a B-glucosidase (e. g., a Bacillus subtilis B-glucosidase, a Bacillus thuringiensis B-glucosidase, a Bacillus cereus B-glucosidase, or a Bacillus clausii osidase). The cellulase preferably comprises a us subtilis endoglucanase.

The endoglucanase can comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 3 l 1.

The endoglucanase can comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 3 l 1.

The endoglucanase can comprise an amino acid sequence having at least 95% identity with SEQ ID NO: 3 l 1.

The endoglucanase can comprise an amino acid sequence having at least 98% identity with SEQ ID NO: 3 l 1.

The endoglucanase can comprise an amino acid sequence having at least 99% identity with SEQ ID NO: 3 l 1.

] The endoglucanase can comprise an amino acid sequence having 100% identity with SEQ ID NO: 3 l 1.

For example, the fusion protein can comprise amino acids 20—35 of BclA (amino acids 20—35 of SEQ ID NO: 1) as the targeting sequence and an amino acid sequence sing SEQ ID NO: 3 11 as the enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source. The fusion protein can further comprise a linker (e.g., a polyalanine linker) between the targeting sequence and the enzyme.

The enzyme that degrades or s a bacterial, fungal, or plant nt source can comprise a lipase (e.g., a us subtilis lipase, a Bacillus thuringiensis lipase, a Bacillus cereus lipase, or a Bacillus clausii lipase).

The enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source can comprise a lignin e. For example, the lignin e can comprise a lignin peroxidase, a laccase, a l oxidase, a ligninase, or a manganese peroxidase.

The enzyme that degrades or s a bacterial, fungal, or plant nutrient source can comprise a se. For example, the protease can comprise a subtilisin, an acid protease, an alkaline protease, a proteinase, a peptidase, an endopeptidase, an exopeptidase, a lysin, a papain, a , a trypsin, a pronase, a carboxylase, a serine protease, a glutamic protease, an aspartate protease, a ne protease, a threonine protease, or a metalloprotease. 13 5 The enzyme that es or modifies a bacterial, fungal, or plant nutrient source can comprise a atase. For example, the atase can comprise a phosphoric monoester hydrolase, a phosphomonoesterase (e.g., PhoA4), a phosphoric diester hydrolase, a phosphodiesterase, a triphosphoric ter hydrolase, a phosphoryl anhydride hydrolase, a pyrophosphatase, a phytase (e.g., a Bacillus subtilis EEl48 phytase or a Bacillus thuringiensis BTOl3A phytase), a trimetaphosphatase, or a triphosphatase.

The enzyme that degrades or modifies a bacterial, , or plant nutrient source can comprise a nitrogenase. For example the nitrogenase can comprise a Nif family nitrogenase (e.g., Paem’bacz’llus massilz'ensz's NifBDEHKNXV).

The enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source can comprise a phospholipase. For example, the phospholipase can se a phospholipase A1, a phospholipase A2, a phospholipase C, a phospholipase D, or a lysophospholipase. The phospholipase preferably comprises a phospholipsae C.

] The phospholipase C can comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 312.

The phospholipase C can comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 312.

The phospholipase C can comprise an amino acid sequence having at least 95% identity with SEQ ID NO: 312.

The phospholipase C can comprise an amino acid sequence having at least 98% identity with SEQ ID NO: 312.

The phospholipase C can se an amino acid sequence having at least 99% identity with SEQ ID NO: 312.

The olipase C can comprise an amino acid sequence having 100% identity with SEQ ID NO: 312.

For example, the fusion protein can comprise amino acids 20—35 of BclA (amino acids 20—35 of SEQ ID NO: 1) as the targeting sequence and an amino acid sequence comprising SEQ ID NO: 312 as the enzyme that degrades or modifies a ial, fungal, or plant nutrient source. The fusion protein can further comprise a linker (e.g., a polyalanine ) between the targeting ce and the enzyme.

B. Proteins 0r peptides that protect plants from pathogens The protein or peptide of interest can comprise a protein or peptide that protects a plant from a pathogen.

The protein or peptide that protects a plant from a pathogen can comprise a plant immune system enhancer protein or peptide.

For example, the plant immune system enhancer protein or peptide can comprise a harpin, a harpin-like n, an (x-elastin, a B-elastin, a systemin, a alanine ammonia-lyase, an elicitin, a defensin, a cryptogein, a flagellin protein, or a flagellin peptide (e.g., flg22).

The protein or peptide that protects a plant from a pathogen can be a protein or peptide that has antibacterial activity, antifungal activity, or both antibacterial and ngal activity. Examples of such proteins and peptides include bacteriocins, lysozymes, lysozyme peptides (e.g., LysM), siderophores, avidins, avidins, non-ribosomal active peptides, conalbumins, albumins, lactoferrins, lactoferrin peptides (e. g., LfcinB), and TasA.

The protein or peptide that protects a plant from a pathogen can be a n or a e that has insecticidal activity, helminthicidal activity, suppresses insect or worm predation, or a ation thereof. For example, the protein or peptide that protects a plant from a pathogen can comprise an insecticidal bacterial toxin (e.g., a VIP insecticidal protein), an xin, a Cry toxin (e. g., a Cry toxin from Bacillus thuringiensis), a protease inhibitor n or peptide (e.g., a trypsin inhibitor or an arrowhead protease inhibitor), a ne protease, or a chitinase. Where the Cry toxin ses a Cry toxin from Bacillus thuringiensis, the Cry toxin can be a CrySB protein or a CryZlA protein. CrySB and Cry21A have both icidal and nematocidal activity.

The protein that protects a plant from a en can comprise an enzyme.

For example, the enzyme can comprise a protease or a lactonase. The proteases and lactonases can be c for a bacterial signaling molecule (e. g., a ial lactone homoserine signaling molecule).

Where the enzyme comprises a lactonase, the lactonase can comprise 1,4- lactonase, 2-pyrone-4,6-dicarboxylate lactonase, 3-oxoadipate enol-lactonase, actinomycin lactonase, deoxylimonate A-ring-lactonase, olactonase L-rhamnono-l,4-lactonase, limonin-D-ring-lactonase, steroid-lactonase, triacetate-lactonase, or xylono-l,4-lactonase.

The enzyme can comprise an enzyme that is specific for a cellular component of a bacterium or fungus. For example, the enzyme can comprise a B-l,3-glucanase, a B-l,4- 2015/050807 glucanase, a B-l,6-glucanase, a chitosanase, a ase, a chitosanase-like enzyme, a se, a peptidase, a proteinase, a protease (e.g., an alkaline protease, an acid protease, or a neutral protease), a mutanolysin, a stapholysin, or a lysozyme.

Where the enzyme comprises a chitosanase, the chitosanase can comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 3 13.

The chitosanase can comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 3 13.

The chitosanase can comprise an amino acid sequence having at least 95% identity with SEQ ID NO: 3 13.

The anase can comprise an amino acid sequence having at least 98% ty with SEQ ID NO: 3 13.

The anase can se an amino acid sequence having at least 99% identity with SEQ ID NO: 3 13.

The chitosanase can comprise an amino acid sequence having at least 100% identity with SEQ ID NO: 3 13.

For example, the fusion protein can comprise amino acids 20—35 of BclA (amino acids 20—35 of SEQ ID NO: 1) as the targeting sequence and an amino acid sequence comprising SEQ ID NO: 3 13 as the enzyme that is specific for a cellular component of a bacterium or fungus. The fusion protein can further comprise a linker (e.g., a polyalanine linker) between the targeting sequence and the enzyme.

] For any of the above proteins or peptides that protect a plant from a en, the pathogen can comprise a protein or a peptide of interest that protects a plant from a bacterial pathogen, a fungal pathogen, a worm pathogen, or an insect pathogen.

For example, the bacterial pathogen can se an u-class Proteobacterium, a B-class Proteobacterium, a y-class Proteobacterium, or a combination thereof; or n the bacterial pathogen comprises Agrobacterz'um tumefacz’ens, Pantoea stewartz'z', Erwinia carotovora, Ralstonia cearum, Pseudomonas syringae, Pseudomonas aeruginosa, Xanthomonas campestris, or a combination thereof.

] The protein or peptide that protects a plant from a pathogen can comprise a protein or peptide protects the plant from predation by a worm or an insect en.

The worm or insect pathogen can comprise an army worm, a black cutworm, a European corn borer, a fall rm, a cutworm, a Japanese beetle, a lesser comstalk borer, a maize billbug, a seed corn maggot, a webworm, a southern comstalk borer, a southern corn 13 8 rootworm, a southern potato wireworm, a stalk borer, a sugarcane , a white grub, a cabbage looper, a boll weevil, a yellow striped armyworm, a cereal leaf beetle, a chinch bug, an aphid, a beet armyworm, a Mexican bean beetle, a soybean , soybean stem borer, or a combination thereof.

C. Proteins 0r QthideS that enhance stress-resistance in giants The protein or peptide of interest can comprise a protein or peptide that enhances stress resistance in a plant.

For example, the protein or peptide that enhances stress resistance in a plant can comprise an enzyme that degrades a stress-related compound. Stress-related compounds include, but are not limited to, aminocyclopropane-l-carboxylic acid (ACC), reactive oxygen s, nitric oxide, oxylipins, and phenolics. Specific reactive oxygen species include hydroxyl, hydrogen peroxide, , and superoxide.

The enzyme that degrades a stress-related compound can comprise a superoxide ase, an oxidase, a catalase, an aminocyclopropane-l-carboxylic acid deaminase, a peroxidase, an antioxidant enzyme, or an antioxidant peptide.

When the enzyme that degrades a stress-related compound comprises a superoxide dismutase, the xide ase can comprise superoxide dismutase 1 (SODAl) or superoxide dismutase 2 (SODAZ).

The superoxide dismutase can comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase can se an amino acid sequence having at least 90% identity with SEQ ID NO: 155 or 156.

The xide dismutase can se an amino acid sequence having at least 95% identity with SEQ ID NO: 155 or 156.

The xide dismutase can comprise an amino acid sequence having at least 98% identity with SEQ ID NO: 155 or 156.

The superoxide ase can comprise an amino acid sequence having at least 99% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase can se an amino acid sequence having 100% identity with SEQ ID NO: 155 or 156.

The protein or peptide that enhances stress resistance in a plant can comprise a protein or peptide that protects a plant from an environmental stress. The environmental stress can comprise, for example, t, flood, heat, freezing, salt, heavy metals, low pH, high pH, or a combination thereof For instance, the n or peptide that protects a plant from an environmental stress can comprises an ice nucleation protein, a ase, a phenylalanine ammonia lyase, an isochorismate synthase, an isochorismate pyruvate lyase, or a choline dehydrogenase.

D. Plant binding proteins or es The protein or peptide of interest can comprise a plant g protein or peptide. The plant binding protein or peptide can be any protein or e that is capable of specifically or non-specifically binding to any part of a plant (e.g., a plant root or an aerial portion of a plant such as a leaf, stem, flower, or fruit) or to plant matter. Thus, for example, the plant binding protein or peptide can be a root binding protein or peptide, or a leaf binding protein or peptide.

Suitable plant binding ns and peptides include adhesins (e.g., rhicadhesin), flagellins, omptins, lectins, expansins, biofilm structural proteins (e. g., TasA or YuaB) pilus proteins, curlus proteins, intimins, invasins, agglutinins, and afimbrial proteins.

E. Enzymes that catalyze the production of nitric oxide Many plant species do not inherently have a high germination rate. For such plants, it would be desirable to increase the germination rate. Nitric oxide is a powerful germinant that when present in proximity to a plant seed, increases germination.

The present invention relates to fusion proteins comprising any of the targeting sequences, exosporium ns, exosporium protein nts, or spore coat ns described herein and an enzyme that catalyzes the tion of nitric oxide synthase. Thus, the protein or peptide of interest can comprise an enzyme that catalyzes the production of nitric oxide. Fusion proteins comprising an enzyme that catalyzes the production of nitric oxide can be expressed in recombinant Bacillus cereus family members or inant spore-forming ia for the purpose of delivering the enzyme that catalyzes the tion of nitric oxide to a plant seed, a plant, a plant growth medium, or an area surrounding a plant or a plant seed, and y stimulating germination.

For example, the enzyme that catalyzes the production of nitric oxide can comprise a nitric oxide synthase (e.g., a Bacillus thuringiensis nitric oxide se or a Bacillus subtilis nitric oxide synthase, for example a nitric oxide synthase from Bacillus thuringiensis BT013A or Bacillus subtilis 168) or an arginase.

For example, the nitric oxide synthase can comprise one of the amino acid ces described below in Table 8.

Table 8. Exem lar nitric oxide s nthase se uences Nitric oxide s nthase —SEQ ID NO.

Bacillus subtilis Nitric Oxide S nthatase us thurin iensis Nitric Oxide S nthatase —261 The nitric oxide synthase can also comprise a sequence having a high degree of sequence identity with the nitric oxide synthase sequences shown in Table 8 above. For example, the nitric oxide synthase can comprise an amino acid sequence haVing at least 85% sequence identity with SEQ ID NO: 260 or 261.

The nitric oxide synthase can comprise an amino acid sequence haVing at least 90% sequence identity with SEQ ID NO: 260 or 261. the nitric oxide synthase can comprise an amino acid sequence haVing at least 95% sequence identity with SEQ ID NO: 260 or 261. the nitric oxide synthase can comprise an amino acid sequence haVing at least 98% sequence identity with SEQ ID NO: 260 or 261. ] the nitric oxide synthase can comprise an amino acid sequence haVing at least 99% sequence ty with SEQ ID NO: 260 or 261. the nitric oxide synthase can comprise an amino acid sequence haVing at least 100% sequence identity with SEQ ID NO: 260 or 261.

When the protein or peptide of interest comprises a nitric oxide synthase, the fusion n can comprise one of the amino acid sequences shown in Table 9 below. In the ces shown in Table 9 below, the targeting sequence is shown in boldface text, a six amino acid alanine linker is indicated by ining, and the sequence of the nitric oxide synthase is shown in plain text. Thus, the fusion protein can comprise SEQ ID NO: 262 or 263.

Table 9. Exem lar fusion s com I risin_ a nitric oxide s nthase Fusion protein Amino Acid ce (SEQ ID NO) Met + Amino acids 20_ MAFDPNLVGPTLPPIPPAAAAAAMEEKEILWNEAKAFIAACYQE 3 5 of BC1A, alanine LGKEEEVKDRLADIKSEIDLTGSYVHTKEELEHGAKMAWRNSNRC IGRLFWNSLNVIDRRDVRTKEEVRDALFHHIETATNNGKIRPTITIF linker and Bacillus ’ PPEEKGEKQVEIWNHQLIRYAGYESDGERIGDPASCSLTAACEELG subtlll's Nltr10 OXIde WRGERTDFDLLPLIFRMKGDEQPVWYELPRSLVIEVPITHPDIEAFS Synthatase DLELKWYGVPIISDMKLEVGGIHYNAAPFNGWYMGTEIGARNLA (SEQ ID NO: 262) DEKRYDKLKKVASVIGIAADYNTDLWKDQALVELNKAVLHSYKK QGVSIVDHHTAASQFKRFEEQEEEAGRKLTGDWTWLIPPISPAATH IFHRSYDNSIVKPNYFYQDKPYE 2015/050807 Fusion protein Amino Acid Sequence (SEQ ID NO) Met + Amino acids 20— MAFDPNLVGPTLPPIPPAAAAAAMSKTKQLIEEASHFITICYKELS 3 5 of 301A, alanine KEHFIEERMKEIQAEIEKTGTYEHTFEELVHGSRMAWRNSNRCIGR LFWSKMHILDAREVNDEEGVYHALIHHIKYATNDGKVKPTITIFKQ linker and Bacillus ’ YQGEENNIRIYNHQLIRYAGYKTEMGVTGDSHSTAFTDFCQELGW thurmglensZS NltI‘IC QGEGTNFDVLPLVFSIDGKAPIYKEIPKEEVKEVPIEHPEYPISSLGA Oxide Synthatase KWYGVPMISDMRLEIGGISYTAAPFNGWYMGTEIGARNLADHDR (SEQ ID NO; 263) YNLLPAVAEMMDLDTSRNGTLWKDKALIELNVAVLHSFKKQGVS IVDHHTAAQQFQQFEKQEAACGRVVTGNWVWLIPPLSPATTHIYH KPYPNEILKPNFFH Nitric oxide synthases from a number s, including Bacillus thuringiensis, Bacillus cereus, Bacillus subtilis and Bacillus mycoia’es can be used as the protein or peptide of interest in the fusion proteins.

F. Nucleic acid binding proteins and peptides The delivery of nucleic acids to plants in the field would be desirable, but has been hampered by the instability of nucleic acids, which degrade y when introduced the environment (e.g., into a plant growth medium such as soil).

The t invention relates to fusion ns comprising any of the targeting sequences, exosporium proteins, exosporium protein fragments, or spore coat proteins described herein and a nucleic acid g protein or peptide. Such fusion proteins stabilize nucleic acids and can be used to deliver nucleic acids to soil and/or to .

Thus, the protein or peptide of interest can comprise a nucleic acid g protein or peptide. For example, the c acid g protein or peptide can comprise an RNA binding protein or peptide or a DNA binding protein or e.

The RNA binding protein or peptide can comprise a non-specific RNA binding protein or peptide or a specific RNA binding protein or peptide.

For example, the RNA binding peptide can se an qu protein (e. g., a Bacillus thuringiensis qu protein).

The DNA binding protein or peptide can comprise a small acid-soluble spore protein (SASP). For example, the SASP can comprise a SASP encoded by an SspA gene, an SspB gene, an SspC gene, an SspD gene, an SspE gene, an SspF gene, an SspG gene, an SspH gene, an SspI gene, an SspJ gene, an SspK gene, an SspL gene, an SspM gene, an SspN gene, an SspO gene, or an SspP gene. For e, the SASP can comprise a SASPd, a SASPB, or a SASPy. The SASP can comprise a Bacillus thuringiensis SASP.

The nucleic acid binding protein can comprise one of the amino acid sequences described below in Table 10.

Table 10. Exemplary SASP and Hf u se uences SEQ ID N0.

SASPa -_—SASP 265 Hf. 266 The nucleic acid binding protein can also comprise a sequence having a high degree of sequence identity with any of the sequences shown above in Table 10. For example, the nucleic acid g n can comprise a nucleic acid sequence haVing at least 85% identity with any of SEQ ID NOs: 264—266.

] The nucleic acid binding protein can se a nucleic acid sequence haVing at least 90% identity with any of SEQ ID NOs: 264—266.

The nucleic acid binding protein can comprise a nucleic acid sequence haVing at least 95% identity with any of SEQ ID NOs: 264—266.

The nucleic acid binding protein can comprise a nucleic acid sequence haVing at least 98% identity with any of SEQ ID NOs: 264—266.

The nucleic acid binding protein can comprise a nucleic acid sequence haVing at least 99% identity with any of SEQ ID NOs: 264—266.

The nucleic acid binding protein can comprise a nucleic acid sequence haVing at least 100% identity with any of SEQ ID NOs: 6.

For example, when the protein or peptide of interest comprises a nucleic acid binding protein or peptide, the fusion n can se one of the amino acid sequences shown in Table 11 below. In the ces shown in Table ll below, the targeting ce is shown in boldface text, a six amino acid alanine linker is indicated by underlining, and the sequence of the nucleic acid binding protein or peptide (SASPd, SASPB, or qu) is shown in plain text. Thus, for example, the fusion protein can comprise SEQ ID NO: 267, 268, or 269.

Table 11. Exemplary fusion ns comprising a nucleic acid binding protein Fusion protein Amino Acid Sequence SEQ ID NO Met + Amino acids 20_35 of 1301A, MAFDPNLVGPTLPPIPPAAAAAAAAMAQQSRSRSNNNN alanine linker and SASPa DLLIPQAASAIEQMKLEIASEFGVQLGAETTSRANGSVGGE ITKRLVRLAQQNMGGQFH (SEQ ID NO: 267) Met + Amino acids 20_35 of 1301A, MAFDPNLVGPTLPPIPPAAAAAAAAMANNNSGNSNNLL alanine linker and SASPy VPGAAQAIDQMKLEIASEFGVNLGADTTSRANGSVGGEIT KRLVSFAQQNMGGGQF (SEQ ID NO: 268) 2015/050807 Fusion protein Amino Acid Sequence (SEQ ID NO) Met + Amino acids 20_35 of 1301A, MAFDPNLVGPTLPPIPPAAAAAAAAMKPINIQDQFLNQIR alanine linker, and qu KENTYVTVFLLNGFQLRGQVKGFDNFTVLLESEGKQQLIY KHAISTFAPQKNVQLELE (SEQ ID NO: 269) Nucleases can also be used to both bind to and cleave nucleic acid molecules. ses have high affinity for RNA and DNA molecules, and exert their enzymatic activity by cleaving RNA and/0r DNA molecules into smaller RNA and/0r DNA fragments. Nucleases can be specific, recognizing and cleaving specific DNA or RNA sequences, or non-specific, cleaving any DNA and/or RNA that they come in contact with. Nucleases can be categorized into exonucleases (nucleases that cleave nucleotides off of the ends ofRNA and/or DNA les), or cleases ases that cleave a phosphodiester bond within a polynucleotide chain).

Each nuclease enzyme has an active site that comprises particular amino acids that act to catalyze the cleavage of the nucleic acid molecule. Mutation of these active sites can inactivate the active site and allow for high affinity binding of the nuclease to its nucleic acid substrate, without cleavage of the substrate. Thus, such mutants can bind to and stabilize the nucleic acid molecule without cleaving the nucleic acid molecule.

] Thus, the nucleic acid binding protein can comprise a se (e. g., a nuclease having an inactivated active site).

When the protein or e of interest comprises a nucleic acid binding protein or e, a nucleic acid molecule can be bound to the nucleic acid binding protein or peptide. The nucleic acid can comprise, for example, a modulating RNA molecule; an RNAi molecule; a microRNA; an aptamer; or a DNA molecule that encodes a modulating RNA molecule, an RNAi le, a microRNA, or an aptamer.

XIII. Recombinant Bacillus cereus family member hosts ] As described above, a Bacillus cereus family member can serve as a host for expression of fusion proteins sing a targeting sequence, an exosporium protein, or an exosporium protein fragment that targets the fusion protein to the exosporium 0f the Bacillus cereus family member; serve as a host for expression of modulator proteins that modulate the expression of a fusion protein; can serve as a host for overexpression of an exosporium enzyme; can be genetically inactivated; or can comprise a mutation or other genetic alteration that allows for collection of free exosporium.

] The recombinant Bacillus cereus family member can coexpress two or more of any of the fusion proteins discussed above. For example, the recombinant Bacillus cereus family member can ess at least one fusion protein that comprises a plant binding protein or peptide, together with a fusion n comprising a plant growth ating protein or peptide, a fusion protein comprising a protein or peptide that ts a plant from a pathogen, a fusion protein comprising protein or e that enhances stress resistance in a plant, a fusion protein sing an enzyme that catalyzes the production of nitric oxide, or a fusion protein comprising a nucleic acid binding protein or peptide.

The recombinant us cereus family member can se any Bacillus species that is capable of producing an exosporium. For example, the recombinant us cereus family member can comprise Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoia’es, Bacillus pseudomycoia’es, Bacillus samanii, Bacillus gaemokensis, Bacillus weihenstephensis, Bacillus nsis, or a combination thereof. In particular, the recombinant Bacillus cereus family member can comprise us thuringiensis or Bacillus mycoides.

To generate a recombinant Bacillus cereus family member expressing a fusion protein, any Bacillus cereus family member can be conjugated, transduced, or transformed with a vector encoding the fusion protein using standard methods known in the art (e. g., by electroporation). The bacteria can then be screened to identify transformants by any method known in the art. For example, where the vector includes an antibiotic resistance gene, the bacteria can be screened for otic resistance. Alternatively, DNA encoding the fusion protein can be integrated into the chromosomal DNA of a B. cereus family member host. The recombinant Bacillus cereus family member can then exposed to conditions which will induce sporulation. Suitable conditions for inducing sporulation are known in the art. For example, the recombinant Bacillus cereus family member can be plated onto agar plates, and ted at a temperature of about 30°C for several days (e.g., 3 days).

Inactivated s, non-toxic strains, or cally manipulated strains of any of the above s can also suitably be used. For example, a Bacillus thuringiensis that lacks the Cry toxin can be used. Alternatively or in addition, once the recombinant B. cereus family member spores expressing the fusion protein have been generated, they can be inactivated to prevent further germination once in use. Any method for inactivating bacterial spores that is known in the art can be used. Suitable methods include, without limitation, heat treatment, gamma irradiation, x-ray irradiation, UV-A irradiation, UV-B irradiation, chemical treatment (e. g., treatment with gluteraldehyde, formaldehyde, hydrogen peroxide, acetic acid, bleach, or any combination thereof), or a combination f Alternatively, spores derived from nontoxigenic s, or genetically or physically inactivated strains, can be used.

Many Bacillus cereus family member strains have inherent beneficial attributes. For example, some strains have plant-growth promoting effects. Any of the recombinant Bacillus cereus family members described herein can comprise a plant-growth promoting strain of bacteria.

The growth promoting strain of bacteria can comprise a strain of bacteria that produces an insecticidal toxin (e. g., a Cry toxin), produces a fungicidal compound (e.g., a B-1,3-glucanase, a chitosanase, a lyticase, or a combination thereof), produces a cidal compound (e.g., a Cry toxin), produces a bacteriocidal compound, is resistant to one or more antibiotics, comprises one or more freely replicating plasmids, binds to plant roots, colonizes plant roots, forms biofilms, solubilizes nutrients, secretes organic acids, or any combination thereof.

For example, where the recombinant Bacillus cereus family member comprises a growth promoting strain of bacteria, the plant growth-promoting strain of bacteria can comprise (a) Bacillus mycoia’es BT155 (NRRL No. 1), (b) Bacillus ’es EEl l8 (NRRL No. B-509l8), (c) Bacillus mycoia’es EEl4l (NRRL No. B-509l6), (d) Bacillus mycoia’es BT46-3 (NRRL No. B-50922), (e) Bacillus cereus family member EEl28 (NRRL No. B-509l7), (f) Bacillus giensis BT013A (NRRL No. B-50924), (g) Bacillus cereus family member EE349 (NRRL No. B-50928), (h) Bacillus cereus family member EE- B00377 (NRRL B-67l 19), (i) Bacillus pseudomycoia’es EE-B00366 (NRRL B-67120), or (j) us mycoia’es EE-B00363 (NRRL B-67121). Each of the strains (a) through (g) was deposited with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS), having the address 1815 North University Street, Peoria, Illinois 61604 USA, on March 10, 2014, and is identified by the NRRL deposit number ed in parentheses.

Bacillus thuringiensis BT013A is also known as Bacillus thuringiensis 4Q7. Each of the strains (h) through (j) were deposited with the USDA ARS on August 19, 2015, and is fied by the NRRL t number provided in parentheses.

These growth promoting strains were ed from the rhizospheres of various us plants and were identified by their 16S rRNA sequences (listed below in Table 12), and through biochemical assays. The strains were identified at least to their genus designation by means of conventional biochemistry and morphological indicators. Biochemical assays for confirmed Gram-positive strains such as Bacillus included growth on PEA medium and nutrient agar, microscopic examination, growth on 5% and 7.5% NaCl medium, growth at pH 5 and pH 9, growth at 42°C and 50°C, the ability to produce acid upon fermentation with cellobiose, e, glycerol, glucose, sucrose, d-mannitol, and starch; fluorescent t production; gelatin hydrolysis; nitrate reduction; catalase tion, starch hydrolysis; oxidase reaction, urease production and motility. Identification of these s and demonstration of their plant-growth promoting effects are described further in the Examples hereinbelow.

Table 12. Partial 16S rRNA sequences for plant-growth promoting Bacillus cereus family members Strain SEQ ID NO. for partial 16S ribosomal RNA se uence Bacillus m c0ia’es EEl 18 270 Bacillus m c0ia’es EEl4l 271 Bacillus m c0ia’es BT46-3 272 Bacillus cereus famil member EEl28 273 Bacillus thurin iensis BTOl3A 274 Bacillus cereus famil member EE349 275 Bacillus m coides BTl55 276 For example, the recombinant us cereus family member comprising a plant-growth promoting strain of bacteria can comprise Bacillus mycoides BTl 55, Bacillus mycoides EEl4l, or us thuringiensis BTOl3A.

The recombinant Bacillus cereus family member can comprises an endophytic strain of bacteria. For example, the endophytic strain of bacteria can comprise Bacillus cereus family member EE349, Bacillus cereus family member EE439, Bacillus thuringiensis EE4l7, Bacillus cereus EE444, or us thuringiensis EE3 l9, us thuringiensis EE-B00184, Bacillus cereus family member EE-B00377; Bacillus pseudomycoides EE-B00366; or Bacillus mycoia’es EE-B00363. us cereus family member EE349 is also a plant growth promoting strain of bacteria and is described above. As discussed further in the Examples below, Bacillus cereus family member EE349 has also been found to be endophytic.

Bacillus cereus family member EE439, Bacillus thuringiensis EE4l7, Bacillus cereus EE444, us giensis EE3 l9, Bacillus thuringiensis EE-B00184, Bacillus cereus family member EE-B00377; Bacillus pseudomycoides EE-B00366; or Bacillus mycoia’es EE-B00363 are described further below in Section XIV.

The endophytic strain of bacteria can comprise Bacillus cereus family member EE439, us giensis EE4l7, Bacillus cereus EE444, Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus cereus family member EE-B00377; Bacillus pseudomycoia’es EE-B00366; or Bacillus mycoia’es 363.

] The recombinant Bacillus cereus family member can comprise a strain of bacteria that is capable of ing an herbicide or a pesticide. As discussed further below in the Examples, Bacillus cereus family member EE349, Bacillus cereus family member EE- B00377, Bacillus pseudomycoia’es EE-B00366, and Bacillus mycoia’es EE-B00363 have been found to be capable of degrading herbicides and/or pesticides. Thus, when the recombinant Bacillus cereus family member comprises a strain of bacteria that is capable of degrading an herbicide, the strain of bacteria that is capable of ing an herbicide can comprise Bacillus cereus family member EE349, Bacillus cereus family member EE-B00377, us pseudomycoia’es EE-B00366, or Bacillus ’es 363.

The strain of bacteria that is capable of degrading an herbicide or a ide can degrade a sulfonylurea herbicide (e. g., sulfentrazone), an aryl triazine herbicide, dicamba, 2,4-D, a phenoxy herbicide, a pyrethrin, a pyrethroid, or a combination thereof.

] The strain of bacteria that is capable of degrading a pesticide can be a strain of bacteria that is capable of degrading a pyrethrin.

The recombinant Bacillus cereus family member can comprise a probiotic strain of bacteria. For example, the probiotic strain of bacteria can comprise Bacillus cereus family member EE349, Bacillus cereus family member EE439, Bacillus thuringiensis EE4l7, or Bacillus cereus EE444.

The inant Bacillus cereus family member can comprise an inactivating on in its BclA gene, its CotE gene, or its CotO gene (e.g., a knock-out of the BclA gene, CotE gene, or CotO gene). For example, the recombinant Bacillus cereus family member can comprise an inactivating mutation in its BclA gene (e.g., a knock-out of the BclA gene). It has been found that expression of fusion proteins in a recombinant Bacillus cereus family member having such a mutation results in increased sion levels of the fusion protein.

XIV. Endophytic ial Strains ] The present invention further relates to endophytic bacterial strains. While many bacteria of the phere have a symbiotic relationship With the plant, only a small subset of these bacteria are capable of being internalized into the plant and growing endophytically. As described further in the Examples hereinbelow, several Bacillus cereus 2015/050807 family member strains and several non-Bacillus cereus family member bacterial strains were isolated from corn seedlings and found to have the ability to grow endophytically in plants.

A. ytic Bacillus cereus family members The present invention relates to ically pure bacterial cultures of bacteria that have the ability to grow endophytically. The bacterial strain in each of these bacterial cultures can be: (a) Bacillus cereus family member EE439 (NRRL B-50979); (b) us thuringiensis EE417 (NRRL B-50974); (c) Bacillus cereus EE444 (NRRL B-50977); (d) Bacillus thuringiensis EE319 (NRRL B-50983), (e) Bacillus thuringiensis EE-B00184 (NRRL B-67122); (f) Bacillus cereus family member EE-B00377 (NRRL B-67119); (g) Bacillus mycoia’es EE-B00366 (NRRL B-67120); or (h) Bacillus mycoia'es EE-B00363 (NRRL B- 67121). Each of strains (a) through (c) was deposited with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS), having the address 1815 North University Street, Peoria, Illinois 61604 USA, on September 10, 2014, and are identified by the NRRL numbers provided in parentheses following the names of each strain. Strain (d) was deposited with the USDA ARS on September 17, 2014 and is identified by the NRRL number provided in parentheses ing the name of the strain. Each of strains (e) through (h) was deposited with the USDA ARS on August 19, 2015 and are identified by the NRRL numbers provided in parentheses following the names of each strain.

The novel s disclosed herein were identified by 16S ribosomal RNA (rRNA) sequencing. Thus, Bacillus cereus family member EE439 has a 16S ribosomal RNA ce having at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 277. Bacillus thuringiensis EE417 has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 278.

Bacillus cereus EE444 has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence ty with the ce of SEQ ID NO: 279. Bacillus thuringiensis EE319 has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence ty with the sequence of SEQ ID NO: 280. Bacillus thuringiensis EE-B00184 has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 301. Bacillus cereus family member EE-B00377 has a 16S ribosomal RNA ce having at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 304. us pseudomycoia’es EE-B00366 has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 303. Bacillus mycoides EE-B00363 (NRRL B-67121) and the bacteria has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 302. The 16S rRNA sequences are listed below in Table 13.

Table 13. Partial 16S rRNA sequences for Bacillus cereus family member endophytic strains rRNA se u uence Bacillus cereus famil member EE439 Bacillus thurin; iensis EE417 Bacillus cereus EE444 Bacillus thuriniensis EE319 Bacillus thuriniensis EE-B00184 Bacillus m coides EE-B00363 Bacillus pseudom coides EE-B00366 Bacillus cereus famil member EE-B00377 The present invention further relates to a biologically pure bacterial culture wherein the bacteria in the bacterial e are mutants of Bacillus cereus family member EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus cereus family member EE-B00377, us mycoia’es EE-B00366, or us mycoia’es EE-B003 63 sing one or more mutations, wherein the bacteria are endophytic.

B. Other endophytic bacterial strains The present invention also relates to other biologically pure bacterial cultures of bacteria (non-Bacillus cereus family members) that have the ability to grow endophytically.

These strains were isolated from corn seedlings, as described in detail below in the Examples.

The bacterial strain in each of these bacterial es can be (a) Bacillus megaterium EE385 (NRRL 0), (b) Bacillus sp. EE387 (NRRL B-50981), (c) Bacillus circulans EE388 (NRRL B-50982), (d) Bacillus subtilis EE405 (NRRL B-50978), (e) bacillusfusiformis EE442 (NRRL B-50975), (f) Lysinibcaillus spliaericus EE443 (NRRL B-50976), or (g) us pumilus EE-B00143 (NRRL B-67123). Each of the strains (a) through (i) was deposited with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS), having the address 1815 North University Street, Peoria, is 61604 USA, on ber 10, 2014, and are identified by the NRRL numbers provided in parentheses following the names of each . ing deposit, us sp. EE3 87 was determined to be a Bacillus pumilus-like strain. Strain (g) was deposited with the USDA ARS on August 19, 2015 and is identified by the NRRL number provided in parentheses following its name.

The novel strains disclosed herein were identified by 16S ribosomal RNA (rRNA) sequencing. Thus, us megaterium EE385 has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with the ce of SEQ ID NO: 281. Bacillus sp. EE3 87 has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with the ce of SEQ ID NO: 282. Bacillus circulans EE388 has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 283. Bacillus subtilis EE405 has a 16S mal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 284. Lysinibacillusfusiformis EE442 has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 285.

Lysinibcaillus sphaericus EE443 has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence ty with the sequence of SEQ ID NO: 286. us pumilus EE-B00143 has a 16S ribosomal RNA ce having at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 305. The 16s rRNA sequences are listed below in Table 14.

Table 14. Partial 16S rRNA sequences for non-Bacillus cereus family member endophytic strains SEQ ID No. for artial 16S rRNA se uence The present invention further relates to a biologically pure bacterial culture wherein the bacteria in the bacterial culture are mutants of us megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus is EE405, Lysinibacillusfusiformis EE442, or Lysinibcaillus sphaericus EE443, comprising one or more mutations, wherein the bacteria are endophytic.

The present invention also relates to a biologically pure bacterial culture wherein the bacteria in the bacterial culture are mutants of Bacillus megaterium EE385, Bacillus sp. EE387, us circulans EE388, us subtilis EE405, Lysinibacillusfusiformis EE442, WO 44661 l or Lysinibcal'llus sphaerl'cus EE443, comprising one or more mutations, n the bacteria are probiotic.

XV. Inoculums The invention further relates to inoculums of any of the biologically pure bacterial strains described above in the preceding section. The inoculums are for application to plants, plant seeds, a plant growth medium, or an area nding a plant or a plant seed, wherein the inoculum comprises an effective amount of any one of the biologically pure bacterial cultures and an agriculturally acceptable r.

The inoculum can comprise an effective amount of a mixture comprising at least two of the biologically pure bacterial cultures described above in the immediately preceding section.

The inoculum can further comprise an effective amount of a rhizobacteria.

The rhizobacteria can be a biologically pure bacterial culture of a rhizobacteria strain. The rhizobacteria can comprise Bradyrhz'zobz’um genus bacteria (e. g., Bradyrhz'zobiumjaponicum), z'um genus bacteria (e. g., Rhizobz'um lz’, z'um leguminosarum, or a ation thereof), or a combination thereof XVI. Plant Seeds Coated with an Enzyme that Catalyzes the Production of Nitric Oxide or with inant Bacteria that Overexpress an Enzyme that Catalyzes the Production of Nitric Oxide A plant seed is also provided which is coated with: (i) an enzyme that catalyzes the production of nitric oxide; (ii) a superoxide dismutase or (iii) a recombinant microorganism that expresses an enzyme that catalyzes the production of nitric oxide or a superoxide dismutase, wherein the expression of the enzyme that catalyzes the production of nitric oxide or the superoxide dismutase is increased as compared to the expression of the enzyme that catalyzes the production of nitric oxide or the superoxide dismutase in a wild-type microorganism under the same ions.

] The enzyme that zes the production of nitric oxide can comprise a nitric oxide synthase or an arginase.

The enzyme that catalyzes the production of nitric oxide can comprise a nitric oxide synthase, such as a nitric oxide synthase from Bacillus thuringiensis BT013A or Bacillus subtilis 168.

For example, the nitric oxide synthase can comprise an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 260 or 261.

The nitric oxide synthase can comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 260 or 261.

The nitric oxide synthase can se an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 260 or 261.

The nitric oxide se can comprise an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 260 or 261.

The nitric oxide synthase can comprise an amino acid sequence having at least 99% ce identity with SEQ ID NO: 260 or 261.

The nitric oxide synthase can se an amino acid sequence having 100% sequence identity with SEQ ID NO: 260 or 261.

The superoxide dismutase can comprise xide dismutase 1 (SODAl) or superoxide dismutase 2 ).

The superoxide dismutase comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase comprises an amino acid ce having at least 95% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase ses an amino acid sequence having at least 98% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase comprises an amino acid ce having at least 100% identity with SEQ ID NO: 155 or 156.

When the plant seed is coated with the recombinant microorganism, the recombinant microorganism can comprise a Bacillus species, Escherechia c0li, an Aspergillus species such as Aspergillus niger, or a Saccliromyces species such as Saccliromyces cerevisiae.

For example, the inant microorganism can comprise a Bacillus cereus family member, Bacillus subtilis, Bacillus licheniformis, or Bacillus megaterium.

Amino acid sequences for exemplary nitric oxide synthetase s are provided above in Table 8. Amino acid sequences for ary xide dismutases are provided above in Table 2.

XVII. Formulations Formulations are provided which comprise a recombinant Bacillus cereus family member as described herein, exosporium fragments derived from spores of a recombinant Bacillus cereus family member as described herein or a recombinant spore-forming bacterium as described herein, and an agriculturally able carrier.

The agriculturally acceptable carrier can se an additive, such as an oil, a gum, a resin, a clay, a polyoxyethylene glycol, a terpene, a viscid organic, a fatty acid ester, a sulfated alcohol, an alkyl ate, a petroleum sulfonate, an alcohol sulfate, a sodium alkyl butane diamate, a polyester of sodium tane dioate, a benzene acetonitrile derivative, a proteinaceous material, or a combination thereof.

The agriculturally acceptable carrier can comprise a thickener, such as a long chain alkylsulfonate of polyethylene glycol, a polyoxyethylene oleate, or a combination thereof; a surfactant such as a heavy petroleum oil, a heavy petroleum distillate, a polyol fatty acid ester, a polyethoxylated fatty acid ester, an aryl alkyl polyoxyethylene glycol, an alkyl amine acetate, an alkyl aryl sulfonate, a polyhydric alcohol, an alkyl phosphate, or a combination thereof; or an anti-caking agent such as a sodium salt (e.g., a sodium salt of monomethyl naphthalene sulfonate, a sodium salt of dimethyl naphthalene sulfonate, a sodium sulfite, a sodium sulfate, or a combination thereof), a calcium carbonate, diatomaceous earth, or a combination thereof.

The additive can comprise a proteinaceous material such as a milk product, wheat flour, soybean meal, blood, albumin, gelatin, a meal, yeast extract, or a combination thereof; The agriculturally acceptable carrier can comprise vermiculite, al, sugar y carbonation press mud, rice husk, carboxymethyl cellulose, peat, perlite, fine sand, calcium carbonate, flour, alum, a starch, talc, polyvinyl idone, or a combination thereof.

The formulation can comprise a seed coating formulation, a liquid formulation for application to plants or to a plant growth medium, or a solid formulation for ation to plants or to a plant growth medium. The seed coating ation can se an aqueous or oil-based solution for application to seeds or a powder or granular formulation for application to seeds. The liquid formulation for application to plants or to a plant grth medium can comprise a concentrated formulation or a ready-to-use formulation. The solid formulation for application to plants or to a plant growth medium can comprise a granular formulation or a powder agent.

The formulation further can comprise a fertilizer, a micronutrient fertilizer material, an insecticide, an herbicide, a plant grth amendment, a fungicide, an insecticide, a molluscicide, an algicide, a bacterial inoculant, a fungal inoculant, or a combination thereof.

The bacterial inoculant can comprise a bacterial inoculant of the genus Rhizobium, a bacterial inoculant of the genus Bradyrhizobium, a ial inoculant of the genus izobium, a bacterial inoculant of the genus Azorhizobium, a bacterial inoculant of the genus Allorhizobium, a bacterial inoculant of the genus izobium, a ial inoculant of the genus Kluyvera, a bacterial inoculant of the genus Azotobacter, a bacterial inoculant of the genus Pseudomonas, a bacterial inoculant of the genus Azospirillium, a bacterial inoculant of the genus Bacillus, a ial inoculant of the genus Streptomyces, a bacterial ant of the genus Paenibacillus, a bacterial inoculant of the genus Paracoccus, a bacterial inoculant of the genus Enterobacter, a bacterial inoculant of the genus Alcaligenes, a bacterial inoculant of the genus Mycobacterium, a bacterial inoculant of the genus Trichoa’erma, a bacterial inoculant of the genus Gli0claa’ium, a bacterial inoculant of the genus Glomus, a bacterial inoculant of the genus Klebsiella, or a combination thereof.

] The bacterial inoculant can se a plant-growth promoting strain of bacteria. The plant-growth promoting strain of ia can produce an insecticidal toxin, produce a fungicidal compound, produce a nematocidal nd, produce a bacteriocidal compound, can be resistant to one or more antibiotics, can comprise one or more freely replicating plasmids, bind to plant roots, colonize plant roots, form biofilms, lize nutrients, secrete c acids, or combinations thereof.

For example, the bacterial ant can comprise Bacillus aryabhattai CAP53 (NRRL No. B-508l9), Bacillus aryabhattai CAP56 (NRRL No. 7), Bacillus flexus BT054 (NRRL No. B-508l6), Paracoccus kona'ratievae NC35 (NRRL No. B-50820), Bacillus mycoia’es BTlSS (NRRL No. B-50921), Enterobacter cl0acae CAPlZ (NRRL No. B- , Bacillus nealsonii BOBA57 (NRRL No. NRRL B-50821), Bacillus mycoia’es EEl l8 (NRRL No. B-509l8), Bacillus subtilis EEl48 (NRRL No. 7), Alcaligenesfaecalis EElO7 (NRRL No. B-50920), Bacillus mycoia’es EEl4l (NRRL NO. B-509l6), Bacillus mycoia’es BT46-3 (NRRL No. B-50922), Bacillus cereus family member EEl28 (NRRL No. B- 50917), Bacillus thuringiensis BT013A (NRRL No. 4), Paenibacillus massiliensis BT23 2015/050807 5 (NRRL No. B-50923), Bacillus cereus family member EE349 (NRRL No. B-50928), Bacillus subtilis EE218 (NRRL No. B-50926), Bacillus megaterium EE281 (NRRL No. B-50925), Bacillus cereus family member EE-B00377 (NRRL B-67119); Bacillus pseudomycoia’es EE- B00366 (NRRL 0), Bacillus mycoia’es EE-B00363 (NRRL B-67121), Bacillus pumilus EE-B00143 mRRL B-67123), or Bacillus giensis EE-B00184 (NRRL B-67122) or a ation thereof. Each of these s was deposited with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS), having the address 1815 North University Street, Peoria, Illinois 61604 U.S.A., on March 11, 2013 (Bacillus aryabhattai CAP53, Bacillus arjyabhattai CAP56, Bacillusflexus BT054, Parac0ccus kona'ratievae NC35, Enterobacter cl0acae CAP12, and us nealsonii ), on March 10, 2014 (Bacillus mycoia’es BT155, Bacillus ’es EE118, Bacillus subtilis EEl48, Alcaligenesfaecalis EE107, Bacillus mycoia’es EEl4l, Bacillus mycoia’es BT46-3, Bacillus cereus family member EE128, Bacillus thuringiensis BT013A, Paenibacillus massiliensis BT23, Bacillus cereus family member EE349, Bacillus subtilis EE218, and Bacillus megaterium EE28l), or on August 19, 2015 (Bacillus cereus family member EE-B00377; Bacillus pseudomycoia’es EE-B00366, Bacillus mycoia’es EE-B00363, Bacillus pumilus EE-B00143, or Bacillus thuringiensis EE- B00184) and is identified by the NRRL numbers provided in parentheses.

These plant-growth promoting strains were isolated from the rhizospheres of various vigorous plants and were identified by their 16S rRNA sequences, and through biochemical assays. The strains were identified at least to their genus designation by means of conventional biochemistry and morphological indicators. Biochemical assays for confirmed Gram-negative strains such as ccus kona'ratievae, Alcaligenesfaecalis, and Enterobacter cl0acae included growth on MacConkey medium and nutrient agar, microscopic examination, growth on 5% and 7.5% NaCl medium, growth at pH 5 and pH 9, growth at 42°C and 50°C, the ability to e acid upon fermentation with cellobiose, lactose, ol, glucose, e, d- mannitol, and starch; fluorescent pigment production; gelatin hydrolysis; nitrate reduction; starch hydrolysis; oxidase reaction, se production, urease production and motility.

Similarly, the biochemical assays for confirmed Gram-positive strains such as Bacillus and Paenibacillus included growth on PEA medium and nutrient agar, microscopic examination, growth on 5% and 7.5% NaCl medium, growth at pH 5 and pH 9, growth at 42°C and 50°C, the y to produce acid upon tation with cellobiose, lactose, glycerol, glucose, sucrose, d- mannitol, and starch; fluorescent pigment tion; gelatin ysis; nitrate reduction; catalase production, starch hydrolysis; oxidase on, urease production and motility.

Identification of these strains and demonstration of their plant-growth ing effects are described further in the Examples hereinbelow. Partial l6S rRNA ces for the strains Bacillus mycoia’es BTlSS, Bacillus mycoia’es EEl l8, Bacillus mycoia’es EEl4l, Bacillus mycoia’es BT46-3, Bacillus cereus family member EEl28, Bacillus thuringiensis BTOl3A, and Bacillus cereus family member EE349 are provided in Table 12 above. Partial l6S rRNA sequneces for the strains Bacillus aryabhattai CAP53, Bacillus aryabhattai CAP56, Bacillus flexus BT054, ccus kona'ratievae NC35, Enterobacter cl0acae CAPl2, Bacillus nealsonii , Bacillus subtilis EEl48, Alcaligenesfaecalis EElO7, Paenibacillus massiliensis BT23, Bacillus subtilis EE2l8, and Bacillus megaterium EE28l are listed in Table 15 below.

Table 15. Partial 16S rRNA sequences for onal plant-growth promoting s of bacteria Strain SEQ ID NO. for artial 16S ribosomal RNA se uence Bacillus ar abhattai CAP53 287 Bacillus ar abhattai CAP56 288 Bacillus exus BT054 289 Paracoccus kona'ratievae NC35 290 Enterobacter cl0acae CAPl2 291 Bacillus nealsonii BOBA57 292 Bacillus subtilis EEl48 293 Alcali enes aecalis EElO7 294 Paenibacillus massiliensis BT23 295 Bacillus subtilis EE2l8 296 Bacillus me aterium EE28l 297 For example, the formulation can comprise a plant-growth ing strain of bacteria comprising Paracoccus kona’ratievae NC35, Bacillus aryabhattai CAP53, or us megaterium EE28 l n the formulation further comprises any of the recombinant Bacillus cereus family members described herein, including any of the recombinant plant- growth promoting Bacillus cereus family member strains herein (e.g., recombinant Bacillus ’es BTlSS, Bacillus mycoia’es EEl4l, or Bacillus giensis BT013A).

The fungal inoculant can comprise a fungal inoculant of the family aceae, a fungal inoculant of the family Claroidoglomeraceae, a fungal inoculant of the family Gigasporaceae, a fungal inoculant of the family Acaulosporaceae, a fungal inoculant of the family Sacculosporaceae, a fungal inoculant of the family Entrophosporaceae, a fungal inoculant of the family Pacidsporaceae, a fungal inoculant of the family Diversisporaceae, a fungal inoculant of the family Paraglomeraceae, a fungal inoculant of the family osporaceae, a fungal inoculant of the family Geosiphonaceae, a fungal inoculant of the family Ambisporaceae, a fungal inoculant of the family Scutellosporaceae, a fungal inoculant of 2015/050807 the family Dentiscultataceae, a fungal inoculant of the family Racocetraceae, a fungal inoculant of the phylum Basidiomycota, a fungal inoculant of the phylum Ascomycota, a fungal inoculant of the phylum Zygomycota, or a combination thereof. {@0631} The spore—forming bacterium, alone or in combination with the insecticide, can further comprise an effective amount of at least one ide. {@9532} Typical fungicidal ingredients also include Captan (N- trichlorornethyl)tliio—4— cyclohexane—E,2~dicar’boximide), Fludioxoni 1 (1H2,2—clifluoro—E,3~ benzodioxol—4~yE)~l~l>l—pyrrol—3~carhonitril; carbendazirn one (commercially available under the tradename RovralRTlViQ), tebuconazole, thiabendazole, azoxystrobin, prochloraz, and Oxadixyl (N—(Zfi—dimethyEphen,yl)~2—rnethoxy~N~(2~oxo—3~ oxazolidinyl) acetamide} if a formulation, plant seed, or inoculuni comprises a fungicide, the fungicide can comprise aldimorph, ampropylfos, ampropylfos potassium, im, anilazine, azaconazole, azoxystrobin, benalaxyl, nil, benomyl, benzamacril, benzamacryl-isobutyl, bialaphos, binapacryl, biphenyl, bitertanol, blasticidin-S, boscalid, bromuconazole, mate, buthiobate, calcium polysulphide, capsimycin, captafol, , carbendazim, carvon, quinomethionate, chlobenthiazone, chlorfenazole, chloroneb, chloropicrin, chlorothalonil, chlozolinate, clozylacon, cufraneb, cymoxanil, cyproconazole, cyprodinil, cyprofuram, debacarb, dichlorophen, diclobutrazole, diclofluanid, diclomezine, dicloran, diethofencarb, dimethirimol, dimethomorph, dimoxystrobin, diniconazole, diniconazole-M, dinocap, diphenylamine, dipyrithione, ditalimfos, dithianon, dodemorph, dodine, drazoxolon, edifenphos, epoxiconazole, etaconazole, ethirimol, etridiazole, famoxadon, fenapanil, fenarimol, fenbuconazole, fenfuram, opan, lonil, fenpropidin, fenpropimorph, fentin acetate, fentin hydroxide, ferbam, ferimzone, fluazinam, flumetover, fluoromide, fluquinconazole, flurprimidol, flusilazole, amide, flutolanil, fol, folpet, l-aluminium, fosetyl- , fthalide, fuberidazole, furalaxyl, furametpyr, furcarbonil, furconazole, furconazole-cis, furmecyclox, guazatine, hexachlorobenzene, nazole, hymexazole, imazalil, imibenconazole, tadine, iminoctadine albesilate, iminoctadine triacetate, iodocarb, iprobenfos (IBP), iprodione, irumamycin, isoprothiolane, isovaledione, kasugamycin, im— methyl, copper preparations, such as: copper hydroxide, copper naphthenate, copper oride, copper sulphate, copper oxide, oxine-copper and Bordeaux mixture, mancopper, mancozeb, maneb, mzone, mepanipyrim, mepronil, metalaxyl, metconazole, methasulfocarb, methfuroxam, metiram, metomeclam, metsulfovax, mildiomycin, utanil, myclozolin, nickel dimethyldithiocarbamate, nitrothal-isopropyl, nuarimol, e, oxadixyl, 8 oxarnocarb, oxolinic acid, oxycarboxirn, oxyfenthiin, utrazole, pefurazoate, azole, pencycuron, phosdiphen, pirnaricin, piperalin, polyoxin, orirn, probenazole, prochloraz, procyrnidone, nocarb, propanosine-sodiurn, propiconazole, propineb, prothiocinazole, pyrazophos, pyrifenox, pyrirnethanil, pyroquilon, pyroxyfur, quinconazole, quintozene (PCNB), sulphur and r preparations, tebuconazole, tecloftalarn, tecnazene, tetcyclasis, tetraconazole, thiabendazole, thicyofen, thifluzarnide, anate-rnethyl, tioxyrnid, tolclofos- methyl, tolylfluanid, rnefon, triadirnenol, triazbutil, triazoxide, trichlarnide, tricyclazole, tridernorph, trifloxystrobin, triflurnizole, triforine, uniconazole, rnycin A, Vinclozolin, Viniconazole, zarilarnide, zineb, zirarn and also Dagger G, OK-8705, OK-880l, a-(l,l- dimethylethyl)—(3-(2-phen0xyethyl)-1H- l ,2,4-triazole- l -eth anol, a-(2,4-dichlor0phenyl)— [3 - flu0r0pr0pyl-l H-- l ,2,4-triazoleethanol, a-(2,4-dichlor0phenyl)- [3 -rnethoxy-a-rnethyl-l H- l,2,4-triazol e-l -ethanol, a-(S-rnethyl -l,3-dioxanyl)-[3-[[4-(triflu0rornethyl) -phenyl]—rnet hylene]—l 4-triazole-l -ethanol, RS)—6-hydr0xy-2,2,7,7-tetramethyl(l H-l,2,4- triazol-l-yl)-3 -octanone, (E)-a-(methoxyirnino)-N-rnethylphen0xy-phenylacetarnide, lisopropyl {2-rnethyl- l -[[[ l -(4-rnethylphenyl)-ethyl]-arnin0]—carbonyl] -pr0pyl} carbarnate, l-(2,4- dichlorophenyl)—2-(l H- l ,2,4-triazol- l -yl)-ethanone-O-(phenyl )-0xirne, nethyl- l - naphthalenyl)-l H-pyrrole-2,5-dione, l-(3,5-dichlorophenyl)(2-propenyl)-2,5- pyrrolidindione, l - [(diiodomethyl)—sulphonyl] rnethyl-benzene, l -[[2-(2,4-dichlor0phenyl)— l , 3-di0xolanyl]-rnethyl]-l H-irnidazole, l-[[2-(4-chlor0phenyl)—3-phenyloxiranyl]—rnethyl]-l H- l ,2,4-triazole, l-[ l -[2- [(2,4-dichlorophenyl)—rnethoxy] -phenyl] -ethenyl] -l H-irnidazole, 1- methyl n0nyl(phenylrnethyl)—3-pyrr01idinole, 2',6'-dibrornornethyl-4'-triflu0r0rnethoxy- flu0r0-rnethyl- l , 3 -thiazole -carb0xanilide, 2,2-dichloro-N—[ l -(4-chlorophenyl)-ethyl]- l - ethylrnethyl-cyclopr0panecarboxarnide, 2,6-dichlor0(rnethylthi0)—4-pyrirnidinylthiocyanate , 2,6-dichloro-N—(4-trifluor0rnethylbenzyl)—benzarnide, 2,6-dichlor0-N—[[4- orornethyl)-phenyl]-rnethyl]—benzarnide, 2-(2,3,3-trii0dopr0penyl)-2H-tetrazole, 2-[(l- methylethyl)—sul phonyl]—5-(trichlor0rnethyl)- l ,3 ,4-thiadiazole, 2-[[6-deoxyO-(4-O-rnethyl- (3 -D-glyc0pyranosyl)-a-D-glucopyran0s yl]-arnino]—4-rnethoxy-l H-pyrrolo [2,3-d]pyri midine- -carb0nitrile, 2-arninobutane, no(br0rn0rnethyl)-pentanedinitrile, 2-chlor0-N—(2,3- dihydro-l,l,3-trirnethyl-l H-indenyl)—3-pyridinecarboxarnide, 2-chlor0-N—(2,6- dimethylphenyl)-N-(is0thi0cyanat0rnethyl)-acetarnide, 2-phenylphenol (OPP), 3,4-dichlor0- l - [4-(difluorornethoxy)—phenyl] -pyrrole-2,5 -dione, 3 ,5 -dichloro-N-[cyan0 [( l -rnethylpr0pynyl)— oxy] -rnethyl] -benzarnide, 3 -(l , l -dirnethylpr0pyl0x0- 1 H-indene-Z-carbonitrile, 3 - [2-(4- chlorophenyl)ethoxyisoxazolidinyl]-pyridine, 4-chlor0cyan0-N,N—dirnethyl-S-(4- methylphenyl)-l azole-l-sulphonamide, 4-methyl-tetrazolo[ l ,5-a]quinazolin-5(4H)—one, 8-(1 , l -dimethylethyl)-N-ethyl-N-propyl- l ,4-dioxaspiro[4, 5]decanemethanamine, 8- hydroxyquinoline sulphate, 9H-Xanthene[(phenylamino)-carbonyl]carboxylic hydrazide, bis-( 1 -methylethyl)-3 -methyl[(3 lbenzoyl)-oxy]—2, 5 -thiophenedicarboxylate, cis- l -(4- chlorophenyl)(l H- l ,2,4-triazol- l -yl)-cycloheptanol, cis[3 - [4-(l l -dimethylpropyl)- phenylmethylpropyl]-2,6-dimethyl-morpholine hydrochloride, ethyl [(4-chlorophenyl)-azo]— cyanoacetate, ium bicarbonate, methanetetrathiol-sodium salt, methyl l-(2,3-dihydro-2,2- dimethyl-inden- l -yl)-l H-imidazole-S -carboxylate, methyl N—(2,6-dimethylphenyl)-N-(5 - isoxazolylcarbonyl)-DL-alaninate, methyl N—(chloroacetyl)-N-(2,6-dimethylphenyl)-DL- alaninate, N—(2,3-dichlorohydroxyphenyl)-l-methyl-cyclohexanecarboxamide, N—(2,6- yl phenyl)methoxy-N-(tetra hydrooxofuranyl)-acetamide, N—(2,6-dimethyl p henyl)methoxy-N-(tetrahydrooxothienyl)-acetamide, N-(2-chloronitrophenyl) methylnitro-benzenesulphonamide, N-(4-cyclohexylphenyl)-l ,4,5 rahydro pyrimidinamine, N—(4-hexylphenyl)—l,4,5,6-tetrahydropyrimidinamine, N—(S-chloro methylphenyl)methoxy-N—(2-oxooxazolidinyl)-acetamide, N—(6-methoxy)—3-pyridinyl)- cyclopropanecarboxamide, N—[2,2,2-trichloro-l-[(chloroacetyl)-amino]-ethyl]-benzamide, N—[3- chloro-4,5-bis(2-propinyloxy)-phenyl]-N'-methoxy-methanimidamide, N—formyl-N-hydroxy- DL-alanine-sodium salt, 0,0-diethyl [2-(dipropylamino)oxoethyl]- ethylphosphoramidothioate, O-methyl S-phenyl phenylpropylphosphoramidothioate, S-methyl l,2,3-benzothiadiazolecarbothioate, and 2H]-l-benzopyrane-2,l'(3'H)-isobenzofuran]— 3'-one, N-trichloromethyl)thio cyclohexane-l,2-dicarboximide, tetramethylthioperoxydicarbonic e, methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)- DL-alaninate, 4-(2,2-difluoro-l,3-benzodioxolyl)-l-H-pyrrolcarbonitril or a combination thereof.

Additionally, suitable fungicides include the following: (1) a compound capable to inhibit the nucleic acid synthesis like xyl, benalaxyl-M, bupirimate, chiralaxyl, clozylacon, irimol, ethirimol, furalaxyl, hymexazol, metalaxyl, metalaxyl-M, ofurace, oxadixyl, oxolinic acid; (2) a compound capable to inhibit the mitosis and cell on like benomyl, carbendazim, diethofencarb, ethaboxam, fuberidazole, pencycuron, thiabendazole thiophanate-methyl, zoxamide; (3) a compound capable to inhibit the respiration for example as CI-respiration inhibitor like diflumetorim; as CII-respiration inhibitor like boscalid, fenfuram, flutolanil, furametpyr, yclox, mepronil, oxycarboxine, penthiopyrad, thifluzamide; as CHI-respiration inhibitor like amisulbrom, trobin, cyazofamid, dimoxystrobin, enestrobin, famoxadone, fenamidone, fluoxastrobin, kresoxim-methyl, metominostrobin, orysastrobin, picoxystrobin, trifloxystrobin; (4) a compound capable of to act as an uncoupler like dinocap, fluazinam, meptyldinocap; (5) a compound capable to inhibit ATP production like fentin acetate, fentin chloride, fentin hydroxide; (6) a compound capable to inhibit AA and protein biosynthesis like andoprim, blasticidin-S, cyprodinil, kasugamycin, kasugamycin hydrochloride hydrate, mepanipyrim, pyrimethanil; (7) a compound capable to t the signal transduction like lonil, quinoxyfen; (8) a compound capable to inhibit lipid and membrane synthesis like biphenyl, chlozolinate, edifenphos, etridiazole, rb, iprobenfos, iprodione, isoprothiolane, procymidone, propamocarb, propamocarb hydrochloride, pyrazophos, tolclofos- methyl, Vinclozolin; (9) a compound capable to t ergosterol biosynthesis like aldimorph, azaconazole, bitertanol, bromuconazole, cyproconazole, diclobutrazole, diniconazole, diniconazole-M, dodemorph, dodemorph acetate, epoxiconazole, etaconazole, fenarimol, onazole, fenhexamid, pidin, fenpropimorph, fluquinconazole, midol, flusilazole, flutriafol, azole, furconazole-cis, hexaconazole, imazalil, imazalil sulfate, imibenconazole, metconazole, utanil, naftif1ne, nuarimol, oxpoconazole, paclobutrazol, pefurazoate, penconazole, prochloraz, propiconazole, prothioconazole, pyributicarb, pyrifenox, nazole, spiroxamine, tebuconazole, afine, tetraconazole, triadimefon, triadimenol, orph, triflumizole, triforine, uniconazole, Viniconazole, nazole; (10) a compound capable to inhibit cell wall synthesis like benthiavalicarb, bialaphos, dimethomorph, flumorph, iprovalicarb, mandipropamid, polyoxins, polyoxorim, validamycin A; (l l) a compound e to inhibit melanine biosynthesis like carpropamid, diclocymet, fenoxanil, phthalide, pyroquilon, lazole; (12) a compound capable to induce a host e like zolar-S-methyl, probenazole, tiadinil; (13) a compound capable to have a multisite action like Bordeaux mixture, captafol, captan, chlorothalonil, copper naphthenate, copper oxide, copper oxychloride, copper preparations such as copper hydroxide, copper sulphate, dichlofluanid, dithianon, dodine, dodine free base, ferbam, fluorofolpet, , guazatine, guazatine acetate, iminoctadine, iminoctadine albesilate, iminoctadine triacetate, mancopper, mancozeb, maneb, metiram, metiram zinc, oxine- copper, propineb, sulphur and sulphur preparations ing calcium polysulphide, tolylfluanid, zineb, ziram; (14) a compound selected in the following list: (2E)—2-(2-{[6-(3-chloro methylphenoxy)fluoropyrimidinyl]oxy}pheny- l)(methoxyimino)-N-methylacetamide, (2E) {2- [( { [( l E)— l -(3 - {[(E)- l -fluorophenylVinyl]oxy} phenyl)ethyliden- e] amino } oxy)methyl]phenyl} (methoxyimino)-N-methylacetamide, l lorophenyl) (lH-l ,2,4-triazol- l -yl)cycloheptanol, l-[(4-methoxyphenoxy)methyl]-2,2-dimethylpropyl- l H- ole-l-carboxylat- e, 2,3,5,6-tetrachloro(methylsulfonyl)pyridine, 2—butoxyiodo -4H-chromenone, 2-chloro-N-(l , l ,3 -trimethyl-2,3 -dihydro- l H-inden yl)nicotinamide, 2-phenylphenol and salts, 3,4,5-trichloropyridine-2,6-dicarbonitrile, 3,4- dichloro-N-(2-cyanophenyl)isothiazolecarboxamide, 3-[5-(4-chlorophenyl)—2,3- dimethylisoxazolidin-3 -yl]pyridine, 5 o(2,4,6-trifluorophenyl)-N-[( l R)— l ,2,2- hylpropyl][l,2,4]t- riazolo[l,5-a]pyrimidinamine, 5-chloro(4-methylpiperidin-l-yl)- 6-(2,4,6-trifluorophenyl) [l,2,4]triazolo[l,5-a]pyrimidine, 5-chloro-N-[(lR)-l,2- dimethylpropyl](2,4,6-trifluorophenyl) [l ,2,4]triazolo[l ,5-a]pyrimidinamine, 8- hydroxyquino line sulfate, benthiazole, bethoxazin, capsimycin, carvone, chinomethionat, cufraneb, cyflufenamid, cymoxanil, dazomet, debacarb, dichlorophen, ezine, dicloran, difenzoquat, difenzoquat methylsulphate, diphenylamine, ferimzone, flumetover, fluopicolide, fluoroimide, flusulfamide, fosetyl-aluminium, l-calcium, fosetyl-sodium, hexachlorobenzene, irumamycin, isotianil, methasulfocarb, methyl (2E) {2-[({cyclopropyl[(4- methoxyphenyl)imino]methyl}thio)methyl]phenyl- }methoxyacrylate, methyl l-(2,2- yl-2,3-dihydro- l H-inden- l -yl)- l H-imidazole-S -carboxylate, methyl isothiocyanate, metrafenone, mildiomycin, N—[2-(l ,3-dimethylbutyl)phenyl]—5-fluoro-l ,3-dimethyl-lH- pyrazolecarbo- xamide, N—(3',4'-dichlorofluorobiphenylyl)—3-(difluoromethyl)- l -meth- yl- l H-pyrazolecarboxamide, N—(3 -ethyl-3 ,5 , 5 -trimethylcyclohexyl)-3 -(formylamino) hydroxybenzamide, N—(4-chloronitrophenyl)—N—ethylmethylbenzenesulfonamide, N-(4- chlorobenzyl)-3 - [3 -methoxy(propyn- l -yloxy)phenyl]propanamide, N— [(4- chlorophenyl)(cyano)methyl][3-methoxy(propyn- l -yloxy)pheny- l]propanamide, N—[(5- bromochloropyridinyl)methyl]-2,4-dichloronicotinamide, N— [ l -(5-bromo-3 -chloropyridin- 2-yl)ethyl] -2,4-dichloronicotinamide, N—[ l -(5-bromo-3 opyridinyl)ethyl]fluoro iodonicotinamide, N—[2-(4- {[3-(4-chlorophenyl)propyn- l -yl]oxy} methoxyphenyl)ethyl] - N&- lt;-(methylsulfonyl)valinamide, N— {(Z)-[(cyclopropylmethoxy)imino] [6- (difluoromethoxy)—2,3-difluorophenyl- ]methyl} phenylacetamide, N— {2-[1 ,l'- bi(cyclopropyl)yl]phenyl} (difluoromethyl)-, l-methyl- l H-pyrazolecarboxamide, N— {2- [3-chloro(trifluoromethyl)pyridinyl]ethyl}(trifluoromethyl)— ide, natamycin, N- N-methyl-N'- {2-methyl(trifluoromethyl)—4- [3 -(trimethylsilyl)pr- poxy]phenyl} imidoformamide, N—ethyl-N-methyl-N'- {2-methyl(difluoromethyl)[3- thylsilyl)propoxy]phenyl}imidoformamide, nickel dimethyldithiocarbamate, nitrothal- isopropyl, O- { l -[(4-methoxyphenoxy)methyl]-2,2-dimethylpropyl} lH-imidazole- l -carboth- ioate, octhilinone, oxamocarb, oxyfenthiin, hlorophenol and salts, phosphorous acid and its salts, piperalin, ocarb fosetylate, propanosine-sodium, proquinazid, pyribencarb, pyrrolnitrine, quintozene, tecloftalam, tecnazene, triazoxide, trichlamide, valiphenal, zarilamid.

The fungicide can se a substituted benzene, a thiocarbamate, an ethylene bis dithiocarbamate, a thiophthalidamide, a copper compound, an organomercury compound, an organotin nd, a cadmium compound, anilazine, benomyl, cyclohexamide, dodine, etridiazole, one, metlaxyl, thiamimefon, triforine, or a combination thereof. if a formulation, plant seed, or incculurn comprises a fungicide, the fungicide. can be a tcliar iiirigicideg Foliar fungicides include copper, eb, penthiopyrad, triazoles, cyproconazole, metconazole, propiconazole, prothioconazole, nazole, azoxystrobin, pyraclastobin, fluoxastrobin, picoxystrobin, trifloxystrobin, , id, thiophanate methyl, chlorothanonil, penthiopyrad, difenconazole, flutriafol, cyprodinil, fluzinam, iprodione, penflufen, cyazofamid, flutolanil, cymoxanil, dimethomorph, pyrimethanil, zoxamide, mandipropamid, metrinam, propamocarb, fenamidone, tetraconazole, chloronab, hymexazol, tolclofos, and fenbuconazole. {00637} if a fern'iulation, plant seed, or incculnm comprises a bacterial ineculant of the genus Bacillus, the bacterial inoculant can comprise Bacillus argri, Bacillus aizawai, Bacillus albolactis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus coagulans, Bacillus endoparasiticus, Bacillus endorhythmos, Bacillus ki, us lacticola, Bacillus lactimorbus, us lactis, Bacillus laterosporus, Bacillus orbus, us licheniformis, Bacillus megaterium, Bacillus medusa, Bacillus metiens, Bacillus natto, Bacillus nigrificans, Bacillus popillae, Bacillus pumilus, Bacillus siamensis, Bacillus sphearicus, Bacillus spp., Bacillus subtilis, Bacillus thuringiensis, Bacillus unifagellatu, or a combination thereof plus those listed in the ry ofBucillus Genus in 's Manual cf Systematic Bacteriology, First Ed. (1986)a hereby incciporated in full by reference. } if a formulation, plant seed, or inoculuni comprises an insecticide, the ii'iscctieide can be a neniatieide, le nematicides include an tihictic nern aticides such as abamectin; carbaniate nematicides such as aceteprole, Bacillus clzimnasporus, chlorcpicrin, benclothiaz, benomyl, Burhalderia ccpacia, uran, ulfan, and cleothocard; dazomel, DECS), DCIP, alanycarb, aldicarb, aldc'xycarb, , diamidafos, fenamiphcs, fosthietan, amidon, cadusafbs, chlorpyrifcs, diclcfenthion, dimethoate, ethoprcphos, fensulfbthion, fostiazate, harping, heterophes, imicyafcs, isamirlefos, isazofos, rnethemyl, mecarphon, fi/Iymtlzccium verrucaria, Paecilamyces lilacinus, phcrate, ocarh, terbufos, thienazin, 2015/050807 triazophos, dazomet, l,Z—dicloropropaiie, l,3—diehloropropene, furfural, iodoniethane, metam, methyl bromide, methyl isothioeyanate, and xylenols. {00639} For example and, without limitation, the nematieide and insecticide can be provided in the form of the commercial product Avieta Duo, which is a mixture of a‘bamectin and thiamethoxam commercially available from Syngenta. {00640} if a formulation, plant seed, or inoculum comprises a bactericide, it may include streptomycin, penicillins, tetracyclines, ampieillin, and oxolinic acid. {00641} The fertilizer can comprise a liquid fertilizer. The micronutrient izer material can comprise boric acid, a borate, a boron frit, copper sulfate, a copper frit, a copper chelate, a sodium tetraborate decahydrate, an iron sulfate, an iron oxide, iron um sulfate, an iron frit, an iron chelate, a manganese sulfate, a manganese oxide, a manganese e, a manganese chloride, a manganese frit, a sodium molybdate, ic acid, a zinc e, a zinc oxide, a zinc carbonate, a zinc frit, zinc phosphate, a zinc chelate, or a combination thereof.

The fertilizer can comprise ammonium sulfate, ammonium nitrate, ammonium sulfate nitrate, ammonium chloride, ammonium bisulfate, ammonium polysulfide, ammonium thiosulfate, aqueous ammonia, anhydrous ammonia, ammonium polyphosphate, aluminum sulfate, calcium nitrate, m um nitrate, m sulfate, calcined magnesite, calcitic one, calcium oxide, m nitrate, dolomitic limestone, hydrated lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, potassium ium e, potassium sulfate, sodium nitrates, magnesian limestone, magnesia, urea, urea-formaldehydes, urea ammonium nitrate, -coated urea, polymer-coated urea, isobutylidene diurea, K2804— 2MgSO4, kainite, sylVinite, kieserite, Epsom salts, elemental , marl, ground oyster shells, fish meal, oil cakes, fish manure, blood meal, rock phosphate, super phosphates, slag, bone meal, wood ash, manure, bat guano, peat moss, compost, green sand, cottonseed meal, r meal, crab meal, fish emulsion, humic acid, or a combination thereof.

A formulation, plant seed, or inoculum can also include at least one biological control agent ed from (1) bacteria, in particular spore-forming bacteria, (2) fungi or yeasts, and (3) isoflavones. Preference is given to combinations comprising as ical control agent a bacterium, in particular a spore-forming, root-colonizing bacterium, or a bacterium useful as biofungicide, selected from the group consisting of [Group (1)]: (1.1) Bacillus agri, (1.2) Bacillus aizawai, (1.3) Bacillus albolactis, (1.4) Bacillus amyloliquefaciens, (1.5) us cereus, (1.6) Bacillus coagulans, (1.7) Bacillus endoparasiticus, (1.8) Bacillus endorhythmos, (1.9), (1.10) Bacillus kurstaki, (1.11) Bacillus lacticola, (1.12) Bacillus lactimorbus, (1.13) Bacillus , (1.14) Bacillus laterosporus, (1.15) Bacillus orbus, (1.16) Bacillus licheniformis, (1.17) us medusa, (1.18) Bacillus megaterium, (1.19) Bacillus metiens, (1.20) Bacillus natto, (1.21) us nigrificans, (1.22) Bacillus ae, (1.23) us pumilus, (1.24) Bacillus siamensis, (1.25) Bacillus sphaericus (products known as VectoLex.sup.S), (1.26) Bacillus is, or B. is var. amyloliquefaciens, (1.27) Bacillus thuringiensis, in particular B. thuringiensis var. israelensis (products known as VectoBac.RTM.) or B. thuringiensis subsp. aizawai strain ABTS-1857 (products known as i), or B. giensis subsp. kurstaki strain HD-l (products known as Dipel ES), (1.28) Bacillus uniflagellatus, (1.29) Delftia acidovorans, in particular strain RAY209 (products known as BioBoost), (1.30) Lysobacter antibioticus, in particular strain 13-1 (Biological Control 2008, 45, 6), (1.31) Lysobacter enzymogenes, in particular strain 3.1T8, (1.32) Pseudomonas chlororaphis, in particular strain MA 342 (products known as Cedomon), (1.33) Pseudomonas proradix (products known as Proradix.RTM.), (1.34) Streptomyces galbus, in particular strain K61 (products known as Mycostop.RTM., cf. Crop tion 2006, 25, 468-475), (1.35) Streptomyces griseoviridis (products known as Mycostop.RTM.).

Preference is further given to combinations comprising as biological control agent a fungus or a yeast selected from the group consisting of [Group (2)]: (2.1) Ampelomyces quisqualis, in particular strain AQ 10 (product known as AQ 10.RTM.), (2.2) Aureobasidium ans, in particular blastospores of strain DSM14940 or blastospores of strain DSM 14941 or mixtures thereof (product known as Blossom Protect.RTM.), (2.3) Beauveria bassiana, in ular strain ATCC 74040 (products known as Naturalis.RTM.), (2.4) Candida oleophila, in particular strain 0 (products known as Nexy), (2.5) porium cladosporioides H39 (cf. Eur. J. Plant Pathol. 2009, 123, 401-414), (2.6), (2.7) Dilophosphora alopecuri (products known as Twist Fungus), (2.8) Gliocladiurn catenulatum, in particular strain J1446 (products known as Prestop), (2.9) cillium lecanii (formerly known as Verticillium lecanii), in particular conidia of strain KV01 (products known as Mycotal.RTM., Vertalec.RTM.), (2.10) Metarhizium anisopliea (products known as B10 1020), (2.11) Metschnikovia fructicola, in particular the strain NRRL Y-30752 (products known as Shemer.TM.), (2.12) Microsphaeropsis ochracea (products known as Microx), (2.13), (2.14) Nomuraea rileyi, , (2.16) Penicillium bilaii, in particular strain ATCC22348 (products known as JumpStart.RTM., PB-50, Provide), (2.17) Pichia anomala, in particular strain WRL- 076, (2.18) Pseudozyma flocculosa, in particular strain PF-A22 UL (products known as Sporodex L), (2.19) Pythium oligandrum DV74 (products known as Polyversum), (2.20) Trichoderma asperellum, in particular strain ICC 012 (products known as Bioten), (2.21) Trichoderma harzianum, in particular T. harzianum T39 (products known e.g. as Trichodex).

Preference is further given to combinations comprising as biological control agent an isoflavone selected from the group consisting of [Group (3)]: (3.1) genistein, (3.2) biochanin A10, (3.3) formononetin, (3.4) daidzein. (3.5) glycitein, (3.6) hesperetin, (3.7) naringenin, (3.8) chalcone, (3.9) in, (3.10) Ambiol (2-methyldimethylaminomethyl hydroxybenzimidazol dihydrochoride) (3.11) ate and (3.12) pratensein and the salts and esters thereof. lfa formulation, plant seed, or uni comprises an insecticide, the insecticide can include pyrethi‘oids, organophosphates, caramoyloximes, pyrazoles, amidines, halogenated hydrocarbons, neonieotinoids, and carbamates and derivatives thereof. ularly suitable classes of insecticides include organcphosphates, phenylpyi'azoles and pyi‘ethoids.

Preferred insecticides are those known as terbufos, eliloipyrifos, chlorethoxyfos, tefluthrin, cai'boturan, and tebupirimfos. Commercially available insecticides include thiomethoxam (commercially available from Syngenta under the tradename Ciuiser.

] The insecticide can comprise an organophosphate, a carbamate, a pyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate, a fluoride, , a haloaromatic substituted urea, a hydrocarbon ester, a biologically-based insecticide, or a combination thereof.

] Suitable icides for use herein also include the following: (1) acetylcholine receptor ts/antagonists such as chloronicotinyls/nconicotinoids, ne, bensultap or . Suitable examples of chloronicotinyls/neonicotinoids include acetamiprid, dinotefuran, nitenpyram, nithiazine, thiacloprid, thiamethoxam, imidaclothiz and (2E)[(2- chloro-1,3-thiazolyl)methyl]-3,5-dimethyl-N—nitro-1,3,5-tri- azinanimine; (2) acetylcholinesterase (ACNE) inhibitors such as carbamates and organophosphates. Suitable examples of carbamates include alanycarb, aldicarb, aldoxycarb, carb, aminocarb, carb, benfuracarb, bufencarb, butacarb, rboxim, butoxycarboxim, carbaryl, carbofuran, carbosulfan, chloethocarb, dimetilan, ethiofencarb, fenobucarb, fenothiocarb, formetanate, furathiocarb, isoprocarb, metam-sodium, methomyl, metolcarb, , phosphocarb, pirimicarb, promecarb, propoxur, thiofanox, triazamate, hacarb, XMC and xylylcarb. Suitable examples of organophosphates include acephate, azamethiphos, azinphos (- , -ethyl), bromophos-ethyl, bromfenvinfos (-methyl), butathiofos, cadusafos, 2015/050807 carbophenothion, chlorethoxyfos, enVinphos, chlormephos, chlorpyrifos (-methyl/-ethyl), hos, cyanofenphos, cyanophos, demeton-S-methyl, n-S-methylsulphon, dialifos, diazinon, dichlofenthion, dichlorvos/DDVP, dicrotophos, dimethoate, dimethylVinphos, dioxabenzofos, oton, EPN, ethion, ethoprophos, etrimfos, famphur, fenamiphos, fenitrothion, fensulfothion, fenthion, flupyrazofos, fonofos, formothion, fosmethilan, fosthiazate, heptenophos, iodofenphos, iprobenfos, isazofos, isofenphos, isopropyl O-salicylate, isoxathion, malathion, mecarbam, methacrifos, methamidophos, methidathion, meVinphos, monocrotophos, naled, omethoate, oxydemeton-methyl, parathion (-methyl/-ethyl), phenthoate, phorate, phosalone, phosmet, phosphamidon, phosphocarb, phoxim, pirimiphos (-methyl/-ethyl), ofos, propaphos, propetamphos, prothiofos, prothoate, ofos, pyridaphenthion, pyridathion, quinalphos, sebufos, sulfotep, sulprofos, tebupirimfos, os, terbufos, tetrachlorVinphos, thiometon, triazophos, triclorfon and vamidothion; (3) sodium channel modulators/voltage-gated sodium l blockers such as pyrethroids and oxadiazines.

Suitable examples of pyrethroids include acrinathrin, rin (d-cis-trans, d-trans), beta- cyfluthrin, bifenthrin, bioallethrin, bioallethrin-S-cyclopentyl-isomer, bioethanomethrin, biopermethrin, bioresmethrin, chlovaporthrin, smethrin, cis-permethrin, clocythrin, rothrin, cyfluthrin, cyhalothrin, cyphenothrin, DDT, deltamethrin, empenthrin (lR- isomer), esfenvalerate, etofenprox, fenfluthrin, fenpropathrin, fenpyrithrin, fenvalerate, flubrocythrinate, flucythrinate, rox, flumethrin, fluvalinate, prox, gammacyhalothrin , imiprothrin, kadethrin, lambda-cyhalothrin, metofluthrin, permethrin (cis-, trans-), phenothrin (lR-trans isomer), prallethrin, profluthrin, protrifenbute, pyresmethrin, resmethrin, RU 15525, silafluofen, tau-fluvalinate, tefluthrin, terallethrin, tetramethrin (lR-isomer), tralocythrin, tralomethrin, transfluthrin, ZXI 8901 and pyrethrins (pyrethrum). Suitable example of oxadiazines includes indoxacarb; (4) acetylcholine receptor tors such as yns.

Suitable example of spinosyns includes spinosad; (5) GABA-gated chloride l antagonists such as cyclodiene organochlorines and fiproles. Suitable examples of iene organochlorines include camphechlor, chlordane, endosulfan, gamma-HCH, HCH, heptachlor, lindane and methoxychlor. Suitable examples of fiproles include acetoprole, and vaniliprole; (6) chloride l activators such as mectins. Suitable examples of mectins include abamectin, avermectin, emamectin, emamectin-benzoate, ivermectin, lepimectin, milbemectin and milbemycin; (7) juvenile e mimetics such as diofenolan, epofenonane, fenoxycarb, hydroprene, kinoprene, methoprene, pyriproxifen, triprene; (8) ecdysone agonists/disruptors such as diacylhydrazines. Suitable es of diacylhydrazines include chromafenozide, halofenozide, yfenozide and tebufenozide; (9) inhibitors of chitinbiosynthesis such as benzoylureas, buprofezin and zine. Suitable examples of benzoylureas include bistrifluron, chlofluazuron, diflubenzuron, fluazuron, flucycloxuron, flufenoxuron, muron, lufenuron, novaluron, noviflumuron, penfluron, teflubenzuron and triflumuron; (lO) tors of oxidative phosphorylation, ATP disruptors such as organotins and diafenthiuron. Suitable examples of organotins include azocyclotin, cyhexatin and fenbutatin oxide; (1 l) decouplers of oxidative phosphorylation by disruption of the H proton gradient such as pyrroles and dinitrophenols. Suitable example of pyrroles includes chlorfenapyr. Suitable examples of dinitrophenols include binapacyrl, dinobuton, dinocap and DNOC; (12) site I on transport inhibitors such as METIs, hydramethylnone and dicofol. Suitable es of METIs include fenazaquin, oximate, pyrimidifen, pyridaben, tebufenpyrad, tolfenpyrad; (13) site II electron transport inhibitors such as rotenone; (14) site 111 electron transport inhibitors such as acequinocyl and fluacrypyrim; (15) microbial disrupters of the intestinal membrane of insects such as Bacillus thuringiensis strains; (16) inhibitors of lipid synthesis such as tetronic acids and tetramic acids. Suitable examples of tetronic acids include spirodiclofen, spiromesifen and spirotetramat. Suitable example of tetramic acids includes cis(2,5- dimethylphenyl)methoxyoxo- l -azaspiro[4.5]decenyl ethyl carbonate (alias: carbonic acid, 3-(2,5-dimethylphenyl)methoxyoxo- l -azaspiro[4.5]decenyl ethyl ester (CAS Reg. No.: 3826088); (l7) carboxamides such as flonicamid; (18) octopaminergic ts such as amitraz; (l9) tors of the magnesium-stimulated ATPase such as propargite; (20) ryanodin receptor agonists such as phthalamides or rynaxapyr. Suitable example of amides includes N.sup .2-[l , l -dimethyl(methylsulphonyl)ethyl]iodo-N.sup. l-[2- methyl-- 4-[ l ,2,2,2-tetrafluoro- l -(trifluoromethyl)ethyl]phenyl]-l ,2-benzenedicarbo- xamide (i.e. flubendiamide, CAS reg. No.: 2724517); (2l) nereistoxin analogues such as thiocyclam en oxalate andthiosultap-sodium; (22) biologics, hormones or pheromones such as achtin, Bacillus spec., Beauveria spec., codlemone, Metarrhizium spec., omyces spec., thuringiensis and illium spec; (23) active compounds having unknown or non- specified mechanisms of action such as fumigants, selective feeding inhibitors, mite growth inhibitors, amidoflumet; benclothiaz, benzoximate, bifenazate, bromopropylate, ezin, chinomethioat, chlordimeform, chlorobenzilate, chloropicrin, azoben, cycloprene, cyflumetofen, dicyclanil, fenoxacrim, fentrifanil, flubenzimine, flufenerim, flutenzin, gossyplure, hydramethylnone, j aponilure, metoxadiazone, petroleum, piperonyl butoxide, potassium oleate, prole, pyridalyl, ole, sulfluramid, tetradifon, tetrasul, triarathene, verbutin, furthermore the compound 3-methylphenyl propylcarbamate (Tsumacide Z), the compound 3-(5-chloropyridinyl)(2,2,2-trifluoroethyl)azabicyclo[3.2.1]octa- ne carbonitrile (CAS reg. No. 3) and the corresponding 3-endo isomer (CAS reg. No. 1859845) (cf. WO 96/37494, WO 98/25923), and also preparations comprising insecticidal effective plant extracts, nematodes, fungi or viruses. Suitable examples of fumigants include aluminium phosphide, methyl bromide and sulphuryl fluoride. le examples of selective feeding inhibitors include cryolite, flonicamid and pymetrozine. Suitable examples of mite growth inhibitors include clofentezine, etoxazole and hexythiazox. {00649} Commercially available nematicidal ients include abamectin (commercially available from Syngenta under the tradename A victa).

H a formulation, plant seed, or inoculurn comprises an herbicide, the herbicide can comprise 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, atrazine, aminopyralid, benefin, bensulfuron, bensulide, bentazon, bromacil, bromoxynil, butylate, carfentrazone, muron, chlorsulfuron, clethodim, clomazone, clopyralid, cloransulam, cycloate, DCPA, desmedipham, dicamba, dichlobenil, diclofop, diclosulam, diflufenzopyr, enamid, diquat, diuron, DSMA, endothall, EPTC, ethalfluralin, ethofumesate, prop, fluazifop-P, flucarbazone, cet, flumetsulam, flumiclorac, flumioxazin, fluometuron, fluroxypyr, fomesafen, foramsulfuron, glufosinate, glyphosate, lfuron, hexazinone, imazamethabenz, imazamox, imazapic, imazaquin, imazethapyr, isoxaben, isoxaflutole, lactofen, linuron, MCPA, MCPB, mesotrione, metolachlor-s, metribuzin, metsulfuron, molinate, MSMA, napropamide, am, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxyfluorfen, paraquat, pelargonic acid, pendimethalin, phenmedipham, picloram, primisulfuron, prodiamine, prometryn, pronamide, propanil, prosulfuron, n, pyrithiobac, quinclorac, quizalofop, rimsulfuron, sethoxydim, n, ne, sulfentrazone, sulfometuron, sulfosulfuron, iuron, terbacil, thiazopyr, sulfuron, thiobencarb, tralkoxydim, ate, triasulfuron, tribenuron, triclopyr, trifluralin, triflusulfuron, or a ation thereof.

The herbicide can comprise a chlorophenoxy compound, a nitrophenolic compound, a nitrocresolic compound, a dipyridyl compound, an acetamide, an aliphatic acid, an anilide, a benzamide, a c acid, a benzoic acid derivative, anisic acid, an anisic acid derivative, a benzonitrile, benzothiadiazinone dioxide, a thiocarbamate, a carbamate, a carbanilate, chloropyridinyl, a cyclohexenone derivative, a dinitroaminobenzene tive, a fluorodinitrotoluidine compound, isoxazolidinone, nicotinic acid, isopropylamine, an 2015/050807 isopropylamine derivative, oxadiazolinone, a phosphate, a ate, a picolinic acid compound, a triazine, a triazole, a uracil, a urea derivative, endothall, sodium chlorate, a sulfonylurea, an aryl triazine, or a combination thereof.

The formulation can se an herbicide and a strain of bacteria that is capable of degrading the ide.

The strain of bacteria that is capable of degrading an herbicide can comprise Bacillus cereus family member EE349 (NRRL No. B-50928), Bacillus cereus family member 377 (NRRL B-67l l9), Bacillus pseudomycoia’es EE-B00366 (NRRL B-67120), or Bacillus mycoia’es EE-B00363 (NRRL B-67121), or a combination thereof.

The ide to be degraded can comprise a sulfonylurea such as sulfentrazone, an aryl triazine, dicamba, a phenoxy ide, 2,4-D, a pyrethrin, a pyrethroid, or a combination thereof {00655} s can be included in the formulations, such as carboxymethylcellulose and natural and synthetic polymers in the form ofpovvders, granules, or s, such as gain Arahic, chitin, polyvinyl alcohol and polyvinyl acetate, as well as natural phospholipids, such as eephalins and ins, and synthetic phospholipids. Binders include those composed ably of an adhesive polyi'ner that can be natural or synthetic without phytotoxic effect on the seed to be coated. Additional binders that can be included, either alone or in combination, include, for example, polyesters, polyether esters, polyanhydrides, polyester urethanes, polyester amides; polyvinyl acetates; polyvinyl acetate copolyrners; polyvinyl alcohols and tylose; polyvinyl alcohol copolymers; polyvinylpyrolidones; polysaccharides, ing starches, modified es and starch derivatives, dextrins, dextrins, alginates, chitosanes and celluloses, cellulose esters, cellulose ethers and cellulose ether esters including ethylcelluloses, inethylcelluloses, hydroxyrnethylcelluloses, hydroxypropylcelluloses and, earhoxymethylcellulose; fats; oils; proteins, including , gelatin and zeins; gum arabics; shellacs; vinylidene chloride and dene chloride copolyrners; lignosultonates, in particular calcium lignosulfonates; polyacrylates, polymethaerylates and acrylic copolymers; nylacrylates; polyethylene oxide; polybutenes, polyisohutenes, polystyrene, polybutadiene, polyethylenearnines, polyethylenamides; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; and polychloroprene. {006565} A variety of colorants maybe ed, including organic chromophores classified as nitroso, nitro, azo, including monoazo, 'bisazo, and polyazo, diphenylmethane, triarylinethane, xanthene, methane, acridine, thiazole, thiazine, indamine, indopl'ienol, azine, e, anthraquinone, and phthalocyanine. {68657} Other additives that can be added include trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum, and zinc.

] One or more preservatives (cg, antimicrobial agents or other biocidal agents} may also be included for preservation and stabilization of the lbrmulation, ples of le 'bactericides include those based on dichlorophene and henzylalcohol hemi formal l® from lCI or Acticide® RS from Thor Chernie and, Kathon® MK from Dow Chemical) and isothiazolinone derivatives such as alkylisothiazolinones and benzisothiazolinones (Aeticideél) MES from Thor Cheniie). As turther examples, suitable vatives include MlT (2~ niethylwél-isothiazolin—3—one}, Bl'l‘ enzisothiazolin-B—one, which can be obtained from Avecia, lnc. as Proxel GXL as a solution in sodium hydroxide and dipropylene glycol), 5— -Z—(‘lachlorobenzyl)—3(i2l€l)—isothiazolone, S-chloro- 2~methyl~2l-l-isothiazol(hone, 5~ chloro—Zninethyl~2H—isothiazol~3—one, S—chloro— hyl~2Hnisothiazol~3“one—hydrochloride, 4,5«dichloro~2~cyclohexyl—4~ isotliiazolin~3~one, 4,5.~dichloro~2~octyl—2l>l—isotliiazol~3~one, 2— niethyl-XH— azol—Smone, 2—inethyl—2H—isothiazol—3~0ne—calciurn chloride complex, 2— octyl— ZH—isotliiazol~3~one and benzyl alcohol liernil'orrnal, {@8659} Examples of suitable thickeners for the formulations include ccharides, organic clays, or a water—soluble polymer that exhibits pseudoplastic properties in an aqueous medium, such as, for example, gum arabic, gum liaraya, guni anth, guar gum, locust bean gum, Kanthan guin, eenan, alginate salt, casein, dextran, pectin, agar, 2- hydroxyethyl starch, '2- arninoethyl starch, 2 hydroxy ethyl cellulose, methyl cellulose, carboxyrnethyl cellulose salt, cellulose sulfa to salt, polyacrylamide, alkali metal salts of the tnaleic anhydride copolyniers, allcali metal salts ofpolymiethlacrylate. {$8669} Suitable antifreeze ients for the formulation include, for example and without limitation, ethylene , l ,Z—propylene glycol, l ,3~ propylene glycol, l ,2— butanediol, l ,S-butanediol, l ,4-butanediol, l ,4~pentanediol, 3—meth_yl~l,5—pentanediol, 2,3— dimethyln2,3nbutanediol, triinethylol propane, inannitol, sorbitol, glycerol, pentaerythritol, 1,4" cyclohexanedirnethanol, xylenol, bisphenols such as bisphenol A or the like. in addition, ether alcohols such as diethylene glycol, triethylene glycol, thylene glycol, polyoxyethylene or polyoxypropylene glycols of molecular weight up to about 4090, diethylene glycol monomethylether, diethylene glycol monoethylether, ylene glycol monoinethylether, butoxyethanol, butylene glycol inonobutylether, dipen taerythritol, tripentaerythritol, tetrapentaerytlu‘itol, di glycerol, triglycerol, tetraglycerol, pentaglycerol, hexaglycerol. lycerol, octaglycerol and combinations thereof.

XVIII. Plant Seeds The present invention further s to plant seeds coated with any of the recombinant Bacillus cereus family s described herein, with any of the recombinant spore-forming bacteria described herein, with any of the biologically pure bacterial cultures described herein, with any of the inoculums described herein, with any enzyme that catalyzes the production of nitric oxide, with any recombinant microorganism that expresses an enzyme that catalyzes the production of nitric oxide, or with any of the formulations other than vaccines as bed herein.

XIX. s Relating to Plants and Plant Seeds, Methods for Delaying Germination of a Spore of a Recombinant Bacillus cereus family member, and Methods for Making and Using rium Fragments The present invention further s to methods for stimulating plant growth, methods for protecting a plant from a pathogen or enhancing stress resistance in a plant, methods for immobilizing recombinant Bacillus cereus family member spores or recombinant spore forming bacteria on a plant, methods for stimulating germination of a plant seed, methods for ring nucleic acids to plants, methods for delaying germination of a spore of a recombinant Bacillus cereus family member, methods for making and using exosporium fragments, and methods for delivering beneficial bacteria to s.

A. MethOds 0r stimulatin lant rowth The present invention relates to methods for stimulating plant growth.

One method for stimulating plant growth of the present invention comprises introducing into a plant growth medium any of the recombinant Bacillus cereus family members described above or any of the formulations comprising a recombinant Bacillus cereus family member described above. atively, any of the recombinant Bacillus cereus family members bed above or any of the formulations comprising a inant Bacillus cereus family member described above can be applied to a plant, a plant seed, or to an area surrounding a plant or a plant seed. In such methods, the recombinant Bacillus cereus family member expresses a fusion protein comprising a plant growth stimulating protein or peptide. The plant growth stimulating protein or peptide can be ally attached to the rium of the recombinant Bacillus cereus family member.

Another method for stimulating plant growth comprises introducing into a plant growth medium any of the recombinant spore-forming bacteria described above or any of the formulations comprising a recombinant spore-forming bacterium described above.

Alternatively, any of the recombinant forming bacteria described above or any of the formulations comprising a recombinant spore-forming bacterium described above can be applied to a plant, a plant seed, or to an area surrounding a plant or a plant seed. In such methods, the recombinant spore-forming ium expresses a fusion protein comprising a plant growth stimulating protein or peptide. The plant growth stimulating protein or peptide can be physically attached to the spore coat of the recombinant spore-forming ium.

Yet another method for stimulating plant growth comprises introducing into a plant growth medium a recombinant Bacillus cereus family member or a formulation comprising a recombinant Bacillus cereus family member. Alternatively, the recombinant Bacillus cereus family member or the formulation can be applied to a plant, a plant seed, or to an area surrounding a plant or a plant seed. The recombinant Bacillus cereus family member expresses an enzyme involved in nutrient solubilization, a protease, a BclA protein, a BclB n, a CotE protein a CotO protein, an 13st protein, an ExsFA/BxpB protein, a CotY protein, an ExsFB n, an 13st protein, an 13st protein, a chA n, a chB protein, a Bch protein, a Bpr protein, a BclE protein, a BetA/BAS3290 protein, an 13st protein, an ExsK protein, an 13st n, a YabG protein, or a Tgl protein, n the expression of the enzyme involved in nutrient solubilization, the protease, a BclA protein, a BclB protein, a CotE protein a CotO protein, an 13st protein, an ExsFA/BxpB protein, a CotY protein, an ExsFB protein, an 13st protein, an 13st protein, a YjcA protein, a Yj cB protein, a Bch protein, a Bpr protein, a BclE protein, a BetA/BAS3290 protein, an 13st protein, an ExsK protein, an 13st protein, a YabG n, or a Tgl protein is increased as ed to the expression of the enzyme involved in nutrient solubilization, the protease, a BclA protein, a BclB protein, a CotE protein a CotO protein, an 13st protein, an ExsFA/BxpB protein, a CotY protein, an ExsFB protein, an 13st protein, an 13st protein, a YjcA protein, a Yj cB protein, a Bch protein, a Bpr n, a BclE protein, a BetA/BAS3290 protein, an 13st protein, an ExsK protein, an 13st protein, a YabG protein, or a Tgl protein in a wild-type Bacillus cereus family member under the same conditions.

Additional s for ating plant , involving the use of exosporium fragments derived from a recombinant Bacillus cereus family member, are described below.

B. Methods for protecting a plant from a pathogen or enhancing stress resistance in a glant The present invention also relates to methods for protecting a plant from a pathogen or enhancing stress resistance in a plant.

One method for ting a plant from a pathogen or enhancing stress resistance in a plant comprises introducing into a plant growth medium any of the recombinant us cereus family members described above or any of the formulations comprising a recombinant Bacillus cereus family member bed above. Alternatively, any of the recombinant Bacillus cereus family members described above or any of the formulations comprising a recombinant Bacillus cereus family member described above can be applied to a plant, a plant seed, or to an area surrounding a plant or a plant seed. In such methods, the recombinant Bacillus cereus family member expresses a fusion protein comprising a protein or peptide that protects a plant from a pathogen or a protein or peptide that enhances stress resistance in a plant. The protein or e that protects a plant from a pathogen or the protein or peptide that enhances stress resistance in a plant can be physically attached to the exosporium of the recombinant Bacillus cereus family member.

Another method for protecting a plant from a pathogen or enhancing stress resistance in a plant comprises introducing into a plant growth medium any of the recombinant spore-forming bacteria described above or any of the formulations comprising a recombinant forming bacterium described above. Alternatively, any of the recombinant spore-forming bacteria described above or any of the ations comprising a recombinant forming bacterium described above can be applied to a plant, a plant seed, or to an area surrounding a plant or a plant seed. In such methods, the recombinant spore-forming bacterium expresses a fusion protein comprising a protein or peptide that protects a plant from a pathogen or a protein or peptide that es stress ance in a plant. The protein or peptide that protects a plant from a pathogen or the protein or peptide that enhances stress resistance in a plant can be physically attached to the spore coat of the recombinant spore-forming bacterium.

In any of the methods for protecting a plant from a pathogen, plants grown in the plant growth medium comprising the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium are ably less susceptible to infection with the pathogen as compared to plants grown under the same conditions in the identical plant growth medium that does not contain the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium.

In any of the methods for enhancing stress resistance in a plant plants grown in the plant growth medium comprising the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium are preferably less susceptible to stress as compared to plants grown under the same ions in the identical plant growth medium that does not contain the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium.

] Another method for enhancing stress resistance in a plant ses ucing into a plant growth medium a recombinant Bacillus cereus family member or a formulation comprising the recombinant Bacillus cereus family member. atively, the recombinant Bacillus cereus or the formulation can be applied to a plant, a plant seed, or to an area surrounding a plant or a plant seed. The recombinant Bacillus cereus family member expresses a superoxide dismutase or an arginase, wherein the expression of the superoxide dismutase or the arginase is increased as compared to the expression of the superoxide dismutase or the arginase in a wild-type us cereus family member under the same conditions. r method for protecting a plant from a pathogen comprises introducing into a plant growth medium a recombinant Bacillus cereus family member or a formulation comprising the recombinant Bacillus cereus family member. Alternatively, the recombinant Bacillus cereus or the ation can be applied to a plant, a plant seed, or to an area nding a plant or a plant seed. The recombinant Bacillus cereus family member expresses a protease, wherein the expression of the protease is increased as compared to the expression of the protease in a wild-type Bacillus cereus family member under the same ions.

Additional methods for ting a plant from a pathogen or enhancing stress resistance in a plant, involving the use of exosporium fragments derived from a recombinant us cereus family member, are described below.

C. Methods for immobilizing recombinant Bacillus cereus family member sgores or recombinant sgore forming bacteria on a glant The t invention further relates to methods for immobilizing recombinant Bacillus cereus family member spores or recombinant spore forming bacteria on a plant.

One method for immobilizing a recombinant Bacillus cereus family member spore on a plant comprises introducing into a plant growth medium any of the recombinant Bacillus cereus family s described above or any of the formulations comprising a recombinant Bacillus cereus family member described above. Alternatively, any of the inant Bacillus cereus family members described above or any of the formulations comprising a recombinant Bacillus cereus family member bed above can be applied to a plant, a plant seed, or to an area surrounding a plant or a plant seed. In such methods, the inant Bacillus cereus family member expresses a fusion protein comprising a plant binding protein or peptide. The plant binding protein or peptide can be physically attached to the exosporium of the recombinant Bacillus cereus family member.

Another method for immobilizing a spore of a recombinant spore-forming bacterium on a plant comprises introducing into a plant growth medium any of the inant spore-forming bacteria described above or any of the formulations comprising a inant spore-forming bacterium described above. Alternatively, any of the recombinant spore-forming bacteria described above or any of the formulations comprising a recombinant spore-forming bacterium described above can be applied to a plant, a plant seed, or to an area surrounding a plant or a plant seed. In such methods, the recombinant spore-forming bacterium expresses a fusion n comprising a plant binding peptide and the plant binding protein or peptide can be physically attached to the spore coat of the recombinant spore-forming bacterium.

The plant binding protein or e preferably selectively targets and ins the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium on a plant. For example, the plant g protein or peptide can selectively target and maintain the recombinant Bacillus cereus family member on at plant roots, substructures of roots, an aerial portion of a plant, or a substructure of an aerial portion of a plant.

D. s for stimulating germination 01a glant seed 1. Methodsfor stimulating germination involving the use ofa recombinant Bacillus cereusfamily member ofa recombinant spore-forming ium The present invention also provides methods for stimulating germination of a plant seed.

One method for stimulating ation of a plant seed comprises introducing into a plant growth medium any of the recombinant Bacillus cereus family s described above or any of the formulations comprising a recombinant Bacillus cereus family member described above. Alternatively, any of the recombinant Bacillus cereus family members described above or any of the formulations comprising a recombinant Bacillus cereus family member described above can be applied to a plant, a plant seed, or to an area surrounding a plant or a plant seed. In such methods, the recombinant us cereus family member expresses a fusion protein comprising an enzyme that catalyzes the production of nitric oxide. The enzyme that catalyzes the production of nitric oxide can be physically attached to the exosporium of the recombinant Bacillus cereus family member.

Another method for stimulating germination of a plant seed comprises introducing into a plant growth medium any of the recombinant spore-forming bacteria described above or any of the ations comprising a recombinant spore-forming bacterium described above. Alternatively, any of the recombinant spore-forming bacteria described above or any of the ations comprising a recombinant spore-forming bacterium described above can be applied to a plant, a plant seed, or to an area surrounding a plant or a plant seed. In such methods, the recombinant spore-forming bacterium expresses a fusion protein comprising an enzyme that catalyzes the production of nitric oxide, and the enzyme that catalyzes the production of nitric oxide can be physically attached to the spore coat of the recombinant spore- g ium.

] The above methods for stimulating germination of a plant seed preferably comprise applying the recombinant Bacillus cereus family member, the recombinant sporeforming bacterium, or the formulation to a plant seed.

Any of the above methods for ating germination of a plant seed can further comprise applying a substrate for the enzyme that catalyzes production of nitric oxide to the plant growth medium, the plant seed, the plant, or the area nding the plant or the plant seed. For example, the method suitably further comprises adding L-arginine to the plant growth medium, the plant seed, the plant, or the area surrounding the plant or the plant seed. For example, the L-arginine can be applied to an aerial portion of the plant. The L-arginine is preferably applied to the plant seed.

The presence of L-arginine enhances the on and leads to a more pronounced output ofNO by the nitric oxide synthase. Furthermore, L-arginine on a plant seed, a plant growth medium, or an area surrounding a plant can serve as a substrate for the production of nitric oxide by native bacterial enzymes.

] In any of the above methods for stimulating germination of a plant seed, seeds in the plant growth medium comprising the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium or seeds to which the recombinant Bacillus cereus family member or the recombinant spore-forming ium has been applied preferably have an increased germination rate as compared to seeds grown under the same ions in the cal plant growth medium that does not contain the recombinant us cereus family member or the recombinant spore-forming bacterium or seeds to which the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium has not been applied, grown under the same conditions.

In any of the above s for stimulating germination of a plant seed, seeds in the plant growth medium comprising the recombinant Bacillus cereus family member or the recombinant spore-forming ium or seeds to which the inant Bacillus cereus family member or the recombinant spore-forming bacterium has been applied preferably have a longer taproot after ation as compared to seeds grown under the same conditions in the identical plant growth medium that does not contain the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium or seeds to which the recombinant Bacillus cereus family member or the recombinant spore-forming ium has not been applied under the same conditions.

Additional methods for stimulating germination of a plant seed, involving the use of exosporium fragments derived from a recombinant Bacillus cereus family member, are described below. 2. Methodsfor stimulating germination by delivering to plants enzymes that ze the production ofnitric oxide or recombinant microorganisms that overexpress such enzymes Yet another method for stimulating ation of a plant seed comprises introducing into a plant growth medium, or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed: (i) an enzyme that catalyzes the production of nitric oxide; (ii) a superoxide dismutase; or (iii) a recombinant microorganism that expresses an enzyme that catalyzes the production of nitric oxide or a superoxide dismutase, wherein the expression of the enzyme that catalyzes the production of nitric oxide or the superoxide dismutase is increased as compared to the expression of the enzyme that catalyzes the production of nitric oxide or the superoxide dismutase in a wild type rganism under the same conditions.

The method preferably comprises applying the enzyme or the microorganism to a plant seed. 2015/050807 The method can further se applying a substrate for the enzyme that catalyzes production of nitric oxide to the plant growth medium, the plant seed, the plant, or the area nding the plant or the plant seed. For example, the method suitably further comprises adding L-arginine to the plant growth medium, the plant seed, the plant, or the area surrounding the plant or the plant seed. For example, the L-arginine can be applied to an aerial portion of the plant. The L-arginine is preferably applied to the plant seed.

Seeds in the plant growth medium comprising the enzyme or the microorganism or seeds to which the enzyme or the microorganism has been applied preferably have an increased germination rate as compared to seeds grown under the same conditions in the identical plant growth medium that does not contain enzyme or the microorganism or seeds to which the enzyme or the microorganism has not been applied, grown under the same conditions.

Seeds in the plant growth medium comprising the enzyme or the microorganism or seeds to which the enzyme or the microorganism has been applied preferably have a longer taproot after germination as compared to seeds grown under the same conditions in the identical plant growth medium that does not contain the enzyme or the microorganism or seeds to which the enzyme or the rganism has not been d under the same conditions.

] The enzyme that catalyzes the production of nitric oxide synthase can comprise a nitric oxide synthase or an arginase. Where the enzyme that catalyzes the tion of nitric oxide comprises a nitric oxide synthase, the nitric oxide synthase can comprise, for example, a nitric oxide synthase from Bacillus thuringiensis BT013A or Bacillus subtilis 168.

For e, the nitric oxide synthase can have at least 85% sequence identity with SEQ ID NO: 260 or 261.

] The nitric oxide synthase can have at least 90% sequence identity with SEQ ID NO: 260 or 261.

The nitric oxide synthase can have at least 95% sequence identity with SEQ ID NO: 260 or 261.

The nitric oxide synthase can have at least 98% sequence identity with SEQ ID NO: 260 or 261.

The nitric oxide synthase can have at least 99% sequence identity with SEQ ID NO: 260 or 261.

The nitric oxide synthase can have 100% sequence identity with SEQ ID NO: 260 or 261. 2015/050807 The superoxide dismutase can comprise a superoxide dismutase 1 (SODAl) or a superoxide dismutase 2 (SODAZ). The superoxide dismutase can comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase can se an amino acid sequence having at least 90% identity with SEQ ID NO: 155 or 156.

The superoxide ase can se an amino acid sequence having at least 95% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase can comprise an amino acid sequence having at least 98% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase can comprise an amino acid sequence having at least 99% identity with SEQ ID NO: 155 or 156.

The superoxide dismutase can comprise an amino acid sequence having at least 100% ty with SEQ ID NO: 155 or 156.

The recombinant microorganism that expresses an enzyme that catalyzes the production of nitric oxide can se a Bacillus s (e. g., a Bacillus cereus family member, Bacillus subtilis, Bacillus licheniformis, or Bacillus megaterium), Escherechia c0li, an Aspergillus species (e. g., Aspergillus niger), or a Saccliromyces species (e. g., Saccliromyces cerevisiae).

In any of the above methods, the enzyme or the recombinant microorganism can be introduced into the plant growth medium, or applied to a plant, a plant seed, or an area nding a plant or a plant seed in a formulation comprising the enzyme or the recombinant microorganism and an agriculturally acceptable carrier. The formulation can comprise any of the agriculturally acceptable carriers and other components discussed herein.

The enzyme that zes the production of nitric oxide can be delivered purified or unpurified, and can be red alone or in combination with other beneficial proteins, inoculants, or chemicals to the plant seed, the plant growth medium, or an area surrounding the plant or the plant seed.

E. Methods for delivering nucleic acids to plants Methods for delivering nucleic acids to plants are also provided by the present invention.

] One method for delivering c acids to a plant comprises introducing into a plant growth medium any of the recombinant Bacillus cereus family members described above or any of the formulations sing a recombinant Bacillus cereus family member described above. Alternatively, any of the recombinant Bacillus cereus family s described above or any of the formulations comprising a inant Bacillus cereus family member described above can be applied to a plant, a plant seed, or to an area surrounding a plant or a plant seed. In such methods, the recombinant Bacillus cereus family member expresses a fusion protein comprising a nucleic acid binding protein. The nucleic acid binding protein or peptide is bound to a nucleic acid le. The nucleic acid binding protein or peptide can be physically attached to the exosporium of the recombinant Bacillus cereus family .

In such methods, the recombinant Bacillus cereus family member can comprise an endophytic strain of bacteria. The endophytic strain of bacteria can comprise Bacillus cereus family member EE349, Bacillus cereus family member EE439, Bacillus thuringiensis EE4l7, us cereus EE444, us thuringiensis EE3 l9, Bacillus giensis EE-B00184, Bacillus cereus family member EE-B003 77, Bacillus pseudomycoia’es EE-B00366, or us mycoia’es EE-B00363. For example, the endophytic strain of bacteria can comprise Bacillus cereus family member EE439, Bacillus thuringiensis EE4l7, Bacillus cereus EE444, Bacillus thuringiensis EE3 l9, Bacillus thuringiensis EE-B00184, Bacillus cereus family member EE-B00377, Bacillus pseudomycoia’es EE-B00366, or Bacillus mycoia’es EE- B00363.

Another method for delivering c acids to a plant comprises introducing into a plant growth medium any of the recombinant spore-forming ia described above or any of the formulations comprising a recombinant spore-forming bacterium described above.

Alternatively, any of the recombinant spore-forming bacteria described above or any of the formulations comprising a recombinant spore-forming ium described above can be applied to a plant, a plant seed, or to an area surrounding a plant or a plant seed. In such methods, the recombinant spore-forming bacterium expresses a fusion protein sing a nucleic acid g protein. The nucleic acid binding protein or peptide is bound to a c acid molecule.

The nucleic acid g protein or peptide can be physically attached to the spore coat of the recombinant spore-forming bacterium.

The inant spore-forming bacterium can comprise an endophytic strain of bacteria. For example, the endophytic strain of bacteria can comprise Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillus fusiformis EE442, Lysinibacillus sphericus EE443, or Bacillus pumilus EE-BOOl43.

In any of the above methods for delivering nucleic acids to a plant, the nucleic acid molecule can se a modulating RNA molecule; an RNAi molecule; a microRNA; an aptamer; or a DNA le that encodes a modulating RNA molecule, an RNAi molecule, a microRNA, or an aptamer.

The nucleic acid molecules to be delivered to the plant can be produced by any means known the art (e.g., chemical synthesis, inant tion by a microorganism, etc.). The nucleic acid les can then be bound to the nucleic acid binding protein or peptide portion of the fusion proteins described herein in preparation for delivery of such nucleic acids to a plant or plants. The c acid binding proteins and peptides immobilize and stabilize the nucleic acids and allow them to be delivered to the plant intact. The nucleic acid les to be red to the plant can be in an active form, or in an inactive form that can be processed into an active form by the plant.

To accomplish the binding of the nucleic acid molecules to the nucleic acid binding protein or peptide, the nucleic acids molecules can be incubated with the any of the recombinant us cereus members or recombinant spore-forming bacteria described herein that express a fusion protein sing a nucleic acid binding protein or e.

Additional methods for delivering c acids to a plant, involving the use of exosporium fragments derived from a recombinant Bacillus cereus family member, are described below.

F. Methods or dela in ermination o a s ore o a recombinant Bacillus cereus family member The present invention further relates to a method for delaying germination of a spore of a Bacillus cereus family member. The method comprises modifying the Bacillus cereus family member to express an inosine-uridine hydrolase or an alanine racemase, wherein the expression of the inosine-uridine hydrolase or the alanine racemase is increased as ed to the expression of the inosine-uridine hydrolase or the alanine racemase in a wild-type Bacillus cereus family member under the same conditions.

G. Inactivation of the Bacillus cereus family member or recombinant sgore-Zorming bacterium grior to use In any of the above methods that use a recombinant Bacillus cereus family member or a recombinant spore forming bacterium, the method can further comprise vating the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium prior to introduction into the plant growth medium or application to a plant, a plant seed, or an area surrounding a plant or a plant seed.

For example, the vating can comprise subjecting the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium to heat treatment; gamma irradiation; x-ray irradiation; UV-A irradiation; UV-B irradiation; treatment with gluteraldehyde, formaldehyde, en peroxide, acetic acid, bleach, chloroform, or phenol, or a combination thereof.

Alternatively or in addition, the inactivating can comprise modifying the recombinant Bacillus cereus family member recombinant or forming bacterium to express a germination spore protease or a non-specific endonuclease, wherein the expression of the germination spore protease or the non-specific endonuclease is sed as compared to the expression of the germination spore protease or the non-specific endonuclease in a wild-type Bacillus cereus family member under the same conditions, and wherein the recombinant spore- forming bacterium comprises a recombinant bacterium of the genus Bacillus.

H. Methods for making and using exosgorium fragments ] The present invention further s to methods for making and using exosporium fragments. These methods relate to the inant Bacillus cereus family members bed in n IV above, i.e., recombinant Bacillus cereus family members that comprise a mutation or another genetic alteration that allows for the collection of free exosporium.

Thus, the present invention relates to a method for removing rium from spores of a recombinant Bacillus cereus family member. The method comprises subjecting a suspension comprising any of the recombinant Bacillus cereus family members bed in Section IV hereinabove to centrifugation or filtration to produce fragments of exosporium that are separated from the spores. The exosporium fragments comprise the fusion protein.

The method for removing exosporium from spores of a recombinant Bacillus cereus family member can comprise subjecting the suspension comprising the spores to centrifugation and collecting the supernatant, wherein the supernatant comprises the fragments of the exosporium and is substantially free of spores. atively, the method for removing exosporium from spores of a recombinant us cereus family member can comprise subjecting the suspension comprising the spores to filtration and collecting the filtrate, wherein the filtrate comprises the fragments of the exosporium and is substantially free of spores.

The suspension of spores can be ed or mechanically disrupted prior to centrifugation or filtration.

The exosporium fragments can also be separated from the spores by gradient centrifugation, affinity purification, or by allowing the spores to settle out of the suspension.

The present invention further s to methods for using the exosporium fragments.

A method for stimulating plant growth is provided. The method comprises introducing exosporium fragments or a ation of comprising the exosporium nts and an agriculturally acceptable carrier into a plant growth medium. Alternatively, the exosporium fragments or the formulation can be applied to a plant, a plant seed, or an area surrounding a plant or a plant seed. The exosporium fragments are derived from spores of a recombinant Bacillus cereus family member described in Section IV hereinabove and comprise the fusion n. The fusion protein comprises a plant growth stimulating protein or peptide.

A method for protecting a plant from a pathogen or enhancing stress resistance in a plant is also provided. The method ses ucing exosporium fragments or a formulation of comprising the exosporium fragments and an lturally able carrier into a plant growth medium. Alternatively, the exosporium nts or the formulation can be applied to a plant, a plant seed, or an area surrounding a plant or a plant seed. The exosporium fragments are derived from spores of a recombinant Bacillus cereus family member described in Section IV hereinabove and comprise the fusion protein. The fusion n comprises a protein or peptide that protects a plant from a pathogen or a protein or peptide that enhances stress resistance in a plant.

When the method is a method for protecting a plant from a pathogen, the fusion protein comprises protein or peptide that protects a plant from a pathogen.

] In the methods for protecting a plant from a pathogen, plants grown in the plant growth medium sing the exosporium fragments are preferably less tible to ion with the pathogen as compared to plants grown under the same conditions in the identical plant growth medium that does not contain the exosporium fragments.

When the method is a method for enhancing stress resistance in a plant, the fusion protein comprises a protein or peptide that enhances stress resistance in a plant.

In the methods for enhancing stress resistance in a plant of, plants grown in the plant growth medium comprising the exosporium fragments are preferably less susceptible to stress as compared to plants grown under the same conditions in the identical plant growth medium that does not contain the exosporium fragments.

A method for immobilizing exosporium fragments on a plant is also provided.

The method comprises introducing exosporium fragments or a formulation of comprising the exosporium fragments and an agriculturally acceptable carrier into a plant growth .

Alternatively, the exosporium fragments or the formulation can be applied to a plant, a plant seed, or an area surrounding a plant or a plant seed. The exosporium fragments are d from spores of a inant Bacillus cereus family member described in n IV hereinabove and se the fusion protein. The fusion protein comprises a plant binding protein or e.

The plant binding protein or peptide preferably ively targets and maintains the exosporium fragments on a plant. For example, the plant binding protein or peptide can selectively target and maintain the exosporium fragments on at plant roots, substructures of roots, an aerial portion of a plant, or a substructure of an aerial portion of a plant.

A method for stimulating germination of a plant seed is also provided. The method comprises introducing exosporium fragments or a formulation of sing the exosporium fragments and an agriculturally acceptable carrier into a plant growth medium.

Alternatively, the exosporium nts or the formulation can be applied to a plant, a plant seed, or an area surrounding a plant or a plant seed. The exosporium fragments are derived from spores of a recombinant Bacillus cereus family member described in Section IV above and comprise the fusion protein. The fusion protein comprises a superoxide dismutase or an enzyme that catalyzes the production of nitric oxide.

In the methods for ating germination, the method preferably ses applying the exosporium fragments to a plant seed.

The methods for stimulating germination can further comprise applying a substrate for the enzyme that catalyzes production of nitric oxide to the plant growth medium, the plant seed, the plant, or the area surrounding the plant or the plant seed. For example, the method suitably further comprises adding L-arginine to the plant growth medium, the plant seed, the plant, or the area surrounding the plant or the plant seed. For example, the L-arginine can be applied to an aerial portion of the plant. The L-arginine is ably applied to the plant seed.

The presence of L-arginine enhances the reaction and leads to a more pronounced output ofNO by the nitric oxide synthase. Furthermore, L-arginine on a plant seed, a plant growth medium, or an area surrounding a plant can serve as a substrate for the production of nitric oxide by native bacterial enzymes.

] In the methods for stimulating ation of a plant seed, seeds in the plant growth medium comprising the exosporium fragments or seeds to which the exosporium fragments have been applied preferably have an increased germination rate as compared to the same seeds grown under the same conditions in the identical plant growth medium that does not contain the exosporium fragments or the same seeds grown under the same ions to which the exosporium fragments have not been applied.

In the methods for stimulating germination of a plant seed, seeds in the plant growth medium comprising the exosporium fragments or seeds to which the exosporium fragments have been applied preferably have a longer taproot after germination as compared to the same seeds grown under the same conditions in the identical plant growth medium that does not contain the exosporium fragments or the same seeds grown under the same conditions to which the exosporium fragments have not been applied.

] A method for ring nucleic acids to a plant is also provided. The method comprises ucing exosporium fragments or a formulation of comprising the exosporium fragments and an agriculturally acceptable carrier into a plant growth medium. Alternatively, the exosporium fragments or the formulation can be applied to a plant, a plant seed, or an area surrounding a plant or a plant seed. The exosporium fragments are derived from spores of a recombinant Bacillus cereus family member bed in Section IV hereinabove and comprise the fusion n. The fusion n comprises a nucleic acid binding n or peptide. The c acid binding protein or peptide is bound to a nucleic acid molecule.

In the method for delivering nucleic acids to a plant, the nucleic acid molecule can comprise a modulating RNA molecule; an RNAi molecule; a microRNA, an aptamer; or a DNA molecule that encodes a ting RNA molecule, an RNAi molecule, a microRNA, or an aptamer.

The nucleic acid molecules to be delivered to the plant can be produced by any means known the art (e.g., chemical synthesis, inant tion by a microorganism, etc.). The nucleic acid molecules can then be bound to the nucleic acid binding protein or peptide n of the fusion proteins described herein in preparation for delivery of such nucleic acids to a plant or plants. The nucleic acid binding proteins and peptides immobilize and stabilize the nucleic acids and allow them to be delivered to the plant intact. The nucleic acid molecules to be red to the plant can be in an active form, or in an inactive form that can be processed into an active form by the plant.

To accomplish the binding of the nucleic acid molecules to the nucleic acid binding protein or peptide, the nucleic acids molecules can be incubated with the exosporium fragments ning a fusion protein comprising a nucleic acid binding protein or peptide.

I. Plant Growth Medium In any of the methods described herein involving the use of a plant growth medium, the plant growth medium can comprise soil, water, an aqueous solution, sand, gravel, a polysaccharide, mulch, compost, peat moss, straw, logs, clay, soybean meal, yeast extract, or a combination thereof.

Furthermore, the plant growth medium can be supplemented with a substrate or a cofactor for an enzyme. For example, the substrate or the cofactor can comprise tryptophan, an adenosine monophosphate, an adenosine diphosphate, an adenosine sphate (e.g., adenosinetriphosphate), indole, a trimetaphosphate, ferrodoxin, acetoin, diacetyl, pyruvate, acetolactate, pectin, cellulose, methylcellulose, starch, , pectin, a protein meal, a cellulose derivative, a phosphate, acetoin, an, an inactive derivative of acetic acid, an inactive derivative of gibberellic acid, a xylan, an oxylan, a fat, a wax, an oil, a phytic acid, a lignin, a humic acid, choline, a choline derivative, proline, a polyproline, a e-rich protein, a proline-rich meal, phenylalanine, mate, L-arginine, NADH, NADPH, ATP, GTP, cytochrome C, cytochrome p450, or a combination thereof.

J. s 0 A lication The methods described herein can se coating seeds with the recombinant Bacillus cereus family , the recombinant spore-forming bacterium, or the exosporium fragments or a formulation containing the recombinant Bacillus cereus family member, the recombinant spore-forming ium, or the or exosporium fragments prior to planting.

The methods described herein can comprise applying the recombinant Bacillus cereus family member, the recombinant forming bacterium, or the exosporium fragments, or a ation containing the recombinant Bacillus cereus family member, the 2015/050807 recombinant spore-forming ium, or the rium fragments to an aerial portion of a plant.

In the methods described herein, introducing the inant Bacillus cereus family member, the recombinant spore-forming bacterium, or the exosporium fragments into the plant growth medium can comprise applying a liquid or solid formulation containing the recombinant Bacillus cereus family member, the recombinant forming bacterium, or the exosporium fragments to the medium. The plant growth medium can comprise soil (e.g., potting soil), t, peat moss, sand, seed starter mix, or a combination thereof The method can comprise applying the formulation to the plant growth medium prior to, concurrently with, or after planting of seeds, seedlings, gs, bulbs, or plants in the plant growth medium.

K. Agrocheml'cals In the methods bed herein, the method can further comprise introducing at least one agrochemical into the plant growth medium or ng at least one agrochemical to plants or seeds.

The agrochemical can comprise a fertilizer (e. g., a liquid fertilizer), a micronutrient fertilizer material (e.g., boric acid, a borate, a boron frit, copper sulfate, a copper frit, a copper chelate, a sodium tetraborate drate, an iron sulfate, an iron oxide, iron um sulfate, an iron frit, an iron chelate, a manganese sulfate, a manganese oxide, a manganese chelate, a manganese chloride, a manganese frit, a sodium molybdate, molybdic acid, a zinc sulfate, a zinc oxide, a zinc carbonate, a zinc frit, zinc phosphate, a zinc chelate, or a combination thereof), an insecticide (e.g., an organophosphate, a carbamate, a pyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate, a fluoride, sulfur, a haloaromatic substituted urea, a hydrocarbon ester, a biologically-based insecticide, or a combination thereof), an herbicide (e. g., a chlorophenoxy compound, a nitrophenolic compound, a nitrocresolic nd, a dipyridyl compound, an acetamide, an aliphatic acid, an anilide, a benzamide, a benzoic acid, a benzoic acid derivative, anisic acid, an anisic acid derivative, a itrile, benzothiadiazinone dioxide, a thiocarbamate, a carbamate, a carbanilate, chloropyridinyl, a cyclohexenone tive, a dinitroaminobenzene derivative, a fluorodinitrotoluidine compound, isoxazolidinone, nicotinic acid, isopropylamine, an isopropylamine derivatives, oxadiazolinone, a ate, a phthalate, a picolinic acid compound, a triazine, a triazole, a uracil, a urea derivative, endothall, sodium chlorate, or a combination thereof), a fungicide (e.g., a substituted benzene, a thiocarbamate, an ethylene bis dithiocarbamate, a thiophthalidamide, a copper compound, an mercury compound, an organotin compound, a cadmium compound, anilazine, benomyl, cyclohexamide, dodine, azole, iprodione, metlaxyl, thiamimefon, triforine, or a combination thereof), a molluscicide, an algicide, a plant growth amendment, a bacterial inoculant (e.g., a bacterial inoculant of the genus Rhizobz'um, a bacterial inoculant of the genus Bradyrhz'zobz’um, a bacterial inoculant of the genus Mesorhz'zobz'um, a bacterial inoculant of the genus Azorhz'zobium, a bacterial inoculant of the genus Allorhz'zobz'um, a bacterial inoculant of the genus Sinorhz'zobz'um, a ial inoculant of the genus Kluyvera, a bacterial inoculant of the genus Azotobacter, a bacterial inoculant of the genus Pseudomonas, a bacterial ant of the genus Azospirillz'um, a bacterial inoculant of the genus us, a bacterial inoculant of the genus omyces, a bacterial inoculant of the genus Paenibacillus, a bacterial ant of the genus ccus, a bacterial inoculant of the genus Enterobacter, a bacterial inoculant of the genus 'genes, a bacterial inoculant of the genus Mycobacterl'um, a bacterial inoculant of the genus Trichoderma, a bacterial inoculant of the genus Gliocladl'um, a bacterial inoculant of the genus Glomus, a bacterial inoculant of the genus 'ella, or a combination thereof), a fungal inoculant (e.g., a fungal inoculant of the family Glomeraceae, a fungal inoculant of the family Claroidoglomeraceae, a fungal inoculant of the family Gigasporaceae, a fungal inoculant of the family Acaulosporaceae, a fungal inoculant of the family osporaceae, a fungal inoculant of the family Entrophosporaceae, a fungal inoculant of the family Pacidsporaceae, a fungal inoculant of the family Diversisporaceae, a fungal inoculant of the family Paraglomeraceae, a fungal inoculant of the family Archaeosporaceae, a fungal inoculant of the family Geosiphonaceae, a fungal inoculant of the family Ambisporaceae, a fungal ant of the family Scutellosporaceae, a fungal inoculant of the family Dentiscultataceae, a fungal inoculant of the family Racocetraceae, a fungal inoculant of the phylum Basidiomycota, a fungal inoculant of the phylum Ascomycota, a fungal inoculant of the phylum Zygomycota, or a combination f), or a combination thereof.

The fertilizer can comprise ammonium sulfate, ammonium nitrate, ammonium sulfate e, ammonium chloride, ammonium bisulfate, ammonium polysulfide, ammonium thiosulfate, s ammonia, anhydrous ammonia, ammonium polyphosphate, aluminum sulfate, calcium nitrate, calcium ammonium nitrate, calcium sulfate, calcined ite, calcitic limestone, m oxide, calcium nitrate, dolomitic limestone, hydrated lime, calcium carbonate, diammonium ate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, potassium magnesium sulfate, potassium e, sodium nitrates, magnesian limestone, magnesia, urea, urea-formaldehydes, urea ammonium e, sulfur-coated urea, polymer-coated urea, isobutylidene diurea, K2804— 2MgSO4, kainite, sylvinite, kieserite, Epsom salts, elemental , marl, ground oyster shells, fish meal, oil cakes, fish manure, blood meal, rock phosphate, super phosphates, slag, bone meal, wood ash, manure, bat guano, peat moss, compost, green sand, cottonseed meal, feather meal, crab meal, fish on, humic acid, or a ation thereof.

The agrochemical can comprise any of the fungicides, bacterial inoculants, or herbicides, described above in section XVII.

L. Plants and seeds [W756] in any ofthe above methods relating to plants, the plant can be a dicotyledon, a monocotyledon, or a gymnosperm. {68757} For example, where the plant is a dicotyledon, the dicotyledon can be selected from the group consisting of bean, pea, tomato, pepper, squash, alfalfa, almond, aniseseed, apple, apricot, arracha, oke, avocado, bambara groundnut, beet, bergamot, black pepper, black wattle, blackberry, blueberry, bitter orange, bok-choi, Brazil nut, breadfruit, broccoli, broad bean, Brussels sprouts, buckwheat, cabbage, camelina, Chinese cabbage, cacao, cantaloupe, caraway seeds, cardoon, carob, , cashew nuts, cassava, castor bean, cauliflower, celeriac, celery, cherry, chestnut, ea, chicory, chili , chrysanthemum, cinnamon, citron, clementine, clove, clover, coffee, cola nut, colza, corn, cotton, cottonseed, , crambe, cranberry, cress, cucumber, currant, custard apple, drumstick tree, earth pea, eggplant, , fennel, fenugreek, fig, filbert, flax, geranium, gooseberry, gourd, grape, grapefruit, guava, hemp, hempseed, henna, hop, horse bean, adish, indigo, e, Jerusalem artichoke, jute, kale, kapok, kenaf, kohlrabi, t, lavender, lemon, lentil, lespedeza, lettuce, lime, liquorice, litchi, loquat, lupine, macadamia nut, mace, in, mangel, mango, , melon, mint, mulberry, mustard, nectarine, niger seed, nutmeg, okra, olive, opium, orange, papaya, parsnip, pea, peach, peanut, pear, pecan nut, persimmon, pigeon pea, pistachio nut, plantain, plum, pomegranate, pomelo, poppy seed, potato, sweet potato, prune, pumpkin, quebracho, quince, trees of the genus Cinchona, quinoa, radish, ramie, rapeseed, raspberry, rhea, rhubarb, rose, rubber, rutabaga, safflower, sainfoin, salsify, sapodilla, a, scorzonera, sesame, shea tree, soybean, spinach, squash, erry, sugar beet, ane, sunflower, swede, sweet pepper, tangerine, tea, teff, tobacco, tomato, trefoil, tung tree, turnip, urena, vetch, walnut, watermelon, yerba mate, wintercress, shepherd’s purse, garden cress, peppercress, watercress, pennycress, star anise, laurel, bay laurel, cassia, jamun, dill, tamarind, peppermint, oregano, rosemary, sage, soursop, pennywort, calophyllum, balsam pear, kukui nut, Tahitian chestnut, basil, huckleberry, hibiscus, passionfruit, star apple, sassafras, cactus, St. John’s wort, loosestrife, hawthorn, cilantro, curry plant, kiwi, thyme, zucchini, ulluco, jicama, waterleaf, spiny monkey orange, yellow mombin, starfruit, amaranth, wasabi, Japanese pepper, yellow plum, mashua, e toon, New Zealand spinach, bower h, ugu, tansy, chickweed, jocote, Malay apple, paracress, sowthistle, Chinese potato, horse parsley, hedge mustard, campion, agate, cassod tree, thistle, bumet, star gooseberry, saltwort, glasswort, sorrel, silver lace fern, collard greens, primrose, cowslip, purslane, knotgrass, nth, tree lettuce, wild betel, West African pepper, yerba santa, on, parsley, chervil, land cress, bumet saxifrage, honeyherb, butterbur, shiso, water pepper, perilla, bitter bean, oca, kampong, Chinese celery, lemon basil, Thai basil, water mimosa, , cabbage-tree, moringa, mauka, ostrich fern, rice paddy herb, yellow sawah lettuce, lovage, pepper grass, maca, bottle gourd, hyacinth bean, water spinach, r, fishwort, Okinawan spinach, lotus sweetjuice, gallant soldier, culantro, arugula, cardoon, , mitsuba, chipilin, samphire, mampat, ebolo, ivy gourd, cabbage thistle, sea kale, chaya, huauzontle, Ethiopian mustard, magenta spreen, good king henry, epazole, lamb’s quarters, centella plumed cockscomb, caper, rapini, napa cabbage, mizuna, Chinese savoy, kai-lan, mustard , Malabar spinach, chard, marshmallow, climbing wattle, China jute, paprika, annatto seed, int, savory, marjoram, cumin, chamomile, lemon balm, allspice, bilberry, cherimoya, erry, , pitaya, durian, erry, feijoa, j ackfruit, j ambul, jujube, physalis, purple mangosteen, rambutan, redcurrant, blackcurrant, salal berry, satsuma, ugli fruit, azuki bean, black bean, black-eyed pea, borlotti bean, common bean, green bean, kidney bean, lima bean, mung bean, navy bean, pinto bean, runner bean, mangetout, snap pea, broccoflower, calabrese, nettle, bell pepper, raddichio, daikon, white radish, skirret, tat soi, broccolini, black radish, burdock root, fava bean, broccoli raab, lablab, lupin, lia, velvet beans, winged beans, yam beans, mulga, ironweed, umbrella bush, tjuntjula, wakalpulka, witchetty bush, wiry wattle, chia, beech nut, candlenut, colocynth, mamoncillo, Maya nut, mongongo, ogbono nut, paradise nut, and cempedak.

Where the plant is a monocotyledon, the tyledon can be selected from the group consisting of corn, wheat, oat, rice, barley, millet, banana, onion, garlic, gus, ryegrass, millet, fonio, raishan, nipa grass, turmeric, saffron, galangal, chive, cardamom, date palm, pineapple, t, leek, scallion, water chestnut, ramp, Job’s tears, , ragi, ss watermeal, arrowleaf elephant ear, Tahitian spinach, abaca, areca, bajra, betel nut, broom millet, broom sorghum, citronella, coconut, cocoyam, maize, dasheen, durra, durum wheat, edo, fique, formio, ginger, d grass, esparto grass, Sudan grass, guinea corn, Manila hemp, henequen, hybrid maize, jowar, lemon grass, maguey, bulrush millet, finger millet, foxtail millet, Japanese millet, proso millet, New Zealand flax, oats, oil palm, palm palmyra, sago palm, redtop, sisal, sorghum, spelt wheat, sweet corn, sweet sorghum, taro, teff, timothy grass, triticale, vanilla, wheat, and yam. {@9759} Where the plant is a gymnosperm, the gymnosperm can be from a family selected from the group consisting of Araucariaceae, Boweniaceae, Cephalotaxaceae, Cupressaceae, Cycadaceae, Ephedraceae, Ginkgoaceae, Gnetaceae, Pinaceae, rpaceae, Taxaceae, Taxodiaceae, Welwitschiaceae, and Zamiaceae. [llthilll The plants and plant seeds described herein may e transgenic plants or plant seeds, such as enic cereals {whean rice), maize, n, potato, cotton, tobacco, oilseed rape and fruit plants (fruit of apples, pears, citrus fruits and grapes. Preferred enic plants include corn, soybeans, potatoes, cotton, tobacco and oilseed rape, } Suitable transgenic plants and seeds can be characterized by the plant’s ion of toxins, especially from the Bacillus lizaringiensis genetic material (eg, by gene CrylA (a), CrylA (b), CiylA (e), CryllA, CrylllA, CrylllBZ or , Crygc, CiyZAb, Cry3Bb, CrylF a combination thereof), The formation of toxins in plants inc “eases the plant’s resistance to insects, arachnids, nematodes and slugs and snails (hereinafter referred to as ”Bt plants"). Bt plants, for example, are connnereially available under the tadenarne YIELD CARD (R9 (for example maize, cotton, soybeans), Knocktlut <R> (for e maize), StarLink ® (for example maize), Bollgard 63,) (cotton), Nucotn ® n) and Newlseaf® (potato) maize varieties, cotton varieties, soybean varieties and, potato varieties. Herbicide tolerance plants include plants under the trade names Roundup Ready ® (a glyphosate tolerance, such as corn, cotton, soybeans), Cleartield (R9 (for example maize), y Link ® (toleran ee with glutosinate, for example oilseed rape), B‘s/ll 69 (with olinone tolerance) and STS ® (tolerance to a sulfonylurea, such as maize). {@8762} Plant seeds as described herein can be genetically ed (eg, any seed, that results in a genetically modified plant or plant part that expresses herbicide tolerance, tolerance to environmental factors such as water stress, drought, Viruses, and en production, or resistance to bacterial, fungi or insect toxins). Suitable genetically modified seeds include those of cole crops, vegetables, fruits, trees, fiber crops, oil crops, tuber crops, coffee, flowers, legume, s, as well as other plants of the monocotyledonous and dicotyledonous s. Preferably, the genetically modified seeds include peanut, tobacco, grasses, wheat, barley, rye, m, rice, rapeseed, sugarbeet, sunflower, tomato, pepper, bean, lettuce, potato, and carrot. Most preferably, the cally ed seeds include cotton, soybean, and corn (sweet, field, seed, or popcorn).

Particularly useful transgenic plants which may be treated according to the invention are plants ning transformation events, or a combination of transformation events, that are listed for example in the databases from various national or regional tory agencies (see for e http://gmoinfo.jrc.it/gmp_browse.aspx and http://www.agbios.com/dbase.php).

XX. Methods for Delivering Beneficial Bacteria and Proteins or Peptides to Animals The present invention further relates to methods for delivering beneficial bacteria and/0r proteins or es to animals.

The administration of bacterial strains that are both tic and are also endophytic to a plant allows for entry of the bacteria into the plant where they divide and multiply. The endophytic and probiotic strains can be red to plants using various methods, e.g., the endophytic and probiotic strains can be delivered Via seed treatment, treatment of the plant growth medium (e.g., soil), irrigation, application to the plant itelf (e.g., foliar application to the aerial portions of a plant). Once inside the plant, the bacteria multiply and colonize the internal tissues of the plant. The plant can then be fed to an animal, which allows for ry of the probiotic bacteria to the . Costs are decreased as to traditional methods for delivering probiotic bacteria to animals, since the endophytic nature of the bacteria allows them to divide and multiply within the plant. By initially delivering a small amount of a probiotic and endophytic strain of baceria to a plant and allowing the bacteria to increase in number inside the plant, the dose increases. In addition, the probiotic and endophytic strain can spread across a target crop prior to harvest and digestion.

Bacterial strains that are capable of colonizing the phylloplane of a plant and are also probiotic can also be used for these purposes. Strains that are capable of colonizing the phylloplane of a plant can be initially red to plants in small doses, and will then divide and colonize the al surfaces of the plants.

Suitable ial strains that are both endophytic or phylloplane- colonizing and probiotic include those strains that can both replicate in the field in or on a plant and that provide benefits to animals upon ingestion. Benefits of probiotic bacteria in animals include but are not limited to tion of the microbiome of the digestive tract of the animal, secretion of enzymes that aid in digestion of plant al, and stimulation of the animals immune system. Examples of digestion-enhancing enzymes that would provide benefit e, but are not limited to cellulases, endoglucanases, exoglucanases, B-glucosidases, amylases, proteases, pectinases, xylanases, xylosidases, lipases, phospholipases, and lignases.

The Bacillus and Lysinibacillus genera are unique in that they n a large number of species that are both endophytic and thus colonize plants, but that can also act as probiotics in vertebrates. Thus, Bacillus and Lysinibacillus species are highly suitable for delivery of probiotics to animals h passaging and growth in plants. Common Bacillus species that can be both endophytic and probiotic include Bacillus subtilis, Bacillusfirmus, Bacillus amyloliquefaciens, Bacillus cereus, us t0y0cerin, Bacillus megaterium, Bacillus pumilus, and Bacillus licheniformis. Lysinibacillus species that are both ytic and probiotic can also be used.

A method for delivering ial bacteria to an animal is ed. The method comprises feeding to an animal a plant d to se a level of an endophytic and probiotic strain of bacteria that is greater than the level of the ytic and probiotic strain of bacteria in the same plant that has not been modified grown under the same conditions.

The plant fed to the animal can comprise a plant grown in a plant growth medium containing the endophytic and probiotic strain of bacteria or a ation sing the endophytic and probiotic strain of bacteria, a plant to which the endophytic and probiotic strain of bacteria was applied, a plant grown from a plant seed to which the endophytic and probiotic strain of bacteria was applied, a plant grown in an area to which the endophytic and probiotic strain of bacteria was applied, or a seed grown in the area to which the endophytic and probiotic strain of bacteria was applied.

The ytic and probiotic strain of bacteria can comprise a Bacillus or Lysinibacillus species. For example, the Bacillus species can comprise Bacillus subtilis, Bacillus firmus, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus t0y0cerin, Bacillus rium, Bacillus pumilus, Bacillus licheniformis, or a combination thereof.

The endophytic and probiotic strain of bacteria can comprise Bacillus cereus family member EE349, us cereus family member EE439, Bacillus thuringiensis EE4l7, Bacillus cereus EE444, us megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillusfusiformis EE442, Lysinibacillus sphericus EE443, Bacillus pumilus EE-B00143, or a combination thereof In addition, proteins or peptides (e.g., enzymes) can be delivered to animals by feeding recombinant Bacillus cereus family members sing a fusion protein containing the protein or peptide, exosporium fragments comprising such fusion ns, or recombinant spore-forming bacteria expressing such fusion ns to the animals. The recombinant Bacillus cereus family member or the recombinant spore-forming bacteria can be an ytic strain of bacteria or a strain of bacteria that is capable of colonizing the phylloplane of a plant, Which allows for delivery of the protein or peptide to the animal via ingestion of a plant that has been colonized by the bacteria. Probiotic recombinant Bacillus cereus family member strains or strains of recombinant spore-forming ia can also be used so that the animal that ingests the recombinant Bacillus cereus family member or the recombinant spore-forming bacteria obtains both the ts of the probiotic bacteria and the benefits of the protein or peptide. Recombinant Bacillus cereus family member strains and strains of recombinant spore-forming bacteria that are both ytic or phylloplane colonizing and probiotic can also be used to r proteins or peptides to s.

Accordingly, a method for delivering proteins or peptides to an animal is also provided. The method comprises feeding to an animal a recombinant Bacillus cereus family member expressing a fusion protein comprising a protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member. atively, the method comprises feeding to an animal exosporium fragments derived from a recombinant Bacillus cereus family member expressing a fusion protein comprising a protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that s the fusion protein to the exosporium of the recombinant Bacillus cereus family member.

The recombinant Bacillus cereus family member can comprise any of the recombinant us cereus family members bed herein that express a fusion protein.

The exosporium fragments can se exosporium nts derived from any of the Bacillus cereus family members described above in Section IV.

The recombinant Bacillus cereus family member can comprise an endophytic strain of bacteria. The endophytic strain of bacteria can comprise Bacillus cereus family member EE349, Bacillus cereus family member EE439, Bacillus thuringiensis EE4l7, Bacillus cereus EE444, us thuringiensis EE3 l9, Bacillus thuringiensis EE-BOOl84, Bacillus cereus family member EE-B00377, Bacillus pseudomycoides EE-B00366, or us mycoia’es 363. For example, the endophytic strain of ia comprises Bacillus cereus WO 44661 family member EE439, Bacillus thuringiensis EE4l7, Bacillus cereus EE444, Bacillus thuringiensis EE3 l9, us thuringiensis EE-BOOl84, Bacillus cereus family member EE- B00377, Bacillus pseudomycoia’es EE-B00366, or Bacillus ’es EE-B00363.

The recombinant Bacillus cereus family member can comprise a probiotic strain of bacteria. The tic strain of bacteria can comprise Bacillus cereus family member EE349 (NRRL No. B-50928), Bacillus cereus family member EE439 (NRRL B-50979), Bacillus thuringiensis EE4l7 (NRRL B-50979), Bacillus cereus EE444 (NRRL B-50977), us thuringiensis BTOl3A (NRRL No. 4), or a combination thereof.

The recombinant Bacillus cereus family member can be comprised within a plant that is fed to the animal.

] Alternatively, the recombinant Bacillus cereus family can comprise a strain of bacteria that is capable of colonizing the phylloplane of a plant. For example, the strain of bacteria that is capable of colonizing the phylloplane of a plant can comprise Bacillus mycoia’es BTlSS, Bacillus mycoia’es EEl l8, Bacillus mycoia’es EEl4l, Bacillus ’es BT46- 3, Bacillus cereus family member EE2l8, Bacillus thuringiensis BTOl3A, Bacillus cereus family member EE-B00377, Bacillus pseudomycoia’es EE-B00366, or Bacillus mycoia’es EE- B00363.

The recombinant Bacillus cereus family member can be present on the phylloplane of a plant that is fed to the animal.

] The targeting sequence, exosporium protein, or exosporium protein nt can comprise: (l) a targeting ce comprising an amino acid sequence haVing at least about 43% identity With amino acids 20—35 of SEQ ID NO: 1, wherein the identity With amino acids 25—35 is at least about 54%; (2) a targeting ce sing amino acids 1—35 of SEQ ID NO: 1; (3) a targeting sequence comprising amino acids 20—35 of SEQ ID NO: 1; (4) a targeting sequence comprising SEQ ID NO: 1 ; (5) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 2; (6) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 1 ; (7) a targeting ce comprising amino acids 5—35 of SEQ ID NO: 1; (8) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 1; (9) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 1; (10) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 1 ; (l l) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 3; (12) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 3; (13) a targeting sequence comprising SEQ ID NO: 3; (14) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 4; (15) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 3; (16) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 3; (17) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 3; (18) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 3; (19) a targeting sequence comprising amino acids 1—38 of SEQ ID NO: 5; (20) a ing sequence comprising amino acids 23—38 of SEQ ID NO: 5; (21) a targeting sequence comprising SEQ ID NO: 5; (22) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 6; (23) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 5; (24) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 5; (25) a ing sequence comprising amino acids 8—38 of SEQ ID NO: 5; (26) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 5; (27) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 5; (28) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 5; (29) a ing sequence comprising amino acids 1—28 of SEQ ID NO: 7; (30) a targeting sequence comprising amino acids 13—28 of SEQ ID NO: 7; (31) a targeting sequence comprising SEQ ID NO: 7; (32) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 8; (33) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 7; (34) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 7; (35) a targeting sequence comprising amino acids 8—28 of SEQ ID NO: 7; (36) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 7; (37) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 9; (38) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 9; (39) a targeting sequence comprising SEQ ID NO: 9; (40) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 10; (41) a targeting ce comprising amino acids 2—24 of SEQ ID NO: 9; (42) a ing sequence comprising amino acids 5—24 of SEQ ID NO: 9; (43) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 9; (44) a ing ce comprising amino acids 1—33 of SEQ ID NO:11; (45) a ing sequence sing amino acids 18—33 of SEQ ID NO: 11 ; (46) a targeting sequence comprising SEQ ID NO: 11 ; (47) an exosporium n comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 12; (48) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 11; (49) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 11; (50) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 11 ; (51) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 11 ; (52) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 11; (53) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 13; (54) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 13 ; (55) a ing sequence comprising SEQ ID NO:13; (56) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 14; (57) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 13; (58) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13 ; (59) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 13; (60) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 13; (61) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 13; (62) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 15; (63) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 15 ; (64) a targeting ce comprising SEQ ID NO:15; (65) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:16; (66) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 15; (67) a ing sequence comprising amino acids 5—43 of SEQ ID NO: 15; (68) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 15; (69) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 15 ; (70) a targeting ce comprising amino acids 15—43 of SEQ ID NO: 15; (71) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 15; (72) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 15; (73) a ing ce comprising amino acids 1—27 of SEQ ID NO: 17; (74) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 17; (75) a targeting sequence comprising SEQ ID NO:17; (76) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:18; (77) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 17; (78) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 17; (79) a targeting sequence sing amino acids 8—27 of SEQ ID NO: 17; (80) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 17; (81) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 19; (82) a ing sequence comprising amino acids 18—33 of SEQ ID NO: 19; (83) a targeting sequence comprising SEQ ID NO:19; (84) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:20; (85) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 19; (86) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 19; (87) a targeting ce comprising amino acids 8—33 of SEQ ID NO: 19; (88) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 19; (89) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 19; (90) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 21 ; (91) a targeting ce comprising amino acids 18—33 of SEQ ID NO: 21; (92) a ing sequence sing SEQ ID NO:21; (93) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:22; (94) a targeting sequence comprising amino acids 2—33 of SEQ ID WO 44661 NO: 21; (95) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 21; (96) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 21; (97) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 21; (98) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 21; (99) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 23; (100) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 23; (101) a targeting sequence comprising SEQ ID NO:23; (102) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:24; (103) a ing sequence comprising amino acids 2—24 of SEQ ID NO:23; (104) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 23; (105) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 23; (106) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 25; (107) a targeting sequence sing amino acids 9—24 of SEQ ID NO: 25 ; (108) a targeting sequence comprising SEQ ID NO:25; (109) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:26; (110) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 25; (111) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 25; (112) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 25; (113) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 27; (114) a ing ce comprising amino acids 15—30 of SEQ ID NO: 27; (115) a targeting sequence comprising SEQ ID NO:27; (116) an exosporium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO:28; (117) a ing ce comprising amino acids 2—30 of SEQ ID NO: 27; (118) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 27; (119) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 27; (120) a targeting sequence sing amino acids 10—30 of SEQ ID NO: 27; (121) a ing sequence comprising amino acids 1—33 of SEQ ID NO: 29; (122) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 29; (123) a targeting sequence comprising SEQ ID NO:29; (124) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO:30; (125) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 29; (126) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 29; (127) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 29; (128) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 29; (129) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 29; (130) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 31; (131) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 31; (132) a targeting sequence comprising SEQ ID NO:31; (133) an rium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO:32; (134) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 31; (135) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31; (136) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 31; (137) a ing sequence comprising amino acids 1—15 of SEQ ID NO: 33; (138) a targeting sequence sing SEQ ID NO:33; (139) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO:34; (140) a targeting sequence comprising amino acids 1—16 of SEQ ID NO: 35; (141) a ing sequence comprising SEQ ID NO:35; (142) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO:36; (143) a targeting ce comprising amino acids 1—29 of SEQ ID NO:43; (144) a targeting sequence comprising amino acids 14—29 of SEQ ID NO: 43; (145) a targeting sequence comprising SEQ ID NO: 43; (146) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 44; (147) a targeting sequence comprising amino acids 2—29 of SEQ ID NO: 43 ; (148) a targeting ce comprising amino acids 5—29 of SEQ ID NO: 43 ; (149) a targeting sequence sing amino acids 8—29 of SEQ ID NO: 43; (150) a targeting sequence comprising amino acids 10—29 of SEQ ID NO: 43; (151) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 45; (152) a targeting sequence comprising amino acids 20—35 of SEQ ID NO: 45 ; (153) a targeting sequence comprising SEQ ID NO: 45 ; (154) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 46; (155) a ing sequence comprising amino acids 2—35 of SEQ ID NO: 45; (156) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 45; (157) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 45; (158) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45; (159) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 45 ; (160) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 47; (161) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 47; (162) a targeting sequence comprising SEQ ID NO: 47; (163) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 48; (164) a targeting sequence sing amino acids 2—43 of SEQ ID NO: 47; (165) a ing sequence comprising amino acids 5—43 of SEQ ID NO: 47; (166) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 47; (167) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 47; (168) a targeting ce comprising amino acids 15—43 of SEQ ID NO: 47; (169) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (170) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 47; (171) a targeting sequence comprising amino acids 1—32 of SEQ ID NO: 49; (172) a targeting sequence comprising amino acids 17—32 of SEQ ID NO: 49; (173) a targeting sequence sing SEQ ID NO: 49; (174) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 50; (175) a targeting sequence sing amino acids 2—32 of SEQ ID NO: 49; (176) a targeting sequence comprising amino acids 5—32 of SEQ ID NO: 49; (177) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (178) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (179) a targeting sequence comprising amino acids 15—32 of SEQ ID NO: 49; (180) a ing sequence comprising amino acids 1—33 of SEQ ID NO: 51; (181) a targeting sequence sing amino acids 18—33 of SEQ ID NO: 51; (182) a targeting sequence comprising SEQ ID NO: 51; (183) an exosporium n comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 52; (184) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 51; (185) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 51; (186) a targeting sequence sing amino acids 8—33 of SEQ ID NO: 51; (187) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 51; (188) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 51; (189) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 53; (190) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 53; (191) a targeting sequence comprising SEQ ID NO: 53; (192) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 54; (193) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 53; (194) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53 ; (195) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53 ; (196) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 53 ; (197) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 53; (198) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 55; (199) a targeting sequence comprising amino acids 15—30 of SEQ ID NO: 55 ; (200) a targeting sequence comprising SEQ ID NO: 55 ; (201) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 56; (202) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 55; (203) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 55; (204) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 55; (205) a targeting sequence sing amino acids 10—30 of SEQ ID NO: 55; (206) a targeting sequence sing amino acids 1—130 of SEQ ID NO: 57; (207) a targeting sequence comprising amino acids 115—130 of SEQ ID NO: 57; (208) a targeting sequence comprising SEQ ID NO: 57; (209) an exosporium protein comprising an amino acid ce haVing at least 85% ty With SEQ ID NO: 58; (210) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 57; (211) a targeting sequence comprising amino acids 5—130 of SEQ ID NO: 57; (212) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (213) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 57; (214) a targeting sequence comprising amino acids 30— 130 of SEQ ID NO: 57; (215) a ing sequence comprising amino acids 40—130 of SEQ ID NO: 57; (216) a targeting sequence comprising amino acids 50—130 of SEQ ID NO: 57; (217) a targeting sequence comprising amino acids 60—130 of SEQ ID NO: 57; (218) a targeting sequence comprising amino acids 70—130 of SEQ ID NO: 57; (219) a targeting sequence comprising amino acids 80—130 of SEQ ID NO: 57; (220) a targeting ce comprising amino acids 90—130 of SEQ ID NO: 57; (221) a targeting sequence comprising amino acids 100—130 of SEQ ID NO: 57; (222) a targeting sequence comprising amino acids 110—130 of SEQ ID NO: 57; (223) an exosporium protein fragment comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 95; (224) a targeting sequence comprising SEQ ID NO: 96; (225) a targeting sequence comprising SEQ ID NO: 97; (226) a ing sequence comprising SEQ ID NO: 98; (227) a targeting sequence comprising SEQ ID NO: 99; (228) a targeting sequence comprising SEQ ID NO: 100; (229) a targeting sequence comprising SEQ ID NO: 101; (230) a targeting sequence sing SEQ ID NO: 102; (231) a targeting sequence comprising SEQ ID NO: 103; (232) a targeting sequence comprising SEQ ID NO: 104; (233) a targeting ce comprising SEQ ID NO: 105; (234) a targeting sequence comprising SEQ ID NO: 106; (235) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (236) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 109; (237) an exosporium n sing an amino acid sequence haVing at least 85% identity With SEQ ID NO: 110; (238) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 111; (239) an rium n comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 112; (240) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 113; (241) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 114; (242) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 115; (243) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 116; (244) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 117; (245) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 118; (246) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 119; (247) an exosporium n comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 120; (248) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 121; (249) a targeting sequence comprising amino acids 22—31 of SEQ ID NO: 1; (250) a targeting sequence comprising amino acids 22—33 of SEQ ID NO: 1; (251) a targeting sequence comprising amino acids 20—31 of SEQ ID NO: 1; (252) a targeting sequence comprising amino acids 14—23 of SEQ ID NO: 3; (253) a targeting sequence comprising amino acids 14—25 of SEQ ID NO: 3; (254) a ing sequence comprising amino acids 12—23 of SEQ ID NO: 3; (255) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 59; (256) a targeting sequence comprising SEQ ID NO: 59; (257) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 60; (258) a targeting ce comprising amino acids 2—30 of SEQ ID NO: 59; (259) a targeting sequence comprising amino acids 4—30 of SEQ ID NO: 59; (260) a targeting sequence comprising amino acids 6—30 of SEQ ID NO: 59; (261) a targeting sequence sing amino acids 1—33 of SEQ ID NO: 61; (262) a targeting sequence sing amino acids 18—33 of SEQ ID NO: 61; (263) a ing sequence comprising SEQ ID NO: 61 ; (264) an rium protein comprising an amino acid ce having at least 85% sequence identity With SEQ ID NO: 62; (265) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 61 ; (266) a targeting sequence sing amino acids 5—33 of SEQ ID NO: 61 ; (267) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 61; (268) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 61; (269) a ing sequence comprising amino acids 1—35 of SEQ ID NO: 63; (270) a targeting sequence comprising SEQ ID NO: 63; (271) an exosporium protein comprising an amino acid sequence having at least 85% ty With SEQ ID NO: 64; (272) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 63; (273) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 63; (274) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 63 ; (275) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 63 ; (276) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 63 ; (277) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 65; (278) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 65; (279) a targeting sequence comprising SEQ ID NO: 65; (280) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 66; (281) a targeting sequence comprising SEQ ID NO: 107; (282) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 65; (283) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 65; (284) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 67; (285) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 67; (286) a targeting sequence comprising SEQ ID NO: 67; (287) an rium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 68; (288) an targeting sequence comprising amino acids 2—27 of SEQ ID NO: 67; (289) a ing sequence comprising amino acids 5—27 of SEQ ID NO: 67; (290) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 67; (291) a targeting sequence sing amino acids 1—38 of SEQ ID NO: 69; (292) a targeting sequence comprising amino acids 23—38 of SEQ ID NO: 69; (293) a targeting sequence comprising SEQ ID NO: 69; (294) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 70; (295) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 69; (296) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 69; (297) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 69; (298) a ing sequence comprising amino acids 15—38 of SEQ ID NO: 69; (299) an rium protein sing SEQ ID NO: 72; (300) a targeting sequence sing SEQ ID NO: 73; (301) an exosporium protein comprising an amino acid ce having at least 95% identity With SEQ ID NO: 74; (302) a targeting sequence comprising amino acids 1—42 of SEQ ID NO: 75; (303) a targeting sequence comprising amino acids 27—42 of SEQ ID NO: 75; (304) a targeting sequence sing SEQ ID NO: 75; (305) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 76; (306) a targeting ce comprising amino acids 2—42 of SEQ ID NO: 75; (307) a targeting sequence comprising amino acids 5—42 of SEQ ID NO: 75; (308) a targeting sequence comprising amino acids 10—42 of SEQ ID NO: 75; (309) a targeting sequence comprising amino acids 15—42 of SEQ ID NO: 75; (310) a targeting sequence comprising amino acids 20—42 of SEQ ID NO: 75; (311) a targeting sequence comprising amino acids 25—42 of SEQ ID NO: 75; (312) a ing sequence comprising amino acids 1—24 of SEQ ID NO: 77; (313) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 77; (314) a ing sequence comprising SEQ ID NO: 77; (315) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 78; (316) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 77; (317) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 77; (318) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 80; (319) a targeting sequence comprising amino acids 1—38 of SEQ ID NO: 81; (320) a targeting sequence comprising amino acids 23—38 of SEQ ID NO: 81; (321) a targeting sequence comprising SEQ ID NO: 81; (322) an exosporium protein comprising an amino acid ce having at least 85% identity With SEQ ID NO: 82; (323) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 81; (324) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 81; (325) a targeting sequence sing amino acids 10—38 of SEQ ID NO: 81; (326) a targeting sequence sing amino acids 15—38 of SEQ ID NO: 81; (327) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 81; (328) a targeting sequence sing amino acids 1—34 of SEQ ID NO: 83; (329) a targeting sequence comprising SEQ ID NO: 83; (330) an exosporium protein comprising an amino acid ce having at least 85% identity With SEQ ID NO: 84; (331) an exosporium protein comprising an amino acid sequence having at least 85% ty with SEQ ID NO: 86; (332) a targeting ce comprising amino acids 1—28 of SEQ ID NO: 87; (333) a targeting sequence comprising amino acids 13—28 of SEQ ID NO: 87; (334) a targeting sequence comprising SEQ ID NO: 87; (335) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 88; (336) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 87; (337) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 87; (338) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 87; (339) a targeting sequence sing amino acids 1—28 of SEQ ID NO: 89; (340) a targeting sequence comprising SEQ ID NO: 89; (341) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 90; (342) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 89; (343) a ing sequence comprising amino acids 5—28 of SEQ ID NO: 89; (344) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 89; (345) a targeting sequence sing amino acids 1—93 of SEQ ID NO: 91 ; (346) a targeting sequence comprising SEQ ID NO: 91 ; (347) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 92; (348) a targeting sequence comprising amino acids 2—93 of SEQ ID NO: 91 ; (349) a targeting sequence comprising amino acids 10—93 of SEQ ID NO: 91 ; (350) a targeting sequence comprising amino acids 20—93 of SEQ ID NO: 91 ; (351) a targeting sequence comprising amino acids 30—93 of SEQ ID NO: 91 ; (352) a targeting sequence comprising amino acids 40—93 of SEQ ID NO: 91 ; (353) a targeting sequence comprising amino acids 50—93 of SEQ ID NO: 91 ; (354) a targeting sequence comprising amino acids 60—93 of SEQ ID NO: 91; (355) a targeting sequence comprising amino acids 1—130 of SEQ ID NO: 93; (356) a targeting sequence comprising SEQ ID NO: 93; (357) an exosporium n comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 94; (358) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 93; (359) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 93; (360) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 93; (361) a targeting sequence comprising amino acids 30—130 of SEQ ID NO: 93; (362) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 122; (363) a targeting sequence ting of amino acids 20—33 of SEQ ID NO: 1; (364) a ing ce consisting of amino acids 21—33 of SEQ ID NO: 1; (365) a targeting sequence consisting of amino acids 23— 31 of SEQ ID NO: 1; (366) a ing sequence consisting of amino acids 1—15 of SEQ ID NO: 96; (367) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 96; (368) a targeting sequence consisting of amino acids 12—25 of SEQ ID NO: 3; (369) a targeting sequence consisting of amino acids 13—25 of SEQ ID NO: 3; (370) a ing sequence consisting of amino acids 15—23 of SEQ ID NO: 3; (371) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 97; (372) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 98; (373) a targeting ce consisting of amino acids 23—36 of SEQ ID NO: ; (374) a targeting sequence consisting of amino acids 23—34 of SEQ ID NO: 5; (375) a targeting sequence consisting of amino acids 24—36 of SEQ ID NO: 5; (376) a targeting sequence consisting of amino acids 26—34 of SEQ ID NO: 5; (377) a targeting sequence consisting of amino acids 13—26 of SEQ ID NO: 7; (378) a targeting sequence consisting of amino acids 13—24 of SEQ ID NO: 7; (379) a targeting sequence consisting of amino acids 14— 26 of SEQ ID NO: 7; (380) a targeting sequence consisting of amino acids 16—24 of SEQ ID NO: 7; (381) a targeting sequence ting of amino acids 9—22 of SEQ ID NO: 9; (382) a targeting sequence consisting of amino acids 9—20 of SEQ ID NO: 9; (383) a ing sequence consisting of amino acids 10—22 of SEQ ID NO: 9; (384) a targeting sequence consisting of amino acids 12—20 of SEQ ID NO: 9; (385) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 105; (386) a ing sequence consisting of amino acids 1—13 of SEQ ID NO: 105; (387) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 11; (388) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 11; (389) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 11; (390) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 98 ; (391) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 98 ; (392) a targeting sequence consisting of amino acids 18— 31 of SEQ ID NO: 13; (393) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 13 ; (394) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 13 ; (395) a targeting sequence consisting of amino acids 21—29 of SEQ ID NO: 13; (396) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 99; (397) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 99; (398) a targeting ce consisting of amino acids 28—41 of SEQ ID NO: 15; (399) a targeting ce consisting of amino acids 28— WO 44661 39 of SEQ ID NO: 15; (400) a targeting ce consisting of amino acids 29—41 of SEQ ID NO: 15; (401) a targeting sequence consisting of amino acids 31—39 of SEQ ID NO: 15; (402) a targeting sequence consisting of amino acids 12—25 of SEQ ID NO: 17; (403) a targeting sequence consisting of amino acids 13—25 of SEQ ID NO: 17; (404) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 100; (405) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 19; (406) a targeting ce consisting of amino acids 18— 29 of SEQ ID NO: 19; (407) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 19; (408) a targeting sequence consisting of amino acids 21—29 of SEQ ID NO: 19; (409) a targeting ce consisting of amino acids 18—31 of SEQ ID NO: 21; (410) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 21; (411) a targeting sequence ting of amino acids 19—31 of SEQ ID NO: 21; (412) a targeting sequence consisting of amino acids 21—29 of SEQ ID NO: 21; (413) a targeting ce consisting of amino acids 1— of SEQ ID NO: 101; (414) a targeting sequence ting of amino acids 1—13 of SEQ ID NO: 101 ; (415) a targeting sequence consisting of amino acids 9—22 of SEQ ID NO: 23; (416) a ing sequence consisting of amino acids 9—20 of SEQ ID NO: 23; (417) a targeting sequence consisting of amino acids 10—22 of SEQ ID NO: 23; (418) a targeting sequence consisting of amino acids 12—20 of SEQ ID NO: 23; (419) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 102; (420) a targeting sequence consisting of amino acids 1— 13 of SEQ ID NO: 102; (421) a targeting sequence consisting of amino acids 9—22 of SEQ ID NO: 25; (422) a targeting sequence consisting of amino acids 9—20 of SEQ ID NO: 25 ; (423) a targeting sequence consisting of amino acids 10—22 of SEQ ID NO: 25; (424) a targeting ce consisting of amino acids 12—20 of SEQ ID NO: 25; (425) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 103; (426) a targeting sequence ting of amino acids 1—13 of SEQ ID NO: 103; (427) a targeting sequence consisting of amino acids 15— 28 of SEQ ID NO: 27; (428) a targeting sequence consisting of amino acids 15—26 of SEQ ID NO: 27; (429) a targeting sequence consisting of amino acids 16—28 of SEQ ID NO: 27; (430) a targeting sequence consisting of amino acids 18—26 of SEQ ID NO: 27; (431) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 104; (432) a targeting sequence ting of amino acids 1—13 of SEQ ID NO: 104; (433) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 33; (434) a targeting sequence consisting of amino acids 1—11 of SEQ ID NO: 33 ; (435) a targeting sequence consisting of amino acids 3—11 of SEQ ID NO: 33 ; (436) a targeting sequence consisting of amino acids 1—14 of SEQ ID NO: 35; (437) a targeting sequence consisting of amino acids 1—12 of SEQ ID NO: 35; (438) a targeting sequence consisting of amino acids 2—14 of SEQ ID NO: 35; (439) a targeting sequence consisting of amino acids 14—27 of SEQ ID NO: 43; (440) a targeting sequence consisting of amino acids 14—25 of SEQ ID NO: 43; (441) a targeting sequence consisting of amino acids 15— 27 of SEQ ID NO: 43; (442) a targeting sequence ting of amino acids 20—33 of SEQ ID NO: 45 ; (443) a targeting sequence consisting of amino acids 20—31 of SEQ ID NO: 45 ; (444) a targeting ce consisting of amino acids 21—33 of SEQ ID NO: 45; (445) a targeting ce consisting of amino acids 1—15 of SEQ ID NO: 106; (446) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 106; (447) a targeting sequence ting of amino acids 28—41 of SEQ ID NO: 47; (448) a targeting sequence consisting of amino acids 28— 39 of SEQ ID NO: 47; (449) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 53; (450) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 53 ; (451) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 53; (452) a targeting sequence comprising amino acids 18—31 of SEQ ID NO: 61; (453) a targeting sequence comprising amino acids 18—29 of SEQ ID NO: 61; (454) a targeting sequence comprising amino acids 19—31 of SEQ ID NO: 61; (455) a targeting sequence sing amino acids 9—22 of SEQ ID NO: 65; (456) a targeting sequence comprising amino acids 9—20 of SEQ ID NO: 65; (457) a targeting sequence comprising amino acids 10—22 of SEQ ID NO: 65; (458) a targeting sequence comprising amino acids 1—15 of SEQ ID NO: 107; (459) a targeting sequence comprising amino acids 1—13 of SEQ ID NO: 107; (460) a targeting sequence comprising amino acids 12—25 of SEQ ID NO: 67; (461) a targeting sequence sing amino acids 12—23 of SEQ ID NO: 67; (462) a targeting sequence comprising amino acids 13—25 of SEQ ID NO: 67; (463) a targeting sequence comprising amino acids 15—23 of SEQ ID NO: 67; (464) a ing sequence comprising amino acids 23—36 of SEQ ID NO: 69; (465) a targeting sequence comprising amino acids 23—34 of SEQ ID NO: 69; (466) a targeting sequence comprising amino acids 24—36 of SEQ ID NO: 69; (467) a targeting ce sing amino acids 26—34 of SEQ ID NO: 69; (468) a targeting sequence sing amino acids 27—40 of SEQ ID NO: 75; (469) a targeting sequence comprising amino acids 27—38 of SEQ ID NO: 75; (470) a targeting sequence comprising amino acids 9—22 of SEQ ID NO: 77; (471) a targeting sequence sing amino acids 9—20 of SEQ ID NO: 77; (472) a targeting sequence comprising amino acids 10—22 of SEQ ID NO: 77; (473) a targeting sequence comprising amino acids 12—20 of SEQ ID NO: 77; (474) a targeting sequence comprising amino acids 23—36 of SEQ ID NO: 81; (475) a targeting ce comprising amino acids 23—34 of SEQ ID NO: 81; (476) a targeting sequence comprising amino acids 24—36 of SEQ ID NO: 81; (477) a targeting sequence 2015/050807 comprising amino acids 26—34 of SEQ ID NO: 81 ; (478) a targeting sequence comprising amino acids 13—26 of SEQ ID NO: 87; (479) a targeting sequence comprising amino acids 13—24 of SEQ ID NO: 87; or (480) a targeting ce sing amino acids 14—26 of SEQ ID NO: For example, the targeting sequence can comprise or consist of an amino acid sequence haVing at least about 50% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 63%.

The targeting sequence can comprise or consist of an amino acid sequence haVing at least about 50% ty with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 72%.

The ing sequence can comprise or t of an amino acid sequence haVing at least about 56% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the ty with amino acids 25—35 is at least about 63%.

The targeting sequence can comprise or consist of an amino sequence haVing at least about 62% ty with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 72%.

The targeting sequence can comprise or consist of an amino acid sequence haVing at least about 68% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 81%.

The targeting sequence can comprise or consist of an amino sequence haVing at least about 75% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 72%.

The targeting sequence can comprise or consist of an amino sequence haVing at least about 75% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 81%.

The targeting sequence can comprise or consist of an amino acid sequence haVing at least about 81% identity with amino acids 20—35 of SEQ ID NO:l, wherein the identity with amino acids 25—35 is at least about 81%.

The targeting sequence can se or consist of an amino acid sequence haVing at least about 81% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 90%.

The targeting sequence can consist of: (a) an amino acid sequence consisting of 16 amino acids and haVing at least about 43% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the ty with amino acids 25—35 is at least about 54%; (b) amino acids 1—35 of SEQ ID NO: 1; (c) amino acids 20—35 of SEQ ID NO: 1; (d) SEQ ID NO: 1; (e) SEQ ID NO: 96; or (f) SEQ ID NO: 120.

The exosporium protein or exosporium protein fragment can comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, , 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, and 122.

The rium protein or exosporium protein fragment can comprise an amino acid sequence haVing at least 95% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, , 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 76, 78, 80, 82,84,86,88,90,92,94,95,108,109,110,111,112,113,114,115,116,117,118,119,120, 121, and 122.

] The exosporium protein or exosporium protein fragment can comprise an amino acid sequence haVing at least 98% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, , 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 76, 78, 80, 82,84,86,88,90,92,94,95,108,109,110,111,112,113,114,115,116,117,118,119,120, 121, and 122.

The exosporium protein or exosporium protein fragment can comprise an amino acid sequence haVing at least 99% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, , 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 76, 78, 80, 82,84,86,88,90,92,94,95,108,109,110,111,112,113,114,115,116,117,118,119,120, 121, and 122.

The exosporium protein or exosporium protein fragment can comprise an amino acid ce haVing at least 100% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 76, 78, 80,82,84,86,88,90,92,94,95,108,109,110,111,112,113,114,115,116,117,118,119, 120, 121, and 122.

The targeting sequence, exosporium protein, or exosporium protein nt can comprise the amino acid sequence GXT at its carboxy terminus, wherein X is any amino acid.

WO 44661 2015/050807 The targeting sequence, exosporium protein, or exosporium protein fragment can comprise an alanine residue at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1.

The targeting sequence, exosporium protein, or exosporium protein fragment can further comprise a methionine, serine, or threonine residue at the amino acid position immediately preceding the first amino acid of the targeting sequence, exosporium protein, or exosporium protein nt or at the position of the ing sequence that corresponds to amino acid 20 of SEQ ID NO: 1.

The fusion protein can further comprise an amino acid linker between the targeting ce, the exosporium protein, or the exosporium protein nt and the protein or peptide of interest. The linker can be any of the linkers described above in Section XI.

The fusion n can be expressed under the control of a sporulation promoter native to the targeting sequence, exosporium n, or exosporium protein fragment of the fusion protein or a n thereof and/or under the control of a high-expression sporulation er. The promoter can be any of the promoters described above in Section III.

A further method for delivering proteins or peptides to an animal is also provided. The method comprises g to an animal a recombinant spore-forming bacterium.

The recombinant spore-forming bacterium can be any of the recombinant spore-forming bacteria described above in Section IX.

The recombinant spore-forming bacterium can be comprised Within a plant that is fed to the animal.

The recombinant spore-forming bacterium can comprise an ytic and probiotic strain of bacteria. For example, the endophytic and probiotic strain of bacteria can comprise Bacillus rium EE385 (NRRL B-50980), Bacillus sp. EE387 (NRRL B-5098l), Bacillus circulans EE388 (NRRL B-50982), Bacillus subtilis EE405 (NRRL 8), bacillusfusiformis EE442 (NRRL B-50975), or Lysinibacillus sphaericus EE443 (NRRL B-50976), us pumilus EE-BOOl43 (NRRL B-67123), or a combination thereof.

In any of the above s, the plant can be processed prior to feeding to the animal.

In any of the above methods involving feeding a plant to an animal, the method can further comprise introducing the endophytic strain of bacteria or a formulation comprising the endophytic strain of bacteria into a plant growth medium. Alternatively, the method can comprise applying the endophytic strain of bacteria or a formulation comprising the 21 l endophytic strain of bacteria to a plant, a plant seed, or an area surrounding a plant or a plant seed. The plant fed to the animal comprises a plant grown in a plant growth medium containing the endophytic and tic strain of bacteria or a formulation comprising the endophytic and probiotic strain of bacteria, a plant to which the endophytic and probiotic strain of bacteria was applied, a plant grown from a plant seed to which the endophytic and probiotic strain of bacteria was applied, a plant grown in an area to which the endophytic and probiotic strain of bacteria was d, or a seed grown in the area to which the endophytic and probiotic strain of bacteria was applied.

In any of the above methods for delivering proteins or peptides to an animal, the protein or peptide of interest comprises an enzyme. For example, the enzyme can comprise a xylanase, a xylosidase, a phytase, a phosphatase, a protease, a cellulase, an endoglucanase, an exogluconase, a glucanase, an amylase (e. g., u-amylase or a B-amylase), a lipase, a phospholipase, a ylase, a galactanase, an actosidase, a B-glucosidase, an amylase, a pectinase, a biotinase, a polygalacturonase, a ligninase, or a combination f. The lipase can comprise a phospholipase A1, a phospholipase A2, a phospholipase C, a phospholipase D, a lysophospholipase, or a ation thereof. The enzyme preferably comprises a xylanase or a phytase.

] In any of the methods comprising feeding a plant to an animal, the plant can be processed prior to feeding to the animal.

In any of the above methods comprising delivery of bacteria, ns, or peptides to an animal, the animal can be a mammal (e. g., a sheep, goat, cow, pig, deer, alpaca, bison, camel, donkey, horse, mule, llama, rabbit, dog, or cat), a bird (e. g., a chicken, turkey, duck, goose, quail, or pheasant), a fish (e.g., salmon, trout, tilapia, tuna, catfish, or a carp), or a crustacean (e.g., a , prawn, lobster, crab, or crayfish).

XXI. Methods for Delivering Beneficial c Acids to Animals, Insects, Worms, Fungi, and Protozoans The ion further relates to methods for delivering a nucleic acid molecule to an , insect, worm, fungus, or protozoan.

The method can comprise feeding to an animal, an insect, or worm a plant modified to se a level of the nucleic acid molecule that is greater than the level of the nucleic acid molecule in the same plant that has not been d, grown under the same conditions.

A further method for delivering a nucleic acid molecule to an animal, insect, or worm is provided. The method can comprise feeding to an animal, insect, or worm a recombinant Bacillus cereus family member expressing a fusion protein comprising a n or e of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion n to the exosporium of the recombinant Bacillus cereus family member. atively, the method can comprise feeding to an animal, insect, or worm a recombinant spore-forming bacterium that expresses a fusion n comprising at least one protein or peptide of interest and a spore coat protein that targets the fusion protein to the surface of a spore of the bacterium. The protein or peptide of interest comprises a nucleic acid binding n or peptide and the nucleic acid molecule is bound to the DNA or RNA binding protein or peptide. The nucleic acid binding protein or peptide can be physically attached to the exosporium of the recombinant Bacillus cereus family member or to the spore coat of the inant spore-forming bacterium. r method for ring a nucleic acid molecule to an , insect, or worm is provided. The method comprises feeding to an animal, insect, or worm exosporium fragments derived from a recombinant Bacillus cereus family member. The exosporium fragments are derived from spores of a inant Bacillus cereus family member described in Section IV hereinabove and comprise the fusion protein. The fusion protein comprises a nucleic acid binding protein or peptide, and wherein the nucleic acid binding protein or peptide is bound to a nucleic acid molecule.

The worm is preferably a nematode.

A method for delivering a nucleic acid molecule to a fungus or a protozoan is provided. The method comprises ting a fungus or a protozoan with a recombinant Bacillus cereus family member expressing a fusion protein comprising a protein or peptide of interest and a ing sequence, exosporium n, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant us cereus family member.

Alternatively, the method comprises contacting a fungus or a protozoan With a recombinant spore-forming bacterium that expresses a fusion protein comprising at least one protein or peptide of interest and a spore coat protein that targets the fusion protein to the surface of a spore of the bacterium. The protein or peptide of interest comprises a nucleic acid binding protein or e and the nucleic acid molecule is bound to the nucleic acid binding n or peptide. 2015/050807 A further method for delivering a nucleic acid molecule to a fungus or a oan is provided. The method comprises contacting a fungus or a protozoan with exosporium fragments. The exosporium fragments are derived from spores of a recombinant Bacillus cereus family member described in Section IV hereinabove and comprise the fusion protein. The fusion n comprises a nucleic acid binding protein or peptide, and wherein the nucleic acid binding protein or peptide is bound to a nucleic acid molecule.

The nucleic acid molecule can comprise a modulating RNA molecule; an RNAi molecule; a NA; an aptamer; or a DNA molecule that s a modulating RNA molecule, an RNAi molecule, a NA, or an aptamer.

The recombinant Bacillus cereus family member can comprise any of the inant Bacillus cereus family members that express a fusion protein.

The fusion protein can comprise any of the fusion ns described herein that include a nucleic acid binding protein.

The spore coat protein comprises a CotB protein, a CotC protein, a CgeA protein, a CotB/H protein, a CotG protein, a spore coat protein X n, or a CotY protein.

The spore coat protein can se an amino acid sequence having at least 85% identity With any of SEQ ID NOs: 252—259.

The spore coat protein can comprise an amino acid sequence having at least 90% identity With any of SEQ ID NOs: 252—259.

The spore coat protein can comprise an amino acid sequence having at least 95% identity With any of SEQ ID NOs: 252—259.

The spore coat protein can comprise an amino acid sequence having at least 98% identity With any of SEQ ID NOs: 252—259.

The spore coat n can comprise an amino acid sequence having at least 99% identity With any of SEQ ID NOs: 252—259.

The spore coat protein can se an amino acid sequence having 100% identity With any of SEQ ID NOs: 252—259.

The above-described methods can be used for numerous purposes. For example, these methods can be used to r RNA or DNA to s for the purpose of decreasing susceptibility of the animal to a disease or treating a disease in the animal (e.g., organic disease such as stroke, diabetes, heart disease, and degenerative diseases). RNAs and DNAs have also been demonstrated to be effective for eliminating or treating disease caused by animal pathogens, such as bacteria, viruses, worms (e.g., nematodes), and fungi. The RNAs and DNAs can act directly on the pathogen, or can work with the animal’s immune system to activate or se the immune response.

] In on, the above methods can be used for eliminating pests, including insects, worms (e.g., nematodes), fungi, and protozoans. Delivery of specific RNAs or DNAs to the pest can lead to decreased y to of the pest to infect a host (e.g., a plant host), decreased feeding on target hosts or plants, direct g through blocking of key genes, or various other effects.

XXII. Vaccines and a method of producing an immunogenic response A vaccine is provided which comprises a pharmaceutically acceptable carrier and recombinant Bacillus cereus family member spores that express a fusion protein as described above in paragraphs [00171], and [00173]—[00176] of Section I wherein the protein or peptide of interest is an antigen or an immunogen.

A further vaccine is provided which comprises a ceutically acceptable carrier and exosporium fragments. The exosporium fragments are derived from spores of a recombinant Bacillus cereus family member described in Section IV hereinabove and se the fusion protein. The fusion protein comprises an antigen.

Yet r vaccine is provided which ses a pharmaceutically acceptable carrier and a recombinant Bacillus cereus family member. The recombinant Bacillus cereus family member is a inant Bacillus cereus family member as described above in Section II.

In the vaccines that comprise exosporium fragments or a recombinant Bacillus cereus family member as described above in Section II, the targeting sequence, rium protein, or exosporium protein fragment can comprise any of the targeting sequences, exosporium proteins, or exosporium n fragments described herein. In particular, the targeting sequence, exosporium protein, or exosporium protein nt can se any of the targeting sequences, exosporium protein, or exosporium protein fragments described above in aphs [00782]—[00801].

The fusion protein can be expressed under the control of a sporulation promoter native to the targeting sequence, exosporium protein, or exosporium protein fragment of the fusion protein or a portion thereof and/or under the control of a high-expression sporulation er. The promoter can be any of the promoters described above in Section III.

When the protein or peptide of st is an antigen, display of the antigen on the outside of the spore or on an exosporium fragment provides an immune system response to achieve vaccination against various pathogens or diseases. Suitable antigens or small molecules are those that are known or expected to illicit a desired immune response that is sufficient to yield a therapeutic or protective effect When expressed on the exterior of a Bacillus spore or displayed on an exosporium fragment. Suitability in large part Will be ined by the folding in the dimensional ure once the recombinant antigen is incorporated into the exosporium, i.e. the nic portion(s) of the recombinant molecule must be available for detection by the immune system.

The pathogens or diseases from Which the antigen can be derived include, but are not limited to, Acintobacter infections, caused by Acinetobacter baumannii; Actinomycosis, caused by Actinomyces israelii, Actinomyces gerencseriae, and Propionibacterium propionicus; African ng sickness, caused by Trypanosoma brucei; Acquired immune deficiency syndrome (AIDS), caused by Human immunodeficiency virus; Amebiasis, caused by Entamoeba histolytica; Anaplasmosis, caused by Anaplasma genus, Anthrax, caused by Bacillus anthracis; Arcanobacterium haemolyticum infection, caused by Arcanobacterium yticum; Argentine hemorrhagic fever, caused by Junin virus; Ascariasis, caused by Ascaris coides, Astrovirus infection, caused by Astroviradae family; Babesiosis, Babesia genus; Bacillus cereus ion, caused by Bacillus cereus; Bacterial pneumonia; ial vaginosis; Bacteroides infection, caused by Bacteriodes genus; Balantidiasis, caused by Balantidium coli; Baylisascaris infection, caused by Baylisascaris genus; BK virus infection, caused by BK virus; Black piedra, caused by Piedraia ; Blastocystis hominis infection, caused by Blastocystis s; Blastomycosis, caused by Blastomyces dermatitidis; Bolivian hemorrhagic fever, caused by Machupo virus; Borrelia infection, caused by Borrelia genus; Botulism (and Infant botulism), caused by the intake of Clostridium botulinum toxin; Brazilian hemorrhagic fever, caused by Sabia; Brucellosis, caused by Brucella genus; Burkliolderia ion, caused by usually Burkholderia cepacia and other Burkliolderia species; Buruli ulcer, caused by Mycobacterium ulcerans; Calicivirus infection (Norovirus and Sapovirus), caused by Caliciviridae family; Campylobacteriosis, caused by Campylobacter genus; iasis iasis; Thrush) usually caused by a albicans and other a species; Cat- scratch disease, caused by ella henselae; Cellulitis, caused by usually Group A Streptococcus and Staphylococcus; Chagas Disease (American trypanosomiasis), caused by Trypanosoma cruzi; Chancroid, caused by Haemophilus ducreyi; Chickenpox, caused by WO 44661 Varicella zoster virus (VZV); Chlamydia, caused by Chlamydia trachomatis; Chlamydophila pneumoniae infection, caused by Chlamydophz’la pneumoniae; Cholera, caused by Vibrz’o ae; Chromoblastomycosis, caused by usually Fonsecaea pedrosoz'; Clonorchiasis, caused by Clonorchz's sinensis; Clostrl'dl'um dzfiicz'le infection, caused by Clostrl'dl'um dl'fiicz'le; ioidomycosis, caused by Coccz'dz'oz'des immitz’s and Coccz'dz'oz'des posadasz'z'; Colorado tick fever (CTF), caused by Colorado tick fever virus ; Common cold (Acute viral rhinopharyngitis; Acute coryza), caused by usually rhinoviruses and coronaviruses; feldt— Jakob disease (CJD), caused by CJD prion; Crimean-Congo hemorrhagic fever , caused by Crimean-Congo hemorrhagic fever virus; Cryptococcosis, caused by Cryptococcus neoformans; Cryptosporidiosis, caused by Cryptosporidium genus; Cutaneous larva migrans (CLM), caused by usually Ancylostoma brazilz'ense and multiple other parasites; Cyclosporiasis, caused by Cyclospora cayetanensz's; Cysticercosis, caused by a solium; Cytomegalovirus infection, caused by Cytomegalovirus; Dengue fever, caused by Dengue viruses (DEN-l, DEN- 2, DEN-3 and DEN-4)-Flaviviruses; Dientamoebiasis, caused by Dientamoebafragz’lis; Diphtheria, caused by Corynebacterium diphtheriae; lobothriasis, caused by Dz'phyllobothrz’um; Dracunculiasis, caused by Dracunculus medinensz's; Ebola hemorrhagic fever, caused by Ebolavirus (EBOV); Echinococcosis, caused by Echinococcus genus; Ehrlichiosis, caused by Ehrlichia genus; Enterobiasis (Pinworm infection), caused by Enterobius vermicularz's; Enterococcus infection, caused by Enterococcus genus; Enterovirus infection, caused by Enterovirus genus; Epidemic typhus, caused by Rickettsz'a prowazekz'z'; Erythema infectiosum (Fifth disease), caused by Parvovirus B19; Exanthem subitum, caused by Human herpesvirus 6 (HHV-6) and Human virus 7 (HHV-7); Fasciolopsiasis, caused by Fascz’olopsz's buskz'; Fasciolosis, caused by Fasciola hepatica and Fascz‘ola gigantica; Fatal familial insomnia (FFI), caused by FFI prion; Filariasis, caused by Filarz'oz'dea superfamily; Food poisoning caused by z'dz'um perfrz’ngens; Free-living amebic infection; Fusobacterz’um infection, caused by Fusobacterz’um genus; Gas gangrene (Clostridial myonecrosz's), caused by usually Clostrz'dz'um perfrz’ngens or other z'dz'um species; Geotrichosis, caused by Geotrichum candidum; Gerstmann-Straussler-Scheinker syndrome (GSS), caused by GSS prion; Giardiasis, caused by Giardia intestinalz’s; Glanders, caused by Burkholderz’a mallet; Gnathostomiasis, caused by Gnathostoma spim'gerum and Gnathostoma hispidum; Gonorrhea, caused by Neisseria gonorrhoeae; Granuloma inguinale (Donovanosis), caused by Klebsz'ella omatz's; Group A ococcal ion, caused by ococcus pyogenes; Group B streptococcal infection, caused by Streptococcus agalactiae; Haemophilus influenzae infection, caused by Haemophilus influenzae; Hand, foot and mouth disease (HFMD), caused by Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV7l); Hantavirus Pulmonary Syndrome (HPS), caused by Sin Nombre virus; Helicobacter pylori infection, caused by Helicobacter pylori; tic-uremic syndrome (HUS), caused by Escherichia coli OlS7:H7; Hemorrhagic fever With renal me (HFRS), caused by Bunyaviridae family; Hepatitis A, caused by Hepatitis A Virus; Hepatitis B, caused by Hepatitis B Virus; Hepatitis C, caused by Hepatitis C Virus; Hepatitis D caused by Hepatitis D Virus; Hepatitis E, caused by Hepatitis E Virus; Herpes x, caused by Herpes simplex virus 1 and 2 (HSV-l and ; Histoplasmosis, caused by lasma capsulatum; Hookworm infection, caused by Ancylostoma duodenale and Necator americanus; Human bocavirus infection, caused by Human bocavirus (HBoV); Human eWingii ehrlichiosis, caused by Ehrlichia i; Human granulocytic anaplasmosis (HGA), caused by Anaplasma phagocytophilum; Human eumovirus infection, caused by Human metapneumovirus (hMPV); Human tic hiosis, caused by Ehrlichia chafleensis; Human papillomavirus (HPV) infection, caused by Human papillomavirus (HPV); Human fluenza virus infection, caused by Human parainfluenza viruses (HPIV); lepiasis, caused by Hymenolepis mum and Hymenolepis diminuta; Epstein-Barr Virus ious Mononucleosis (Mono), caused by Epstein-Ban Virus (EBV); Influenza (flu), caused by Orthomyxoviridae family; Isosporiasis, caused by Isospora Belli; Kawasaki disease (cause unknown but evidence supports that it is infectious); Keratitis; Kingella kingae infection, caused by Kingella kingae; Kuru, caused by Kuru prion; Lassa fever, caused by Lassa virus; Legionellosis (Legionnaires' e), caused by Legionella pneumophila; Legionellosis (Pontiac fever), caused by Legionella pneumophila; Leishmaniasis, caused by Leishmania genus; Leprosy, caused by Mycobacterium leprae and Mycobacterium atosis; Leptospirosis, caused by pira genus; Listeriosis, caused by Listeria monocytogenes; Lyme disease (Lyme borreliosis), caused by usually Borrelia burgdorferi and other Borrelia species; Lymphatic filariasis (Elephantiasis), caused by Wuchereria bancrofti and Brugia malayi; Lymphocytic choriomeningitis, caused by Lymphocytic choriomeningitis virus (LCMV); Malaria, caused by Plasmodium genus; Marburg hemorrhagic fever (MHF), caused by Marburg virus; Measles, caused by Measles virus; Melioidosis (Whitmore's disease), caused by Burkholderia pseudomallei; Meningitis; Meningococcal disease, caused by Neisseria itidis; Metagonimiasis, caused by usually Metagonimus yokagawai; Microsporidiosis, caused by Microsporidia ; Molluscum contagiosum (MC), caused by Molluscum contagiosum virus (MCV); Mumps, caused by 2015/050807 Mumps Virus; Murine typhus (Endemic typhus), caused by Rickettsia typhi; Mycoplasma pneumonia, caused by Mycoplasma pneumoniae; Mycetoma, caused by numerous species of bacteria omycetoma) and fungi (Eumycetoma); Myiasis, caused by parasitic dipterous fly larvae; Neonatal conjunctivitis (Ophthalmia neonatorum), caused by most commonly Chlamydia matis and Neisseria gonorrhoeae; (New) Variant Creutzfeldt-Jakob disease (VCJD, nVCJD), caused by VCJD prion; Nocardiosis, caused by usually Nocardia asteroia’es and other Nocardia species; Onchocerciasis (River blindness), caused by Onchocerca volvulus; Paracoccidioidomycosis (South American blastomycosis), caused by Paracoccidioides brasiliensis; Paragonimiasis, caused by usually Paragonimus westermani and other nimus species; Pasteurellosis, caused by Pasteurella genus; losis capitis (Head lice), caused by lus s capitis; Pediculosis corporis (Body lice), caused by Pea’iculus humanus corporis; Pediculosis pubis (Pubic lice, Crab lice), caused by Phthirus pubis; Pelvic inflammatory disease (PID); Pertussis (Whooping cough), caused by Bordetella pertussis; Plague, caused by Yersinia ; Pneumococcal infection, caused by Streptococcus pneumoniae; Pneumocystis pneumonia (PCP), caused by Pneumocystisjirovecii; Pneumonia; Poliomyelitis, caused by Poliovirus; Prevotella infection, caused by ella genus; Primary amoebic meningoencephalitis (PAM), caused by usually Naegleriafowleri; Progressive multifocal leukoencephalopathy, caused by JC virus; Psittacosis, caused by Chlamydophila psittaci; Q fever, caused by Coxiella burnetii; Rabies, caused by Rabies virus; te fever, caused by Streptobacillus moniliformis and Spirillum minus; Respiratory syncytial virus infection, caused by Respiratory syncytial virus (RSV); Rhinosporidiosis, caused by Rhinosporidium seeberi; Rhinovirus infection, caused by Rhinovirus; Rickettsial infection, caused by Rickettsia genus; tsialpox, caused by Rickettsia akari; Rift Valley fever (RVF), caused by Rift Valley fever virus; Rocky mountain d fever (RMSF), caused by Rickettsia rickettsii; Rotavirus infection, caused by Rotavirus; Rubella, caused by Rubella virus; Salmonellosis, caused by Salmonella genus; SARS (Severe Acute Respiratory Syndrome), caused by SARS coronavirus; Scabies, caused by Sarcoptes i; Schistosomiasis, caused by Schistosoma genus; Sepsis; losis (Bacillary dysentery), caused by Shigella genus; Shingles (Herpes zoster), caused by Varicella zoster virus (VZV); Smallpox (Variola), caused by a major or Variola minor; Sporotrichosis, caused by hrix schenckii; Staphylococcal food poisoning, caused by Staphylococcus genus; Staphylococcal infection, caused by Staphylococcus genus; Strongyloidiasis, caused by Strongyloides stercoralis; Syphilis, caused by Treponema pallia’um; Taeniasis, caused by Taenia genus; Tetanus (Lockjaw), caused by Clostria’ium tetani; Tinea barbae (Barber's itch), caused by usually Trichophyton genus; Tinea capitis (Ringworm of the Scalp), caused by usually Trichophyton tonsurans; Tinea corporis orm of the Body), caused by usually Trichophyton genus; Tinea cruris (Jock itch), caused by y Epidermophytonfloccosum, Trichophyton rubrum, and Trichophyton mentagrophytes; Tinea manuum (Ringworm of the Hand), caused by Trichophyton rubrum; Tinea nigra, caused by usually Hortaea werneckii; Tinea pedis (Athlete's foot), caused by usually Trichophyton genus; Tinea unguium (Onychomycosis), caused by usually Trichophyton genus; Tinea versicolor (Pityriasis versicolor), caused by Malassezia genus; Toxocariasis r Larva Migrans (OLM)), caused by T0x0cara canis or T0x0cara cati; Toxocariasis (Visceral Larva Migrans (VLM)), caused by T0x0cara canis or T0x0cara cati; Toxoplasmosis, caused by T0x0plasma gondii; Trichinellosis, caused by Trichinella spiralis; Trichomoniasis, caused by Trichomonas vaginalis; Trichuriasis (Whipworm infection), caused by Trichuris ura; Tuberculosis, caused by usually Mycobacterium tuberculosis; mia, caused by Francisella tularensis; Ureaplasma urealyticum infection, caused by Ureaplasma urealyticum; Venezuelan equine encephalitis, caused by Venezuelan equine encephalitis virus; Venezuelan hemorrhagic fever, caused by Guanarito virus; Viral pneumonia; West Nile Fever, caused by West Nile virus; White piedra (Tinea ), caused by Trichosporon beigelii; Yersinia pseudotuberculosis ion, caused by Yersinia pseudotuberculosis; Yersiniosis, caused by Yersinia enterocolitica; Yellow fever, caused by Yellow fever virus; Zygomycosis, caused by Mucorales order (Mucormycosis) and Entomophthorales order (Entomophthoramycosis).

When the n or peptide of interest is an antigen, any us cereus family member can be used to express the fusion n. Bacillus thuringiensis or Bacillus mycoides are preferred.

To prepare a vaccine, the antigen of interest is incorporated into the fusion protein by known methods such as PCR splicing by pping extension, ction endonuclease digestion and on, or de novo gene synthesis. The fusion protein gene is then uced into a recombinant Bacillus cereus family member by transfection, transformation, conjugation, electroporation or other known methods. The recombinant Bacillus cereus family member is then grown in culture media (e.g., minimal liquid media) and allowed to sporulate.

Preferably, sporulation continues to completion before the spores are collected and stored.

Spores can be collected by either centrifugation or ng of spores off of growth plates and introduction into liquid media (e.g., PBS or water) followed by centrifugation and washing of the resulting spore pellet in liquid media. Prior to use, the spore pellet can be resuspended in liquid media to a desired tration for use or injection. Where the vaccine is to comprise exosporium nts, the rium fragments can be prepared using any of the methods described in section XIX.H above.

The desired concentration of recombinant Bacillus cereus family member spores or exosporium nts in a vaccine is based on the size of the subject, the amount of active antigen on the surface of the spores, and the presence and concentration of adjuvants in the vaccine formulation. A vaccine of the invention can contain tional adjuvants including pharmaceutically acceptable carriers.

A method of producing an immunogenic response in a subject is provided.

The method comprises administering a vaccine containing recombinant Bacillus cereus family member spores expressing fusion proteins or exosporium fragments comprising fusion proteins as described herein to the subject.

The vaccine as described herein is suitable for intravenous, intrarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, topical, oral, intranasal, intradermal, transepithelial administration or by inhalation.

The vaccine can be administered to a subject which is human, murine, avian, porcine, bovine, ovine, feline, canine, equine, caprine, reptilian or a man primate. The subject is preferably mammalian and most preferably human.

XXIII. Remediation ] When the n or peptide of interest is a ation protein or e, a toxic substance is catalytically converted by the remediation protein or peptide to a non-toxic or less toxic substance.

When the remediation protein or e comprises an enzyme, the enzyme is displayed and stabilized on the e of the spore and can be delivered into contaminated soil or contaminated water in a form Which is active t a target pollutant or target chemical.

Suitable enzymes depend upon the pollutant or al being targeted for remediation.

To prepare a remediation composition, the enzyme of interest is incorporated into the fusion protein by known methods such as PCR splicing by overlapping extension, restriction endonuclease digestion and ligation, or de novo gene synthesis. The fusion protein gene is then introduced into a recombinant Bacillus cereus family member by transfection, transformation, conjugation, electroporation or other known s. The recombinant Bacillus cereus family member is then grown in culture media (e.g., l liquid media) and d to sporulate. Preferably, sporulation continues to completion before the spores are ted and stored. Spores can be collected by either centrifugation or swabbing of spores off of growth plates and introduction into liquid media (e.g., PBS or water) followed by centrifugation and washing of the resulting spore pellet in liquid media. Prior to use, the spore pellet can be resuspended in liquid media to a desired concentration for use. Alternatively, the spore pellet can be formulated into granules at a desired concentration for use and application to the contaminated environment. Where exosporium fragments are to be used for remediation, the rium nts can be prepared using any of the methods described in section XIX.H above.

A method of reducing contaminants in an nment is provided. The method ses exposing a contaminated environment to a recombinant Bacillus cereus family member spore that express the fusion protein as described above in paragraphs [00171], —[00175] and [00177] of Section I wherein the protein or peptide of interest comprises a remediation enzyme.

A further method for reducing contaminants in an environment is provided.

The method comprises ng a contaminated environment to exosporium fragments. The exosporium fragments are derived from a recombinant Bacillus cereus family member described in Section IV hereinabove and comprise the fusion protein. The fusion protein comprises a remediation enzyme.

Yet r method for ng contaminants in an environment is provided.

The method comprises exposing a contaminated environment to spores of a recombinant Bacillus cereus family member. The recombinant Bacillus cereus family member is a recombinant Bacillus cereus family member as desecribed above in Section II.

In the methods for reducing contaminants that comprise exposing a contaminated nment to exosporium fragments or to a recombinant Bacillus cereus family member as described above in Section II, the targeting sequence, exosporium protein, or exosporium protein fragment can be any of the targeting sequences, exosporium proteins, or exosporium protein nts described herein. In particular, the targeting sequence, exosporium protein, or exosporium protein fragment can comprise any of the targeting sequences, rium protein, or exosporium protein fragments described above in aphs —[00801].

The fusion protein can be expressed under the control of a sporulation promoter native to the targeting sequence, exosporium protein, or exosporium protein fragment of the fusion protein or a portion thereof and/or under the control of a high-expression sporulation promoter. The promoter can be any of the promoters described above in Section 111.

When the protein or peptide of interest is a remediation enzyme, any Bacillus cereus family member can be used to express the fusion n. Bacillus thuringiensis, Bacillus cereus, or Bacillus mycoia’es are preferred.

The recombinant Bacillus cereus family member spores can comprise an ytic strain of bacteria for phytoremediation, such as Bacillus cereus family member EE349, Bacillus cereus family member EE439, us thuringiensis EE4l7, Bacillus cereus EE444, or Bacillus thuringiensis EE3 19.

The contaminated environment to be treated can be gas, liquid, semi-liquid, gel, film, semi-solid, or solid. The solid environment can be soil such as surface soil and subsurface soil, compost, crop residue, , mulch, cut trees, a biofilm, a slime layer, mold, sludge, sand, slag, sediment, sewage, waste rock, nuclear waste, munitions and ce, hospital waste, junked auto parts, metal cuttings, insulation waste, food waste, os, batteries, industrial scrap, landfill waste, wood waste, textile waste, glass waste, leather waste, rubber waste, plastic waste, electronic component waste, agricultural waste, photographic waste, c waste, pharmaceutical waste, wax, spent catalysts, or a combination thereof.. The liquid environment can be drinking water, groundwater, surface water, brine, storage tanks, lagoons, an aquatic system, industrial wastewater, acid mine drainage, spent autofluid, spent plating baths, degreasing solutions, dry cleaning solutions, e coolants, drilling fluid waste, cutting fluid waste, lic fracturing fluid waste, lubricant waste, paint, greywater, oily wastewater, pulp mill effluent, a water treatment system, a septic , a sewer system, a precipitation lagoon, a g pond, a lake, a river, or ations thereof. The gaseous environment can be air, a flue gas such as emissions from power plants, waste incinerators, crematoria or refineries, a process exhaust stream, landfill gas, natural gas, propane gas, or a ation thereof The contaminated environment can be inated by s contaminants including, but not limited to, a chemical warfare agent sing sarin (GB; o—isopr‘opyl methylphosphonofluoridate); soman (GD; o—pinacolyl methylphosphonofluoridate); cyclosarin {GR (rcyclohexyl methylphosphonoiluoridate); VX (O—eth'yl S~[2— (diisopropylamino)ethyl]methylphosphonothioate); ta’bun (GA; N,N—dimethylethyl phosphoroamidocyanidate), DFP (diisopropyl phophorofluoridate), or a mustard agent; an WO 44661 2015/050807 inorganic compound comprising arsenic, antimony, barium, beryllium, cadmium, chromium, copper, iron, lead, manganese, mercury, nickel, selenium, silver, tin, um, uranium, zinc or a combination thereof; an organic compound comprising a polycyclic aromatic hydrocarbon (PAH), a chlorinated aromatic compound, a chlorinated aliphatic compound, a nitroaromatic compound (NAC), a phenolic compound, a cyano compound, dioxin, or a combination thereof; a crude oil, a refined oil, a fuel oil, a diesel oil, a gasoline, a hydraulic oil, and kerosene, or a volatile constituent thereof such as benzene, toluene, ethylbenzene, xylene, or naphthalene; an explosive, a fertilizer, a pesticide, an insecticide, or an herbicide The concentration of recombinant spores or exosporium fragments needed to treat a contaminated enVironment is based on factors including the volume or area to be treated, the extent of the target chemical, ant or organic matter present, the amount of time ble for treatment, and amount of active enzyme on the surface of the spores.

] The inant Bacillus cereus family member spores or exosporium fragments can contact the contaminated environment by incorporating the spores or exosporium fragments into a stream ning the contaminant, ting a stream containing the contaminant With an immobilization material containing the spores or exosporium fragments (e.g., a filter, membrane, sponge or te), incorporating the spores or exosporium fragments into granules to be mixed With the contaminated enVironment, spraying the spores or exosporium fragments onto or into the contaminated enVironment, injecting the spores or exosporium fragments into the contaminated enVironment, or drenching the contaminated enVironment With the spores or rium fragments.

The spores can be combined With bacterial inoculants, als, ts, and other products that can expedite the decomposition process.

The ation enzyme includes, but is not limited to, a phosphate binding protein, a protease, a carbohydrate hydrolyase, a lipase, a phospholipase, a se, a nutrient binding protein, a cellulase, an oxidoreductase, a monooxygenase, a diooxygenase, a laccase, a lignin peroxidase, a manganese peroxidase, a peroxidase, a dehalogenase, a catalase, an amylase, a reductase, an oxidase, an amidase, a ligninase, a xylanase, a pectinase, a xylosidase, an endoglucanase, an exoglucanase, a glucosidase, a m inhibitory peptide, an herbicide-degrading enzyme, a pesticide-degrading enzyme (e. g., a pyrethrinase), or a combination f.

Where the enzyme comprises an herbicide-degrading enzyme or a pesticide-degrading enzyme, the recombinant Bacillus cereus family member suitably comprises a strain of bacteria that is e of degrading an herbicide or a pesticide. For example, the strain of bacteria that is capable of degrading an herbicide or a pesticide can comprise us cereus family member EE349 (NRRL No. 8), Bacillus cereus family member EE- B00377 (NRRL B-67l l9); Bacillus pseudomycoia’es EE-B00366 (NRRL B-67120); or Bacillus mycoia’es EE-B00363 (NRRL B-67121).

A method for phytoremediation of contaminated soil is also provided. The method ses introducing recombinant Bacillus cereus family member spores into contaminated soil; or applying the recombinant Bacillus cereus family member spores to a plant planted in contaminated soil, or a plant seed for planting in contaminated soil, or an area of contaminated soil surrounding a plant or a plant seed; wherein the recombinant Bacillus cereus family member spores express a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, rium n, or exosporium protein fragment that targets the fusion n to the exosporium of the recombinant Bacillus cereus family member spore, wherein the fusion protein is the fusion protein as described above wherein the protein or peptide of st comprises a remediation enzyme, and wherein the recombinant Bacillus cereus family member comprises an endophytic strain of bacteria or a root colonizing strain of bacteria. For example, the recombinant spore-forming bacterium can comprise an endophytic strain of bacteria.

A further method for phytoremediation of contaminated soil is provided.

The method comprises expressing a remediation enzyme in a Bacillus cereus family member spore, wherein the expression of the remediation enzyme in the recombinant Bacillus cereus family member spore is increased as ed to the expression of the remediation enzyme in a wild-type Bacillus cereus family member spore. r method for phytoremediation of contaminated soil is also provided.

The method comprises introducing a recombinant spore-forming bacterium into contaminated soil; or applying the recombinant spore-forming bacterium to a plant planted in contaminated soil, or a plant seed to be planted in contaminated soil, or an area of contaminated soil surrounding a plant or a plant seed. The inant spore-forming bacterium ses a fusion n comprising at least one protein or peptide of interest and a spore coat protein that s the fusion protein to the surface of a spore of the bacterium. The spore coat protein comprises a CotB protein, a CotC protein, a CgeA protein, a CotB/H protein, a Cot G protein, a spore coat protein X protein, or a CotY protein. The recombinant spore-forming bacterium comprises an endophytic strain of bacteria or a root colonizing strain of bacteria. The protein or peptide of interest comprises a remediation enzyme. r method for phytoremediation of contaminated soil is also provided.

The method comprises introducing rium fragments into contaminated soil or applying exosporium fragments to a plant planted in contaminated soil, or a plant seed to be planted in contaminated soil, or an area of contaminated soil surrounding a plant or a plant seed. The exosporium fragments are derived from spores of a recombinant Bacillus cereus family member described in n IV herein above and comprise the fusion protein. The fusion protein comprises a remediation enzyme.

Yet another method for emediation of contaminated soil is provided.

The method comprises introducing spores of a recombinant Bacillus cereus family member into contaminated soil. Alternatively, the method comprises applying spores of a recombinant Bacillus cereus family member to a plant planted in contaminated soil, or a plant seed to be planted in contaminated soil, or an area of contaminated soil nding a plant or a plant seed.

The recombinant us cereus family member is a recombinant Bacillus cereus family member as described above in Section II, and the fusion protein comprises a remediation enzyme.

] In the methods for phytoremediation of contaminated soil that involve the use of exosporium fragments or a recombinant Bacillus cereus family member as described above in Section II, the ing sequence, exosporium protein, or exosporium protein fragment can comprise any of the targeting sequences, exosporium ns, or exosporium protein fragments described herein. In particular, the targeting sequence, exosporium protein, or exosporium protein fragment can comprise any of the targeting sequences, exosporium proteins, or exosporium n fragments described above in paragraphs [00782]—[00801].

] The fusion protein can be expressed under the control of a sporulation promoter native to the targeting sequence, exosporium protein, or exosporium protein fragment of the fusion protein or a portion thereof and/or under the control of a high-expression sporulation er. The promoter can be any of the promoters bed above in Section 111.

The remediation enzyme is displayed on the outside of the spores and within the plant so that both the plant and spores can convert the target contaminant. The plant can take up the target contaminant While the spores convert the contaminant into a non-toxic or less toxic form Within the plant or its root system.

The recombinant Bacillus cereus family member spores can comprise an endophytic strain of bacteria, such as Bacillus cereus family member EE349, Bacillus cereus family member EE439, Bacillus thuringiensis EE4l7, Bacillus cereus EE444, or Bacillus thuringiensis EE3 l9, Bacillus thuringiensis EE-B00184, Bacillus cereus family member EE- B00377, us pseudomycoia’es EE-B00366, or Bacillus mycoia’es EE-B00363.

The spores or the exosporium fragments can be applied to the plant or the plant seed, and the plant or plant grown from the plant seed is tolerant to a target contaminant to be ated from the contaminated soil In the method for phytoremediation, recombinant Bacillus cereus family members undergo sporulation within the plant.

The recombinant Bacillus cereus family member spores can be introduced into the plant growth medium by various methods such as soil drench at the time of planting.

The spores can also be coated onto the plant seed as a seed ent.

Preferably, the plant to be treated with the remediation enzyme is tolerant to the target contaminant so that the plant is not injured by the target contaminant.

The concentration of recombinant spores needed for the phytoremediation method is based on factors including volume or area to be treated, the ability of the ytic strains to colonize the plant roots, the extent that the target contaminant is present, and the amount of active enzyme on the surface of the spores.

A further method for reducing contaminants in an environment is provided.

The method comprises exposing a contaminated environment to spores of a Bacillus cereus family member strain that is capable of degrading an herbicide or a pesticide. The contaminants in the environment comprise an herbicide, a pesticide, or a combination thereof. The Bacillus cereus family member strain that is e of degrading an herbicide or a pesticide comprises us cereus family member EE349 (NRRL No. B-50928), Bacillus cereus family member 377 (NRRL B-67l l9); Bacillus mycoia’es EE-B00366 (NRRL B-67120); us mycoia’es EE-B00363 (NRRL B-67121), or a combination thereof The Bacillus cereus family member strain that is capable of degrading an ide or a pesticide can comprise a recombinant Bacillus cereus family member that expresses_a fusion protein comprising at least one protein or peptide of interest and a ing sequence, exosporium protein, or exosporium protein nt that targets the fusion protein to the exosporium of the inant Bacillus cereus family member. The protein or peptide of interest preferably comprises an herbicide-degrading enzyme, a pesticide-degrading enzyme, or a combination thereof.

In this way, dual pesticide or herbicide degrading activity can be obtained since both the Bacillus cereus family member strains and the herbicide-degrading or pesticide- degrading enzymes in the fusion protein will exert pesticide- and/or herbicide-degrading activity. The herbicides and/or ides that are ed by the Bacillus cereus family strain that is capable of degrading an herbicide or a pesticide can be the same as or different from the herbicides and/or pesticides that are degraded by the herbicide-degrading enzyme or the pesticide-degrading enzyme. Thus, where an environment is contaminated with a single type of herbicide or pesticide, dual degrading action against that single herbicide or pesticide can be obtained. Alternatively, where an environment is contaminated with more than one type of herbicide or pesticide, dual degrading action against two or more different herbicides or pesticides can be obtained.

In the methods of reducing inants involving the use of one of the Bacillus cereus family member strains described herein that is e of degrading an herbicide or a pesticide, the targeting sequence, exosporium n, or exosporium protein nt can comprise any of the targeting sequences, exosporium proteins, or exosporium n fragments described herein. In particular, the targeting sequence, exosporium n, or rium protein fragment can comprise any of the targeting sequences, exosporium proteins, or exosporium protein fragments described above in aphs [00782]—[0080l].

XXIV. Breaking Emulsions or Gels in a Hydraulic Fracturing Fluid A method of treating a hydraulic fracturing fluid to break an emulsion or gel within the fluid is provided. The method comprises adding spores of a recombinant us cereus family member spores to a hydraulic fracturing fluid. The recombinant Bacillus cereus family member expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or rium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member spore. Such a fusion protein is described above wherein the n or e of st comprises an enzyme le for breaking the emulsion or gel.

The recombinant Bacillus cereus family member can comprise any of the recombinant Bacillus cereus family members described herein that express a fusion protein.

WO 44661 A further method of treating a hydraulic fracturing fluid to break an emulsion or gel within the fluid is provided. The method comprises adding rium fragments to a hydraulic fracturing fluid. The exosporium nts are derived from a recombinant Bacillus cereus family member described in Section IV hereinabove and comprise the fusion protein. The fusion protein comprises an enzyme suitable for breaking the emulsion or gel.

The enzyme is selected based upon the target emulsion or gel to be treated and the pH of the hydraulic fracturing fluid. Enzymes e, but are not limited to, a llulase, an amylase, a pectinase, a carbohydrate hydrolyase, a cellulase, an agarase, a polygalacturonase, an endoglucanase, or a combination thereof.

The emulsion or gel contains a polymer or other component which the enzyme can digest. The emulsion or gel can comprise a polymer, Arabica gum, agar, xanthan gum, cellulose, carboxymethylcellulose, carboxymethylhydroxyethyl ose, hydroxyethyl methylcellulose, guar, a guar derivative, or a combination f.

When the protein or peptide of interest is an enzyme for breaking an on or gel, any Bacillus cereus family member can be used to express the fusion n. Bacillus thuringiensis or Bacillus mycoia’es are preferred.

The spores or exosporium fragments can be ed into a well that is in contact with a subterranean hydrocarbon-containing formation such as a sandstone reservoir or a carbonate reservoir.

The concentration of spores or exosporium nts needed is based on factors including the size of the well to be treated, the type of emulsion or gel, the amount of active enzyme on the surface of the spores or exosporium fragments, and the presence and concentration of adjuvants delivered with the enzymes.

The enzymes can digest polymers or other components within the emulsion or gel, or can dissolve such components so that the hydraulic ring fluid can be pumped out of the well.

In the methods of treating a hydraulic fracturing fluid to break an emulsion or gel within the fluid, any of the targeting sequences, exosporium proteins, or exosporium protein fragments bed herein can be used. In particular, the targeting sequence, exosporium protein, or exosporium protein fragment can comprise any of the targeting sequences, exosporium protein, or exosporium protein fragments described above in paragraphs ]— The fusion protein can be expressed under the control of a ation promoter native to the targeting ce, exosporium protein, or rium protein fragment of the fusion protein or a portion thereof and/or under the control of a high-expression sporulation promoter. The er can be any of the promoters bed above in Section III.

XXV. Feedstock Processing Feedstock is generated from plants that are ted for their biomass, and processed into feed (bailing, silage, extrusion, pelleting, etc). The plant biomass that constitutes the feedstock is often difficult to digest due to the fibrous nature of the material. The presence of enzymes can greatly assist in the degradation of this fibrous material, leading to a more digestible and easier to process material. Enzymes are traditionally added after the feedstock has been processed and upon delivery to the organism that is ingesting the feedstock. Enzymes delivered in feedstock can e health and weight gain of target animals, as well as reduce the environmental impact of the waste products of animals fed such enzyme-supplemented feed.

These same systems can be utilized to pretreat feedstock that is destined for biofuel production, including processing into bioethanol, biodiesel, or other biofuels.

Many species of spores have the ability to persist on foliar surfaces, such as leaves, stems, and fruit, for long s of time. By using spore display technologies as described herein to display the enzymes on these spores, active enzyme is provided to the feedstock that will be present as the feedstock is harvested. These target enzymes can also be delivered to the feedstock plant at planting, either through delivery of inant spores on the plant seeds, or delivery of the recombinant spores to the plant growth medium or area around the plant.

A method for delivering enzymes to a plant is provided. The method comprises ucing into a plant growth medium a inant Bacillus cereus family member that expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that s the fusion protein to the exosporium of the recombinant Bacillus cereus family member or a formulation comprising a recombinant Bacillus cereus family member as described herein; or applying to a plant, a plant seed, or an area nding a plant or a plant seed the recombinant Bacillus cereus family member or the ation comprising a recombinant Bacillus cereus family member. The protein or e of interest comprises an enzyme. The enzyme can be ally attached to the exosporium of the recombinant Bacillus cereus family member.

Another method for delivering enzymes to a plant is provided. The method comprises introducing into a plant growth medium a recombinant spore-forming bacterium or a formulation sing the recombinant spore-forming bacterium; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed the recombinant spore-forming bacterium or a formulation comprising the recombinant forming ium. The recombinant spore- forming bacterium expresses a fusion n comprising at least one protein or peptide of interest and a spore coat protein that targets the fusion protein to the surface of a spore of the bacterium. The spore coat protein comprises a CotB protein, a CotC protein, a CgeA protein, a CotB/H protein, a Cot G protein, a spore coat protein X protein, or a CotY protein. The recombinant spore-forming ium comprises an endophytic strain of bacteria. The protein or peptide of st comprises an enzyme, and the enzyme is ally attached to the spore coat of the recombinant spore-forming bacterium Yet another method for delivering enzymes to a plant is provided. The method comprises introducing exosporium fragments or a formulation containing the exosporium fragments into a plant growth medium; or applying exosporium fragments or a formulation containing the exosporium fragments to a plant, a plant seed, or an area nding a plant or a plant seed. The rium fragments are derived from spores of a recombinant Bacillus cereus family member described in Section IV hereinabove and comprise the fusion protein. The protein or peptide of interest comprises an enzyme.

Where the method for delivering enzymes to a plant comprises the use of exosporium fragments, the method can further comprise treating the plant with a penetrating agent, a surfactant, a detergent, an oil, or a combination f Optimal bacteria strains for these methods include, but are not limited to, us cereus family members, including Bacillus cereus, Bacillus mycoia’es, Bacillus thuringiensis, and Bacillus pseudomycoia’es, as well as other Bacillus spore formers, ing Bacillus megaterium, Bacillusfirmus, Bacillusflexus, Bacillus subtilis clade members, Bacillus pumilus, us licheniformis, and Bacilllus subtilis.

Application can be directly onto the plant material, optionally in conjunction with nts, such as nonionic or other surfactants. The recombinant Bacillus cereus family member can be d to foliage of the plant prior to harvest such as by spraying onto the foliage. 23 1 Application to the plant seed is generally performed as a seed dip, a slurry, or a polymer-based seed coating. Optionally, the application can be done in ction with seed applied inoculants, fungicides, insecticides, or nematocides.

Application to the plant growth medium or area around the plant can be performed prior to planting, at planting, or post planting of seeds, optionally in conjunction with fertilizers, fungicides, herbicides, or insecticides.

The enzyme is suitable for degrading biomass, ing cellulosic material, aiding digestion in a digestive system of a target animal to which the plant can be fed, or for biofuel production (e. g., for production of bioethanol or biodiesel).

The enzyme includes, but is not limited to, a nonspecific se, a metalloprotease, a cellulase, a xylanase, a phosphatase, an endoglucanase, an exoglucanase, a B- idase, an amylase, a pectinase, a xylosidase, a lipase, a phospholipase, or a combination thereof.

The selection of s may depend on the feedstock and the intended use of the feedstock. The enzymes are preferably degradative enzymes.

Enzymes of interest in the protease family include cific proteases, such as serine proteases, histidine proteases, aspartate proteases, as well as metalloproteases.

Enzymes of interest in the cellulase family would include exoglucanases, endoglucanases, B-l,3 glucosidases, cellulases, hemicellulases, a-glucosidases.

Enzymes of interest in the ses family include xylosidases, endoxylanases, exoxylanases, pectinases, methyl pectinases, polygalacturonase. s of interest in the phosphatases include acid phosphatases, alkaline phosphatases, polyphosphatases, phytases, osphatases, and diphosphatases.

Many of these s are also beneficial to plant growth.

] These enzymes can not only “predigest” some of the feedstock to increase absorption of key nts by a target animal to which the feedstock is fed, but can also aid ion in the digestive system of the target animal.

The “predigestion” of cellulosic material at harvest can te free cellulose during sing for bioethanol and biofuel production, as well as preprocessing of oils destined for biofuel production.

The bacterium can be an endophytic bacterium. Selection of endophytic recombinant bacteria will allow for the bacteria to enter into the plant, but also ze and grow inside the plant tissues. This will establish a growing number of recombinant spore forming sms inside the plant as it grows from use of a relatively minor amount of recombinant spores on the seed or with the seed at ng. Upon harvest of the plant biomass material, the ial will undergo sporulation, creating new enzymes in planta, which are active on the feedstock as it is harvested, transported, and ed, for example, either as animal feed or for biofuel production. This can cantly reduce the input cost of degradative enzymes as compared to ng techniques. This is a unique method of delivering digestive enzymes to the biomass prior to industrial processing.

While the optimal bacterial strains are as described above, ion of endophytic strains will increase efficacy. Preferably, the endophytic bacteria comprises Bacillus cereus family member EE349, Bacillus cereus family member 439, Bacillus thuringiensis EE4l7, Bacillus cereus EE444, Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillusfusiformis EE442, Lysinibacillus sphaericus EE443, or a combination thereof.

The plant can be a crop ed from corn, alfalfa, wheat, a pasture crop, a forage crop, soybean, switchgrass, jicama, sweet sorghum, sugarcane, or a combination thereof, and other biofuel and bioethanol feedstocks.

For the methods for delivering enzymes to a plant, any of the targeting sequences, exosporium proteins, or exosporium protein fragments described herein can be used.

XXVI. Use of Spores in Altering Properties of Target Plants The inant Bacillus cereus family members and recombinant spore- forming bacterium as described herein allow for the interaction of surface displayed signaling les impacting biochemical pathways, and a number of other proteins that benefit plant health. The ce of the spore displayed proteins or peptides can lead to alteration in the metabolism of the target plant, leading to changes in the composition of the plant, its fruit, or other properties or characteristics.

The sion of fusion ns can be directly used to alter the composition of the target plant. Selection of different enzymes leads to varying effects on the target plant.

A method for altering a property of a plant is provided. The method comprises introducing into a plant growth medium a recombinant Bacillus cereus family member that expresses a fusion protein comprising at least one protein or peptide of st and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member or a formulation comprising a recombinant Bacillus cereus family member as described herein; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed the recombinant Bacillus cereus family member or the formulation comprising a recombinant us cereus family member. The protein or peptide of interest comprises a plant signaling molecule or an enzyme that affects plant composition, and the protein or peptide of interest can be physically attached to the exosporium of the recombinant Bacillus cereus family member.

Another method for altering a property of a plant is ed. The method comprises introducing into a plant growth medium a recombinant spore-forming bacterium or a formulation comprising the recombinant spore-forming bacterium; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed the recombinant spore-forming bacterium or a formulation comprising the recombinant spore-forming bacterium. The recombinant spore- g bacterium expresses a fusion protein comprising at least one protein or peptide of interest and a spore coat protein that targets the fusion protein to the surface of a spore of the bacterium. The spore coat protein ses a CotB protein, a CotC protein, a CgeA protein, a CotB/H protein, a Cot G n, a spore coat protein X protein, or a CotY protein. The inant spore-forming bacterium comprises an ytic strain of bacteria. The protein or peptide of interest comprises a plant signaling molecule or an enzyme that affects plant composition, and the protein or e of st can be physically attached to the spore coat of the recombinant spore-forming bacterium Yet another method for altering a property of a plant is provided. The method ses introducing exosporium fragments or a formulation containing the exosporium fragments into a plant growth medium; or applying exosporium fragments or a formulation containing the exosporium fragments to a plant, a plant seed, or an area surrounding a plant or a plant seed. The exosporium fragments are d from spores of a recombinant Bacillus cereus family member described in n IV hereinabove and comprise the fusion protein. The protein or e of interest comprises a plant signaling molecule or an enzyme that affects plant composition.

Where the method for altering a property of a plant ses the use of exosporium nts, the method can further comprise treating the plant With a penetrating agent, a surfactant, a detergent, an oil, or a combination thereof The target bacterium preferably survives or thrives in the environment and on the roots of the target plant. Optimal bacteria strains for these s include, but are not limited to, us cereus family member EE349, Bacillus cereus family member 439, Bacillus thuringiensis EE4l7, Bacillus cereus EE444, Bacillus megaterium EE385, Bacillus sp. EE3 87, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillusfusiformis EE442, 0r Lysinibacillus sphaericus EE443.

The plant signaling les or enzymes can also be delivered to the plant at ng, either through delivery of inant spores on the plant seeds, or delivery of the recombinant spores to the plant grth medium or area around the plant.

Application can be directly onto the plant material, optionally in conjunction with adjuvants, such as nonionic or other surfactants. The recombinant Bacillus cereus family member, the recombinant spore-forming bacterium, or the exosporium fragments can be applied to foliage of the plant prior to harvest such as by spraying onto the foliage.

Application to the plant seed is lly performed as a seed dip, a slurry, or a polymer-based seed coating. Optionally, the application can be done in conjunction with seed applied inoculants, fungicides, insecticides, or nematocides.

Application to the plant growth medium or area around the plant can be performed prior to planting, at planting, or post planting of seeds, optionally in ction with fertilizers, fungicides, ides, or insecticides.

The enzyme includes, but is not limited to, comprises endoglucanases, ses, phospholipases, aminocarboxy- l -propanedeaminase, aminocyclopropane-l- carboxylic acid deaminases, lipases, or a combination thereof.

] The plant signaling molecules include, but are not limited to, flg22 and flagellin peptides, cryptogein, harpins, -like proteins, enzymes that degrade or modify a bacterial, fungal, or plant nutrient source, or a combination thereof.

The enzymes or plant ing molecules can cause desired metabolic changes to the host plant, including increasing the macronutrient and micronutrient uptake or content of the plant s through enlargement of the root systems, sing the protein content of plants such as grains, cereals, and fruit through modifications to metabolism and sed nitrogen uptakes, and modifications to oil content in rapeseed, canola, soybeans and sunflower, sugar t (sucrose) in grapes, sugar cane, switchgrass, sweet sorghum and other biofuel feedstock, medicinal compound content, and cannabinoid content in marijuana. These tions not only increase the value of the plants of interest, but also se the utility of these plants in various industries such as biofuel formation, sugar production, and feedstock production. 23 5 For the methods for altering a property of a plant, any of the targeting sequences, exosporium proteins, or exosporium protein fragments described herein can be used.

XXVII. ection A method of disinfecting a surface is provided. The method comprises exposing a surface to a recombinant Bacillus cereus family member that expresses a fusion protein as described above in paragraphs [00172]—[00175] and ] of Section I, wherein the protein or peptide of interest comprises an antibacterial protein or e.

A further method of disinfecting a surface is ed. The method comprises exposing a surface to rium fragments. The exosporium fragments are derived from a recombinant Bacillus cereus family member described in Section IV hereinabove and comprise the fusion protein. The fusion protein comprises an antibacterial protein or peptide.

Yet another method of disinfecting a e is provided. The method comprises exposing a surface to a recombinant Bacillus cereus family member. The recombinant Bacillus cereus family member is a recombinant us cereus family member as described above in Section II.

In the methods for disinfecting a surface that comprise exposing a surface to exosporium fragments or to a recombinant Bacillus cereus family member as described above in Section II, the targeting sequence, exosporium protein, or exosporium protein nt can be any of the targeting sequences, exosporium proteins, or exosporium protein fragments described herein. In particular, the targeting sequence, exosporium protein, or exosporium protein fragment can comprise any of the targeting sequences, exosporium protein, or exosporium n fragments bed above in paragraphs [00782]—[00801].

The fusion protein can be expressed under the l of a sporulation promoter native to the targeting sequence, exosporium protein, or exosporium n fragment of the fusion protein or a portion thereof and/or under the l of a high-expression sporulation promoter. The promoter can be any of the ers described above in Section 111.

The antibacterial protein or peptide minimizes or prevents viral agents, bacteria, amoebas, pests, or molds from forming on or binding to the e.

The antibacterial protein or e includes, but is not d to, proteases, ses, antimicrobial peptides, LysM, LfcinB, lysostaphin, albumin, defensins, bacteriocins, lipopeptides, innate immune system peptides, lysozyme, lyticase, or a combination thereof.

The recombinant Bacillus cereus family member spores can be used in conjunction with other antimicrobial agents, including disinfectants, rs, antibiotics, ngals, and antivirals.

Although any of the Bacillus cereus family can be utilized to express the fusion proteins, either Bacillus thuringiensis or Bacillus mycoia’es is preferred.

For these s, any of the targeting sequences, exosporium ns, or rium protein fragments described herein can be used.

XXVIII. Other Uses The fusion proteins wherein the protein or peptide of interest is an enzyme or recombinant Bacillus cereus members wherein the protein or peptide of interest is an enzyme that can be used for grease, oil, or fat ent or degumming; leather hide processing; biofuel, biodiesel, or bioethanol formation; sugar sing or sion; starch treatment; paper or linen processing; animal or fungal byproduct treatment or amino acid recovery; targeted digestion of facility wastes; feed or food additives; dietary supplements; animal nutrition; industrial cleaning; grain processing; cosmetic manufacturing; odor control; food or beverage processing; brewing enhancement or additives; detergent additives; or textile or yarn processing.

By displaying an enzyme on the outside of the spore or on exosporium fragments, the enzyme can be stabilized, immobilized, and able to be reused.

Industrial processes generally involve harsh ions, including high temperatures, presence of solvents, and large s of organic matter. These conditions hinder ional enzymes. Expression of the target enzyme on the surface of the spore or on exosporium fragments allows for ance to high temperatures and harsh conditions, and allows for the enzymes to be reisolated and reused.

Key enzymes of interest for such uses include: B-lactamases, proteases, lipases, phospholipases, cellulases, endoglucanases, conases, pectinases, ligninases, amylases (e.g., u-amylases, B-amylases, or glucoamylases), polygalacturonases, glucosidases, osidases, carbohydrate hydrolyases, cell wall hydrolases, nucleases, hemicellulases, xylanases, mannases, laccases, lactases, esterases (e.g., pectin methyl esterases), phytases, phosphatases, ases, glucose oxidases, catalases, lyticases, acetolactate decarboxylass, and ureases.

Preferred enzymes for grease, oil, or fat treatment or degumming, or for ic cturing include lipases, olipases, esterases, and proteases.

Preferred enzymes for leather hide processing include lipases, proteases, peptidases, collagenases, and phospholipases.

Preferred enzymes for l, biodiesel, or bioethanol formation can e, but are not limited to, lipases and esterases.

Preferred enzymes for sugar processing or conversion, for grain sing, and for textile or yarn processing include carbohydrate hydrolases, amylases, es, glucoamylases, invertases, cellulases, hemicellulases, pectinases, pectin methyl esterases, xylanases, ucanases, exoglucanases, glucosidases, galactosidases, laccases, lactases, catalases, and glucose oxidases.

Preferred enzymes for starch ent include es and mylases.

Preferred enzymes for paper or linen processing include cellulases, hemicellulases, xylanases, endoglucanases, laccases, ases, exoglucanases, phytases, catalases, and glucosidases. red enzymes for animal or fungal byproduct treatment or amino acid recovery include proteases, peptidases, lipases, lyticases, cell wall hydrolases, phospholipases, ucanases, cellulases, glucanases and carbohydrate hydrolases.

Preferred enzymes for targeted digestion of facility wastes, industrial cleaning, detergent additives, and odor control e s, phospholipases, proteases, peptidases, amylases, lyticases, cell wall hydrolases, glucoamylases, cellulases, hemicellulases, xylanases, esterases, glucosidases, galactosidases, laccases, lactases, ureases, phytases, phosphatases, and carbohydrate hydrolases.

Preferred enzymes for feed or food additives, dietary supplements, animal nutrition, brewing additives, beverage additives, or wine processing include es, laccases, lyticases, ses, peptidases, carbohydrate hydrolases, pectinases, pectin methyl esterases, esterases, lipases, cellulases, hemicellulases, xylanases, phytases, phosphatases, invertases, glucosidases, galactosidases, lactases, catalases, glucanases, endoglucanases, acetolactate decarboxylase, and glucose oxidases.

Although any of the Bacillus cereus family can be utilized to s the fusion proteins for these uses, either Bacillus thuringiensis or Bacillus mycoia’es is preferred.

A use of fusion proteins comprising an enzyme as the protein or peptide of interest or a recombinant Bacillus cereus family member expressing a fusion protein comprising 23 8 an enzyme as the n or peptide of interest is provided. The fusion protein can be any of the fusion proteins described above in aphs [0017l]—[00175], [00177], and [00179] of Section I. The use can be a use for grease, oil, or fat treatment or degumming; leather hide processing; biofuel, biodiesel, or bioethanol formation; sugar processing or conversion; starch treatment; paper or linen sing; animal or fungal uct treatment or amino acid recovery; targeted digestion of facility wastes; feed or food additives; dietary supplements; animal nutrition; industrial ng; grain sing; cosmetic manufacturing; odor control; food or beverage processing; brewing enhancement or additives; detergent additives; or textile or yarn processing.

A use of exosporium fragments is also provided. The use can be for grease, oil, or fat treatment or degumming; leather hide processing; biofuel, biodiesel, or bioethanol ion; sugar processing or conversion; starch treatment; paper or linen processing; animal or fungal uct treatment or amino acid recovery; targeted digestion of facility wastes; feed or food additives; dietary supplements; animal nutrition; industrial cleaning; grain processing; ic cturing; odor control; food or beverage processing; brewing enhancement or additives; detergent additives; or textile or yarn processing. The exosporium fragments are derived from a recombinant Bacillus cereus family member described in Section IV hereinabove and comprise the fusion protein. The fusion protein comprises an .

A r use of a recombinant Bacillus cereus family member is provided.

The recombinant Bacillus cereus family member is a recombinant Bacillus cereus family member as described above in Section II. The use can be for grease, oil, or fat treatment or degumming; leather hide processing; biofuel, biodiesel, or bioethanol formation; sugar processing or conversion; starch treatment; paper or linen processing; animal or fungal uct treatment or amino acid recovery; targeted digestion of facility wastes; feed or food additives; dietary supplements; animal ion; industrial cleaning; grain processing; ic manufacturing; odor control; food or beverage processing; brewing enhancement or additives; detergent additives; or textile or yarn processing. The fusion protein comprises an enzyme.

In the uses of exosporium fragments or the recombinant us cereus family members as described above in Section II, the targeting sequence, exosporium protein, or exosporium protein fragment can be any of the targeting sequences, exosporium ns, or exosporium protein nts described herein. In particular, the targeting sequence, exosporium protein, or exosporium protein fragment can comprise any of the targeting sequences, exosporium protein, or exosporium protein fragments described above in paragraphs —[00801].

The fusion protein can be expressed under the control of a sporulation promoter native to the targeting sequence, exosporium protein, or exosporium protein fragment of the fusion protein or a portion thereof and/or under the control of a high-expression ation promoter. The promoter can be any of the promoters bed above in Section III.

Having described the invention in detail, it will be nt that modifications and variations are possible without departing from the scope of the invention defined in the appended claims.

EXAMPLES The ing miting examples are provided to further illustrate the present invention.

Example 1. Use of a recombinant Bacillus cereus family member displaying a lipase or an endoglucanase to stimulate plant growth in soybeans.

The Bacillus subtilis lipase and endoglucanase genes were amplified via polymerase chain reaction (PCR) using the following primers shown below in Table 16: TABLE 16 ggatccatggctgaacacaatcc atgaaacggtcaatc (SEQ ID NO: 39) (SEQ ID NO: 37) ggatccttaattcgtattctggcc ggatccttactaatttggttctgt SEQ ID NO: 38 SEQ ID NO: 40 To create fusion constructs, genes were fused to the native bclA promoter of Bacillus giensis DNA encoding the first 35 amino acids of BclA (amino acids 1—35 of SEQ ID NO: 1) using the splicing by overlapping extension (SOE) que. Correct amplicons were cloned into the E. coli/Bacillus shuttle vector pHPl3, and correct clones screened by DNA sequencing. Correct clones were oporated into Bacillus thuringiensis (Cry—, plasmid—) and screened for chloramphenicol resistance. Correct transformants were grown in brain heart infusion broth overnight at 30°C, plated onto nutrient agar plates, and incubated at 30°C for 3 days. Spores expressing the fusion construct (BEMD spores) were collected off of the plates by washing in phosphate buffered saline (PB S) and purified by centrifugation and additional washes in PBS. Non-transformed control Bacillus thuringiensis (B.t.) spores were created identically.

Soybeans (strain Jake 01104) were d 2.54 cm deep in 10 cm deep pots filled with standard loam l. Spores were diluted to a concentration of lxlO4/ml in 50 ml of water and applied to each seed at planting. Plants were grown under ideal light using T5 lamps, 54 watts, and exposed to 11 hours of light a day under controlled temperature conditions between 15.5—25.50C. Plants were watered to saturation every three days over a two week trial.

At the end of two weeks, the height of each plant was measured and measurements were normalized to control Bacillus thuringiensis spores. Two independent trials were performed. s are shown in Table 17, together with the standard error of the mean.

In both trials, soybeans grown in the presence ofBEMD spores displaying either lipase or endoglucanase grew significantly taller than control B.t. spore treated soybeans (statistical analysis assayed via a t-test).

TABLE 17 _——— Treatment Avg. Height, Comparison to SEM cm Control Trial #1 14034 100.0% .521 Lipase, BEMD 17.93 127.8% .395 Endocellulase, BEMD 16.31 116.2% .411 Trial #2 15.39 100.0% Lipase, BEMD 19.15 124.4% Endocellulase, BEMD 17.65 114.7% .313 e 2. Use of a recombinant Bacillus cereus family member displaying an endoglucanase to stimulate plant growth in corn.

] BEMD spores sing endoglucanase were created in an identical fashion as described above in Example 1. Field corn was planted 3.8 cm deep in 10 cm deep pots filled with standard loam topsoil. Spores, l and BEMD expressing endoglucanase, were diluted to a tration of 1x104/ml in 50 ml of water and applied to each plant at planting. A water- only control was also included. Plants were grown under ideal light using T5 lamps, 54 watts, and exposed to 11 hours of light a day under controlled temperature ions between 15.5— . Plants were watered to saturation every three days over the one week trial. At the end of one week, the height of each plant was measured, and measurements were normalized to control Bacillus thuringiensis spores.

Results are shown in Table 18, together with the standard error of the mean.

Corn grown in the presence ofBEMD spores displaying endoglucanase grew significantly taller than both control B.t. spore d ns and water-only control plants (statistical analysis assayed via a t-test).

TABLE 18.

Height, Comparison SEM 18.92 % 0.645 BEMDEndo 22.71 143.40% 0.616 e 3. Use of a recombinant Bacillus cereus family member displaying an endoglucanase or a protease to stimulate plant growth in wheat.

BEMD spores sing endoglucanase were created in an identical fashion as described above in Example 1. BEMD spores expressing E. coli protease PtrB were created using similar methods to those described above in Example 1 and the following primers: ggatccatgctaccaaaagcc (forward, SEQ ID NO: 41) and ggatccttagtccgcaggcgtagc (reverse, SEQ ID NO: 42).

Winter hard Wheat was planted 2.54 cm deep in 10 cm deep pots filled with standard loam topsoil. , control and BEMD expressing endoglucanase or se, were d to a concentration of 1x104/ml in 50 ml of water and applied to each plant at planting. A water-only control was also included. Plants were grown under ideal light using T5 lamps, 54 watts, and exposed to 11 hours of light a day under controlled temperature conditions n .5—25.5°C. Plants were watered to tion every three days over the one week trial. At the end of one week, the height of each plant was measured, and measurements were normalized to control water only plants.

Results are shown in Table 19, together with the standard error of the mean.

Wheat grown in the presence ofBEMD spores displaying endoglucanase or protease grew significantly taller than control B. t. spore treated or water l soybeans (statistical analysis assayed via a t-test).

TABLE 19.

Height, Comparison SEM H20 18.11 100% 0.721 Bt Control 19.96 110.33% -0.752 BEMD Endo BEMD 22.35 123.40% 0.354 Protease Example 4. Use of recombinant Bacillus cereus family members displaying an endoglucanase to stimulate plant growth in ryegrass.

BEMD spores expressing endoglucanase were created in an identical fashion as described above in Example 1. ial ryegrass was planted 6.4 mm deep in 10 cm deep 2015/050807 pots filled with standard loam topsoil. Spores, both l and BEMD expressing endoglucanase, were diluted to a concentration of 1x104/ml in 50 ml of water and d to each plant at planting. A water-only control was also included. Plants were grown under ideal light using T5 lamps, 54 watts, and exposed to 11 hours of light a day under controlled temperature conditions between 5.50C. Plants were watered to saturation every three days over the two week trial. At the end of two weeks, the height of each plant was measured, and measurements were normalized to control water only plants. s are shown in Table 20, together with the standard error of the mean.

Ryegrass grown in the presence ofBEMD spores displaying endocellulase grew significantly taller than control B.t. spore treated or water l ryegrass (statistical analysis assayed via a t- test).

TABLE 20.

H20 11.43 100.0% -0.137 Br l BEMD 12.78 111.9% 0.137 Endo Example 5. Use of recombinant Bacillus cereus family members displaying enzymes involved in the synthesis or activation of plant hormones to ate plant growth.

The BEMD system can also be used to display enzymes involved in the synthesis of plant es. For example, the plant hormone indoleacetic acid is a potent growth stimulator in plants. Indoleacetic acid is synthesized in viva from tryptophan by the enzymes tryptophan monooxygenase and indoleacetamide hydrolase. Indoleacetic acid and other auxin hormones can also be synthesized in viva from tryptophan and/or indole by the s nitrilase, tryptophan aminotransferase, indoleacetaldehyde dehydrogenase, indole pyruvate oxylase, amine oxidase, tryptophan decarboxylase, and tryptophan side chain oxidases.

The BEMD system can also be used to display enzymes involved in the modification of plant growth hormones into bioactive or inactive forms. For example, nitrilase can be expressed on the BEMD system to catalyze the conversion of indoleacetonitrile into the bioactive indoleacetic acid. Additionally, inactive forms of plant hormones, such as indoleacetonitrile can be added into the plant growth media with the BEMD-expressed nitrilase to e a gradual release of active hormone into the plant growth media. Many other inactive or less active forms of plant hormones can be modified using their corresponding enzymes.

Related plant growth hormones (auxins) e indolepyruvic acid, indoleacetaldoxime, indoleacetamide, indoleacetonitrile, indoleethanol, indole pyruvate, indolebutyric acid, phenylacetic acids, 4-chloroindoleacetic acid, and acetaldoxime. These hormones are synthesized from tryptophan and/or indole in vivo via the enzymes tryptophan monooxygenase, indoleacetamide hydrolase, nitrilase, nitrile hydrolase, acetolactate synthetase, alpha acetolactate decarboxylase, tryptophan aminotransferase, - 3- dehyde dehydrogenase, indolepyruvate decarboxylase, amine oxidase, phan decarboxylase, and tryptophan side chain oxidases.

Growth hormones of the cytokinin family can also be synthesized by enzymes expressed in the BEMD system. Examples of cytokinins include kinetin, zeatin (cis and trans), 6-benzylaminopurine, dihydroxyzeatin, —isopentenyl) e, ribosylzeatin, N6-(D2- isopentenyl) adenosine, 2 methylthio-cis-ribosylzeatin, cis ribosylzeatin, ribosylzeatin-S- monosphosphate, N6-methylaminopurine, N6-dimethylaminopurine, 2’-deoxyzeatin riboside, 4- ymethyl-transbutenylaminopurine, topolin, meta-topolin, benzyladenine, methyltopolin, and meta-methyltopolin. These plant growth stimulating compounds are synthesized in vivo from mevalonate or adenosine mono/di/triphosphate by enzymes including adenosine phosphate tenyltransferases, phosphatases, adenosine kinases, e phosphoribosyltransferase, CYP735A, 5 ucleotide phosphohydrolase, adenosine nucleosidases, zeatin cis-trans isomerase, zeatin O-glucosyltransferases, B-glucosidases, cis- hydroxylases, CK cis-hydroxylases, CK N—glucosyltransferases, 2,5-ribonucleotide phosphohydrolases, ine nucleosidases, purine nucleoside phosphorylases, and zeatin reductases.

Using methods similar to those described above in Example 1, any of these enzymes can be incorporated into the BEMD system for display on BEMD spores by creating a fusion construct comprising the enzyme and a targeting sequence that targets the expressed enzyme to the exosporium when the fusion construct is expressed in a Bacillus cereus family member. A recombinant Bacillus cereus family member expressing such a construct can then be added to the soil or other plant growth medium or applied directly to plant foliage using methods similar to those described above in Example 1 for stimulation of plant growth.

The plant growth medium can be supplemented with precursors or substrates for the enzymes. For e, the plant growth medium can be supplemented with tryptophan, adenosine monophosphates, adenosine diphosphates, adenosine triphosphates, or indole.

Suitable concentrations of these substrates are n 100 nM and 100 uM.

Example 6. Use of recombinant Bacillus cereus family members displaying proteases or peptidases that cleave proteins, peptides, proproteins, or preproproteins into bioactive peptides for stimulation of plant growth. ses and peptidases can be expressed in the BEMD system that can enzymatically cleave available proteins in the plant growth media to bioactive peptides that can act on the plant directly or indirectly. Examples include the enzymatic ge of n meal, yeast t, or other n rich meals added to the plant growth medium into active peptides that can directly stimulate plant growth. Bioactive peptides generated by enzymatic cleavage of protein meals include RHPP and RKN l6DlO, potent stimulators of plant root development. Additionally, proproteins or preproproteins can be cleaved into active forms by BEMD-expressed proteases and peptidases to their bioactive forms. Inactive proproteins or preproproteins can be added in the plant growth medium to facilitate their gradual cleavage by BEMD proteases and slow release of bioactive proteins.

Using methods similar to those described above in Example 1, any of these proteases and peptidases can be incorporated into the BEMD system for display on BEMD spores by creating a fusion construct comprising the protease or peptidase and a targeting sequence that s the expressed enzyme to the exosporium when the fusion construct is expressed in a Bacillus cereas family member. A recombinant Bacillus cereas family member expressing such a construct can then be added to soil or other plant growth medium supplemented with soybean meal, yeast extract, or another-protein-rich meal for ation of plant growth. The soybean meal, yeast extract, or other protein-rich meal is ly added to the plant growth medium in the form of a liquid composition comprising about lOug/L to about 100 mg/L of the protein meal, yeast extract, or other n-rich meal.

Example 7. Use of BEMD spores expressing the protease PtrB for stimulation of plant growth.

BEMD spores expressing E. coli protease PtrB were created as described above in Example 3. Soybean seeds were planted 2.54 cm deep in 10 cm deep pots filled with standard loam topsoil. Spores, both control and BEMD expressing protease, were diluted to a concentration of lxlO4/ml in 50 ml of water and d to each plant at planting. A water-only control was also ed. Soybean meal at 25 mg/pot was added in water at planting. Plants were grown under ideal light using T5 lamps, 54 watts, and exposed to 13 hours of light a day under controlled temperature conditions between 15.5—25.5°C. Plants were watered to saturation every three days over the one week trial. At the end of two weeks, the height of each plant was measured, and measurements were normalized to control water only plants.

Results are shown in Table 21, together with the standard error of the mean as a percentage of water l. Soy grown in the presence ofBEMD spores displaying protease grew significantly taller than l B. t. spore treated or water control soybeans stical analysis assayed via a ). The addition of soybean meal to water control or B. thuringiensz’s control plants had little effect. By contrast, in the presence of the soybean meal and the BEMD protease system, the soybean plants responded significantly over all other treatments.

TABLE 21. ent Soybean Meal Height (cm) Normalized to water SEM, as tage of water Water only 25 mg/pot 12.43 102.7% B. thuringiensis 12.52 103.5% BEMD Protease 12.97 107.2% BEMD Protease 25 mg/pot 14.44 119.3% Example 8. Use of recombinant Bacillus cereus family members displaying proteins or peptides involved in the stimulation of plant growth.

The BEMD system can also be used to display proteins or peptides that are directly involved in the promotion of plant growth. For example, plant peptide hormones or non- hormone peptides that stimulate plant growth can be expressed in the BEMD . For example, non-hormone peptides that ly bind to and active plant receptors can be expressed in the BEMD system to directly act on receptors in the plant and roots of target plants. Such peptide hormones and non-hormone peptides include phytosulfokine, va 3 (CLV3), systemin, RKN 16D10, Hg-Syv46, eNOD40, NOD family proteins, ZmlGF, SClVSPll family proteins and peptides, RHPP, POLARIS, and KTI. These peptides and related es can be sed in the BEMD system and delivered to plant growth medium or directly applied to foliage to stimulate plant growth.

] Using methods similar to those described above in Example 1, any of these proteins or peptides can be incorporated into the BEMD system for display on BEMD spores by creating a fusion construct comprising the enzyme and a targeting sequence that targets the expressed enzyme to the exosporium when the fusion construct is expressed in a Bacillus cereus family member. A recombinant us cereus family member expressing such a construct can then be added to the soil or other plant growth medium or applied directly to plant foliage using methods similar to those bed above in Example 1 for ation of plant growth.

Example 9. Use of BEMD spores expressing POLARIS or KTI for stimulation of plant growth.

BEMD spores expressing the plant e POLARIS and soy peptide KTI were created by synthesizing genes coding for the POLARIS or KIT peptides linked to the targeting sequence of SEQ ID NO: 96. The genes were then introduced genes into Bacillus thuringiensis and spores were made as described in Example 1. Soybean seeds were planted 2.54 cm deep in 10 cm deep pots filled with standard loam topsoil. BEMD spores expressing POLARIS or KTI were diluted to a concentration of lxlO4/ml in 50 ml of water and applied to each plant at planting. A water-only control was also included. Pure POLARIS and KTI peptides were also tested for their effects on soybeans at 0.05 mg/pot. Plants were grown under ideal light using T5 lamps, 54 watts, and d to 13 hours of light a day under controlled ature conditions between l5.5—25.5°C. Plants were watered to saturation every three days over the two week trial. At the end of two weeks, the height of each plant was measured, the roots measured, and ements were normalized to control water only plants.

Results are shown in Table 22, together with the rd error of the mean as a percentage of water control. Soy grown in the presence ofBEMD spores displaying POLARIS grew taller and had a slight increase in root development than water control soybeans. The presence of free KTI peptide led to a significant stunting of the plants, losing between 6—8% of their heights, but adding 15% to the length of the roots. Expression of KTI on the BEMD system led to the root growth benefit, but without the stunting effect on the plant height. Importantly, the presence of the Bacillus giensis control spores with the free KTI peptide did not prevent the ng effect of KTI, while the BEMD with KTI displayed no such stunting.

TABLE 22.

Treatment Peptide Roots SEM Height, Normalized Normalized to to Water Water Water 100% 100% Water KTI, 0.05 115% 8.4% 91.8% m_/Pot BEMD N0 106.3% 7.9% 107.3% POLARIS BEMD KTI 113.3% 99.4% B. thuringiensis KTI, 0.05 115% 7.7% 93.4% m_./ot Example 10. Use of inant Bacillus cereus family members displaying enzymes that degrade or modify a bacterial, fungal, or plant nutrient source to stimulate plant growth and/or s nutrients.

The BEMD system can also be used to display enzymes that degrade or modify beneficially a bacterial, fungal, or plant nutrient source present in soil or another plant growth medium. Such enzymes degrade products t in the soil or other plant growth medium into forms that can easily be taken up by plants and/or the beneficial bacteria and/0r fungi of the rhizosphere. Such enzymes include, for example, ide hydrolases to e complex carbohydrates, ases to degrade cellulose; lipases to degrade lipids, including oil, fats, and waxes; 1ignin oxidases to degrade 1ignin and humic acids; proteases to degrade polypeptides; phospholipases to degrade membranes; amidases and nitrogenases to r en; amylases to process starches; nucleases to recover nucleotides, pectinases to break down pectin, sulfatases to recover , and xylanases to break down xylans and oxylans. The resultant products, including simple sugars, amino acids, fatty acids, and other nutrients will be readily available for direct uptake by plants and/or for stimulating beneficial bacteria and/0r fungi to grow and thrive in the rhizospheres of the plants.

In addition, enzymes and other biological molecules can be utilized to release or sequester phosphate, nitrogen, and other key elemental nutrients for plant uptake from their s organic and inorganic forms in soil. For example, phosphatases can be used to degrade phosphates in the environment into usable inorganic phosphates for plant use. The phosphates can be naturally occurring phosphates present in a plant growth medium. Alternatively or in addition, the plant growth medium can be supplemented with phosphates such as trimetaphosphate, a common agricultural ent. Examples of useful atases include phosphoric monoester hydrolases, phosphomonoesterases, phosphoric diester hydrolases, phosphodiesterases, triphosphoric monoester hydrolases, phosphoryl ide hydrolases, pyrophosphatases, phytase, trimetaphosphatases, and triphosphatases. For example, the enzymes trimetaphosphatase, triphosphatase, and pyrophosphatase sequentially break down trimetaphosphate into usable inorganic phosphate.

The nitrogenase family of enzymes converts atmospheric nitrogen (NZ) into ammonia, thereby converting nitrogen that would otherwise be inaccessible to plants into a usable form. Suitable enzymes belong to the Nif family of nitrogenases.

Chemical energy can also be directly added into the plant growth medium as adenosine triphosphate, ferrodoxin, or additional enzymes that create such energy into the BEMD system. These are cofactors for the nitrogenases and are limited in soil. Thus, such cofactors can be added to soil to enhance the reactions described above.

Other ments that can be added to the plant growth medium e starches, cellulose and cellulose derivatives, pectins, xylans and arabinoxylans, fats, waxes, oils, phytic acids, lignins, humic acids, and other nutrient sources that the above enzyme classes exert activity upon.

Using methods r to those bed above in Example 1, any of these enzymes can be incorporated into the BEMD system for display on BEMD spores by creating a fusion construct comprising the enzyme and a targeting ce for ing the fusion construct to the exosporium of a Bacillus cereus family member. The fusion construct can then be expressed in a Bacillus cereus family , and this recombinant Bacillus cereus family member can be added to soil or another plant growth medium using methods similar to those described above in Example 1 for stimulation of plant .

Example 11. Use of BEMD spores expressing a phosphatase for stimulation of plant growth.

BEMD spores expressing Bacillus subtilis Phosphatase A4 (PhoA4) were created by synthesizing a gene coding for PhoA4 linked to the targeting sequence of SEQ ID NO: 96. This gene was then introduced into Bacillus thuringiensis and spores were made as in Example 1. Corn was d 2.54 cm deep in 10 cm deep pots filled with standard loam topsoil.

BEMD spores sing PhoA4, were d to a tration of lxlO4/ml in 50 ml of water and applied to each plant at planting. A water-only control was also included. osphate was added to pots in liquid at a rate of 0.5 mg/pot. Plants were grown under ideal light using T5 lamps, 54 watts, and exposed to 13 hours of light a day under controlled temperature conditions between 15.5—25.50C. Plants were watered to saturation every three days over the two week trial. At the end of two weeks, the height of each plant was measured, and measurements were normalized to control water only .

Results are shown in Table 23. Corn grown in the ce ofBEMD spores displaying PhoA4 exhibit enhanced growth, especially in the ce of added polyphosphate.

This effect was greater than the effect of the polyphosphate alone.

TABLE 23.

Additive Growth, Com arison to Water 100% Pol oohoshate 110.8% BEMD PhoA4 108.3% BEMD PhoA4 Pol oohoshate 114.8% Example 12. Use of recombinant Bacillus cereus family members displaying enzymes ed in the synthesis of 2,3-butanediol or the synthesis or activation of gibberellic acid for stimulation of plant growth.

The BEMD system can also be used display enzymes involved in the synthesis of the plant-growth promoting compound 2,3-butanediol. In vivo, tanediol is synthesized by beneficial bacteria and fungi in the rhizosphere from acetoin, yl, acetolactate, or pyruvate by the s acetolactate synthetase, (x-acetolactate decarboxylase, pyruvate decarboxylase, diacetyl reductase, butanediol dehydrogenases, and acetoin reductase.

The BEMD system can also be used to display enzymes involved in the synthesis or activation of the plant-growth promoting nd gibberellic acid. Gibberellic acid can be produced from inactive or less active forms Via the action of enzymes, including but not limited to ylamine reductases, 2—oxogluturate dioxygenases, gibberellin 2B/3B hydrolases, gibberellin 3-oxidases, and gibberellin 20-oxidases.

Any of these enzymes can be incorporated into the BEMD system for display on BEMD spores using methods similar to those described above in Example 1. A fusion construct can be ed that ses the enzyme and a targeting sequence that targets the enzyme to the exosporium when the fusion construct is expressed in a Bacillus cereus family member. The fusion construct is then expressed in a Bacillus cereus family member, and the Bacillus cereus family member is added to soil or another plant growth medium for stimulation of plant growth.

] To increase the effect of the enzymes displayed on BEMD, the soil can be supplemented with substrates for the enzymes. For example, the soil or other plant growth medium can be supplemented with acetoin, which is a substrate for acetoin reductase; pyruvate, which is a substrate for pyruvate decarboxylase; diacetyl, which is a substrate for diacetyl reductase; and/or acetolactate, which is a ate for acetolactate decarboxylase. Alternatively or in addition, the soil or other plant growth medium can be supplemented with less potent or inactive forms of gibberellic acid, which will converted into more active forms by the enzymes bed above in the soil or other plant growth medium. e 13. Use of recombinant Bacillus cereus family members displaying proteases for protecting plants from pathogens ] The BEMD system can also be used display proteases that protect plants from one or more pathogens. For example, certain bacterial pathogens can communicate between individual members via secretion of bacterial lactone homoserines or related ing molecules. Thus, proteases c for bacterial lactone homoserine signaling molecules can protect plants from such bacterial pathogens by disrupting communication between bacteria, a step essential for the bacteria to secrete toxins and upregulate virulence factors. Suitable proteases specific for bacterial e homoserine signaling molecules include endopeptidases and exopeptidases.

Proteases specific for bacterial lactone homoserine ing les can be incorporated into the BEMD system using methods similar to those described above in Example 1. A fusion construct can be prepared that comprises the protease and a targeting sequence that targets the protease to the exosporium when the fusion construct is expressed in a Bacillus cereus family member. The fusion construct is then expressed in a Bacillus cereus family member, and the Bacillus cereus family member is added to soil or another plant growth medium. The protease can then degrade the bacterial lactone homoserine signaling molecules, blocking a key step in the virulence of these organisms and thereby helping to protect the plant from these ens. Other proteases and peptidases work effectively in this capacity on the BEMD system as demonstrated above in Example 6 and 7. e 14. Use of recombinant Bacillus cereus family members displaying antimicrobial proteins and peptides for protecting plants from ens.

The BEMD system can also be used display enzymes that exhibit antibacterial and/or antifungal activities that can help protect plants from one or more pathogens.

For example, antimicrobial proteins and peptides such as bacteriocins, lysozymes (e.g., LysM), siderophores, avidins, streptavidins, conalbumin, n, lactoferrins (e.g., LfcinB), or TasA can all be sed in the BEMD system to exert their effect on bacterial and fungal ens of plants. Bacteriocins, albumin, conalbumin, lysozymes, and lactoferrin exert direct antimicrobial action on their targets, whereas phores, avidins, and streptavidins bind l essential nutrients that pathogens require for virulence. For example, the peptide LfcinB of lactoferrin, when expressed on the surface of the BEMD system would lyse bacteria cells that are susceptible to the lactoferrin peptides in the plant growth medium. These proteins and peptides have specific action on select microbes, and can selectively target a group of pathogens without obstructing all es in the plant growth medium.

Any of these proteins or es can be incorporated into the BEMD system for display on BEMD spores using methods similar to those described above in Example 1. A fusion construct can be prepared that comprises the enzyme and a targeting sequence that targets the enzyme to the rium when the fusion uct is expressed in a Bacillus cereus family member. The fusion construct is then expressed in a Bacillus cereus family member, and the us cereus family member is added to soil or another plant growth medium for protection of plants from one or more pathogens. e 15. Use of BEMD spores expressing antimicrobial peptides for protecting plants from bacteria.

Genes were sized that coded for either of two antimicrobial peptides, LfcinB (derived from bovine lactoferrin) and LysM (derived from chicken lysozyme), linked to a BclA targeting sequence (SEQ ID NO: 96), under the l of the BclA promoter (SEQ ID NO: 215). The genes were introduced into us thuringiensis BT013A and spores were made by growing an overnight culture of the transformed Bacillus in brain heart infusion broth, plating onto nutrient agar plates at 30°C and allowing to grow for 3 days. Spores were washed off the plates and rinsed 3X in PBS. Staphylococcus epidermidis cultures were grown overnight in TSB broth at 37°C. The overnight culture was then pelleted, washed in PBS, and resuspended in PBS at an Abs595 = 0.2. lxlO4 BEMD expressing the LysM or LfcinB peptides was incubated in the PBS with the S. midis for 3 hours at 37°C, with shaking. A control sample of S. epidermidis was left untreated (no BEMD ). After the 3 hour incubation, dilution plates of the S. epidermidis were made and incubated at 37°C overnight. S. epidermidis cultures were counted the next day, and percent killing quantified. In Table 24 below, a record of the killing activity was recorded. The BEMD sed peptides killed a significant number of S. epidermidis cells. This would directly translate into killing of bacteria on the rhizosphere, seed, or other plant material. The selection of peptides specific to n classes of bacteria can also skew the population of the microorganisms near the plant in a beneficial way, or can selectively target key pathogens.

Table 24.

% Killed 100% BEMD L sM BEMD LfcinB Example 16. Use of recombinant Bacillus cereus family members displaying enzymes for protecting plants from pathogens.

The BEMD system can also be used y enzymes that protect plants from one or more pathogens. For example, yeast and mold cell walls are degraded by enzymes such as B-l,3-glucanases, B-l,4-glucanases, B-l,6-glucanases, chitosanases, chitinases, chitosanase- like proteins, and lyticases. Bacteria cell walls are degraded by s selected from proteinases, ses, mutanolysin, stapholysin, and lysozymes. Each of these cell wall degrading s can be expressed on the BEMD system and added to plant growth medium for selective inhibition of pathogenic microbes in the rhizosphere.

The BEMD system can also be used to display enzymes or ns that protect plants from insect or worm pathogens, for example by suppressing insect and/or worm predation of d plants. Examples of such ns and enzymes of interest include endotoxins, Cry toxins, other insecticidal protein toxins, protease inhibitors, cysteine proteases, the CrySB protein, the Cry 21A protein, chitinase, se inhibitor proteins, protease inhibitor peptides, trypsin inhibitors, and arrowhead protease inhibitors.

] Any of these proteins or peptides can be incorporated into the BEMD system for display on BEMD spores using s similar to those described above in Example 1. A fusion construct can be prepared that comprises the enzyme and a targeting sequence that targets the enzyme to the exosporium when the fusion uct is expressed in a Bacillus cereus family member. The fusion construct is then expressed in a Bacillus cereus family member, and the Bacillus cereus family member is added to soil or r plant growth medium for protection of plants from pathogens.

Example 17. Use of BEMD spores expressing an antifungal enzyme for protecting plants, and demonstration of efficacy against Sacchromyces.

A gene was sized that encoded an antifungal enzyme, B-l,3-glucanase from Bacillus subtilis, linked to a BclA targeting sequence (SEQ ID NO: 96) under the control of the BclA promoter (SEQ ID NO: 215). The gene was and introduced into Bacillus thuringiensis BT013A and pores were made by growing an overnight culture of the transformed Bacillus in brain heart infusion broth, plating onto nt agar plates at 30°C, and allowing to grow for 3 days. Spores were washed off the plates and rinsed 3X in PBS. Saccharomyces cerevisiae es were grown overnight in YZ broth at 37°C. The overnight culture was then pelleted, washed in PBS, and resuspended in PBS at an Abs595 = 0.2. 1x104 BEMD expressing B-1,3-glucanase was ted in the PBS with the Saccharomyces for 1 hour at 37°C, with shaking. A l sample of Saccharomyces was left untreated (no BEMD spores). After the 3 hour incubation, dilution plates of the romyces were made and incubated at 37°C overnight. Saccharomyces cultures were d the next day, and percent killing quantified. In Table 25 below shows the killing activity of the BEMD spores expressing glucanase. The BEMD-expressed enzyme killed a significant number of Saccharomyces cells. This would directly translate into killing of fungal microorganisms on the rhizosphere, seed, or other plant material. The selection of proteins specific to certain classes of fungi can also skew the population of the microorganisms near the plant in a beneficial way, or can selectively target key fungal pathogens.

Table 25.

% Killed 100% 0% BEMD B_—1,3-lucanase 17% Example 18. Use of recombinant Bacillus cereus family members displaying plant immune system atory peptides or proteins for protecting plants from pathogens.

The BEMD system can also be used display plant immune system enhancer peptides and proteins. These proteins can be expressed on the outside of the BEMD spore and delivered into the plant growth medium to stimulate the plant immune system to allow the plant to protect itself from plant pathogens. Example proteins and peptides include harpin, u-elastins, B-elastins, systemins, phenylalanine ammonia-lyase, elicitins, defensins, cryptogein, and flagellin proteins and peptides. Exposure of plants to these proteins and peptides will stimulate resistance to many plant pathogens in .

Any of these proteins or peptides can be orated into the BEMD system for display on BEMD spores using methods similar to those described above in Example 1. A fusion construct can be ed that comprises the enzyme and a targeting ce that targets the enzyme to the exosporium when the fusion construct is expressed in a Bacillus cereus family member. The fusion construct is then expressed in a Bacillus cereus family member, and the Bacillus cereus family member is added to soil or r plant growth medium for protection of plants from pathogens.

Example 19. Use of recombinant Bacillus cereus family s ying a root or leaf binding protein or peptide to immobilize the recombinant Bacillus cereus family member on a root system of a plant or on plant .

Root and leaf binding proteins and peptides can also be incorporated into the BEMD system to allow the BEMD spores to be immobilized on a root system or on leaves of a plant. Display of such root or leaf g ligands on the BEMD spores allows for targeting of the spores to the root system of a plant or to uctures of the root system or to the leaves or to substructures of leaves to maintain the BEMD spores at an optimal location for other displayed biological les and enzymes to be effective.

For example, rhicadhesin is a root binding ligand that binds to root hairs.

Thus, display of rhicadhesin on the BEMD spores thus targets the spores to root hairs.

Additional proteins that could be utilized for selective binding to plant roots or leaves include adhesins, flagellin, omptins, lectins, pili proteins, curlus proteins, intimins, invasins, agglutinin, afimbrial proteins, TasA, or YuaB.

Such root or leaf g proteins and es can be incorporated into the BEMD system using methods similar to those described above in Example 1. A fusion construct can be prepared that comprises the root or leaf binding protein or peptide and a targeting sequence that targets the protein or peptide to the exosporium when the construct is expressed in a Bacillus cereus family member. The fusion construct containing the root or leaf binding ligand is then expressed in a Bacillus cereus family member. Such fusion constructs can be coexpressed with one or more additional fusion constructs comprising any of the beneficial s discussed herein (e.g., an enzyme involved in the sis of a plant e, an enzyme that degrades a nutrient source, or a proteases that protects a plant from a pathogen).

The recombinant Bacillus cereus family member is added to soil or another plant growth medium, or applied to the leaves of a plant. The root or leaf binding ligand targets the Bacillus cereus family member to the root system of the plant or to the leaves of the plant and immobilizes it there, thus ng the coexpressed fusion construct to exert its effects in close proximity to the root or leaf system.

Example 20. Use of recombinant Bacillus cereus family members displaying proteins or enzymes to enhance stress resistance of plants.

Proteins, peptides, and s that e stress resistance in a plant can be incorporated into the BEMD system and delivered to target plants via addition to roots, leaves, or the plant growth medium. During s of stress, plants release stress-related compounds, including aminocyclopropane-l-carboxlic acid (ACC), reactive oxygen species, and 5 others, resulting in a negative impact on plant growth. The BEMD system can be used to display enzymes that e such stress-related compounds, such as aminocyclopropane-l-carboxylic acid deaminase, superoxide dismutases, es, catalases, and other enzymes that act on reactive oxygen species. Such enzymes reduce the amount of these -related nds and allow plants to continue to grow and even thrive under stressed conditions.

Any of these ns or peptides can be incorporated into the BEMD system for display on BEMD spores using methods similar to those described above in Example 1. A fusion construct can be prepared that comprises the enzyme and a targeting sequence that targets the enzyme to the exosporium when the fusion construct is expressed in a Bacillus cereus family member. The fusion construct is then sed in a Bacillus cereus family member, and the Bacillus cereus family member is added to soil or to another plant growth medium or applied to the leaves of a plant for enhancing the stress resistance of a target plant.

Example 21. Preparation of BEMD spores expressing the protective enzyme catalase.

A gene was synthesized that encoded the protective enzyme se from Bacillus cereus linked to a BetA ing sequence (SEQ ID NO: 97) under the control of the BetA promoter (SEQ ID NO: 197). This gene was and introduced into Bacillus giensis . Spores were made by growing an overnight culture of the transformed Bacillus and wildtype strain in brain heart infusion broth, plating onto nutrient agar plates at 30°C, and allowing to grow for 3 days. Spores were washed off the plates and rinsed 3X in PBS. 3 drops of hydrogen peroxide was added to each spore pellet. The enzyme catalase converts the hydrogen peroxide into water and 02 gas. The control spores did not bubble, while the BEMD- catalase spores y did, demonstrating enzyme activity on the surface of the spores. Other protective enzymes can be displayed in a similar fashion and delivered to the plant to act upon free radicals produced during stress by the plants.

Example 22. Use of recombinant Bacillus cereus family members displaying proteins or enzymes that protect seeds or plants from an environmental stress. ns, peptides, and enzymes that protect a plant from an environmental stress can be incorporated into the BEMD system and red to target plants via addition to roots, leaves, fruit, or the plant grth medium. During periods of freezing, plants can be damaged by the effect of ice. The BEMD system can be used to display peptides, proteins, or enzymes that protect plants from such effects. For example, the BEMD system can be used to display choline dehydrogenases, which act by producing protective ts that protect the plant or seed from frost. Substrates for these s (e.g., choline and/or choline derivatives) can also be added to the plant growth medium. Addition of such substrates can enhance the amount of protectant ne and related chemistries) produced in the plant environment by the BEMD expressed enzymes. Betaine derivatives are known to protect seeds from cold stress. 6] Any of these proteins or peptides can be incorporated into the BEMD system for display on BEMD spores using s similar to those described above in Example 1. A fusion construct can be prepared that comprises the enzyme and a targeting sequence that s the enzyme to the exosporium when the fusion construct is expressed in a Bacillus cereus family member. The fusion construct is then expressed in a Bacillus cereus family member, and the Bacillus cereus family member is added to soil or to another plant growth medium or applied to the leaves of a plant for protecting the plant from environmental stresses and s.

Example 23. Enhanced expression of fusion constructs 0n the BEMD system by use of ed or alternative promoter elements.

The BEMD system can y a wide range of proteins, peptides, and enzymes using one or more of the targeting sequences described herein. Some of these targeting sequences have a high affinity for the exosporium which would be cial for fusion protein expression, but their low fusion protein sion level limits their use on the BEMD system.

For such fusion proteins and sequences, alternative high-expression sporulation promoters can be used instead of the native promoters.

For e, SEQ ID NO: 13 (amino acids 1—39 ofB. weihenstephensis KBAB4 gene 3572) provides a very effective N—terminal sequence for the delivery of proteins to the exosporium ofBacillus cereus family members, as shown in Table 26 below. All genes were synthesized in their complete form (including promoter s and regions coding for fusion proteins) as described herein. When the native promoter elements for B. weihenstephensis KBAB4 gene 3572 (SEQ ID NO: 217) were used to express a fusion protein comprising the targeting sequence of SEQ ID NO: 13 fused to a ctosidase enzyme (from E. c0li), a low level of fusion protein was expressed, leading to a reduction in enzyme activity on the surface of the spore. Enzyme activity was measure by the conversion of 0.5M o-nitrophenylgalactoside in solution over 10 minutes. Enzyme conversion was measured with a spectrophotometer at ABS540. Replacement of the native promoter elements of the B. weihenstephensis KBAB4 gene 3572 with the high-expression promoters of SEQ ID NO: 197 (B. cis BetA/BAS3290) or SEQ ID NO: 218 (B. weihenstephensis KBAB4 YVTN B-propeller protein) led to a dramatic se in the enzymatic activity of the spores. On the other hand, replacement of the native promoter elements for B. weihenstephensz’s KBAB4 gene 3572 with the er native to B. anthracis Sterne BAS1882 (SEQ ID NO: 216) led to a decrease in the tic activity of the spores. The expression level of the targeting sequence of SEQ ID NO: 13 fused to B- galactosidase was much lower (0.3 8X) when driven by the promoter of BAS 1 882 (SEQ ID NO: 216), and was greatly improved when driven from the BetA promoter (SEQ ID NO: 197) or YVTN n promoter (SEQ ID NO: 218).

TABLE 26.

Promoter Fusion Protein B-galactosidase activity on Fold Change BEMD system, ized SEQ IDNO:217 SEQ IDNO: 13—[3- 100% alactosidase alactosidase alactosidase alactosidase Example 24. Isolation and identification of plant-growth promoting bacterial strains.

Soil samples from rhizospheres of the iest and most resistant potato (Solanum tuberosum), yellow summer squash (Cucurbita pepo), tomato um lycopersicum), and pole bean (Phaseolus coccineus) plants were collected, diluted in e water, and spread onto nutrient agar plates. Bacterial isolates that demonstrated high growth rates and were able to be passaged and propagated were selected for further study. The selected strains were grown in minimal media (KH2P04 3 g, NazHPO4 6 g, NH4Cl 1 g, NaCl 0.50 g, MgSO4 7H20 0.15 g, CaClz 2H20 0.013 g, and glucose 1 g, per L dry weight). Overnight cultures (30°C) of selected strains were spun down, media decanted off, and resuspended in an equal amount of led water. Ten lettuce seeds per treatment were planted at a depth of 1 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were inoculated at planting in 4 cm pots with 0.5 ul of resuspended bacteria in water mixed into 10 ml of H20.

Ten ml of H20 was sufficient to deliver the bacteria into the 3 in3 (7.62 cm3) of soil as well as saturate the soil for proper ation of seeds. Plants were grown at temperatures between 65— 75°F (18—24°C) with 11 hours of light/day, and 5 ml of watering every 3 days. After one week, plant heights and leaf diameters, as well as l health of the plants were collected. Initial screening of rhizosphere isolates resulted in obtaining greater than 200 distinct species of bacteria and fungi from the phere of the four plants. Some of the bacterial species are 2015/050807 8 described in Table 27. Identified strains are indicated by their proper bacterial identifications.

Other strains are indicated by their unknown identification . Inoculants giving results near control (+/— 2%) were not included in the table.

TABLE 27 —ButterheadLettuce—— Bacterial Inoculant Avg. Height (cm) Uninoculated Paracoccus kondratz'avae 2 1 1 1.1% .05 NC35 B. aryabhattaz‘ CAP53 202-8% Bflexus BT054 136-1% Bacillus mycoz'des strain 2.17 120.4% .21 BT155 B. aryabhattaz‘ CAP56 3- nealsom'z' BOBA57 E- cloacae CAP12 'nknown 8 'nknown 122 'nknown 39 'nknown 402 ———I_("(“(“(“(“(“(“(“ 0] Bacterial strains that produced the greatest effect on the overall plant health and plant height in the initial lettuce trial were subjected to further identification. Bacterial strains were grown overnight in Luria Bertani broth at 37°C, and overnight cultures were spun down in a centrifuge. Media was decanted and the remaining bacterial pellet was subjected to chromosomal DNA isolation using the Qiagen ial Chromosomal DNA Isolation kit.

Chromosomal DNA was subjected to PCR cation of the 16S rRNA coding regions using the primers E338F 5’-ACT CCT ACG GGA GGC AGC AGT-3’ (SEQ ID NO: 298), E1099R A ’-GGG TTG CGC TCG TTG C-3’ (SEQ ID NO: 299), and E1099R B 5’-GGG TTG CGC TCG TTA C-3’ (SEQ ID NO: 300). PCR amplicons were purified using a Promega PCR purification kit, and the resultant amplicons were diluted and sent to the sity of Missouri DNA Core for DNA sequencing. DNA sequences were compared to the NCBI BLAST database of bacterial isolates, and genus and species were identified by direct comparison to known s. Top fied species are indicated in Table 27. In many cases, 16S rRNA DNA sequences were only able to delineate the genus of the selected bacterial strain. In cases where a direct identification was not forthcoming, additional biochemistry analyses, using methods standard in the field, were performed to differentiate strains at the species and strain levels, and are listed in Table 28.

TABLE 28 CAP12 cloacae kondratiavae aryabhattai mycoides aryabhattai nealsom' E. P. B. CAP53 B. BT155 B. CAP56 B. BOBA57 Urease Catalase Oxidase Nitrate Growth, 5% NaCl Growth, 7.5% NaCl Growth, 42°C Growth, 50°C Growth, H 5 , H 9 Acid, Cellobiose Acid, e Acid, Starch Example 25. Isolation and identification of additional growth promoting bacterial Soil samples from agricultural fields near Gas, Kansas were collected, diluted in sterile water, and spread onto nutrient agar plates. Bacterial isolates that demonstrated high growth rates and were able to be passaged and propagated were selected for further study. The selected strains were grown in minimal media 4 3 g, NagHPO4 6 g, NH4Cl l g, NaCl 0.50 g, MgSO4 7H20 0.15g, CaClz 2H20 0.013g, and glucose 1 g, per L dry weight). Overnight cultures (30°C) of selected s were spun down, media decanted off, and resuspended in an equal amount of distilled water. Corn seeds were coated with cial seed polymer mixed with water alone (1 .6 ul per seed total) or cial seed polymer containing selected bacterial strains (1 .6 ul per seed total). Coated seeds were planted in (3 inch) 7.62 cm diameter pots at a depth of 1 inch (2.54 cm) in loam top soil (Columbia, MO) that was sieved to remove large debris. Plants were grown at temperatures between l8—24°C °F) with ll hours of light/day, and 50 ml of watering at planting and every 3 days. After two weeks, plant heights and leaf diameters, as well as overall health of the plants were collected. For germination assays and determining 3 day root length, seeds were coated as indicated above and evenly dispersed at seeds per paper towel. The paper towels were wetted with 10 mls of water, rolled up, placed in a small plastic bag and incubated at 30°C or placed on a germination heat mat at 27—30°C 0F). Root measurements were recorded after 3 days. Initial ing of rhizosphere isolates resulted in obtaining greater than 100 distinct species of bacteria and fungi from the rhizosphere. Some of the bacterial species are described in Table 29. Identified strains are indicated by their proper bacterial fications.

TABLE 29 ——Treatments Avg. Height Avg. Root Length (2 weeks) (3 days) normalized to normalized to polymer control polymer control Bacterial Inoculant (%) (%) Polymer control B. des EEl 18 B. subtilis EEl48 'genesfaecalz’s EElO7 B. mycoz'des EEl4l B. mycoz'des BT46-3 B. cereus family member EEl28 B. thuringiensis BTOl3A Paenibacillus massill'ensz's BT23 B. cereus family member EE349 B. subtilis EE218 B. megaterz'um EE28l Bacterial strains that produced the greatest effect on plant health are bed in Table 29. Bacterial strains were grown overnight in Luria Bertani broth at 37°C, and overnight es were spun down in a centrifuge. Media was decanted and the remaining bacterial pellet was subjected to chromosomal DNA isolation using the Qiagen Bacterial somal DNA Isolation kit. Chromosomal DNA was subjected to PCR amplification of the 16S rRNA coding regions using the primers E338F 5’-ACT CCT ACG GGA GGC AGC AGT-3’ (SEQ ID NO: 298), ElO99R A 5’-GGG TTG CGC TCG TTG C-3’ (SEQ ID NO: 299), and ElO99R B 5’-GGG TTG CGC TCG TTA C-3’ (SEQ ID NO: 300). PCR amplicons were purified using a Promega PCR purification kit, and the resultant amplicons were diluted and sent to the sity of Missouri DNA Core for DNA sequencing. DNA sequences were compared to the NCBI BLAST database of ial es, and genus and species were identified by direct comparison to known strains. Top identified species are indicated in Table 16. In many cases, 168 rRNA DNA ces were only able to delineate the genus of the selected ial strain. In cases where a direct identification was not forthcoming, additional biochemistry analyses, using methods standard in the field, were performed to differentiate strains at the species and strain levels, and the entiated strains are listed in Table 30.

TABLE 30 148 EE281 family BT23 BT46-3 EE118 thurmgtenszs. EE349 EE family EE128 EE141 13A cereus B BTO subtilis megaterium mycoides es cereus mycoides B. member B. 3- B. Paenibacillus massiliensis B. Alcaligenesfaecalis B. B. member B.

Rhizoid Colon — Growth, 7.5% Wk - - NaCl Growth, 42°C — Growth, 50°C — Growth, H 5 - wk —_IIIIIIIIIIIIIIIIIlI-IIIIII —_---- wk = weak growth or low growth Example 26. Testing of plant-growth promoting bacterial strains on alfalfa.

The selected s were grown in minimal media (KH2P04 3 g, NagHPO4 6 g, NH4Cl l g, NaCl 0.50 g, MgSO4 7H20 0.15 g, CaClz 2H20 0.013 g, and glucose 1 g, per L dry weight). Overnight cultures (30°C) of selected strains were spun down, media decanted off, and bacteria resuspended in an equal amount of distilled water. Ten Zeba-coated alfalfa seeds were planted for each treatment at a depth of 0.6 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were inoculated at planting with 0.5 ul of resuspended bacteria in water mixed into 10 ml of H20. Ten ml of H20 was sufficient to deliver the bacteria into the 3 in3 (7.62 cm3) of soil as well as saturate the soil for proper germination of seeds.

Plants were grown at atures between 65—75°F (l8—24°C) with ll hours of light/day, and ml of watering every 3 days. a was allowed to grow for 1 week to analyze emergence and initial outgrowth of plants under described conditions. Identified strains ted by their proper bacterial identifications and final height data are listed in Table 3 1.

TABLE 31 ———_ Bacterial ant Avg. Height ison SEM (cm) ulated "— B. aryabhattaz’CAP56 101.20% B. nealsom'z'BOBA57 101.70% E. cloacae CAP12 116.23% Example 27. Testing of plant-growth promoting bacterial strains on cucumbers.

The selected strains were grown in minimal media (KH2P04 3 g, NagHPO4 6 g, NH4Cl l g, NaCl 0.50 g, MgSO4 7H20 0.15 g, CaClz 2H20 0.013 g, and glucose 1 g, per L dry weight). Overnight cultures (30°C) of ed strains were spun down, media decanted off, and resuspended in equal amount of distilled water. Ten cucumber seeds were d for each treatment at a depth of 1 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were inoculated at planting with 0.5 ul ofresuspended bacteria in water mixed into ml of H20. Ten ml of H20 was sufficient to deliver the bacteria into the 3 in3 (7.62 cm3) of soil as well as saturate the soil for proper germination of seeds. Plants were grown at temperatures between 65—75°F (l8—24°C) with ll hours of light/day, and 5 ml of ng every 3 days. Cucumbers were allowed to grow for 2 weeks to analyze emergence and initial outgrowth of plants under described conditions. Identified strains indicated by their proper bacterial identifications and final height data are listed in Table 32.

TABLE 32 —Cucumbers_— Bacterial Inoculant Avg. Comparison SEM Height (cm) ——__ W0 2016/044661 Example 28. Testing of plant-growth promoting bacterial strains on yellow squash.

The ed strains were grown in minimal media (KH2P04 3 g, NazHPO4 6 g, NH4Cl 1 g, NaCl 0.50 g, MgSO4 7H20 0.15 g, CaClz 2H20 0.013 g, and glucose 1 g, per L dry weight). Overnight cultures (30°C) of selected strains were spun down, media decanted off, and resuspended in an equal amount of distilled water. Ten yellow squash seeds were d for each treatment at a depth of 1 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were inoculated at ng with 0.5 ul of resuspended bacteria in water mixed into 10 ml of H20. Ten ml of H20 was sufficient to deliver the bacteria into the 3 in3 (7.62 cm3) of soil as well as saturate the soil for proper germination of seeds. Plants were grown at temperatures between 65—75°F (18—24°C) with 11 hours of light/day, and 5 ml of watering every 3 days. Squash was allowed to grow for 2 weeks to analyze emergence and l outgrowth of plants under described conditions. Identified strains indicated by their proper ial fications, final height data, and final leaf diameter (by span of the two leaves) data are listed in Table 33.

TABLE 33 —-_---Su uash Bacterial Inoculant Avg. Comparison SEM Leaf Comparison Height Diameter cm cm Uninoculated 10-16 _-_ B. aryabhattaz’CAP53 11.75 115.60% % B. flexus BT054 11.88 116.90% 125.20% Bacillus mycoz'des 11.92 117.20% .051 6.33 124.60% BT155 B. aryabhattaz’CAP56 11.95 117.60% 124.60% B. nealsom'z'BOBA57 11.89 117.00% 126.40% E. cloacae CAP12 11.42 % 134.40% e 29. Testing of plant-growth promoting bacterial strains on ryegrass.

The selected strains were grown in minimal media (KH2P04 3 g, NazHPO4 6 g, NH4Cl 1 g, NaCl 0.50 g, MgSO4 7H20 0.15 g, CaClz 2H20 0.013 g, and glucose 1 g, per L dry weight). Overnight cultures (30°C) of selected strains were spun down, media decanted off, and resuspended in an equal amount of led water. Thirty ryegrass seeds were planted for each treatment at a depth of 0.3 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were ated at planting with 0.5 ul of resuspended bacteria in water mixed into 10 ml of H20. Ten ml of H20 was sufficient to deliver the bacteria into the 3 in3 (7.62 cm3) of soil as well as saturate the soil for proper germination of seeds. Plants were grown at temperatures between 65—75°F (18—24°C) with 11 hours of light/day, and 5 ml of ng every 3 days. Ryegrass was allowed to grow for 1.5 weeks to analyze emergence and initial outgrowth of plants under bed conditions. Identified strains indicated by their proper bacterial identifications and height data are listed in Table 34.

TABLE 34 —Rerass — Bacterial Inoculant Avg. Height Comparison SEM (cm) Uninoculated —_.023 B. ar abhattaz' CAP53 124.70% .012 B. exusBT054 137.30% .034 Bacillusm coidesBT155 % .049 B. ar abhattaz'CAP56 % .009 B. nealsom'z'BOBA57 —142.40% .045 E. cloacae CAP12 % .015 Example 30. Testing of growth promoting ial strains on corn.

The selected strains were grown in minimal media (KH2P04 3 g, 4 6 g, NH4Cl 1 g, NaCl 0.50 g, MgSO4 7H20 0.15 g, CaClz 2H20 0.013 g, and glucose 1 g, per L dry weight). Overnight cultures (30°C) of selected strains were spun down, media decanted off, and resuspended in an equal amount of distilled water. Ten corn seeds were planted for each treatment at a depth of 2.5 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were inoculated at planting with 0.5 ul ofresuspended bacteria in water mixed into ml of H20. Ten ml of H20 was sufficient to deliver the bacteria into the 3 in3 (7.62 cm3) of soil as well as saturate the soil for proper germination of seeds. Plants were grown at temperatures between 65—75°F (18—24°C) with 11 hours of light/day, and 5 ml of watering every 3 days. Corn was allowed to grow for 2 weeks to analyze emergence and initial outgrowth of plants under described conditions. Identified strains indicated by their proper ial identifications and final height data are listed in Table 35.

TABLE 35 BacterialInoculant Avg. Height Comparison SEM Uninoculated _— B. aryabhattaz’ CAP53 11.01 123.60% .081 B. flexus BT054 w112.00% .095 Bacillus mycoides strain BT155 _ 107.90% .041 B. aryabhattaz’ CAP56 107.10% .088 B. m'l' BOBA57 103.70% .077 Example 31. Testing of growth promoting bacterial strains on soybeans.

The selected strains were grown in minimal media (KH2P04 3 g, NazHPO4 6 g, NH4C1 1 g, NaCl 0.50 g, MgSO4 7H20 0.15 g, CaClz 2H20 0.013 g, and glucose 1 g, per L dry weight, or for Bradyrhizobium or Rhizobium on yeast mannitol . Overnight cultures (30°C) of selected strains were spun down, media decanted off, and resuspended in equal amount of distilled water. Ten soybean seeds were planted for each treatment at a depth of 2.5 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were inoculated at planting with 0.5 ul of resuspended bacteria in water mixed into 10 ml of H20.

When g two bacterial strains, 0.5 ul of each resuspended bacteria was mixed into 10 ml of H20. Ten ml of H20 was sufficient to deliver the bacteria into the 3 in3 (7.62 cm3) of soil as well as te the soil for proper germination of seeds. Plants were grown at temperatures between 65—75°F (18—240C) with 11 hours of light/day, and 5 ml of watering every 3 days. Soybeans were allowed to grow for 2 weeks to analyze emergence and initial outgrowth of plants under described conditions. Identified strains indicated by their proper bacterial identifications and final height data are listed in Table 36. culation of bacteria strains in the present invention with members of the Bradyrhizobium sp. or ium sp. lead to an increase in plant growth compared to either inoculant alone.

TABLE 36 Bacterial Inoculant Avg. Comparison SEM Height ———089 -146 -177 -117 -133 -077 B. arjyabhattai CAP53 and 16.72 119.9% .182 Brad rhizobium So.

B. aryabhattai CAP53 and Rhizobium sp. 17.32 124.2% .086 Bradyrhizobium Sp. 14.25 102.2% ium sp. 14.75 105.8% Example 32. Bacillus cereus family s with plant growth promoting utes.

Bacillus mycoia’es strain BT155, Bacillus mycoia’es strain EE118, Bacillus mycoia’es strain EE141, Bacillus ’es strain BT46-3, Bacillus cereus family member strain EE349, Bacillus thuringiensis strain BT013A, and Bacillus megaterium strain EE281 were W0 2016/044661 grown in Luria Bertani broth at 37°C and overnight cultures were spun down, media decanted off, and ended in equal amount of distilled water. 20 com seeds were planted for each treatment at a depth of 2.5 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were ated at planting with 0.5 ul ofresuspended bacteria in water mixed into 50 ml of H20. Fifty ml of H20 was sufficient to deliver the bacteria into the 29 in3 (442.5 cm3) of soil as well as saturate the soil for proper germination of seeds. Plants were grown at temperatures between 65—72°F with 13 hours of light/day, and 5 ml of watering every 3 days.

Seedlings were allowed to grow for 2 weeks to analyze emergence and initial outgrowth of plants under described conditions. Identified strains indicated by their proper bacterial identifications and final height data are listed in Table 37.

TABLE 37 Bacteria] Inoculant Avg. Percentage Height, cm, Corn 11.41 100% 12.43 108.9% 12.84 112.5% 11.81 103.5% Bacillus thuringiensis 12.05 105.6% BT013A member EEl28 12.85 112.6% 11.99 105.1% All plant growth ing bacteria tested had a ial effect on corn height at two weeks under the described conditions. The Bacillus cereus family member EEl28 strain had the greatest effect in this trial, giving a greater than at 14% boost in corn height.

Example 33. Enhanced selection of Bacillus cereus family members to screen for plant growth-promoting and other beneficial activities as BEMD sion host.

The BEMD system can be used to y a wide range of proteins, peptides, and enzymes using any of the targeting sequences described herein to provide beneficial agricultural effects. Additional beneficial effects can be obtained by selecting an expression host (a Bacillus cereus family member) having inherent beneficial attributes. Many strains of members of the us cereus family have plant-growth promoting benefits. Additionally, many us cereus family member strains provide have protective effects, through direct idal, insecticidal, nematocidal, or other tive activities. By using such strains these as the sion host for the BEMD system, the end spore product would have a combination of positive benefits in agriculture.

Table 38 provides results for an experiment n a fusion protein was expressed in various Bacillus cereus family member strains. All strains are expressed a fusion protein comprising amino acids 1—35 of SEQ ID NO: 1 and the phosphatase PhoA4 from Bacillus is, a beneficial enzyme for enhanced phosphate uptake in corn. The gene was synthesized, cloned into the pMK4 vector, and introduced into each of the Bacillus spp. indicated in Table 38 below. Strains were taken into sporulation by incubation at 30°C on nutrient agar plates ning chloramphenicol 10 ug/ml for three days. Spores were collected, washed, and applied to corn at planting at a rate of 1x105 CFU/ml in 50 ml of water per 7.62 cm diameter pot with 5 mg polyphosphate per pot. Corn was grown in silt loam soil for two weeks.

Plants were grown under ideal light using T5 lamps, 54 watts, and exposed to 13 hours of light a day under controlled temperature conditions between 15.5—25.50C. Plants were watered to saturation every three days over a two week trial. At the end of two weeks, the height of each plant was measured and measurements were normalized to control Bacillus thuringiensis spores. sion of the SEQ ID NO: 1 — Phosphatase fusion protein led to an increase in corn height at 2 weeks regardless of the expression host strain selected. As shown in Table 38, use of a plant-growth promoting Bacillus cereus family member r increased corn height TABLE 38.

Bacillus Species Strain Fusion Protein Height at 2 weeks, Normalized B. thuringiensis Strain BT013A 100% B. thuringiensis Strain BT013A SEQ ID NO: 1-Phosphatase 117.4% B. 'es Strain EE141 107.3% B. mycoia’es Strain EE141 SEQ ID NO: 1-Phosphatase 123.3% B. cereusfamily Strain EE 128 None 124.1% member B. cereusfamily Strain EE128 SEQ ID NO: 1-Phosphatase 131.7% member B. mycoia'es Strain BT155 104.8% B. ’es Strain BT155 SEQ ID NO: 1-Phosphatase 121.9% Example 34. Use of various targeting sequences to s B-galactosidase on the surface of Bacillus thuringiensis.

A wide variety of ing sequences that that have a high degree homology with amino acids 20—35 of BclA (amino acids 20—35 of SEQ ID NO: 1) can be used to display s, ns, and peptides on the surface of Bacillus cereus family members. Several targeting sequences were compared by making fusion proteins ning the targeting sequences linked to Bacillus subtilis lipase. Fusion constructs were synthesized using the promoters native to the targeting sequence, cloned into the replicating plasmid pMK4, and introduced into Bacillus thuringiensis BT013A. Strains were taken into sporulation by incubation at 30°C on nutrient agar plates containing chloramphenicol 10 ug/ml for 3 days.

Spores were collected, washed, and resuspended in PBS at a rate of 1x108/ml. 1 X 105 spores for each fusion construct spores were suspended in 400 ul deO. The reactions were warmed with the reaction components to the desired reaction temperature (40°C). 200 ul g buffer was added (9:1 Solution A: Solution B). Solution A was 50 mM Tris pH 10 and 13.6 mM deoxycholic acid and Solution B was 3 mg/ml p-nitrophenyl palmitate in panol. The reaction was ted at 40°C for 10 minutes and placed on ice, centrifuged to remove spores, and absorbance at 420 nm was recorded. The results are shown in Table 39 below. ty was normalized to a control fusion protein comprising amino acids 1—35 of SEQ ID NO: 1 fused to Bacillus subtilis lipase.

TABLE 39.

Strain Enz me Relative activit B. giensis Lipase 100% BT013A SEQ ID \0: 1 B. thuringiensis Lipase 92.5% BT013A SEQ ID \0: 3 B. thuringiensis Lipase 13.5% BT013A SEQ ID \0: 7 B. thuringiensis Amino acid 1—24 of Lipase 24.8% BT013A SEQ ID \0: 9 B. thuringiensis Amino acid 1—33 of Lipase 98.5% BT013A SEQ ID\O: 13 B. thuringiensis Amino acid 1—33 of Lipase 107.8% BT013A SEQ ID\O:21 B. thuringiensis SEQ ID \0: 96 Lipase 137.1% BT013A B. thuringiensis Lipase 146.3% BT013A B. thuringiensis Lipase 115.7% BT013A B. thuringiensis Lipase 81.5% BT013A Several targeting sequences linked to lipase result in higher expression levels and activity of enzyme on the surface of spores. In particular, SEQ ID NOs. 96, 98, and 100, each containing a shorter targeting sequence, resulted in enhanced fusion sion on the surface of the BEMD spores. All the fusion proteins containing targeting sequences tested resulted in surface display of lipase.

Example 35. Use of various exosporium ces to express lipase 0n the surface of Bacillus thuringiensis and demonstration of fusion protein localization to the exosporium surface.

A wide variety of exosporium proteins can be used to display enzymes, proteins, and peptides on the surface of Bacillus cereas family members. Several different exosporium proteins were compared by making fusion proteins containing the rium proteins linked to Bacillus sabtz’lz’s lipase as described in Example 34. Fusion constructs were synthesized using the promoter native to the exosporium protein indicated in Table 40 below, cloned into the replicating plasmid pMK4, and introduced into Bacillus tharz'ngz'ensz’s BT013A.

Spores displaying the various exosporium protein-Bacillus sabtz’lz’s 168 lipase fusions were made by g the ormed bacteria in brain heart infusion broth with selective pressure from ug/ml chloramphenicol, plating onto nt agar plates, and incubating at 30°C for 3 days.

After 3 days, the spores were washed off the plates, purified by centrifugation, and resuspended in PBS at 1 x 108 CFU/ml. 1 X 105 spores for each fusion construct were resuspended in 400 ul deO.

The ons were warmed with the reaction components to the d reaction temperature (40°C). 200 ul of working buffer was added (9:1 on A: Solution B). on A was 50 mM Tris pH 10 and 13.6 mM deoxycholic acid and Solution B was 3 mg/ml ophenyl palmitate in isopropanol. The reaction was incubated at 40°C for 10 minutes and placed on ice, centrifuged to remove spores and absorbance at 420 nm was recorded. s are shown in Table 40 below. Activity was normalized to SEQ ID NO: 109 linked to lipase.

TABLE 40.

Strain Exosorium roteinmEnz me Relative activi BT013A BT013A B. thuringiensis SEQ ID NO: 113 Lipase 17.8% BT013A BT013A BT013A Use of the exosporium ns of SEQ ID NOS. 109 and 110 resulted in the highest enzyme activity on the spore. All the fusion proteins containing exosporium proteins resulted in surface display of active Bacillus subtilis 168 lipase, albeit at different levels. 8] Additional exosporium proteins were demonstrated to result in targeting of fusion proteins to the exosporium using the fluorescent reporter mCherry. Fusion constructs were created that contained the rium proteins of SEQ ID NOs. 111, 120, and 110 linked to the mCherry reporter. Spores were grown for 1.5 days, collected, and resuspended as described above. 7 ul of fluorescent spores were put under a Nikon E1000 microscope and imaged during late sporulation. Circular localization in a ring is tive of outer spore layer localization, and the appearance matches that of an exosporium n. Fluorescent microscopy s are shown in Figure 2. Panels A, B, and C of Figure 2 are fluorescent microscopy images of spores expressing fusion proteins comprising the exosporium proteins of SEQ ID NOs. 111, 120, and 110, respectively, and the y reporter. All three s demonstrated high levels of fluorescence and exosporium localization, demonstrating their potential utility for the expression of foreign proteins on the surface of the exosporium.

Example 36. Use of various targeting sequences and exosporium proteins to express phosphatase in Bacillus subtilis spores and effects of the phosphatase-expressing spores in soybeans.

BEMD spores expressing Bacillus subtilis EEl48 Phosphatase A4 (PhoA4) were created by gene synthesis of the genes coding for s ing sequences and rium proteins under the control of their native promoters linked to PhoA4. The sized genes were cloned into pMK4 and introduced into Bacillus thuringiensis BT013A.

Spores displaying the various exosporium protein-Bacillus subtilis EEl48 PhoA4 fusions were made by growing the transformed bacteria in brain heart infusion broth with selective pressure from 10 ug/ml chloramphenicol, plating onto nutrient agar plates, and incubating at 30°C for three days. After three days, the spores were washed off the , purified by centrifugation, and resuspended in PBS at 1 x 108 .

Soybeans were planted 2.54 cm deep in 10 cm deep pots filled with standard loam topsoil. BEMD spores expressing PhoA4 were diluted to a concentration of 1 X 104/ml in 50 ml of water and applied to each plant at planting. A water-only control was also included. osphate was added to pots in liquid at a rate of 0.5 mg/pot. Plants were grown under ideal light using T5 lamps, 54 watts, and exposed to 13 hours of light a day under controlled ature conditions between 15.5—25.50C. Plants were watered to saturation every three days over the two week trial. At the end of two weeks, the height of each plant was measured, and measurements were normalized to control water-only plants.

Results are shown in Table 41. Soy grown in the presence ofBEMD spores expressing fusion proteins containing PhoA4 linked to various targeting sequences and exosporium proteins with different fusion partners with PhoA4 all ted enhanced growth, but the extent of the effect varied depending on the targeting sequence or rium protein used.

TABLE 41.

Bacillus species Targeting sequence or Height at 2 weeks, exosporium protein Normalized linked to Ph0A4 H20 (No bacteria) N/A 100% Bacillus thuringiensis Amino acids 1—35 of 100% Strain BT013A SEQ ID \0: 1 Bacillus thuringiensis Amino acids 1—28 of 117.4% Strain BT013A SEQ ID \0: 3 Bacillus thuringiensis Amino acids 1—33 of 107.3% Strain BT013A SEQ ID \0: 21 Bacillus thuringiensis SEQ ID \0: 96 123.3% Strain BT013A Bacillus thuringiensis SEQ ID \0: 98 124.1% Strain BT013A Bacillus giensis SEQ ID \0: 109 131.7% Strain BT013A Bacillus thuringiensis SEQ ID \0: 110 104.8% Strain BT013A Example 37. Co-application of BEMD spores and seed treatments, liquid fertilizers, and other additives.

BEMD spores expressing fusion proteins were tested for compatibility with s seed treatments. The BEMD spores expressed fusion proteins comprising the targeting sequence of amino acids 1—35 SEQ ID NO: 1 linked to a phosphatase (PhoA4) from Bacillus subtilis EE148 or the POLARIS peptide. The synthesized genes were cloned into pMK4 and introduced into Bacillus thuringiensis BT013A. Spores displaying the various exosporium protein-Bacillus subtilz's EE148 PhoA4 or POLARIS s were made by growing the transformed ia in brain heart on broth with selective pressure from 10 ug/ml chloramphenicol, plating onto nutrient agar plates, and incubating at 30°C for three days. After three days, the spores were washed off the plates, purified by centrifugation, and ended in PBS at 1 x 108 CFU/ml.

Plants were grown under ideal light using T5 lamps, 54 watts, and exposed to 13 hours of light a day under controlled temperature conditions between 15.5—25.50C. Plants were watered to tion every three days over the two week trial. At the end of two weeks, the height of each plant was measured, and measurements were normalized to control water only plants. Results are shown in Table 42 below. Drench = applied to soil at 50 ml per pot. Polymer = ACCELERON seed g polymer only. BEMD spores were added at 1 x 104 cells/50 ml for drench applications. BEMD spores were added at 1.3X10 4/cells/seed for seed coating applications. 100 and 66 are standard commercial starter fertilizer compositions. 10 0 is liquid ammonium phosphate. 66 is low salt liquid phosphate fertilizer with an ortho/poly formulation. Colorant = Becker ood red seed coating coloring agent. MACHO, APRON, and CRUISER are commercial fungicides used on seeds. MACHO contains the active ingredient imidacloprid, APRON ns the active ingredient mefenoxam, and CRUISER contains a mixture of the active ingredients thiamethoxam, mefenoxam, and fludioxonil. The spores were found to be compatible with many seed applications and retained their y to stimulate plant growth in corn.

TABLE 42.

BEMD treatment Chemical Corn height at 2 weeks, normalized None None (Water Drench) 100% None Polymer Only 101 .3% BEMD PhoA4 N/A (Drench) 111.3% BEMD S N/A (Drench) 106.7% BEMD PhoA4 Polymer 109.3% BEMD POLARIS Polymer 107.3% BEMD PhoA4 Polymer + Colorant 102.3% BEMD treatment Chemical Corn height at 2 weeks, normalized BEMD PhoA4 Polymer -- MACHO 107.9% BEMD PhoA4 Polymer -- APRON 112.3% BEMD PhoA4 Polymer -- CRUISER 116.8% BEMD PhoA4 Polymer -- Colorant + 113.7% MACHO + APRON + CRUISER None 100 Starter 108.5% Drench BEMD PhoA4 100 Starter 114.7% Fertilizer Drench None 66 Starter 102.6% Fertilizer Drench BEMD PhoA4 66 Starter 112.9% Fertilizer Drench BEMD spores were found to be compatible with all seed coating amendments tested. There was a slight decrease in activity when BEMD PhA4 spores were combined with nt and polymer alone, but the spores regained full activity with colorant in ation with other fungicides. BEMD spores also worked well with liquid fertilizers. Starter fertilizers contributed to plant growth most likely through direct nutrient supplementation. BEMD spores worked with both starter fertilizers, suggesting that phosphatase activity can still lead to increased plant growth in the ce of excess nutrients. Combinations ofBEMD spores with fungicides exhibited greater increases in plant growth than BEMD spores alone, likely due to protection given to young corn plants during early growth.

Example 38. The use of the BEMD spores as a foliar addition for reducing stress inhibition of growth on corn.

The BEMD spore display system can be used to deliver enzymes that can ate some stress from growing plants in the field or greenhouse. To accomplish this, enzymes were selected that selectively act upon ve oxygen species in soil. ve oxygen species are a key marker of stress in plants.

BEMD spores expressing fusion proteins comprising the targeting sequence of amino acids 1—35 of SEQ ID NO: I linked to chitosanase, superoxide ase, catalase, or [31,3 glucanase from Bacillus thuringiensis BT013A were ted. The synthesized genes were cloned into pMK4 and introduced into Bacillus thuringiensis BT013A. Spores displaying the various protein fusions were made by growing the transformed bacteria in brain heart infusion broth with selective re from 10 ug/ml chloramphenicol, plating onto nutrient agar plates, and incubating at 30°C for three days. After three days, the spores were washed off the plates, purified by centrifugation, and resuspended in PBS at l x 108 CFU/ml.

Three week old corn plants at the V5 stage were grown under ideal light using T5 lamps, 54 watts, and exposed to 13 hours of light a day under controlled temperature conditions between 15.5—25.50C. Plants were watered to saturation every three days over the course of the trial. As the plants reach V5, BEMD spores or positive l chemicals were sprayed on the leaves at either 1 X 105 BEMD spores/ml or at the recommended rates for the chemicals. A total of 1 ml of spray was applied to each plant individually. Plant heights were taken just prior to the application of the foliar sprays. The corn plants were then stressed by warming to 322°C and decreasing ng to once per week. Plants were kept under stressed conditions for two weeks. At the end of the two weeks, plant heights were again measured, and visual appearance ed. Under these stressed ions, plant growth was minimal in control treatments. The ability to continue to grow under stressed conditions was ed by an increase in plant height over the two week span as compared to the only control.

Results are shown in Table 43 below.

TABLE 43.

Treatment Change in plant Height over 2 week stress Bacillus thuringiensis l ml/plant —l.6% BT013A s oores BEMD Chitosanase l ml/plant BEMD Chitosanase l ml/plant and 5 mM 4.7% and an BEMD Superoxide l ml/plant 8.3% Dismutase BEMD Bl,3 l ml/plant 4.9% Glucanase Salicylic Acid 1 ml/plant Benzothiadiazole l ml/plant 7.3% BEMD Catalase l nt Several destressing enzymes were applied to corn using the BEMD system, as shown in in Table 43 above. Control spores had no significant effect (decrease in plant height of —l .6%. The BEMD chitosanase enzyme had a positive effect when combined with its substrate, chitosan. The two best ming enzymes were BEMD B-l,3-glucanase and BEMD superoxide dismutase. BEMD B-l,3-glucanase has a primarily ngal activity, but can also have direct effects on plants. Salicylic acid and BTH were positive ls for the foliar assay, and ve responses were seen for both. This foliar delivery method can be used for delivering destressing enzymes to the plants at various times of the .

Example 39. Expression levels of fusion proteins using various Sigma-K containing promoters.

As shown in Example 23 above, replacing native promoter of a targeting sequence, exosporium protein, or exosporium protein nt can greatly affect the level of fusion protein expressed on the exosporium of a Bacillus cereus family spore. For example, replacing the native BclA promoter with the BclB promoter greatly reduces the level of fusion protein on the surface of Bacillus cereus family member spores. Alternatively, replacement of native BclB promoter with the BclA er increases fusion protein levels on the exosporium dramatically.

Relative promoter expression levels for various exosporium proteins under the control of their native sporulation promoters were obtained from microarray data from Bergman et al., 2008. The relative expression levels were determined during late sporulation timing (300 minutes after the start of the experiment), when sigma K promoters are most active.

Sigma K promoters are key promoters for expression of exosporium localized genes and ated ns. Relative expression is the increase in a gene’s expression level when compared to the average of all other genes of the chromosome at all given times. Table 44 below shows the relative expression levels of a variety of sigma K driven genes in Bacillus cereus family s.

TABLE 44.

Protein (Promoter SEQ ID NO.) (Fold increase in mRNA) Coto SEQ ID NO: 226 se SEQ ID NO: 225 EcIC SEQ ID NO: I79 SI-ma K SEQ ID NO: 227 BclA adjacent US Glycosyl transferase promoter 1 72.25 SEQ ID NO: 229 BclA adjacent DS yl transferase promoter 2 73.96 SEQ ID NO: 230 BclA SEQ ID NO: 215 77.44 Est SEQ ID NO: 220 32.49 Protein (Promoter SEQ ID NO.) Relative Expression (Fold increase in mRNA) Y'cA SEQ ID NO: 222 Y'cB SEQ ID NO: 223 70.56 BXoB/ExsFA SEQ ID NO: 224 30.25 InhA SEQ ID NO: 228 34.25 e 40. Preparation and testing of BEMD spores expressing a fusion n comprising a nitric oxide se, and use of such spores for stimulating germination of plant seeds.

BEMD spores expressing a fusion protein containing amino acids 20—35 of BclA, a 6-alanine linker, and the nitric oxide synthase enzyme from Bacillus subtilis 168 were generated. The nitric oxide synthase (NOS) enzyme from Bacillus subtilis 168 was gene synthesized in fusion to the BclA er, mal g site (RBS), start codon and amino acids 20—35 of BclA. A six-alanine linker region was included to separate the BclA targeting sequence from the NOS enzymes. The amino acids sequences of these fusion proteins, including the methionine encoded by the BclA start codon, amino acids 20—35 of BclA, the six- amino acid linker, and the NOS enzyme, are provided above in Table 9. These clones were subcloned in the shuttle vector pHPl3 via digestion with XhoI and ligation into the SalI site of pHPl3. Correct constructs were ced and verified, transformed into E. c0li cells. The resultant plasmids were transformed into Bacillus thuringiensis BT013A and Bacillus mycoia’es EEl 5 5.

The recombinant Bacillus thuringiensis BT013A and Bacillus mycoia’es EEl 55 transformed with the plasmids encoding the NOS fusion proteins were then induced to sporulate by swabbing the bacteria onto nutrient agar plates and incubating the plates at 30°C for 72 hours. After 72 hours, the bacterial spores were collected from the plate by swabbing into sterile phosphate buffered saline (PB S), and were purified by density centrifugation three times.

The spores were then applied to commercial corn and soy hybrid seeds at rates of 1x105 spores/seed. The soybean hybrid variety was BECK 335NR, which contains the cyst nematode protection gene, the ROUNDUP READY glyphosate resistance gene, and the K- gene for Phytophthora resistance. The corn hybrid variety was BECK 5540RR, which contains the P READY sate resistance gene. The seeds were then lightly dusted with L- arginine. A control set of seeds was dusted with L-arginine, but with no spores. Seeds were then placed between two paper towels, which were then wetted with 25 ml of H20. The paper towels were then rolled, placed into a small sandwich bag, and sealed y. These bags were then placed in a 30°C incubator and allowed to germinate for 24, or 48 hours. The number of seeds germinated at each timepoint was measured, and the results compared to untreated and control seeds. The results of these experiments are shown in Tables 45 and 46 below.

Table 45. se in germination rate in hybrid soybean seeds d with spores of recombinant Bacillus cereus family members expressing a fusion protein containing nitric oxide s nthase.

Germination Da 1 % Germination Da 2 % Naked so bean seed 15.0% 92.3% So bean seed olus L-Arinine 20.5% 94.9% Soybean seed plus B. 28.9% 97.5% thuringiensis BTO 1 3A sing B. subtilis NOS fusion orotein Soybean seed with L-arginine 30.0% 97.5% and B. mycoia’es EEl 55 ex ressin B. subtilis NOS Table 46. Increase in germination rate in hybrid corn seeds treated with spores of inant Bacillus cereus family members expressing a fusion protein ning nitric oxide s nthase.

Germination Da 1 % Germination Da 2 % Naked corn seed 0.0% 77.5% Corn seed plus nine 4.1% 80.5% Corn seed plus B. thuringiensis 6.5% 82.5% BT013A expressing B. subtilis NOS fusion rotein Corn seed with L-arginine and B. 4.3% 95.0% mycoia’es EEl55 expressing B. subtilis NOS As can be seen from Tables 45 and 46, treatment of seeds with L-arginine and a recombinant Bacillus cereus family member expressing a fusion protein sing a nitric oxide synthase enzyme led to an increase in the number of germinated seeds, in both soybeans and corn. e 41. Preparation and testing of BEMD spores expressing a fusion protein sing nucleic acid binding proteins BEMD spores expressing a fusion protein containing amino acids 20—35 of BclA, an eight-alanine linker, and the non-specific DNA binding protein SASPOL from Bacillus subtilis 168 or the non-specific DNA binding protein SASPy from Bacillus subtilis 168. DNA encoding SASPOL and SASPy was gene synthesized in frame with the BclA promoter, RBS, start codon BclA and amino acids 20—35 of BclA. An eight alanine linker region was included n the BclA targeting sequence and the A binding proteins. The linker allows for greater flexibility and protein folding of the fusion proteins. The amino acid sequences for these fusion proteins, including the methionine encoded by the BclA start codon, amino acids 20—35 of BclA, the eight-amino acid , and the SASPu or SASPy protein are provided above in Table ll. The synthesized genes were ed with Xhol, and ligated into the Sall site of pHPl3 to generate the plasmids pHPl3-BclA20—35- SASPu and pHPl3-BclA20—25- SASPy. pHPl3 is a well characterized 5.5 kbp shuttle vector plasmid having chloramphenicol and erythromycin resistance cassettes. It was constructed by the ligation of plasmids pE 1 94, pC 1 94, and pUC9.

Correct clones were subjected to DNA sequencing and transformed into the SCSl 10 strain of E. coli. The plasmid DNA was then purified, and transformed into the us giensis BT013A. These bacteria were then induced to sporulate by swabbing onto nutrient agar plates for 72 hours at 30°C. The spores were collected and purified as described above in the immediately preceding e.

To assess the ability of the recombinant spores to bind nucleic acids, the recombinant Bacillus cereus family members transformed with the plasmids encoding the SASPOL and SASPy fusion proteins were then incubated in PBS with random DNA primers that contained a cein tag on the 5’ ends. A control using non-recombinant spores was also included in the experiment. The spores were incubated for ten minutes with 50 mM tagged DNA, and then washed by centrifugation for one minute at 10,000 rpm. The supernatant was removed, and the spores were resuspended in 1 ml of PBS. The spores were again pelleted and the atant removed after centrifugation, and then subjected to analysis. The fluorescein- labeled DNA treated spores were examined under an E600 Nikon fluorescent microscope and DNA binding was determined by the change in the total fluorescence overall as compared to the control spores that did not n the DNA-binding fusion proteins. The results this assays are shown in Table 47 below.

Table 47. DNA g to recombinant Bacillus cereus family member spores expressing a fusion rotein com I risin_ a DNA bindin_ rotein Treatment DNA Bindin_ Normalized B. thuringiensis BTOl3A 100% s oores non-recombinant B. thuringiensis BTOl3A 341.2% spores expressing BclA- SASPOL fusion orotein B. thuringiensis BTOl3A 250.1% spores expressing BclA- SASP fusion orotein In addition, Figure 3 shows DNA binding to spores as measured by fiuorescein-labeled DNA binding. In Figure 3, ol” refers to non-recombinant B. thuringiensz’s BTOl3A spores (non-recombinant), “SASPa” refers to B. thurz’ngz’ensz’s BTOl3A spores sing BclA-SASPu fusion protein, and SASPc refers to B. thuringz’ensz‘s BT013A spores expressing BclA-SASPy fusion protein. 9] As can be seen from the data shown in Table 47 and Figure 3, the spores expressing the SASPu or SASPy fusion proteins bound a significantly greater amount ofDNA than the non-recombinant spores, demonstrating a strong affinity of these spores for DNA.

Example 42. Preparation and testing of BEMD spores expressing a fusion protein comprising a nuclease 0] In addition to the non-specific DNA and RNA g proteins discussed above in the immediately preceding example, nucleases can also be used to both bind to and cleave nucleic acid molecules. BEMD spores expressing a fusion protein containing amino acids —35 of BclA and an endonuclease enzyme were generated and assayed for their ability to bind to and cleave DNA.

The Bacillus subtilis endonuclease l was PCR ed and fused in frame to the BclA promoter, RBS, start codon and amino acids 20—35 of BclA. This construct was then cloned into the pHPl3 plasmid to create the plasmid pHPl3-BclA20—35-endonuclease. This construct was ced and transformed into and propagated in E. coli. The plasmid DNA was then isolated from the E. coli and introduced into Bacillus thuringiensis BTOl3A. Spores were created and purified as described in Example 40 above.

Endonuclease ty was assayed by incubating recombinant spores expressing the endonuclease fusion protein and non-recombinant control spores in PBS at a concentration of 1x108 spores/ml in PBS with 300 ng of salmon sperm DNA and l ug/ml DAPI (4’,6-diamidinophenylindole) DNA stain. The reaction was allowed to proceed continue for minutes at 37°C. After 10 minutes, the atant was assayed for cleaved DNA using a fiuorometer. As DNA is cleaved, the DAPI stain is released from the individual freed nucleotides, and thus cleavage can be determined by loss of DAPI staining over time. The results of this assay are shown in Table 48 below. 2015/050807 Table 48. Nuclease Activity and DNA binding by BEMD spores expressing an endonuclease fusion rotein Treatment Construct Loss of DNA Spore-bound DNA signal (fluorescence on su n ernatant BT013A S o ores BT013A S o ores The data provided above in Table 48 show that the clease fusion protein was expressed on the exosporium of the Bacillus thuringiensis BT013A spores, and was able to cleave the salmon sperm DNA as evidenced by the loss of DAPI signal in the atant. Surprisingly, a portion of the endonuclease bound the DNA tightly without cleaving it, retaining the DAPI fluorescence signal on the spores, even after washing the spores to remove excess DNA. This demonstrates that not all the DNA was processed, and that nucleases expressed on the outside of the spore can bind DNA tightly. To increase this effect, a nuclease having an inactivated active site could be used in the fusion protein, which would lead to less cleavage of the DNA and even more binding DNA on the spores.

Example 43. Agricultural use of spores expressing fusion proteins ning nucleic acid binding proteins or peptides The recombinant Bacillus cereus family or recombinant spore-forming bacteria members expressing fusion proteins comprising nucleic acid binding proteins or es can be used in agriculture to deliver nucleic acids to a plant growth medium (e.g., soil) and/or to plants. For example, the recombinant Bacillus cereus family members or recombinant spore-forming bacteria can be delivered to plants via seed treatment, in furrow/soil drench treatment, or foliar treatment. Furthermore, the fusion ns comprising nucleic acid binding ns or es can be expressed in any of the endophytic Bacillus cereus family s or any of the other endophytic Bacillus s described , enabling delivery of nucleic acids bound to the nucleic acid binding proteins internally to the plant, where they would be more effective in reaching their target cells. For example, the fusion proteins comprising nucleic acid binding proteins can be expressed in the endophytic strain Bacillus cereus family member EE349. Expression of another fusion protein (comprising ucanase as the protein of interest) in this strain is described in Example 51 below, demonstrating that the fusion proteins expressed in this endophytic strain are delivered internally to plants. Thus, expression of the fusion proteins comprising SASPu, SASPy, qu, or a nuclease having an inactivated active site in endophytic Bacillus cereus family member strains such as Bacillus cereus family member EE349 can provide a means to deliver RNA and DNA (e.g., RNAi or rDNA) internally to a plant. Other ecific binding nucleic acid binding proteins or peptides could also be used in the fusion proteins for this purpose.

Example 44. Preparation of BEMD spores that express a fusion protein and also overexpress a protein that modulates sion of fusion proteins.

Overexpression of various exosporium ns (referred to herein as “modulator ns”) in a recombinant us cereus family member expressing any of the fusion proteins described herein can modulate (increase or decrease) the expression level of the fusion protein. These modulator proteins include Est, ExsFA/BxpB, CotY, CotO, ExsFB, InhAl, InhA2, Est, Est, chA, Yj cB, Bch, AcpC, InhA3, alanine racemase l, alanine racemase 2, BclA, BclB, BxpB, BclE, BetA/BAS3290, CotE, Est, ExsK, Est, YabG, Tgl, superoxide dismutase l (SODAl), and superoxide dismutase 2 (SODAZ).

The ability to control the expression level of the fusion protein allows for control of the amount of the protein or e of interest of the fusion protein that is displayed on the outside of the spore of the inant Bacillus cereus family member. For example, when the protein or peptide of interest of the fusion protein comprises a plant growth stimulating protein or peptide (e.g., an enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source), the recombinant Bacillus cereus family member expressing the fusion protein produces a spore that when d to a seed, plant, or plant growth medium, has a beneficial effect on the plant due to the action of the plant growth stimulating protein or peptide. Modulation of the expression level of the fusion n results in modulation of the level of the e or protein of interest that is displayed on the outside of the recombinant Bacillus cereus family member spore. In some cases, increasing the level of fusion protein expression would be beneficial (e. g., where there is a desire to increase the sion of an enzyme and thereby se the amount of enzyme per spore that can be delivered to a plant). In other cases, decreasing the level of fusion protein expression would be beneficial (e.g., where there is a desire to decrease the expression of a n and thereby decrease the amount of n per spore that is delivered to a plant, for example, where high levels of the protein would have detrimental effects on the plant).

To generate plasmids for expression of fusion proteins in Bacillus cereus family members, PCR fragments were generated that contained the BclA er (SEQ ID NO: 85), start codon, and amino acids 20—35 of BclA fused in frame to either Bacillus subtilis 168 endoglucanase or the B-galactosidase gene from E. c0li DHSu. These PCR fragments were digested with XhoI and ligated into the SalI site of the pSUPER plasmid to generate the plasmids pSUPER-BclA 20—35-Endoglucanase and pSUPER-BclA 20—35-Bgal, respectively.

The pSUPER plasmid was generated through fusion of the pUC57 plasmid (containing an ampicillin resistance cassette) with the pBCl6-l plasmid from Bacillus (containing a tetracycline resistance). This 5.5 kbp plasmid can replicate in both E. c0li and us spp.

The pSUPER-BclA Endoglucanase and pSUPER-BclA 20—35-Bgal ds were transformed into and propagated in dam methylase negative E. c0li strains. The sequences of the pSUPER-BclA 20—35-Endoglucanase and pSUPER-BclA 20—35-Bgal plasmids were verified by DNA sequencing.

The -BclA 20—35-Endoglucanase and pSUPER-BclA 20—35-Bgal plasmids were transformed into the host strains Bacillus thuringiensis BT013A (for pSUPER- BclA 20—35-Endo) or Bacillus ’es BTlSS R-BclA 20—35-Bgal). These transformed strains expressed either the B-galactosidase enzyme or the endoglucanase enzyme on the outside of the spore.

To generate plasmids for overexpression of modulator proteins, PCR fragments containing the native er s for and genes ng ExsFA/BXpB, CotO, ExsFB, chB, Bch, AcpC, BclA, BclB, BxpB, and CotE were generated, digested with SalI, and ligated into the pHPl3 plasmid. The tide sequences for the native promoter regions are ed above in Table 3. The pHPl3 plasmid is a multicopy plasmid and therefore results in high sion levels of the encoded modulator proteins when the plasmids are transformed into a Bacillus cereus family member host cell. The pHPl3 plasmids containing the promoter regions and genes encoding ExsFA/BxpB, CotO, ExsFB, chB, Bch, AcpC, BclA, BclB, BxpB, BclE, BetA/BAS3290, and CotE are referred to herein as pHPl3-ExsFA/BxpB, pHPl3- CotO, pHPl3-ExsFB, pHPl3-chB, pHPl3-Bch, pHPl3-AcpC, pHPl3-BclA, pHPl3-BclB, pHP l 3-BxpB, and pHP l , tively.

The pHPl3 plasmids containing the promoter regions and genes encoding the modulator proteins were transformed into and propagated in E. c0li strains. The sequences of these plasmids were verified by DNA sequencing.

The pHPl3 plasmids encoding the modulator ns were transformed into Bacillus thuringiensis BT013A containing pSUPER-BclA 20—35-Endoglucanase or Bacillus mycoia’es BTlSS containing pSUPER-BclA 20—35-Bgal. The resultant recombinant bacteria were plated onto nutrient agar plates containing 10 ug/ml chloramphenicol to select for the pHPl3 plasmids and 10 ug/ml tetracycline to select for the pSUPER plasmids. Bacteria containing both plasmids were then grown in brain heart infusion broth overnight with both tetracycline and chloramphenicol. The overnight cultures were then swabbed onto nt agar, and bacteria were allowed to sporulate at 30°C for 72 hours. After 72 hours, the bacterial spores were collected from the plate by swabbing into sterile PBS, and were purified by density centrifugation three times. The pure spores were then diluted to 1x108 CFU/ml, and assayed for enzyme actiVity using on a population of 1x108 colony forming units (CFU). e 45. Enhanced or diminished expression of fusion proteins on the BEMD system by pression of a protein that modulates sion of the fusion uct The recombinant Bacillus mycoz'des EElSS spores generated as described above in the immediately ing example were assayed for B-galactosidase actiVity, and the recombinant Bacillus thuringiensis BT013A spores generated as bed above in the ate preceding example were assayed for endoglucanase activity.

B-galactosidase activity was assayed by measuring hydrolysis of the chromogenic substrate ortho-Nitrophenyl-B-galactoside (ONPG). A commercial source of B- galactosidase was used to prepare standards (0.2 ug, 0.4 ug, and 0.8 ug from a 100 ug/mL stock). 250 ul of spore preparation was pelleted and the spores were resuspended in 50 uL of enzyme dilution buffer (10 mM TRIS, pH 7.6, 0.2 M NaClz, 5% glycerol). 600 ul of prewarmed 37°C substrate mixture (10 mM KCl,l mM MgSO4°7 H20, 100 mM NaHzPO4, pH 7.5) containing 1.14 mg/mL ONPG was added to each sample and standard. Each reaction was incubated at room temperature for 2 s. 250 ul of 1M sodium carbonate was added to stop the reaction. The solution was centrifuged for 5 min at 14,000 X g to remove the spores from the absorbance reading. The absorbance was determined at 420 nm using an IMPLEN nanospectrophotometer model P330. Samples were performed in cate with a blank for each reaction. The results of this assay are shown below in Table 49.

Table 49. Effects of overexpression of exosporium proteins on the expression levels of BclA —35-5 al. rotein modulator rotein (Normalized) .SUPER-BclA 20B_al——100% SUPER-BclA 1-a1 SUPER-BclA 201-a1 SUPER-BclA 201-a1 pSUPER-301A 20Bga1 Plasmid encoding fusion Plasmid encoding Enzyme Activity rotein modulator rotein (Normalized) o SUPER-BCIA 20B_a1oHP13-BclB —132.6% l SUPER-BclA 20 3 _a1 oHP13-AcC 76.1% pSUPER-BclA 20-3 5-Bga1 pHP13-BxpB 103% As can be seen from results shown in Table 49, overexpression of CotO, CotE, BclA, BclB, and BxpB increased the sion of the fusion protein containing B- galactosidase, resulting in increased enzyme ty on the spores. By contrast, overexpression of YjGB or AcpC decreased the expression of the fusion protein ning B-galactosidase, resulting in decreased enzyme activity on the spores. 6] The assay for endoglucanase activity was performed by determining ase activity using a carboxymethylcellulose (CMC) ate and a osalicylic acid (DNS reagent). A commercial source of ce11u1ase enzyme was used to prepare standards in 50 mM citrate buffer, pH 4.8. 1% CMC (carboxymethylcellulose sodium salt) was prepared in 50 mM citrate buffer, pH 4.8 to serve as the substrate for the reaction. 250 ul of spore preparation was pelleted and the spores were resuspended in 150 uL of 50 mM citrate buffer, pH 4.8. The reaction was carried out with a reagent composed of 1% DNS, 1% NaOH, 0.05% , 0.2% phenol, and 18.2% Rochelle salts. 150 ul of the sample was mixed with 250 ul of the 1% CMC substrate and incubated in a water bath at 50°C for 15 minutes. 300 ul of DNS reagent was added and the samples boiled at 100°C for 10 minutes and then cooled on ice. The solution was centrifuged for 5 minutes at 14,000 X g to remove the spores from the absorbance reading. The absorbance was determined at 540 nm using an IMPLEN nanospectrophotometer model P330.

Samples were performed in triplicate with a blank for each reaction. The results from this assay are shown in Table 50 below.

Table 50. Effects of overexpression of rium proteins on the sion levels of BclA —35-Endo_lucanase.

Plasmid encoding modulator Enzyme Activity n rotein n rotein Normalized — — 0% pSUPER—BclA 20 — 100% endo lucanase pSUPER—BclA 20 pHP13-C0tO 215.7% endo se pSUPER—BclA 20 pHP13-CotE 125.5% endo lucanase pSUPER—BclA 20 pHP13-chB 89.3% endo lucanase pSUPER—BclA 20 pHP13-BclB 193.0% endo lucanase n rotein n rotein (Normalized) endo lucanase endo lucanase pSUPER—BclA 20 pHPl3-Bch 3.2% endo lucanase As shown in Table 50, overexpression of CotO, CotE, BclB, and BxpB increased expression of the fusion protein containing endoglucanase, resulting in increased enzyme ty on the spores. Overexpression of YjcB, AcpC, or Bch, on the other hand, decreased expression of the fusion protein, resulting in sed enzyme activity on the .

In sum, overexpression of CotO, CotE, BclB, or BxpB increased expression of both fusion proteins, ing in increased activity of both B-galactosidase and endogloconase on spores expressing the BclA 20—35-Bgal or BclA endoglucanase fusion proteins, respectively. pression of YjcB or AcpC on the other hand, decreased expression of both fusion proteins, resulting in decreased activity of B-galactosidase and endogloconase on spores expressing the BclA 20—35-Bgal or BclA 20—35-endoglucanase fusion proteins, respectively.

Overexpression ofBch and BclA20—35 tagged eGFP also decreased expression of the BclA —35-endoglucanase fusion protein, while overexpression of BclA increased expression of the BclA 20—35-Bgal fusion protein.

Example 46. Effects of BEMD spores sing a fusion protein and overexpressing a modulator protein on corn growth Application of recombinant Bacillus thuringiensis BT103A and Bacillus mycoia’es BT155 spores expressing a fusion protein comprising Bacillus subtilis 168 endoglucanase to corn results in increased seedling Vigor and growth response over the course of two weeks. Alternations in the expression level of the fusion protein comprising endoglucanase induced by overexpression of a modulator protein in such spores as described above in the immediately preceding e results in corresponding alterations in the s of the BEMD spores on corn .

To demonstrate this, pSUPER—BclA 20—35-Endoglucanase and the pHPl3- CotO or pHPl3-BclB were coexpressed in Bacillus thuringiensis BT013A. Spores were created on nutrient agar as described above in e 40. The spores were diluted to a concentration of 1x104 spores/50 ml water, and the 50 m1 of water was added to commercial hybrid corn seed in potting soil at planting. The corn hybrid variety was BECK 5540RR, which contains the P READY glyphosate resistance gene. The corn seeds were coated with a fungicide and a biological inoculant. 1] Plants were grown under artificial light for 14 hours a day and plant growth over a ten day period was determined. Plants were d every three days over the course of the experiment. After ten days, the plants were measured for height and normalized against the height of untreated corn plants. The results of these experiments are shown in Table 51 below.

Table 51. s of BEMD spores expressing a fusion protein comprising an endoloconase and overex n ressin_ a modulator rotein on h brid corn _rowth Plasmid encoding Plasmid Expression Strain Corn Growth fusion n encoding (Normalized to modulator pSUPER—BclA 20-35 protein Endoglucanase alone control) -Endolucanase BT013A -Endolucanase BT013A -Endolucanase BT013A As shown in Table 51, overexpression of the exosporium proteins CotO and BclB increased the effects of the BclA 20—35-endoglucanase fusion protein on corn seedling growth and vigor at 10 days. These effects ate with the expression levels of the fusion protein in BEMD spores expressing BclA 20—35-endoglucanase and pHPl3-CotO or pHPl3- BclB, indicating that the effects on seedling growth and vigor are attributable to the alteration of fusion protein expression levels by the modulator proteins e 47. Genetic inactivation of Bacillus cereus family s and use of such inactivated Bacillus cereus family members for expression of fusion proteins As described above, overexpression of germination spore protease (GPR) in its active form in the ore of a Bacillus cereus family member during sporulation results in proteolytic cleavage of ns in the forespore and inactivation of the spore. Similarly, overexpression of a non-specific endonuclease in the forespore during sporulation destroys the DNA in the spore, leading to an inactivated spore particle in a percentage of the spore population.

A plasmid encoding a non-specific endonuclease under the control of a sigma G promoter was generated. The non-specific clease 1 from Bacillus subtilis 168 and a sigma G promoter (SEQ ID NO: 235) were gene synthesized and ligated into the pHP13 plasmid using the Sall site to generate the d pHP13-SigG-nuclease. Correct clones were sequenced and transformed into and propagated in E. c0li cells. Plasmid DNA was isolated from the E. c0li cells and transformed into Bacillus thuringiensis BT013A. Correct clones were verified by PCR.

The amino acid sequence for Bacillus subtilis 168 clease l is provided above in Table 4.

Bacillus thuringiensis BT013A cells sing the sigma G endonuclease were created and purified on nutrient agar plates as described above in Example 40. Spores were quantified visually using a hemocytometer, d, and dilution plated onto nutrient agar plates.

The ratio of live spores to killed spores was calculated by determining the change from visual counting to plate counts. Control spores (untreated) were included in each assay. Additionally, lxlO8 spores were UV irradiated for 10 minutes using a handheld UV lamp, and the assay repeated. The visual count and plate count were again compared to assess spore killing. The results from these assays are shown in Table 52 below.

Table 52. Viability of Bacillus cereus family member spores expressing a non-specific nuclease under the control of a sima G romoter UV Live Ratio Bacillus thurin iensis BTOl3A 100% 61.3% Bacillus thuringiensis BT013A 70.4% 24.5% ex .ressin: SiG-endonuclease As can be seen from Table 52, expression of clease 1 under the l of a sigma G promoter sed cell viability by about 30% in spores that were not exposed to UV irradiation and by about 75% in spores that were exposed to UV irradiation.

Co-expression of both a ation spore protease and a nonspecific endonuclease under the control of sigma G promoters would be expected to further decrease spore viability.

Example 48. Preparation of exosporium fragments from inant Bacillus cereus family s comprising a knockout 0f the CotE gene.

The plasmid pUCpE was constructed that contained the pUCl9 backbone which is able to replicate in E. c0li, as well as the origin of replication omycin resistance cassette from pE194. This construct is able to replicate in both E. c0li and Bacillus spp. A 1 kb DNA region that corresponding to the upstream region of the CotE gene and a 1 kb region ponding to the downstream region of the gene CotE were PCR amplified from Bacillus anthracis ASteme. The two 1 kb regions were then spliced together using splicing by overlapping extension via 15 bp homologous overhangs that corresponded to the opposing PCR amplicons. This 2 kb fragment was digested with Xhol (in external primers) and ligated into the Sall site of pUCpE. This plasmid construct was d by ion and DNA sequencing. A Gram-positive omega-kanamycin resistance gene was digested with BamHI and placed between the two l-kb regions. The final construct was again PCR verified and sequenced, and the final plasmid was introduced into Bacillus anthracis ASteme. Correct clones were screened by looking for both erythromycin resistance and kanamycin resistance.

Clones were passaged under high temperature (40°C) in brain heart infusion broth in the presence of kanamycin (25 ug/ml) and were routinely struck for isolation onto LB agar plates containing kanamycin and grown at 30°C. Individual colonies were icked onto LB agar plates containing omycin 5 ug/ml and grown at 30°C. Clones that maintained kanamycin resistance but lost erythromycin resistance (signifying loss of the d but recombination and removal of the CotE gene) were grown in brain heart infi1sion broth plus kanamycin, and chromosomal DNA was isolated using a Qiagen Chromosomal DNA isolation kit. Proper deletion of the CotE gene was ined by PCR cation of the CotE gene region and loss of CotE, and gain of the cin resistance cassette.

A construct was generated (pHPl3-AcpC-eGFP) that encoded the rium protein Ach (acid phosphatase) fused in frame to the fluorescent reporter protein eGFP (enhanced green fluorescent protein). The pHP13-Ach-eGFP construct included the native Ach promoter, ribosomal binding site, and coding sequence for Ach (from B. anthracis ASteme), fused in frame to eGFP (from pGFPuv). This construct was generated by PCR cation of the individual AcpC and eGFP genes with corresponding primers that contained a 15 bp overlapping region corresponding to the alternate ons. . The two PCR ons were then purified, and combined into a second PCR reaction using external primers that contained XhoI sites. The two amplicons prime each other with their compatible ends, and create a filsion PCR ons, that were purified and digested with XhoI for 1 hour at 37°C.

The spliced PCR product was cloned into the SalI site of pHP13, and correct clones were ce verified and transformed into SCSI 10 E. coli. The plasmid DNA was subsequently isolated from the E. coli and introduced into B. anthracis ASteme CotE::Kan ted as described above, which was grown in brain heart infusion broth containing 10 ug/ml chloramphenicol overnight at 30°C. One iter of this culture was inoculated into nutrient broth (50 ml) in a baffled flask and grown at 30°C for 3 days. Spores were collected via centrifugation at 10,000 X g for 5 minutes, and the supernatant (containing the broken exosporium fragments) was filtered through a 100,000 Da membrane filter to obtain purified exosporium fragments containing the fusion proteins. 1] A transmission electron micrograph showing the CotE knockout spores is provided in Figure 4. The closed arrows indicate fragments of exosporium that have been separated from the spores, and the open arrow indicates a spore from which the exosporium has been removed.

The purification of the exosporium fragments was performed as follows: CotE: :kan spores were grown in brain heart infusion broth overnight at 30°C and swabbed onto nutrient agar plates and grown at 30°C for 3 days. After 3 days, the spores were collected by swabbing the plates with cotton swabs wetted with PBS and resuspended into 1 ml of PBS in a microcentrifuge tube. The spores were separated from the culture by centrifugation, and supernatant ning the exosporium fragments filtered through a 0.22 uM filter to remove any residual spores. The filtrate was then filtered through a 100 kDa filter to t exosporium nts but allow free proteins to pass through the filter. The 100 kDa filter was washed, and the ted exosporium fragments boiled in SDS buffer for 5 s and separated by SDS- PAGE electrophoresis. Figure 5 provides a photograph of an SDS-PAGE gel showing the purified exosporium fragments (lane 2) and a protein marker standard (lane 1). The exosporium fragments shown in lane 2 represent the individual ns that tute the exosporium fragments. Only a subset of bands that would normally be seen in a whole spore SDS-PAGE preparation are apparent.

Ten microliters of the exosporium fragment preparation containing the AcpC- eGFP fusion protein was tested for activity in a phosphatase assay against pNPP (p-nitrophenyl polyphosphate). Acid phosphatase activity was detected by spectrophotometry based on release of p-nitrophenol from phosphate through phosphatase activity. Briefly, 1 ml of 10 mM pNPP in phosphate buffer at pH 6.0 was incubated with exosporium nts in a 1 ml microcentrifuge tube and allowed to incubate at 37°C for 10 minutes. After 10 minutes, the tube was centrifuged for 1 minute to remove excess spores, and the supernatant read on a spectrophotometer at 420 nm for free p-nitrophenol. It was found that the purified exosporium fragments were able to effectively release the phosphate groups from pNPP, trating that the Ach was present in the exosporium fragments. The results of this assay are shown in Figure 6. In Figure 6, “CotE l spores” refers to CotE knock-out spores alone (not expressing the GFP fusion protein), “CotE Acp-eGFP” refers to the CotE knock-out spores expressing the GFP fusion n, and “CotE AcpC-eGFP fragments” refers to the exosporium fragments obtained as described above from the CotE knock-out spores expressing the AcpC-eGFP fusion protein.

These results demonstrate that mutations that disrupt the exosporium, such as a out mutation in the CotE gene, can be used to generate exosporium fragments that are substantially free of spores, and demonstrates that these exosporium fragments contain fusion proteins that are targeted to the exosporium.

Example 49. Expression of fusion proteins in recombinant Bacillus cereus family members that are capable of ing herbicides, and use of such recombinant Bacillus cereus family members for stimulation of plant growth Recombinant Bacillus cereus family members expressing fusion proteins can have potent effects on plant health and growth, as illustrated, for example, in Examples 1—4, 7, 9, ll, 33, 36, 37, and 38 above. The fusion proteins comprising a targeting sequence, an exosporium protein, or an exosporium protein fragment described herein can be used in a number of different species and strains within the Bacillus cereus , which includes Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoia’es, us pseudomycoia’es, Bacillus samanii, Bacillus gaemokensis, Bacillus weihenstephensis, and Bacillus t0y0iensis. Many members of the Bacillus cereus family are potent degraders of organic and inorganic material in the environment, and some us cereus family members have the y to degrade herbicides. Expression of the fusion proteins in such strains would be advantageous since this would provide herbicide degrading activity, thereby alleviating the stress on plants that can be caused by the use of herbicides, in addition to the ability to stimulate plant growth or confer other benefits to plant health, depending on the peptide or protein of interest selected for inclusion in the fusion n.

Bacillus cereus family member EE349 was isolated, identified, and terized as described above in e 25, and was found to have the ability to ate plant . This strain has further been found to have the ability to degrade multiple herbicides, including sulfonylureas and aryl triazines.

To trate the ability of Bacillus cereus family member EE349 to degrade ides, 1x105 Bacillus cereus family member EE349 spores were coated onto lentils planted into soil containing various concentrations of sulfentrazone. The seeds were allowed to grow at 24°C for 3 weeks on a 13 hour day/night cycle, with watering every 3 days. After 3 weeks, the plants were measured for root . A control set of seeds Without us cereus family member EE349 was planted under cal conditions.

The results of this experiment can be seen in Figure 7. In Figure 7, “protected” refers to seeds treated with Bacillus cereus family member EE349, and WO 44661 “unprotected” refers to untreated seeds. The y-axis shows the root length normalized against a water-only control. Figure 7 shows that as the concentration of the herbicide was increased, the inhibition of root growth also increased. However, application of Bacillus cereus family member EE349 to seeds ated the majority of this inhibition, even at full strength of the herbicide in soil. Thus, as can be seen from Figure 7, Bacillus cereus family member EE349 can act as a safener.

Moreover, the ability of Bacillus cereus family member EE349 to express fusion proteins is demonstrated in Example 51 below. Thus, Bacillus cereus family member EE349 can be used as a dual-purpose safener and host for sion of the fusion proteins comprising a targeting ce, an exosporium protein, or an exosporium protein fragment that targets the fusion n to the exosporium.

Example 50. Preparation of recombinant Bacillus cereus family members that overexpress exosporium enzymes and effects of such recombinant Bacillus cereus family members on plants The exosporiums ofBacillus cereus family members naturally contain various l enzymes that can have beneficial effects on plants. For e, the exosporiums ofBacillus cereus family members contain enzymes involved in nutrient solubilization (e.g., acid phosphatases such as AcpC), inosine uridine hydrolases, proteases (e.g., metalloproteases such as InhAl , InhA2, and InhA3), enzymes that catalyze the degradation of free ls (e. g., superoxide dismutases such as SODAl and , arginases, and alanine racemases. Overexpression of such enzymes in us cereus family s can provide recombinant Bacillus cereus family s that will have beneficial effects when applied to seeds, plants, a plant growth media, or an area surrounding a plant or a plant seed.

The metalloproteases InhA2 and InhA3, acid phosphatase (AcpC), and superoxide dismutase l and 2 were PCR amplified with their native promoters with primers that ned XhoI sites (amino acid sequences for InhA2, InhA3, AcpC, SODAl and SODA 2 are provided above in Tables 1 and 2, and nucleotide sequences for the native promoters for these proteins are provided above in Table 3). The PCR products were digested with XhoI, and cloned into the E. c0li/Bacillus shuttle vector pHPl3 via its SalI site. Correct clones were verified by PCR and DNA sequencing. The plasmids were introduced into Bacillus giensis BTOl3A and Bacillus mycoia’es EElSS. Correct clones were screened by plating onto LB agar plates containing chloramphenicol. Overnight cultures of correct clones were grown in brain heart infusion broth containing chloramphenicol, and 1 ml of this overnight culture was inoculated into 50 ml of nutrient broth and cultured for 3 days at 30°C. Sporulation was verified via light microscopy. Spores were then subjected to enzymatic assays.

Bacillus mycoia’es EElSS spores pressing AcpC (i.e., spores containing the pHPl3-AcpC (acid phosphatase) d) were assayed for phosphatase activity. One milliliter of the sporulation culture pelleted and the pellet was resuspended in 1 ml of PBS, and tested for activity in a phosphatase assay t pNPP (p-nitrophenyl polyphosphate) as described above in Example 48. The AcpC overexpressing spores had a much higher phosphatase activity, as illustrated in Figure 8. In Figure 8, the y-axis shows units of phosphatase activity, indicated by the release of p-nitrophenol.

The increased acid phosphatase activity observed for the Bacillus mycoia’es EEl 55 spores modified to press AcpC can solubilize nts in the environment upon the addition of such spores to a plant growth medium or application of such spores to a plant seed, a plant, or an area surrounding a plant or a plant seed. Since phosphate is a very important nutrient for plant growth and development, this can increase plant growth and provide beneficial effects on plant health.

Similarly, superoxide ase is a very powerful antioxidant protein.

Overexpression of a superoxide dismutase in a Bacillus cereus family member would provide spores having the ability to degrade free radicals, which exert stress on plants. Removal of the free ls would alleviate some of this stress and lead to increased plant vigor under stressful conditions. Bacillus thuringiensis BT013A spores overexpressing SODAl and SODA2 (i.e., spores transformed with the pHPl3-SODAl and pHPl3-SODA2 plasmids, tively) can be subjected to tic analysis. One iter of the sporulation culture can be pelleted and the pellet and resuspended in 1 ml of deO containing xanthine. Xanthine oxidase can then be added to the reaction mixture, as well as cytochrome C. Inhibition of the degradation of cytochrome C in this assay indicates activity of the superoxide dismutase.

] Bacillus ’es EElSS spores pressing a zinc metalloprotease (i.e., spores transformed with the pHPl3-InhA2 d) were subjected to enzymatic analysis. One milliliter of the sporulation culture was pelleted and the pellet was resuspended in 1 ml of PBS.

The spores were then reacted with 0.5% ein, a protease substrate, for 5 minutes. These reaction mixtures were precipitated with TCA (trichloroacetic acid) to remove undigested casein, and the absorbance of the remaining free azo dye was read at ABS595. The spores overexpressing InhA2 generated 211% more protease activity as compared to non-recombinant Bacillus mycoia’es EEl 55 spores.

Examples 3 and 7 above illustrate that expression of a protease on the exosporium of a Bacillus cereus family member can provide beneficial s on . The Bacillus giensis BT013A spores lnhAl, lnhA2, or lnhA3 would have similar effects upon introduction into a plant growth medium, or application to plant seeds, plants, or an area surrounding a plant or a plant seed.

Example 51. Expression of fusion proteins in an endophytic Bacillus cereus family strain Bacillus cereus family member EE349 was found to have the ability to grow endophytically and to be capable as serving as a host strain for the BEMD system. To demonstrate the ability of Bacillus cereus family member EE349 to grow endophytically and to serve as a host strain for the BEMD system, Bacillus cereus family member EE349 was transformed with the pSUPER—BclA 20endoglucanase plasmid (described above in Example 44). Spores were made and purified as described above in Example 40.

These spores were diluted to a concentration of 1x105 spores/50 ml water, and the 50 ml of water was then added to commercial hybrid corn seed in g soil at planting.

The corn seeds were coated with a ide and a biological inoculant. The corn hybrid variety was BECK 5475RR, which contains the ROUNDUP READY glyphosate resistance gene and AQUAMAX drought resistance gene. Plants were grown under artificial light for 14 hours a day and plant growth over a ten day period was determined. Plants were watered every three days over the course of the experiment. After ten days, the plants were ed for height and normalized against the height of untreated corn plants. The results of these ments are shown in Table 53.

Table 53. Effects of an endophytic Bacillus cereus family member expressing the BclA 20- olucanase fusion rotein on corn seedlin_ _rowth sion Strain Corn Growth Normalized 100% EE349 endo lucanase EE349 As can be seen from the data shown in Table 53, expression of the pSUPER— BclA 20endoglucanase in the endophytic strain Bacillus cereus family member EE349 resulted in increased corn growth as compared to untreated plants, or plants treated with Bacillus cereus family member EE349 alone.

Bacillus cereus family member 349 expressing the BclA 20 endoglucanase was then isolated from the inside of the corn plants. The ten day old plants were extracted from the soil and washed to remove excess debris. The plants were then inverted, d to 5% bleach for ten minutes, washed in water, exposed to hydrogen peroxide (10%) for ten minutes, washed again in water, and the stalks split with a sterile razor blade. The split halves of the stalks were placed face down onto nutrient agar plates for two hours. After two hours, the stalks were removed, and the agar plates incubated at 30°C for 48 hours. After 48 hours, the plates were examined for colony morphology, and Bacillus cereus family member es found internal to the plant were toothpicked onto nutrient agar and nutrient agar plus tetracycline plates (to select for bacteria containing the pSUPER35 BclA-endoglucanase plasmid). The resultant increase in Bacillus cereus family member 349 colony numbers is indicated shown in Table 54. These s demonstrate the ability of the BEMD system to be introduced into the target plant by expression in an endophytic strain of the Bacillus cereus family.

Table 54: Endo n h tic assa on Bacillus cereus famil member EE349 Treatment Endophytic Bacillus cereus Tetracycline resistant Bacteria family bacteria Bacillus cereus family (all strains) members Bacillus cereus family member EE349 transformed with 35 BclA- endoglucanase Tetracycline resistant Bacillus clones were grown overnight at 30°C in brain heart on broth plus tetracycline, and spun down at xg for 5 minutes. The supernatant was removed, and the pellet frozen overnight at -20C. Chromosomal DNA was then extracted from each clone, and the presence of the pSUPER—20-35 BclA-endoglucanase plasmid determined by transformation of the somal DNA ining the plasmid) into DHSu E. c0li cells and plating on LB plus ampicillin plates. Correct clones were subjected to DNA ce analysis, which verified that Bacillus cereus family member 349 was internal to the plant (endophytic) and contained the plasmid.

Many endophytic bacteria were found in the corn seedlings, with a number of different strains and species within the Bacillus cereus family found inside both the control and the EE349 treated plants. The tetracycline resistant us cereus family members (indicating the presence of the pSUPER35 BclA-endoglucanase plasmid) were only found in the treated corn seedlings, and all had the same colony morphology of the original expression host, Bacillus cereus family s EE349. The presence of the pSUPER 20-35 BclA-endoglucanase d was verified by PCR amplification using unique primers.

Example 52. ion, fication, and characterization of endophytic Bacillus cereus family bacterial strains 3] In addition to the endophytic strain Bacillus cereus family member 349 discussed above in the immediately preceding example, several other Bacillus cereus family members that have the ability to grow ytically were also identified: Bacillus cereus family member EE439, Bacillus thuringiensis EE4l7, Bacillus cereus EE444, Bacillus giensis EE3 l9, Bacillus thuringiensis EE-B00184, Bacillus mycoia’es EE-B00363, Bacillus mycoia’es EE-B00366, and Bacillus cereus family member EE-B00377.

To obtain these additional Bacillus cereus family members, commercial hybrid corn seed was planted in potting soil and allowed to grow. The corn seeds were coated with a fungicide and a biological inoculant. Plants were grown under artificial light for 14 hours a day and plant growth over a 14 day period was determined. Plants were watered every three days over the course of the experiment. After 14 days, the plants were extracted from the soil and washed to remove excess debris. The plants were then inverted, exposed to 5% bleach for ten minutes, washed in water, exposed to hydrogen peroxide (10%) for ten minutes, washed again in water, and the stalks split with a sterile razor blade. The split halves of the stalks were placed face down onto nutrient agar plates for two hours. After two hours, the stalks were removed, and the agar plates incubated at 30°C for 48 hours. After 48 hours, the plates were ed for colony morphology, and Bacillus cereus family member colonies found internal to the plant were toothpicked onto nutrient agar. These were then were grown overnight at 30°C in brain heart infusion broth, and spun down at 10,000 X g for 5 minutes. The supernatant was removed, and the pellet frozen overnight at —20°C. Chromosomal DNA was then extracted from each clone, and the identity of each colony verified by PCR using 168 rRNA primers and amplicons were sent for DNA sequencing and identification. The 168 rRNA ces for these strains are provided above in Table 13. e 53. Isolation, identification, and characterization of additional endophytic bacterial strains (non-Bacillus cereus family members) The endophytic bacterial s Bacillus megaterium EE385, Bacillus sp.

EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillusfusiformis EE442, Lysinibacillus spp. EE443, and Bacillus pumilus EE-B00143 were isolated from corn seedlings.

Two week old corn ngs were first sterilized. The plants were extracted them from the soil and washed them to remove excess debris. The plants were then ed, exposed to 5% bleach for ten minutes, washed in water, exposed to hydrogen peroxide (10%) for ten minutes, and washed again in water. The stalks were then split with a sterile razor blade. The split halves of the stalks were placed face down onto nutrient agar plates for two hours. After two hours, the plant stems were removed from the plates, and the plates were then incubated at 30°C for 48 hours. Bacilli colonies that were endophytic were selected for further analysis. These strains were grown up in brain heart infusion broth overnight at 30°C, and the cultures subjected to extraction ofDNA using a Qiagen Chromosomal DNA Kit. The DNA was PCR amplified to obtain the 16S rRNA gene, which was sent for DNA sequencing. The resultant ces BLAST searched using the NCBI databases to establish the identity of the Bacilli species. The 16S rRNA sequences are provided above in Table 14.

Example 54. Expression of fusion proteins comprising a spore coat protein in endophytic Bacillus bacterial strains The endophytic bacterial strains Bacillus thuringiensis EE319, Bacillus firmus A30, and Bacillus lichenformis A4 were transformed to n plasmids encoding s spore coat proteins fused to endoglucanase. The plasmids pHP13-CotC-endoglucanase and pHP13-CgeA-endoglucanase were d. Each of these plasmids encoded the spore coat protein (CotC or CgeA) fused in frame with to a polyalanine linker ning eight alanine residues and endoglucanase. The polyalanine linker and ucanase were fused to the carboxy terminus of the spore coat ns. 7] To create the plasmids encoding the fusion proteins, the ucanase gene from Bacillus subtilis 168 was PCR amplified. The genes encoding the spore coat proteins CotC and CgeA were also PCR amplified from the chromosomal DNA ofBacillus subtilis 168 (CotC) or Bacillus amyloliquefaciens (CgeA). Correct amplicons were then subject to splicing by overlapping extension PCR to generate the fusion protein DNA fragment h annealing of homologous 15 bp overhangs. External primers were each engineered to contain XhoI sites. The ons were cleaned up with a Promega PCR clean up kit, and the DNA digested with XhoI and ligated into the SalI site of pHP13. The plasmid DNAs were then sequenced, transformed into E. c0li cells, and the DNA introduced into the various endophytic Bacillus strains.

Spores of each of the recombinant Bacillus s expressing the fusion ns were generated by swabbing overnight cultures onto nutrient agar plates, which were then incubated at 30°C for 72 hours. After 72 hours, ial spores were ted from the plates by swabbing into sterile PBS. Spores were purified by density fugation three times, diluted to 1x108 CFU/ml, and d for endogloconase activity as bed above in Example 45. The s of this assay are shown in Table 55 below and in Figure 9.

Table 55. Endogloconase activity in Bacillus spores expressing fusion proteins CotC- endo ' lucanase or C ' eA-endo ' lucanase Expression Strain Enzyme Reading /Activit Bacillusfirmus A30 .201 Spore Control Bacillus thuringiensis .206 BT013A Bacillusfirmus A30 ,818 endo lucanase Bacillus thuringiensis 1.738 endo lucanase EE3 19 Bacillus licheniformis 0.414 endo lucanase A4 In Figure 9, CotCl, CotC2, and CotC3 are three separate experimental sporulation cultures of Bacillus thuringiensis EE3 19 with pHPl3-CotC-Endo.

Example 55. Effects of Bacillus spores expressing fusion proteins CotC-endoglucanase, CotB—endoglucanase, or CgeA-endoglucanase on growth of corn and soy seeds Spores of the recombinant Bacillus species expressing the fusion ns sing a spore coat protein and endoglucanase (e.g., the ndoglucanase, CotB- endoglucanase, or CgeA-endoglucanase fusion proteins described above in the immediately preceding example) can be tested for their effects on the growth of plants (e. g., corn and soy) as follows. Spores can be generated as described above in the immediately preceding example, washed, diluted to 1x108 CFU/ml in water, and applied to plant seeds (e. g., corn and soy seeds) at a rate of 1x10577 spores/seed. The spores can then be applied either as a seed treatment or as a soil drench. The plants can be planted 1” deep in 4” pots, and grown at 183°C with a 13 hour light/dark cycle. After two weeks, plant height and root length can be determined.

Example 56. Delivery of probiotic bacteria to animals by feeding plants comprising such bacteria to the animal Probiotic bacteria can be delivered to animals (e.g., livestock, fish, or other animals) by applying the probiotic bacteria to a plant seed, to a plant growth medium (e.g., by in furrow application to soil), to a plant (e.g., by foliar application) or to an area surrounding a plant or a plant seed, and subsequently feeding such plants or plants grown from the plant seeds to the animal. Bacteria can be applied to plant leaves or stems while plants are growing, and will colonize the phylloplane (leaf and stem surface). The plants can be subjected to sing into animal feed prior to feeding to the .

The use of endophytic strains of bacteria in such methods allows the ia to survive and persist in plant tissue, such that they will be ingested in significant numbers by the animal upon ingestion of plant matter from the plant. For example, the strains Bacillus cereus family member EE349, us cereus family member EE439, Bacillus thuringiensis EE4l7, Bacillus cereus EE444, Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillusfusiformis EE442, Lysinibacillus spp.

EE443, and Bacillus pumilus EE-BOOl43 are t to be tic and are endophytic and can be used in these methods.

Any of these strains or other probiotic and endophytic strains can be grown and spores ted as described above in Example 40. The spores can then be applied to a plant growth medium, a plant seed, a plant, or an area surrounding a plant or a plant seed. Plants grown in the plant growth , plants grown from the plant seeds, plants to which the bacteria were applied, or plants or plant seeds grown in an area to which the bacteria were applied can grow and subsequently be fed to an animal. Endophytic bacteria can ze the internal tissue of the plant, and replicate to great numbers inside the plant. The bacteria will sporulate upon the use of traditional harvesting methods, allowing for prolonged storage of plant matter (e.g., as hay or silage) that can later be fed to a target animal.

Only a small amount of bacteria needs to be used in these methods, since the endophytic bacteria will naturally ze and proliferate on and in the plants.

Example 57. Delivery of beneficial enzymes to animals by g to the animals plants comprising a recombinant Bacillus cereus family member or other recombinant bacteria expressing a fusion protein sing the beneficial enzyme The recombinant Bacillus cereus family members expressing a fusion protein sing a protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium that are described herein can also be used to deliver beneficial enzymes to animals. The recombinant Bacillus cereus family s can be fed directly to the animals (e. g., by mixing a recombinant Bacillus cereus family member into animal feed that is subsequently fed to the animal).

Alternatively, the methods bed above in the immediately preceding example for delivering bacteria to animals can be used in connection with recombinant Bacillus cereus family member expressing a fusion protein that comprises a protein or peptide that has beneficial effects in an animal (e.g., an enzyme that aids digestion of plant ).

Enzymes present in feed for livestock, fish, and other animals can impact the nutrient , yield, and health of the animal that s the enzymes. Enzymes that are beneficial for animal health include, for example, xylanases, phytases, phosphatases, proteases, cellulases, endoglucanases, glucanases, amylases, lipases, phospholipases, glycosylases, anases, (x-galactosidases, es, pectinases, biotinases, and polygalacturonases, among others. The BEMD system can be used to express such enzymes on the surface of the exosporium. Recombinant Bacillus cereus family members sing a fusion protein comprising one of these enzymes can be applied to a plant growth medium, a plant seed, a plant, or an area surrounding a plant or a plant seed. rly, the recombinant bacteria that express a fusion protein comprising one of these enzymes and a spore coat protein that targets the fusion n to a surface of a spore of the bacterium can be used in these s. The recombinant bacteria can be applied to a plant growth medium, a plant seed, a plant, or an area surrounding a plant or a plant seed. Plants grown in the plant growth medium, plants grown from the plant seeds, plants to which the bacteria were applied, or plants or plant seeds grown in an area to which the bacteria were applied can be grown and subsequently fed to an , and the beneficial enzyme thereby delivered to the animal. The bacteria will sporulate upon the use of traditional harvesting methods, allowing for prolonged storage of plant matter (e.g., as hay or silage) that can later be fed to a target animal.

Endophytic strains ofBacillus cereus family members can be used as hosts for expression of the fusion ns comprising a protein or peptide of interest (e.g., an enzyme having beneficial effects in animals) and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium. For example, the ytic s Bacillus cereus family member EE349, Bacillus cereus family member EE439, Bacillus thuringiensis EE4l7, us cereus EE444, and Bacillus thuringiensis EE3 19 described herein can be used as hosts.

Additional Bacillus cereus family members can be selected to be applied to the aerial portions of the plant, as these bacteria do not have to be endophytic to colonize the phylloplane. For example, Bacillus mycoia’es BTlSS, Bacillus mycoia’es EEl l8, Bacillus mycoia’es EEl4l, Bacillus mycoia’es BT46-3, Bacillus cereus family member EE2l8, Bacillus thuringiensis BTOl3A, Bacillus thuringiensis 184, Bacillus mycoia’es EE-B00363, Bacillus pseudomycoides 366, or Bacillus cereus family member EE-B00377 can be used for this purpose.

Similarly, endophytic strains of recombinant ia can be used as hosts for the expression of fusion proteins comprising a protein or peptide of interest and a spore coat protein that targets the fusion protein to a surface of a spore of the ium. For example, the endophytic strains Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillusfusiformis EE442, Lysinibacillus spp. EE443, or Bacillus pumilus EE-B00143 can be used as hosts.

The use of ytic strains of bacteria in these methods allows the bacteria to survive and persist in plant tissue, such that both the bacteria and the fusion proteins expressed by the bacteria will be ingested in significant numbers by the animal upon ingestion of plant matter from the plant. Thus, through a simple addition of the recombinant Bacillus cereus family member or other recombinant bacteria at planting, beneficial s can be spread throughout the plant tissue and delivered to animals upon ingestion of plant matter.

Example 58: Use of Various Targeting ces to s Endoglucanase 0n the Surface of Bacillus cereus Family Member Spores, and Use of Such Spores for ing Plant Growth 1] The pSUPER plasmid was modified by cloning of a PCR generated fragment through homologous recombination that fused the BclA promoter, start codon, and amino acids -35 of BclA (amino acids 20—35 of SEQ ID NO: 1) in frame with Bacillus subtilis 168 endoglucanase (pSUPER-BclA 20Endo) as described above in Example 44. This plasmid was then subjected to inverse PCR to amplify the entire plasmid backbone, but leaving out the sequence corresponding to amino acids 20-35 of BclA. This inverse PCR product was combined with a PCR product that amplified the equivalent region from each of SEQ ID NOs. 5, 15, 25, 81, 85, 87, or amino acids 20—33 of SEQ ID NO: 1. Thus, constructs were created that contained each of the following targeting sequences fused in frame with Bacillus subtilis 168 endoglucanase: (1) amino acids 20—35 of SEQ ID NO: 1; (2) amino acids 23—38 of SEQ ID NO: ; (3) amino acids 28—43 of SEQ ID NO: 15; (4) amino acids 9—24 of SEQ ID NO: 25; (5) amino acids 23—38 of SEQ ID NO: 81; (6) amino acids 13—28 of SEQ ID NO: 85; (7) amino acids 13—28 of SEQ ID NO: 87; and (8) amino acids 20—33 of SEQ ID NO: 1. Each construct ned the wildtype BclA promoter and a methionine at the start codon, ed by the targeting sequence fused in frame to the Bacillus subtilis endoglucanase gene. Each of these constructs was transformed into E. c0li and plated to obtain single colonies on Luria plates plus ampicillin (100 ug/ml). Plasmids from each single colony were grown up in overnight cultures in Luria broth plus ampicillin, and purified using a WIZARD SV miniprep kit, and sequences were verified by Sanger sequencing. DNA was also quantified Via spectrophotometry, and the DNA was introduced into Bacillus thuringiensis BT013A. In addition, the pSUPER-BclA35 Endo construct was introduced into Bacillus thuringiensis BT013A which had the native BclA protein removed from its genome through homologous recombination (BclA knockout, “BclA KO”). Correct colonies were screened by plating on nutrient broth plate containing otic (tetracycline at 10 ug/ml). Each positive colony was grown up in brain heart infusion broth at °C ght at 300 rpm, with antibiotic, and genomic DNA was purified and re-sequenced to verify genetic purity. Verified colonies were grown ght in brain heart infusion broth with ug/ml tetracycline, and induced to sporulate through sporulation in a yeast t-based media.

Each of the production runs in the yeast extract-based media were collected at 48 hours post production of spores, and ted to enzyme comparison of the resultant spores using the methodology described above in Example 45. The absorbance was ined at 540 nm using an IMPLEN nanophotometer model P330. There were three samples and a blank for each reaction. The results from the enzyme readings are shown in Table 56.

For corn, 1 ul of each of the whole broth for each of the ucts was placed onto each seed. For summer squash, 2 ul of whole broth for each construct was placed onto each seed. To accomplish this, 50 seeds were placed in a 50 ml l bottom polypropylene tube and vortexed lightly using a vortex mixer. To this swirling of seeds, 50 ul (for corn) or 100 ul (for squash) of broth containing the recombinant spores was slowly pipetted into the tube, and the vortexing action coated the seeds with an even coating of the whole cell broth from each construct. These seeds were then planted at 1” deep into native soil using a 39.6 cm3 (15.6 in3) ng pot, with two seeds per pot. The pots were then watered to saturation, and the plants allowed to germinate. The plants were grown in a controlled growth room, set to 70°F during the day, and 60°F during the evening, with a light period of 14 hours/day, under artificial light conditions, for 14 days. After 14 days, the plants were measured for height, and results were normalized to a control group that received only water as treatment on the seeds.

Table 56. Enz me levels and lant _r0wth henot es.

Targeting Sequence Sequence Squash Average Plant Sequence ty to AA Identity to AA Growth Growth Phenotype -35 of BclA 25735 of BclA Phenotype Phenotype Change l H20 0 mU/ml N/A N/A 100% 100% —100% SEQ ID NO: 1 SEQ ID \0: 5 SEQ ID \O:15 SEQ ID \0: 25 SEQ ID \0: 81 SEQ ID \0: 85 SEQ ID \O:87 AA 2033 of 30.5 N/A 100% 104.6% 100.7% 102.7% SEQ ID NO: 1 AA 20735 of 100.8 100% 100% ND ND ND SEQ ID NO:1 in BT013A BclA AA=amino acids ND=not determined The above data Show that each of these constructs was able to stimulate plant growth and Show that the use of different targeting sequences allows for control of the expression level of the enzyme on the outside of the Spore.

Use of amino acids 20—35 of SEQ ID NO: 1 or AA 13—28 of SEQ ID NO: 85 as the targeting sequence resulted in the highest levels of enzyme production. This iS sing considering the low degree of identity between these targeting sequences (43.8% identity over the entire length of the targeting sequence). Use of amino acids 28—43 of SEQ ID NO: 15 or amino acids 9—24 of SEQ ID NO: 25 resulted in the t plant response across the two plant types. Expression of the fusion protein containing amino acids 20—25 of SEQ ID NO: 1 as the targeting sequence in the BT013A BclA KO host led to very large (263.8%) increase in the amount of enzyme activity on the surface of the Spores as compared to expression of the same fusion n in the wild-type strain.

Example 59: Use of Various Targeting Sequences and Exosporium Proteins to Express Phospholipase, Lipase, and Endoglucanase on the Surface of Bacillus cereus Family Member Spores Plant Growth , and Use of Such Spores for ing 6] The pSUPER plasmid was modified by cloning of a PCR generated fragment (Xhol digestion and ligation) that fused the BclA promoter, start codon, and amino acids 20—35 of BclA (amino acids 20—35 of SEQ ID NO: 1) followed by a six alanine linker sequence in frame with either Bacillus thuringiensis atidylcholine-specific phospholipase C gene (PC-PLC) (pSUPER-BclA 20PL) or Bacillus subtilis lipase LipA (pSUPER-BclA35- Lipase), or us subtilis endoglucanase eng (pSUPER-BclA35-Endo) as described above in Example 44. These ds were then subjected to inverse PCR to amplify the entire plasmid backbone, but leaving out the sequence corresponding to the amino acids 20-35 of BclA. This e PCR product was combined with a PCR product that amplified the lent region from each of SEQ ID NOS. 5 (i.e., amino acids 23—38 of SEQ ID NO: 5), 15 (i.e., amino acids 28—43 of SEQ ID NO: 15), and 25 (i.e., amino acids 9—24 of SEQ ID NO: 25; the full-length exosporium proteins of SEQ ID NOs. 120, 111, 121, 108, and 114; or amino acids 20—33, 20—31, 21—33, 23—33, or 23— 31 of SEQ ID NO: 1. Each e constructs contained the wild-type BclA promoter, a methionine at the start codon, followed by the targeting sequence or exosporium protein fused in frame to the Bacillus cereus atidylcholine-specific phospholipase C, Bacillus subtilis 168 Lipase LipA, or Bacillus subtilis 168 eng endoglucanase gene. Each of these constructs was screened for correct transformants as described in Example 58 above.

Each of the production runs in the yeast extract-based media were collected at 48 hours post production of spores, and subjected to enzyme comparison of the resultant spores.

Determination of enzyme data for endoglucanase was med as described above in Example 58. For the olipase C enzyme assay, 1 ml of recombinant spores was pelleted at 10,000 X g for 3 s, and supernatant removed and discarded. The spore pellet was then resuspended in 500 pl reaction buffer (0.25 mM Tris-HCL, 60% glycerol, 20 mM o-nitrophenyl phosphorylcholine, pH 7.2). A negative control for enzyme assays contained BT013A spores with no enzyme expression. Each sample was incubated at 37°C for 18 hours, centrifuged again to remove the spores, diluted 1:1 in water, and the Abs540 read using a spectrophotometer. This was compared to a standard curve against commercially sed phospholipase and lipase controls to establish the U/ml of activity. The results from the enzyme readings are shown in Tables 57 and Table 58.

Table 57. End0_lucanase Enz me Levels Endoglucanase Levels (mU/ml) 0 mU/ml 38-2 .7 29.7 24.4 24.0 11.0 .5 tin Se uence, Ex 1 eriment #2 End0_lucanase Levels (mU/ml) 48.22 60.86 19.93 45.31 54.1 AA=Amino acids Many of the targeting sequences and rium proteins were able to display a large amount of active enzymes on the surface of the SporeS, including SEQ ID NOS. 108, 111, 114, 120, and 121. Amino acids 20—31,21—33, and 23—31 of SEQ ID NO: 1 provided similar enzyme expression levels to amino acids 20—35 of SEQ ID NO: 1, indicating that smaller fragments are te for the display of enzymes on the surface of the SporeS. Only amino acids 23—33 of SEQ ID NO: 1 exhibited a diminished enzyme display level on the SporeS.

Table 58. Phospholipase Enzyme levels Targeting PC-PLC Enzyme Levels Lipase Enzyme Levels Se uence 0.0 00 ID NO: 1 SEQ ID NO: 5 SEQ IDNO: 15 IDNO: 25 SEQ ID NO: 114 .446 SEQ ID NO: 120 3.612 SEQ ID NO: 111 .738 AA=Amino acidS Similar to the results shown above in Table 57, the t levels of olipase or lipase on the spore surface were observed when amino acids 20—35 of SEQ ID NO: 1, amino acids 23—38 of SEQ ID NO: 5, or the exosporium protein sequence of SEQ ID NO: 120 were used.

The effects of these spores expressing l of these constructs on nodulation in soybeans are shown below in Table 59.

Table 59. Phos holi n ase Plant Res n onses Tar__etin Se u uence Nodulation er Plant So bean Control H20 ‘- Strain Control Bacillus ; iensis BTOl3A Bacillus thuringiensis BT013A expressing a fusion 14.0 protein ofAA 20-35 of SEQ ID NO: 1 and .oohosholiase ns plants were coated as above, but the assay was run out to 3 weeks’ time. Plants were carefully removed, dirt washed gently off of the roots, and nodules counted for each plant. As shown in Table 59, addition of spores displaying olipase onto the seeds of soybean allows for an accelerated number of nodules on the plants, which is a positive indication for both early growth as well as eventual increases in yield in ns.

Example 60: g ofMIR3I9 RNA and Random RNA 1 to us cereus Spores Expressing a Fusion Protein Containing a Nucleic Acid Binding Protein, and Use of Such Spores to Deliver RNA to Plants DNA and RNA can be bound to Bacillus cereus family member spores that express fusion proteins containing a ing sequence and a nucleic acid binding n or peptide on their exosporium, as described in the above Examples and in the Description. The spores act as a delivery mechanism, delivering the target nucleic acid (e.g., a miRNA) to the target plant. To demonstrate this ability of the recombinant Bacillus cereus family member spores, a common miRNA, MIR319 was delivered to soybeans using spores expressing a fusion protein containing amino acids 20—35 of SEQ ID NO: 1 fused in frame to the known DNA binding gene SspC. MIR319 has different effects on plant phenotype in different plants, and even within different parts of the same plant. For example, in some species, treatment of leaves with MIR319 leads to curling of leaves, whereas in other species, application of MIR3 19 leads to stress resistance. MIR319 is ubiquitous across plant genomes, is a global regulator of pathways, and its delivery into various plants leads to various phenotypes.

Table 60: RNAs used in this stud MIR319 UUGGACUGAAGGGUGCUCCC GAGCUCUCUUCAGUCCACUC (SEQ (SEQ ID NO: 306) ID NO: 307) or AGAGCGUCCUUCAGUCCACUC SEQ ID NO: 308 Random GAGCCCATGGTTGAATGAGT ACTCATTCAACCATGGGCTC (SEQ RNA #1 SEQ ID NO: 309 ID NO: 310 Synthetic MIR319 microRNAfrom Glycine max (soybean) was designed to match the MIR319 sequence available in miRBase (miRBase.org, central repository for microRNA sequences). Two partially complementary single stranded sequences were synthesized by Integrated DNA Technologies (IDT, Iowa) to represent the 3 ’ and 5 ’ mature gene products known to exist in viva (two different versions of the 5’ ce were used). Likewise, two single stranded RNAs were synthesized with random sequences not matching anything in the soy genome as a l. The double stranded (ds) gene products were made by combining the two single stranded (ss) ts at 95°C for 10 min and then cooling slowly at room ature to allow for ing. Bacillus giensis expressing a fusion protein containing the BclA promoter, a methionine residue as the start codon, and amino acids 20—35 of SEQ ID NO: 1 fused in frame to the known DNA binding gene SspC (an (1/B type SASP, Small Acid- soluble Spore Protein C ofBacillus thuringiensis ) was engineered by standard cloning procedures as described above in Example 58. This construct (SspC—BclA) was created in E. coli, transformed into Bacillus thuringiensis BT013A and clones d by DNA sequencing. B. thuringiensis spores expressing SspC—BclA were obtained by an overnight growth of transformed bacteria in brain heart infusion broth (BHI) for 2 days in a yeast extract-based media until a y of 2 x 108 spores per milliliter (ml) was achieved with less than 1% vegetative cells.

DNA was extracted from an t of the parent BHI culture and sent for sequencing to confirm incorporation of the SspC—BclA plasmid. To prepare spores for seed treatments, 1 ml of spore culture in the yeast extract-based media was pelleted by centrifugation and resuspended in 100 pl of water. This concentrated suspension was d and spores were used at 6 x 108 spores/ml. For each soy seed, 1 ul of spores was combined with 10 ul ofRNA at 10 uM and incubated at 30°C for 2 hours (scaled up for multiple seeds). After this incubation spores were pelleted (carrying bound RNA) and unbound excess RNA in the atant was discarded and the pellet was resuspended in 10 ul of water. Samples were applied to the seeds as follows: 39.6 cm3 (15.6 in3) of Timberline brand commercial top soil was prepared in each pot and a 1 inch indentation was made where 2 ml of water was applied and a single seed was set on top. The 10 ul spore+ bound RNA sample was applied by micropipetting directly on to the top of the seed.

Seeds were allowed to sit for 30 min and then the adjacent soil was pushed to loosely cover the seed. The seeds were d to germinate for 4 days in an ial light plant growth room with a 13/11 hour light/day cycle, and at 21°C day/15°C night temperature range. On day 14 soy plants were uprooted, photographed and measured. Heights were normalized to water control treated plants (See Table 61).

Example 41 above describes the ability of the SspC-BclA recombinant Bacillus cereus family member spores to bind to and hold DNA. To assess RNA binding ability of the SspC-BclA expressing spores, biotin labeled random RNA sequences were sized by IDT and incubated with the spores exactly as was done for the treatments described above (1 ul of spores at 6 x 108 spores/ml + 10 ul of 10 uM RNA for 2 hours at 30°C, pelleted and resuspended in 10 ul of water). Avidin conjugated to Fluorescein (FITC) (Life Technologies) was added to the 10 ul spore+RNA sample at 20 ug/ml final concentration and incubated for 1 hour at room temperature in the dark. Avidin is known to bind biotin and FITC is a fluorescent tracer. Spores were ed once again to remove excess d avidin-FITC and resuspended in 4% paraformaldehyde made in PBS and stored at 4°C overnight in the dark. Spores were inspected for fluorescence and photographed (See Table 62). In addition, as shown in Figure 10, the Sspc-BclA tagged spores were able to bind and retain both ssRNA and dsRNA, as shown by the FITC-avidin labeling of spores in the ce of the ssRNA or dsRNA bound with biotin.

To te the results shown in Figure 10, spores were incubated with either double or single stranded RNA (of a random sequence) tagged with biotin and detected with avidin conjugated to fluorescein (FITC). No fluorescence was detected on spores incubated with water only.

Brightfield and corresponding fluorescent images were taken with 40X objective and 10X ocular lenses.

As can be seen in Table 61 below, the major effect ofMIR319 as a seed treatment on soybeans is on root growth and overall . Curly roots were defined as having at least two 180° turns. Heights were measured along the main stalk. When n plants were uprooted and assayed for the presence of “curly roots”, a phenotype observed by our group c to soybeans, no evidence of curly roots was found in the water control, the BT013A strain control, the double stranded (dsRNA) RNA alone control, or the spores alone (carrier control). The only evidence of curly roots is noted when both the SspC-BclA spores (the carrier) was delivered to the seed with the dsRNA (60% curly roots) (also see Figure 10). Figure 11 also shows the phenotypic changes in the soybean plants when exposed to SspC-BclA spores combined with ds MIR319 RNA. When the spores are used to deliver the RNA, the impact of the RNA is amplified, leading to an sed stunting and curly root phenotype in Figure 11. To generate the results shown in Figure 11, soy seeds were treated with double stranded (ds) M1R319 with or without prior g to B. thurz'ngz'ensz's spores sing SspC—BclA.

Application of dsM1R319 resulted in slightly taller plants on e; r, ation of dsM1R319 bound to spores resulted in “curly” roots defined as having at least two 180° turns and l less height. The median sample from each experimental condition is shown. Images were taken using a digital camera with plants together in a single image.

As an RNA control, a random set of ssRNA (single-stranded) and dsRNA was applied to soybeans. In these experiments, the random ssRNA had no effect when applied alone, while the dsRNA had a ng effect on the height of the plants when delivered to the seeds. In both cases, when the spores (carrier) were used in conjunction with either the random ssRNA or the dsRNA version, the stunting phenotype was increased significantly (33% and 27.8% stunted, respectively). This stunting is not evident in the spore (carrier control) alone samples. These data, when taken together, demonstrate the ability of the spores to amplify and specifically deliver ssRNA and dsRNA to plants by application to the seed, and demonstrate the ability of two different RNAs (Random #1 and M1R319) to affect phenotype when delivered via Bacillus cereus spores expressing a fusion protein containing a DNA/RNA binding protein.

Table 61: Root and Hei ' ht effect of MIR3I9 on so bean develo n ment Seed Treatment % Curly Roots Height re licates each Normalized to Control Water (Control) 0 100% Water + Spores (Control) 0 105.21% Random ssRNA #1 no 0 102.62% Stores Random ssRNA #1 + spores 0 69.62% dsM1R319 no spores 0 125.30% dsM1R319 + spores 60% 67.10% Random dsRNA #1 no 0 82.40% SIOFeS Random dsRNA #1 + spores 0 54.61% Table 62: scence detection on SspC-BclA expression spores with bound biotin labeled RNA Spore Treatment Fluoresence Detected on S ores Sores -- Water control or back round sIore uorescence Not detected Spores -- Water + -Fitc (controlfor ound spore Not Detected +FITC uorescence Sores -- biotin labeled ssRNA + Avidin-FITC Sores -- biotin labeled dsRNA + Avidin-FITC As can be seen in Table 62, no fluorescence was detected on the spores without the presence of RNA. Both single stranded (ss) and double stranded (ds) RNA was detected on the spores.

Example 61: Delivery of nucleic acids to Caenorhabditis elegans nematodes by ingestion of recombinant us thuringiensis spores expressing a fusion protein Delivery ofRNA and DNA to nematodes has a great deal of applications in both plant science, animal health, and in basic research. Nematodes cause a great deal of damage and yield loss to commercial and non-commercial growing operations for key crops, and parasitic nematodes cause high morbidity in humans and other animals in many impoverished areas of the world. Delivery ofRNA and DNA has the potential to alleviate and treat many nematode problems, and delivery ofRNA and DNA ucts has been demonstrated to be useful in impacting target nematodes. This example illustrates the utility of the RNA/DNA delivery mechanism described above in Example 60 in ring spores to nematodes.

Wild type C. elegans nematodes were purchased from Carolina Biological (North Carolina) and maintained at 23°C on NGM-Lite agar plates coated with OPSO E. coli for food. Two different Bacillus giensis BT013A strains were engineered by standard cloning procedures to express amino acids 20—35 of SEQ ID NO: 1 fused in frame to green cent n (GFP) or mCherry to trace the presence of spores in the gut. These green or red fluorescently tagged spores were obtained by an overnight growth in BHl (brain heart infusion) medium, ed by three days in a yeast extract-based media until a density of imately 2 x 108 spores per milliliter (ml) was achieved with less than 1% vegetative cells. To prepare spores for feeding to nematodes, 1 ml of spore culture in media was pelleted by centrifugation and resuspended in 100 pl of water to remove excess media. This concentrated suspension was counted and d to l x 108 spores/ml. To feed spores to the worms, 1 ul of the spore suspension containing both the red and green fluorescently tagged spores was added to a 60 mm NGM-lite agar plate with 10 ul of PBS (phosphate ed saline) to aid in spreading. No other 3 10 food source was made available. Twenty wild type nematodes of various ages were transferred to the plates immediately. Living nematodes were checked 5 hours later for ingestion of spores using standard fluorescence microscopy.

As can be seen in Figure 12 and Table 63, the centralized gut of the des fluoresced when fed recombinant Bacillus cereus family member spores expressing the fusion protein containing the targeting sequence and GFP, whereas the gut did not fluoresce when fed OPSO E. c0li (standard food). Images were taken of live nematodes with 4X objective and 10X ocular lenses. This trates the ability of these spores to be both ingested and delivery of a “cargo” of target proteins, exemplified by the green fluorescence protein. Other Exosporium proteins and targeting proteins can also be used interchangeably with a targeting sequence to deliver RNA and DNA to a nematode or other target organism. Other recombinant Bacillus cereus family member spores can also be used due to the high degree of conserved nature of the exosporium and its creation on the surface of the spore.

Table 63: C. eleans fluorescence detected in the ut Green Fluorescence Detected in GFP expressing B. Yes (High) thurin. iensis BT013A Example 62: Construction, Purification, and Uses of Exosporium nts 1] Knock out (KO) Mutants: To make est and c0tE ut (KO) mutant strains of Bacillus thuringiensis BT01 3A, the plasmid pKOKI shuttle and integration vector was constructed that contained the pUC57 backbone, which is able to replicate in E. c0li, as well as the origin of replication erythromycin resistance cassette from pEl94. This construct is able to replicate in both E. c0li and Bacillus spp. A 1 kb DNA region that corresponded to the am region of the c0tE gene and a 1 kb region that ponded to the downstream region of the gene c0tE were PCR ed from Bacillus thuringiensis BT013A. A second construct was made that ned the 1 kb DNA region that corresponded to the upstream region of the est gene and a 1 kb region that ponded to the downstream region of the gene est, both of which were PCR amplified from Bacillus thuringiensis BT013A. For each construct, the two 1 kb s were then spliced together using homologous recombination with overlapping regions with the pKOKI plasmid. This plasmid construct was verified by digestion and DNA sequencing. Clones were screened by looking for omycin resistance. 31 1 Clones were passaged under high ature (40°C) in brain heart infusion broth. Individual colonies were icked onto LB agar plates containing erythromycin 5 ug/ml, grown at 30°C, and screened for the presence of the pKOKI plasmid as a free plasmid by colony PCR. Colonies that had an integration event were continued through passaging to screen for single colonies that lost erythromycin resistance fying loss of the plasmid but recombination and removal of the est or c0tE gene). Verified deletions were confirmed by PCR amplification and cing of the target region of the some. The pSUPER-BclA -35 Endo plasmid (described above in Example 58) was transformed into each of the est and c0tE KO mutants. As described above in Example 48, the c0tE KO mutant was also transformed with the pSUPER BclA 20-35 eGFP d (made as described above in Example 44, but with endoglucanase swapped for eGFP by homologous recombination).

Dominant Negative Mutants: To create a nt negative mutant, we PCR amplified the N—terminal half and the C-terminal half of CotO (Seq ID NO: 126), containing the amino acids 1-81 and 81-199 respectively, and cloned these fragments into the pHP13 vector using homologous recombination (the pHPl3 vector is described above in e 1). Correct clones were verified by Sanger sequencing. Each of the two CotO dominant negative mutants was uced into Bacillus thuringiensis BT013A that contained the pSUPER-BclA 20-35 Endo construct, which produces endoglucanase on the e of the spore as illustrated above in Example 58.

Exosporium Fragment Creation: For each of the two KO mutants, and both of the dominant negative mutants, an overnight culture was grown in BHI media at 30°C, 300 rpm, in baffled flasks with antibiotic selection. One milliliter of this ght culture was ated into a yeast extract-based media (50 ml) in a baffled flask and grown at 30°C for 3 days. An aliquot of spores was removed, 1% Tween was added, and the spores were agitated by vortexing for one minute. The spores were collected via centrifugation at 10,000 X g for 5 minutes, and supernatant containing the exosporium fragments was filtered through a 0.22 uM filter to remove any residual spores. The supernatant (containing the broken exosporium fragments) was filtered through a 100,000 Da membrane filter to obtain purified exosporium fragments containing the fusion proteins. Smaller MW proteins were removed by passaging h the 100 kDa filter. No spores were found in the filtrate or retentate of the supernatant.

Transmission electron micrographs are provided in Figure 15 g intact spores of Bacillus thuringiensis BT013A (panel A) surrounded by attached exosporium, and spores of the Bacillus thuringiensis BT013A CotE knock-out mutant (panel B), from which the 3 12 exosporium has detached. Arrows in panel A of Figure 15 indicate the exosporium of intact , while arrows in panel B of Figure 15 indicate exosporium that has detached from the spores. Panel C of Figure 15 shows a ission on micrograph of a purified exosporium fragment ation of derived from the Bacillus thuringiensis BT013A CotE knock-out (prepared as described above by vortexing, centrifugation, and filtration of the supernatant), visualized by negative staining. Images were taken on a JEOL JEM 1400 transmission electron microscope. No visible exosporium fragments were observed when control spores lus thuringiensis BT013A without the CotE knockout, sing the BclA 20-35 Endo fusion protein, data not shown) were subjected to same ing, centrifugation, filtration procedures described above. ce of BclA 20-35 Endoglucanase in Exosporium nts collection from the CotE and Est Knockout and CotO Dominant Negative Mutants: Exosporium fragments were created and purified as described above that contained the pSUPER BclA 20Endo plasmid that creates an exosporium that contains the endoglucanase enzymes on the surface of the spores. Exosporium fragments containing this construct were created from the cotE knockout mutant spores, est knockout mutant spores, CotO N—terminal dominant mutant spores, or CotO C-terminal dominant mutant spores. In each of these experiments, the amount of ty for the endoglucanase on the exosporium fragments was fied as a percentage of the total enzyme levels. These results were compared against a wildtype construct that did not contain any s, but did contain the pSUPER BclA 20Endo plasmid.

Effects of Exosporium Fragments on Plant Growth: These rium fragments were then delivered as a seed treatment onto soybean seeds (as described in Example 59 above). A wild-type control (B. thuringz‘ensz‘s BT013A expressing the BclA 20-35 Endo construct) was also coated onto soybeans seeds. For each experiment, 1 ul of exosporium fragments from each uct, or a 1:2, a 1:4, or a 1:8 dilution of the fragments was applied to each seed.

Table 64: Exos orium Fra ' ment Enz me Activi and Plant Growth Res I onse Mutation Construct Endoglucanase Soy Plant Soy Plant Soy Plant Presence Activity, Growth Growth Growth of Exosporium Response, Response, Response, ? Fragments 1:2 1:4 1:8 (mU/ml) dilution dilution dilution Wild-type BclA 20-35 10.3 93.1% 92.2% 83.4% No BT013A Endo cotE KO BclA 20-35 269.0 121.4% 110.7% 90.7% No Endo Mutation Construct ucanase Soy Plant Soy Plant Soy Plant Presence Activity, Growth Growth Growth of Exosporium Response, Response, se, Spores? Fragments 1:2 1:4 1:8 dilution on dilution CotO NTD 22.4 99.6% N/A dominant Endo CotO CTD 27.5 95.8% N/A dominant Endo These s demonstrate that mutations that disrupt the exosporium, such as a knock-out mutation in the cotE or est gene, or a dominant negative mutation in the CotO protein, can be used to generate exosporium fragments that are substantially free of spores, and demonstrates that these exosporium nts contain fusion proteins that are targeted to the exosporium. These fragments can be utilized to promote plant growth and in other applications.

There was a small amount of background endoglucanase activity in the exosporium fragment preparation from the BT013 strain having no mutations and expressing the BclA 20—25 Endo construct (BT013A BclA 20—35 Endo). This was unexpected and may represent a low level of unstable exosporium that is being released from spores and captured during the exosporium fragment collection process. CotE and 13st KO strains contain the highest amount of enzyme in the exosporium fragment fraction. The CotO dominant negative mutants that express a fusion protein also have an ed level of enzyme in the exosporium fragment fraction as well.

The exosporium fragments from the CotE and Est s (not expressing BclA 20-35 Endo) applied directly to plants had a negative effect on growth and were removed from this experiment. When the exosporium fragments from BT013A BclA 20—35 Endo were applied to soybeans, there was a negative growth phenotype. When exosporium fragments from the CotE or Est mutants expressing the BclA 20-35 Endo fusion n were added to soybeans, a substantial increase in growth rate occurred (+28.3% and +14.8% over BT013A BclA 20—35 Endo fragments). The CotE mutant exosporium fragments were still active at the 1:4 dilution, but the Est exosporium fragments were no longer giving a growth benefit to the soybeans at this dilution. The CotO dominant negative mutants expressing the BclA 20-35 Endo fusion protein gave a small increase in n growth compared to the nts from BT013A BclA 20—35 Endo, giving +6.5% and +2.7% growth, respectively. 3 14 Example 63: Additional Demonstration of the utility of endophytic Bacillus cereus family members and other recombinant Bacillus species to deliver es, ns, and enzymes endophytically to the plant Bacillus thuringiensis EE4l7, Bacillus thuringiensis EE-B00184, Bacillus cereus EE439, and Bacillus sp. EE3 87 were found to have the y to grow endophytically and to be capable as serving as a host strain for the BEMD system (See Examples 52 and 53).

To demonstrate the ability of these Bacilli to grow endophytically and to serve as a host strain for the BEMD , each ofthese strains was transformed with the pMK4-BclA 20eGFP plasmid (described above in Example 62). Spores were made and purified as described above in Example 40.

These spores were diluted to a concentration of lxlOS/ml, and 1 ul of whole cell broth was then added to commercial hybrid corn seed in potting soil at planting. The corn seeds were coated with a fungicide and a biological inoculant. The corn hybrid variety was BECK 6l75YE, which contains the ROUNDUP READY glyphosate resistance gene and AQUAMAX t resistance gene. Plants were grown under artificial light for 14 hours a day and plant growth over a ten day period was ined. Plants were watered every three days over the course of the experiment.

Bacillus thuringiensis EE4l7, Bacillus thuringiensis EE-B00184, Bacillus cereus EE439, and Bacillus sp. EE3 87, expressing the BclA 20eGFP were then isolated from the inside of the corn plants. The ten day old plants were extracted from the soil and washed to remove excess . The plants were then inverted, washed in water, exposed to 5% bleach for ten minutes, washed in water, exposed to 70% l for ten minutes, washed again in water, and the stalks split with a sterile razor blade. The split halves of the stalks were placed face down onto nutrient agar plates for two hours at 30°C. After two hours, the stalks were removed, and the agar plates incubated at 30°C for 48 hours. After 48 hours, the plates were examined for colony morphology, and Bacillus colonies found internal to the plant were toothpicked onto nutrient agar and nutrient agar plus chloramphenicol plates (to select for bacteria containing the pMK435 BclA-eGFP plasmid). Results are shown in Table 65.

These results demonstrate the ability of the BEMD system to be introduced into the target plant by expression in an endophytic strain of the Bacillus cereus family. Figure 13 also demonstrates the ability of Bacillus thuringiensis EE-B00184 to express eGFP on the , as evidenced by fluorescent microscopy. In Figure 13, arrows denote single spores. Figure 14 demonstrates the ability of the isolated bacterial colonies from plants to ce green, trating that they do in fact deliver the protein of interest n eGFP) inside the plants. Figure 14 shows 3 l 5 fluorescence of colonies of endophytic bacteria isolated from inside corn plants on plates, illuminated with a GFP filtered lamp.

Table 65: Endoh tic deliver of “caro” roteins Strain Endophytic “Cargo” % Bacillus % Bacillus colonies + for colonies + for lasmid eGFP Bacillus thuringiensis Yes BclA 20-35 29.8% 29.8% EE4l7 eGFP EE-B00184 eGFP eGFP EE439 eGFP To further demonstrate the ability of these ytic strains to express proteins on the surface of the spores, the following constructs were introduced into Bacillus sp.

EE3 87: pHPl 3 plasmid with endoglucanase fused to either: BclA 20-35, CotB, CotG, CotC, CgeA, InhA, InhA2, InhAl, CotY, or AcpC (amino acids 20—25 of SEQ ID NO: 1 or SEQ ID NOs. 252, 256, 253, 254, 108, 121, 114, 111, and 120, respectively). The pSUPER 0-35 Endo construct bed above in Example 58 was also introduced into Bacillus thuringiensis EE-BOOl84, another endophytic strain. Transformed cells were screen by PCR and Sanger sequencing. Spores for each of these constructs was made by growing up an overnight culture in BHI plus selection (chloramphenicol), and 500 ul of each culture was d onto nutrient broth agar plates and allowed to incubate at 30°C for 3 days. After 3 days, the spores were swabbed off into PBS, diluted to a concentration of lxlOS/ml, spun down to recover the spores, and enzyme measurement of the spores was performed as described above in Example 58. The enzyme concentration was calculated as mU/ml for each uct. The ability ofBacillus sp.

EE387 to express fusion proteins on its spore surface is ted by the levels of enzyme.

Bacillus sp. EE3 87 was able to express all of the spore fusion proteins on its surface, but AcpC (SEQ ID NO: 120) was a or fusion protein for this strain. This finding was sing since Bacillus sp. EE3 87 is not a Bacillus cereus family member strain and does not have an exosporium, yet exhibited surface expression of fusion proteins containing exosporium proteins or targeting sequences d from exosporium proteins (e.g., CotY, AcpC, and amino acids —35 of SEQ ID NO: 1). 2015/050807 3 16 Table 66: Endophytic s Bacillus sp. EE387 (EE387) and Bacillus thuringiensis EE- B00184 (EE-B00184) exressin_ fusion roteins Exosporium Protein or Host Endophytic Endoglucanase activity Targeting Seguence Strain gmU/ml[ Fusion Partner CotB SEQ ID NO: 252 EE387 4.0 CotG SEQ ID NO: 256 EE387 4.2 CotC SEQ ID NO: 253 EE387 4.4 C_eA SEQ ID NO: 254 EE387 4.1 AA 20—35 of SEQ ID NO: 1 EE387 16.3 InhA SEQ ID NO: 108 EE387 7.5 InhA2 SEQ ID NO: 121 EE387 CotY SEQ ID NO: 111 EE387 4.9 ACoC SEQ ID NO: 120 EE387 36.0 InhAl SEQ ID NO: 114 EE387 4.5 AA 20—35 of SEQ ID NO: 1 184 95.8 These endophytic strains can also be administered to the plant through addition into the plant growth medium, including soil, irrigation, and granular formulations.

Endophytic s can also enter the target plant through the aerial portions of the plants. These create a unique and effective delivery ism for delivering proteins and es of interest into the plant, or in the case of DNA and RNA binding proteins, delivering RNA and DNA into the plant.

These data, in Bacillus sp. EE3 87 also demonstrate demonstrates that amino acids 20—35 of BclA (SEQ ID NO: 1), and SEQ ID NOs. 108, 121, and 120 all have noticeably positive data in Bacillus strains outside of the Bacillus cereus family. Bacillus thuringiensis EE- B00184 is also an exceptional host expression system. These levels are both noticeable and ve, indicating a conserved mechanism for attachment may be present in other Bacillus species for these proteins.

Spore Surface Expression of us thuringiensis EE-B00184. Bacillus thuringiensis EE-B00184 was transformed with pSUPER BclA 20-35 eGFP, and allowed to sporulate as described above. Spores were pelleted, washed, and subjected to cence microscopy to demonstrate the spore surface laden with eGFP ns in Figure 13.

Example 64: Expression of fusion proteins in herbicide— and ide-degrading Bacillus cereus family member strains Examples 49 and 51 above demonstrate the ability of the herbicide-degrading strain Bacillus cereus family member EE349 in both degrading herbicides and serving as a host strain for expression of a fusion protein attached the exosporium of its spores. To further 3 l7 demonstrate the ability of ide-degrading strains to produce enzyme laden exosporium on their spores, we introduced the pHPl3 CotC-Endo (SEQ ID NO: 253), pSUPER AcpC-Endo (SEQ ID NO: 120), pSUPER InhA2-Endo (SEQ ID NO: 121) and pSUPER 23—38 SEQ ID NO:5-Endo) into Bacillus cereus family member EE-B00377. A description ofpHP13 CotC- Endo can be found in Example 54, a description ofpSUPER AcpC-Endo and pSUPER InhA2- Endo can be found in Example 59, and a description ofpSUPER 23—38 SEQ ID NO:5-Endo can be found in Example 58. Bacillus cereus family member EE-B00377 was identified as a potent degrader of pyrethrin, dicamba, and 2,4-D. Herbicide and pesticide degradation was verified by both growth on the herbicide or ide as a nutrient source, as well as by ion of dicamba and 2,4-D in the presence of the herbicide or pesticide- degrading strain. Plasmids were made and cells transformed identically to Example 48 above. Each construct was verified by Sanger sequencing. Spores were created by using the sporulation media and conditions outlined in e 48. Enzyme activity was also performed as in Example 58 above.

Table 67: Enzyme expression levels of fusion proteins in pesticide degrading strain Bacillus cereus famil member EE-B00377. ucanase activit mU/ml CotC-Endo SEQ ID NO: 253 AcC—Endo SEQ ID NO: 120 SUPER 23—38 SEQ ID Nos-Endo 8] As can be seen in Table 67, Bacillus cereus family member EE-B00377 is able to produce endoglucanase and display the endoglucanase on its exosporium using several different exosporium proteins or targeting sequences. Of the constructs tested, amino acids 23— 38 of SEQ ID NO: 5 or SEQ ID NO: 253 gave the highest enzymes levels in this strain.

This example demonstrates the ability of the spore displayed system to be sed in herbicide- and pesticide-degrading strains. This system can be used to express other target proteins on the surface of the spores, including those that act on herbicides or pesticides themselves, such as herbicide-degradation enzymes, ide-degradation enzymes, metabolic enzymes, reductases, oxidases, and other useful enzymes for the breakdown of pesticides alone or in the presence of plants.

Example 65: Use of Free Nitric Oxide Synthase (NOS) and spore-bound NOS to enhance plant germination 0] Example 40 trates the ability of nitric oxide synthatase (NOS) from Bacillus subtilis I 68 to stimulate germination when attached to the exosporium of us cereus family s, and ring that NOS — spore protein fusion to seeds or in the 3 18 Vicinity of seeds. In this example, free NOS from Bacillus thuringiensis BT013A (SEQ ID NO: 261) and free eNOS (epithelial NOS from bovine neutrophils, Sigma-Aldrich, Cat No N1533) can also help induce ation, or increased outgrowth of seeds exposed to NOS. The pHP13 T NOS, pHP13 BclA-BS NOS, and pHP13 BclA-SODA (superoxide dismutase) plasmids were made in identical fashion to pHP13 BclA-BS NOS that described in e 40 and were transformed into Bacillus thuringiensis BT013A. pHP13 BclA- BT NOS contains the BclA promoter, start codon, BclA amino acids 20-35, a 6 e linker, and the Bacillus thuringiensis BT013A NOS gene (see Table 9, SEQ ID NO: 263). pHP13 S NOS contains the BclA promoter, start codon, BclA amino acids 20-35, a 6 alanine linker, and the Bacillus subtilis 168 NOS gene (see Table 9, SEQ ID NO: 264). pHP13 BclA-SODA contains the BclA promoter, BclA amino acids 20-35, a 6 alanine linker, and the Bacillus cereus superoxide dismutase 1 (SODAl) gene (SEQ ID NO: 155).

Table 68 shows the results of a soil germination assay. In this assay, commercial variety BECK’S 294NR (ROUNDUP READY) was coated with either 1 ul water (control) or 1 ul of water spiked with 34.2 mU of Bovine Neutrophil eNOS. 50 seeds of each were then planted and grown as described in Example 58, but with 4 seeds per pot. After 7 days, the plants were measured for height. As can be seen in Table 68, the presence of the eNOS allowed for increased outgrowth of the seeds, g to a 30.7% increase in shoot height of the treated soybeans.

Table 68: Influence of free eNOS 0n lant heitht in so beans.

Hoi-ht ized to Control H20, 1 l/seed 100.0% H20 with 34.2 mU eNOS/seed 130.7% In addition to the soil germination test described above, standard germination assays were med as described in Example 40. For soybeans, we choose 2 year old soybean seed with a lower germination rate, and coated 1 ul on each of 50 seeds with the ents.

Treatments were HZO control (water), L-arginine, Bacillus thuringiensis BT013A (strain control), Bacillus thuringiensis BT013A with pHP13 BclA -BT NOS, and Bacillus thuringiensis BT013A with pHP13 BclA BS NOS. The s for soybeans are shown in Table 69 below.

Table 69: Influence of s n ore-dis la ed NOS on _erminati0n rate in so beans.

H20, 1 l/seed H20 l with L-Ar-inino Bacillus thurin; iensis BT013A strain control Bacillus thurin iensis BT013A with BS NOS Bacillus thuringiensis BT013A with BT NOS W0 2016/044661 3 19 Standard germination assays were also performed as described above for commercial hybrids of sorghum. Each sorghum seed was coated with 0.5 ul on each of 50 seeds with the treatments. Treatments were H20 l (water), L-arginine, Bacillus thuringiensis BT013A (strain control), Bacillus thuringiensis BT013A with pHP13 S NOS, Bacillus thuringiensis BT013A with pHP13 T NOS. After 4 days, the seeds were measured for shoot height and root length to examine increased outgrowth of the seeds, and all data were normalized to the water control. The results are shown in Table 70 below. The addition of either BT NOS or BS NOS led to a greatly sed root length and shoot growth, with the difference most evident in the BS NOS treatment.

Table 70: S n ore bound NOS and increased sor ' hum outrowth Treatment Root Growth gNormalized1 Shoot Growth Normalized H20, 1 l/seed 100.0% 100.0% H20 Control with nine 109% 89% Bacillus thuringiensis BT013A 75% 145% strain control Bacillus thuringiensis BT013A 163% 293% with BS NOS Bacillus thuringiensis BT013A 141% 190% with BT NOS The sorghum ment above was repeated, but with slightly different treatments. Treatments were H20 control (water), Bacillus thuringiensis BT013A (strain control), Bacillus thuringiensis BT013A with pHP13 BclA —SODA, or free bovine eNOS. After 4 days, the seeds were measured for shoot height and root length to look at increased outgrowth of the seeds, and all data normalized to the water control. The results are shown in Table 71 below. The addition of either BT SODA or free NOS (eNOS) leads to a greatly sed root length and shoot growth.

Table 71: S n ore bound SODA and free NOS and sed sor ' hum outrowth Treatment Root Growth gNormalized1 Shoot Growth Normalized H20, 0.5 l/seed 100.0% 100.0% strain control with oHP13 BclA-SODA 1 Free eNOS, 34.2 mU/seed 123% 311% Taken together, these s demonstrate that overexpression of nitric oxide synthatases from multiple sources can be added to seeds and increase their germination rate and outgrowth of seeds, in both soil and traditional germination methods. This effect can is also found when adding free NOS to seeds. The addition of superoxide dismutase with the spores also leads to an increase in the outgrowth of seeds. L-arginine ed in the germination rate increases when utilized alone, or assisted in a lesser extent when mixed with NOS enzymes.

The NOS genes are ent in a y of microorganisms, and these microorganisms can be genetically modified to enhance their ability to express NOS on the seed, or in the vicinity of the seed in plant growth media. sion ofNOS on a spore leads provides a superior delivery system, as vegetative microorganisms are more fragile and do not survive on the seed for long periods of time. Expression on spores using the targeting sequences, exosporium proteins, exosporium protein fragments, and spore coat proteins described herein would all be viable ways of delivery the NOS to seeds.

Example 66: Modulation of enzyme expression and plant growth As demonstrated in Examples 44, 45, and 46, overexpression of a modulator protein in a recombinant Bacillus cereus family member that co-expresses a fusion n can lead to increased and sed levels of that fusion protein being incorporated into the exosporium. Fusion proteins and constructs were made and spores made as described above in Examples 44 and 45. Growth assays were performed as bed above in Example 46.

As can be seen in Table 72, expression of the pSUPER BclA 20-35 Endo fusion proteins on the surface of the Bacillus thuringiensis BT013A spores using amino acids —35 of SEQ ID NO: 1 as the targeting sequence led to increased growth in corn, soy, and squash. This effect can be sed when a second exosporium protein is overexpressed. Each of the CotO, BxpB, and YjcB overexpression s had a pronounced effect on corn, soy, and/or squash growth, with increases most prominent in corn.

Table 72: Spore bound SODA and free NOS and increased sorghum outgrowth Treatment Corn Growth fly Sguash Growth Growth HzO, 1.0 l/seed 100.0% 100.0% 100.0% Bacillus thuringiensis 103.8% 108.8% 105.8% BT013A with pSUPER BclA -35 Endo Base Base with oHP13 BclA-CotO 109.6% 106.4% 105.2% Base With pHPl3 BClA- 106.8% 117.2% 113.9% BXoB Base with oHP13 BclA-Y'cB 110.4% 122.4% 106.7% Overexpression of other tor proteins can also modulate fusion protein expression levels as well as plant growth effects, including those described herein and in es 44 and 45 above. Each of these can be used to alter or tailor the enzyme levels to desired effective levels.

Example 67: Overexpression of Exosporium Proteins and Effects of on Plants Overexpression of lly occurring spore and exosporium proteins can impact the effect that plant growth promoting, endophytic, and other Bacillus cereus family members have on plants. Expression of various exosporium proteins as part of a fusion protein or as free enzyme can have beneficial effects on plants, as illustrated above for phosphatases (Examples 11 and 36), nitric oxide synthatase (Example 65), and proteases such as InhA les 3, 6, 7, 13). Other rium and spore proteins, such as alanine racemase and inosine uridine preferring hydrolases, can prevent or delay germination of spores, and their pression will make spore less prone to quick germination, an unwanted side effect in the use ofmany types of spores. Lastly, spores that overexpress certain exosporium proteins can alter the overall assembly of the exosporium, leading to alterations in the binding of spores to . An e of this can be seen in Table 73 below.

Spores were created as described for Bacillus thuringiensis BT013A in Example 58. Growth assays were performed by ent of 1 ul of whole cell broth from each construct per corn seed, or 2 ul per squash seed. Treatment of seeds, planting, and data ing was med as in Example 58.

Bacillus mycoia’es strain EE155, a plant growth promoting strain of the Bacillus cereus family, was transformed with overexpression plasmids as described in Example 44. Overexpression of exosporium proteins in this strain directly led to an increase in the binding of the spores to the plant, and leads to higher plant growth promotion. Specifically, overexpression of BclB, BclA, CotO, CotE led to enhanced plant growth promotion. Other exosporium proteins can be pressed that can lead to alterations in the structure of the exosporium, including Est, ExsFA/BxpB, CotY, CotO, ExsFB, InhAl, InhA2, Est, Est, chA, chB, Bch, AcpC, InhA3, alanine racemase l, alanine racemase 2, BclA, BclB, Bpr, BclE, BetA/BAS3290, CotE, Est, ExsK, Est, YabG, Tgl, superoxide dismutase l (SODAl), and superoxide dismutase 2 (SODAZ). Overexpression or on of any of these genes will lead to alterations of exosporium structure, and lead to potentiating the plant growth s associated with members of the Bacillus cereus family.

WO 44661 Table 73: Overexression of exos n orium roteins in Bacillus m coides EE155 Bacteria Overexpression Squash Growth Corn Growth protein on plasmid (Normalized to (Normalized to nHP13 l) control) Bacillus mycoz'des N/A (Control) B l 5 5 B l 5 5 B l 5 5 B l 5 5 B l 5 5 e 68: Plant tissues binding through use of rium displayed binding proteins Spores that are useful for the display of exogenous and endogenous proteins can be utilized as fusion partners to enhance spore binding to es, including plant tissue. To demonstrate this attribute, Bacillus thuringiensis BT013A spores were transformed with plasmids pSUPER BclA 20-35 TasA, pSUPER BclA 20-35 Expansin, pSUPER BclA 20-35 Endo, and pSUPER BclA 20-35 l. TasA and expansin are plant g proteins. The control plasmid ned the BclA promoter, a start codon and amino acids 20-35 of SEQ ID NO: 1, but did not include a fusion partner. These constructs were prepared as in identical fashion to the others described in above in Example 58.

To perform the tissue binding assay, 2 week old corn plants and 3 week old soybean plants were grown as described in Example 58, but without any seed treatment. The primary leaf and first trifoliate of the plants was then swabbed with 1 ml of spores containing each of the above constructs. The leaves were allowed to dry, clipped from the plants and placed into a 50 ml conical tube with 10 ml of water, and vortexed heavily. The spores that were released from the leaf into the water were counted on a hemacytometer, and the counts compared to those expected if no spores bound to the leaves. This experiment was repeated in ten times, and a second experiment was performed which ed plating of the water onto antibiotic plates (tetracycline plus nutrient agar) overnight at 30°C. The final counts are shown in Table 74.

Table 74: Plant tissue _ is increased with bindin_ rotein ex n ression on s n ores Treatment Crop l Binding Change in Binding % Change in (Construct) % Binding from (plate assay) Binding from control control -35 l Endolucansae Ex 0 ansin TasA Control (BclA Soy -35 Control BclA 20-3 5 Soy Endolucansae BclA 20-3 5 Soy Ex 0 ansin BclA 20-3 5 Soy TasA As can be seen from Table 74, the control BT013A spores have a high affinity for the BT013A spores for soybeans, with 58.3% and 65.2% of the spores bound for the controls. Despite this, expression of endoglucanase, expansin, or TasA on the surface of the spores led to an increase in binding of spores to the soy leaves, with many spore preparations approaching 100% bound to the leaves. In corn, there was much less binding for the control spores, especially in the plate assay. The results from the plate assay are the most striking, with an increase in each of the expression constructs, with TasA at 100% of spores bound in that assay.

These binding proteins can also be utilized in any of the recombinant spore forming microorganisms, utilizing any of the expression systems or fusion rs described herein. This system would also be useful in ction with the exosporium strips, to create a n ry system that is both cell free and binds tightly to leaves.

Example 69: Use of Recombinant Spore-Forming Bacteria Expressing Fusion Proteins Containing Cot/Cge Proteins and an Enzyme for Promoting Plant Growth Coat proteins form protein layers that are found on all Bacillus species spores described to date, as well as d genera Virginibacillus, Lysinibacz'llus, Clostrz'dz'a, and Paenz’bacz’llus. Fusion of proteins or es of interest to the coat proteins allows sion of foreign proteins on the surface of the spore, and delivery of these proteins or peptides of interest to plants. To demonstrate the ability of the coat proteins to deliver enzymes to plants, a series of constructs were created. The pHP13 plasmid from the Bacillus Genetic Stock Culture collection was used to clone each of the constructs described below into the le cloning site using homologous recombination utilizing their native promoter elements.

CotB, CotG, and CotC from Bacillus is M01099 or CgeA from Bacillus amyloliquefaciens was fused in frame with the endoglucanase eng gene from Bacillus subtilis 168, the lipA lipase gene from Bacillus subtilis 168, or the pc-plc gene from Bacillus thuringiensis BT013A. These ucts were cloned into pHP13 via homologous recombination, verified by Sanger sequencing, and transformed into Bacillus subtilis EE405, Bacillus subtilis A09, Bacillus cereus family member EE439, Bacillus sp. EE398, or Bacillus thuringiensis EE-B00184. Each ormant was also screened for correct clones by Sanger sequencing. After confirmation of the clones, each clone was grown up in brain heart infusion broth (BHI) plus tetracycline (10 ug/ml) overnight at 30°C, and 100ul of the ght culture was swabbed onto nutrient agar plates plus tetracycline. These plates were incubated at 30°C for 3 days, and spores were collected by swabbing with a water-wettened cotton swab and resuspended in water.

Spores for endoglucanase assays were then d to 1x108 CFU/ml in water, and assayed for enzyme activity by utilizing the chromophore 4 chloro 2 nitrophenyl cellotetrose (4C2NC, 3 mM in water). For this method, 50 ul of spores was placed into a 96 well plate, and 50 ul of a 300nM 4C2NC solution added to each plate. The plate was then incubated at 30°C and absorbance at 410 nm read after 0.5 hours. In all cases, the respective strain l absorbance was subtracted out of the total absorbance of each clone to negate any background activity.

Spores for s assays were diluted to 1x108 CFU/ml in water, and assayed for enzyme activity in a second method utilizing the chromophore 4-nitrophenyl palmitate (4NP, 3 mM in water). For this method, 50 ul of spores was placed into a 96 well plate, and 50 ul of a 300nM 4NP solution added to each plate. The plate was then ted at 30°C and absorbance at 410 nm read after 0.5 hours. In all cases, the respective strain control absorbance was subtracted out of the total absorbance of each clone to negate any ound activity.

Spores for phospholipase assays were d to 1x108 CFU/ml in water, and d for enzyme activity as described above for phospholipase in Example 58. In all cases, the respective strain control absorbance was subtracted out of the total absorbance of each clone to negate any background activity.

Plant growth responses and treatments were applied and collected as described for squash in Example 58 above. All heights were ized against a strain control with no enzyme displayed on the spores.

Table 75: Coat n fusions and their enzyme expression levels.

Treatment Fusion Endoglucanase Lipase Phospholipase Plant (Construct) partner Enzyme Enzyme Enzyme growth for endo Activity, Activity, Activity, response (Absorbance (Absorbance (Absorbance above strain minus control) minus minus control, control control S u uash Bacillus subtilis N/A 100% A09 Strain Control 101.5% 101.0% mm99.4% 0-13 -_— ND Bacillus subtilis N/A 100% EE405 Strain Control EE405 1.84 104.7% B. thuringiensis N/A 100% BE 1 84 strain control .262 95.5% —_——-_105% 119.4% —-——-_110.1% Bacillus sp. N/A ND 100% EE387 strain control 108.0% 105.6% 106.3% 108.5% N/A = not able ND = not determined The data in Table 75 demonstrate that the coat proteins work broadly on a number of plant benefiting enzymes, in both Bacillus cereus family s , EE439) and non-Bacillus cereus family members (EE405, A09, and EE3 87 from here and Example 65).

Addition of spore displayed enzymes, endoglucanase in this e, leads to an increased plant growth phenotype in most cases.

EMBODIMENTS 4] For further illustration, onal non-limiting embodiments of the present disclosure are set forth below.

Embodiment 1 is a fusion protein comprising at least one n or peptide of interest and: (1) a targeting sequence comprising amino acids l—30 of SEQ ID NO: 59; (2) a targeting sequence sing SEQ ID NO: 59; (3) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 60; (4) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 59; (5) a targeting ce comprising amino acids 4—30 of SEQ ID NO: 59; (6) a targeting sequence comprising amino acids 6—30 of SEQ ID NO: 59; (7) a ing sequence comprising amino acids 1—33 of SEQ ID NO: 61; (8) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 61 ; (9) a targeting sequence comprising SEQ ID NO: 61 ; (10) an exosporium protein comprising an amino acid sequence haVing at least 85% ce ty With SEQ ID NO: 62; (11) a targeting ce comprising amino acids 2—33 of SEQ ID NO: 61 ; (12) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 61; (13) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 61; (14) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 61; (15) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 63; (16) a targeting sequence comprising SEQ ID NO: 63; (17) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 64; (18) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 63; (19) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 63 ; (20) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 63 ; (21) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 63; (22) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 63; (23) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 65; (24) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 65; (25) a ing sequence comprising SEQ ID NO: 65; (26) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 66; (27) a targeting sequence comprising SEQ ID NO: 107; (28) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 65; (29) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 65 ; (30) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 67; (31) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 67; (32) a targeting sequence comprising SEQ ID NO: 67; (33) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 68; (34) an targeting sequence comprising amino acids 2—27 of SEQ ID NO: 67; (35) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 67; (36) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 67; (37) a targeting sequence comprising amino acids 1—38 of SEQ ID NO: 69; (38) a ing sequence comprising amino acids 23—38 of SEQ ID NO: 69; (39) a targeting ce comprising SEQ ID NO: 69; (40) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 70; (41) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 69; (42) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 69; (43) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 69; (44) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 69; (45) an exosporium protein comprising SEQ ID NO: 72; (46) a targeting sequence comprising SEQ ID NO: 73; (47) an exosporium protein sing an amino acid sequence haVing at least 95% identity With SEQ ID NO: 74; (48) a targeting ce comprising amino acids 1—42 of SEQ ID NO: 75; (49) a targeting sequence comprising amino acids 27—42 of SEQ ID NO: 75; (50) a targeting sequence comprising SEQ ID NO: 75; (51) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 76; (52) a ing sequence comprising amino acids 2—42 of SEQ ID NO: 75 ; (53) a targeting sequence comprising amino acids 5—42 of SEQ ID NO: 75 ; (54) a targeting sequence comprising amino acids 10—42 of SEQ ID NO: 75; (55) a targeting sequence comprising amino acids 15—42 of SEQ ID NO: 75; (56) a targeting ce comprising amino acids 20—42 of SEQ ID NO: 75; (57) a targeting sequence sing amino acids 25—42 of SEQ ID NO: 75 ; (58) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 77; (59) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 77; (60) a targeting sequence comprising SEQ ID NO: 77; (61) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 78; (62) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 77; (63) a targeting ce comprising amino acids 5—24 of SEQ ID NO: 77; (64) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 80; (65) a ing sequence sing amino acids 1—38 of SEQ ID NO: 81; (66) a ing sequence comprising amino acids 23—38 of SEQ ID NO: 81; (67) a targeting sequence comprising SEQ ID NO: 81; (68) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 82; (69) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 81; (70) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 81; (71) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 81; (72) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 81; (73) a targeting sequence sing amino acids 20—38 of SEQ ID NO: 81; (74) a targeting sequence comprising amino acids 1—34 of SEQ ID NO: 83; (75) a targeting 2015/050807 sequence comprising SEQ ID NO: 83; (76) an rium protein comprising an amino acid ce having at least 85% ty with SEQ ID NO: 84; (77) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 86; (78) a targeting sequence comprising amino acids 1—28 of SEQ ID NO: 87; (79) a targeting sequence comprising amino acids 13—28 of SEQ ID NO: 87; (80) a targeting sequence comprising SEQ ID NO: 87; (81) an exosporium n comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 88; (82) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 87; (83) a targeting sequence sing amino acids 5—28 of SEQ ID NO: 87; (84) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 87; (85) a targeting sequence comprising amino acids 1—28 of SEQ ID NO: 89; (86) a targeting sequence comprising SEQ ID NO: 89; (87) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 90; (88) a targeting ce comprising amino acids 2—28 of SEQ ID NO: 89; (89) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 89; (90) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 89; (91) a targeting sequence comprising amino acids 1—93 of SEQ ID NO: 91; (92) a targeting ce comprising SEQ ID NO: 91 ; (93) an exosporium protein comprising an amino acid sequence having at least 85% ty with SEQ ID NO: 92; (94) a targeting sequence comprising amino acids 2—93 of SEQ ID NO: 91; (95) a targeting sequence comprising amino acids 10—93 of SEQ ID NO: 91; (96) a targeting sequence comprising amino acids 20—93 of SEQ ID NO: 91 ; (97) a targeting sequence comprising amino acids 30—93 of SEQ ID NO: 91; (98) a targeting sequence comprising amino acids 40—93 of SEQ ID NO: 91; (99) a targeting sequence comprising amino acids 50—93 of SEQ ID NO: 91; (100) a targeting sequence comprising amino acids 60—93 of SEQ ID NO: 91; (101) a targeting sequence comprising amino acids 1—130 of SEQ ID NO: 93; (102) a targeting sequence comprising SEQ ID NO: 93; (103) an exosporium protein sing an amino acid sequence having at least 85% identity with SEQ ID NO: 94; (104) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 93; (105) a ing sequence comprising amino acids 10—130 of SEQ ID NO: 93 ; (106) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 93 ; (107) a targeting sequence comprising amino acids 30—130 of SEQ ID NO: 93 ; or (108) an exosporium protein comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 122.

Embodiment 2 is a fusion protein of embodiment 1, n the fusion protein comprises: (1) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 59; (2) a targeting sequence sing amino acids 4—30 of SEQ ID NO: 59; (3) a targeting sequence WO 44661 comprising amino acids 6—30 of SEQ ID NO: 59; (4) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 61; (5) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 61; (6) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 61; (7) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 61; (8) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 63; (9) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 63 ; (10) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 63; (11) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 63 ; (12) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 63; (13) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 65; (14) a ing sequence comprising amino acids 5—24 of SEQ ID NO: 65; (15) an targeting ce comprising amino acids 2—27 of SEQ ID NO: 67; (16) a ing sequence comprising amino acids 5—27 of SEQ ID NO: 67; (17) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 67; (18) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 69; (19) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 69; (20) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 69; (21) a targeting sequence sing amino acids 15—38 of SEQ ID NO: 69; (22) a targeting sequence comprising amino acids 2—42 of SEQ ID NO: 75; (23) a targeting sequence comprising amino acids 5—42 of SEQ ID NO: 75; (24) a targeting sequence comprising amino acids 10—42 of SEQ ID NO: 75; (25) a targeting sequence comprising amino acids 15—42 of SEQ ID NO: 75; (26) a targeting sequence comprising amino acids 20—42 of SEQ ID NO: 75; (27) a targeting sequence comprising amino acids 25—42 of SEQ ID NO: 75; (28) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 77; (29) a ing sequence comprising amino acids 5—24 of SEQ ID NO: 77; (30) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 81; (31) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 81; (32) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 81; (33) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 81; (34) a ing sequence comprising amino acids 20—38 of SEQ ID NO: 81; (35) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 87; (36) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 87; (37) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 87; (38) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 89; (39) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 89; (40) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 89; (41) a targeting sequence sing amino acids 2—93 of SEQ ID NO: 91; (42) a targeting sequence sing amino acids 10—93 of SEQ ID NO: 91; (43) a targeting ce comprising amino acids 20—93 of SEQ ID NO: 91 ; (44) a ing sequence comprising amino acids 30—93 of SEQ ID NO: 91; (45) a targeting sequence comprising amino acids 40—93 of SEQ ID NO: 91; (46) a ing sequence comprising amino acids 50—93 of SEQ ID NO: 91; (47) a targeting sequence comprising amino acids 60—93 of SEQ ID NO: 91; (48) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 93; (49) a targeting sequence sing amino acids 10—130 of SEQ ID NO: 93; (50) a targeting sequence sing amino acids 20—130 of SEQ ID NO: 93; or (51) a targeting sequence comprising amino acids 30—130 of SEQ ID NO: 93.

Embodiment 3 is a fusion protein comprising at least one protein or e of interest and: (1) a targeting sequence consisting of amino acids 20—33 of SEQ ID NO: 1; (2) a targeting ce consisting of amino acids 21—33 of SEQ ID NO: 1; (3) a targeting sequence consisting of amino acids 23—31 of SEQ ID NO: 1; (4) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 96; (5) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 96; (6) a targeting sequence consisting of amino acids 12—25 of SEQ ID NO: 3; (7) a targeting sequence consisting of amino acids 13—25 of SEQ ID NO: 3; (8) a targeting sequence consisting of amino acids 15—23 of SEQ ID NO: 3; (9) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 97; (10) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 98; (11) a targeting sequence consisting of amino acids 23—36 of SEQ ID NO: 5; (12) a targeting sequence consisting of amino acids 23—34 of SEQ ID NO: 5; (13) a targeting sequence consisting of amino acids 24—36 of SEQ ID NO: 5; (14) a targeting sequence consisting of amino acids 26—34 of SEQ ID NO: 5; (15) a ing sequence consisting of amino acids 13—26 of SEQ ID NO: 7; (16) a targeting sequence consisting of amino acids 13—24 of SEQ ID NO: 7; (17) a targeting sequence consisting of amino acids 14—26 of SEQ ID NO: 7; (18) a targeting sequence consisting of amino acids 16—24 of SEQ ID NO: 7; (19) a targeting sequence consisting of amino acids 9—22 of SEQ ID NO: 9; (20) a targeting sequence consisting of amino acids 9—20 of SEQ ID NO: 9; (21) a targeting sequence consisting of amino acids 10—22 of SEQ ID NO: 9; (22) a targeting sequence consisting of amino acids 12—20 of SEQ ID NO: 9; (23) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 105; (24) a targeting sequence ting of amino acids 1—13 of SEQ ID NO: 105; (25) a ing sequence consisting of amino acids 18—31 of SEQ ID NO: 11 ; (26) a targeting sequence ting of amino acids 18—29 of SEQ ID NO: 11 ; (27) a targeting sequence consisting of amino acids 19— 31 of SEQ ID NO: 11; (28) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 98 ; (29) a targeting ce ting of amino acids 1—13 of SEQ ID NO: 98 ; (30) a targeting sequence ting of amino acids 18—31 of SEQ ID NO: 13; (31) a targeting sequence 33 1 consisting of amino acids 18—29 of SEQ ID NO: 13; (32) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 13; (33) a targeting sequence consisting of amino acids 21— 29 of SEQ ID NO: 13; (34) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 99; (35) a targeting ce consisting of amino acids 1—13 of SEQ ID NO: 99; (36) a targeting sequence consisting of amino acids 28—41 of SEQ ID NO: 15; (37) a targeting sequence consisting of amino acids 28—39 of SEQ ID NO: 15; (38) a targeting sequence consisting of amino acids 29—41 of SEQ ID NO: 15; (39) a targeting sequence consisting of amino acids 31— 39 of SEQ ID NO: 15; (40) a targeting sequence ting of amino acids 12—25 of SEQ ID NO: 17; (41) a ing sequence consisting of amino acids 13—25 of SEQ ID NO: 17; (42) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 100; (43) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 19; (44) a targeting sequence ting of amino acids 18—29 of SEQ ID NO: 19; (45) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 19; (46) a targeting sequence consisting of amino acids 21— 29 of SEQ ID NO: 19; (47) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 21; (48) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 21; (49) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 21 ; (50) a targeting sequence consisting of amino acids 21—29 of SEQ ID NO: 21 ; (51) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 101 ; (52) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 101; (53) a targeting sequence consisting of amino acids 9—22 of SEQ ID NO: 23; (54) a targeting sequence ting of amino acids 9—20 of SEQ ID NO: 23 ; (55) a targeting sequence consisting of amino acids 10—22 of SEQ ID NO: 23 ; (56) a targeting ce consisting of amino acids 12—20 of SEQ ID NO: 23; (57) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 102; (58) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 102; (59) a ing sequence consisting of amino acids 9—22 of SEQ ID NO: 25; (60) a targeting sequence consisting of amino acids 9—20 of SEQ ID NO: 25; (61) a targeting sequence consisting of amino acids 10—22 of SEQ ID NO: ; (62) a targeting sequence consisting of amino acids 12—20 of SEQ ID NO: 25 ; (63) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 103; (64) a targeting sequence ting of amino acids 1—13 of SEQ ID NO: 103 ; (65) a targeting sequence consisting of amino acids 15—28 of SEQ ID NO: 27; (66) a targeting sequence consisting of amino acids 15—26 of SEQ ID NO: 27; (67) a targeting sequence consisting of amino acids 16— 28 of SEQ ID NO: 27; (68) a targeting ce consisting of amino acids 18—26 of SEQ ID NO: 27; (69) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 104; (70) a ing sequence consisting of amino acids 1—13 of SEQ ID NO: 104; (71) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 33; (72) a targeting sequence consisting of amino acids 1—11 of SEQ ID NO: 33; (73) a targeting sequence consisting of amino acids 3—11 of SEQ ID NO: 33; (74) a targeting sequence consisting of amino acids 1—14 of SEQ ID NO: 35; (75) a targeting sequence consisting of amino acids 1—12 of SEQ ID NO: ; (76) a targeting sequence consisting of amino acids 2—14 of SEQ ID NO: 35; (77) a targeting sequence consisting of amino acids 14—27 of SEQ ID NO: 43; (78) a ing sequence consisting of amino acids 14—25 of SEQ ID NO: 43; (79) a targeting sequence consisting of amino acids 15—27 of SEQ ID NO: 43; (80) a targeting sequence consisting of amino acids 20— 33 of SEQ ID NO: 45; (81) a ing ce consisting of amino acids 20—31 of SEQ ID NO: 45 ; (82) a targeting sequence consisting of amino acids 21—33 of SEQ ID NO: 45 ; (83) a targeting sequence ting of amino acids 1—15 of SEQ ID NO: 106; (84) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 106; (85) a targeting sequence consisting of amino acids 28—41 of SEQ ID NO: 47; (86) a targeting sequence consisting of amino acids 28—39 of SEQ ID NO: 47; (87) a targeting sequence consisting of amino acids 18— 31 of SEQ ID NO: 53; (88) a targeting sequence ting of amino acids 18—29 of SEQ ID NO: 53 ; (89) a targeting sequence ting of amino acids 19—31 of SEQ ID NO: 53 ; (90) a targeting sequence comprising amino acids 18—31 of SEQ ID NO: 61; (91) a targeting sequence comprising amino acids 18—29 of SEQ ID NO: 61; (92) a targeting sequence sing amino acids 19—31 of SEQ ID NO: 61; (93) a targeting sequence comprising amino acids 9—22 of SEQ ID NO: 65; (94) a targeting sequence comprising amino acids 9—20 of SEQ ID NO: 65 ; (95) a targeting sequence sing amino acids 10—22 of SEQ ID NO: 65; (96) a targeting sequence sing amino acids 1—15 of SEQ ID NO: 107; (97) a targeting sequence comprising amino acids 1—13 of SEQ ID NO: 107; (98) a targeting ce comprising amino acids 12—25 of SEQ ID NO: 67; (99) a targeting sequence comprising amino acids 12—23 of SEQ ID NO: 67; (100) a targeting sequence comprising amino acids 13—25 of SEQ ID NO: 67; (101) a targeting sequence comprising amino acids 15—23 of SEQ ID NO: 67; (102) a targeting sequence comprising amino acids 23—36 of SEQ ID NO: 69; (103) a targeting sequence comprising amino acids 23—34 of SEQ ID NO: 69; (104) a targeting sequence comprising amino acids 24—36 of SEQ ID NO: 69; (105) a targeting sequence comprising amino acids 26—34 of SEQ ID NO: 69; (106) a targeting sequence comprising amino acids 27—40 of SEQ ID NO: 75; (107) a targeting sequence comprising amino acids 27—38 of SEQ ID NO: 75; (108) a targeting sequence comprising amino acids 9—22 of SEQ ID NO: 77; (109) a targeting sequence comprising amino acids 9—20 of SEQ ID NO: 77; (110) a targeting sequence comprising amino acids 1044—22 of SEQ ID NO: 77; (111) a targeting sequence comprising amino acids 12—20 of SEQ ID NO: 77; (112) a targeting sequence comprising amino acids 23—36 of SEQ ID NO: 81; (113) a targeting sequence comprising amino acids 23—34 of SEQ ID NO: 81; (114) a targeting sequence comprising amino acids 24—36 of SEQ ID NO: 81; (115) a targeting sequence comprising amino acids 26—34 of SEQ ID NO: 81; (116) a targeting sequence comprising amino acids 13—26 of SEQ ID NO: 87; (117) a targeting sequence comprising amino acids 13—24 of SEQ ID NO: 87; or (118) a targeting sequence comprising amino acids 14—26 of SEQ ID NO: 87. ment 4 is a fusion protein comprising an enzyme that catalyzes the tion of nitric oxide or a nucleic acid binding protein or peptide, and: (1) a ing sequence comprising an amino acid sequence having at least about 43% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity With amino acids 25—35 is at least about 54%; (2) a ing sequence comprising amino acids 1—35 of SEQ ID NO: 1; (3) a targeting sequence comprising amino acids 20—35 of SEQ ID NO: 1; (4) a targeting sequence comprising SEQ ID NO: 1; (5) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 2; (6) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 1; (7) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 1; (8) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 1 ; (9) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 1; (10) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 1; (11) a targeting ce comprising amino acids 1—27 of SEQ ID NO: 3; (12) a targeting ce sing amino acids 12—27 of SEQ ID NO: 3; (13) a targeting sequence comprising SEQ ID NO: 3; (14) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 4; (15) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 3; (16) a targeting sequence sing amino acids 5—27 of SEQ ID NO: 3; (17) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 3; (18) a targeting sequence sing amino acids 10—27 of SEQ ID NO: 3; (19) a targeting sequence comprising amino acids 1—38 of SEQ ID NO: 5; (20) a targeting sequence comprising amino acids 23—38 of SEQ ID NO: 5; (21) a targeting sequence comprising SEQ ID NO: 5; (22) an exosporium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO: 6; (23) a ing ce comprising amino acids 2—38 of SEQ ID NO: 5; (24) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 5; (25) a targeting ce comprising amino acids 8—38 of SEQ ID NO: 5; (26) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 5; (27) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 5; (28) a targeting sequence sing amino acids 20—38 of SEQ ID NO: 5; (29) a targeting sequence sing amino acids 1—28 of SEQ ID NO: 7; (30) a targeting sequence comprising amino acids 13—28 of SEQ ID NO: 7; (3 l) a targeting ce comprising SEQ ID NO: 7; (32) an exosporium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO: 8; (33) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 7; (34) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 7; (35) a targeting sequence comprising amino acids 8—28 of SEQ ID NO: 7; (36) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 7; (37) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 9; (38) a targeting sequence sing amino acids 9—24 of SEQ ID NO: 9; (39) a targeting sequence comprising SEQ ID NO: 9; (40) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 10; (41) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 9; (42) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 9; (43) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 9; (44) a targeting sequence comprising amino acids 1—33 of SEQ ID NO:ll; (45) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 11 ; (46) a targeting sequence comprising SEQ ID NO: 11 ; (47) an exosporium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO: 12; (48) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 11 ; (49) a targeting ce comprising amino acids 5—33 of SEQ ID NO: 11 ; (50) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 11 ; (5 l) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 11; (52) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: ll; (53) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 13; (54) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 13; (55) a targeting sequence comprising SEQ ID NO: 13; (5 6) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 14; (57) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 13; (58) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13; (59) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 13; (60) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 13; (61) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 13; (62) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 15; (63) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 15; (64) a targeting sequence comprising SEQ ID NO:l5; (65) an exosporium protein comprising an amino acid ce having at least 85% identity with SEQ ID NO:16; (66) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 15; (67) a targeting sequence comprising amino WO 44661 33 5 acids 5—43 of SEQ ID NO: 15 ; (68) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 15; (69) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 15; (70) a ing sequence comprising amino acids 15—43 of SEQ ID NO: 15; (71) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 15; (72) a targeting ce comprising amino acids 25—43 of SEQ ID NO: 15; (73) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 17; (74) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 17; (75) a targeting sequence comprising SEQ ID NO:17; (76) an exosporium n comprising an amino acid sequence having at least 85% identity with SEQ ID NO:18; (77) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 17; (78) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 17; (79) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 17; (80) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 17; (81) a targeting ce comprising amino acids 1—33 of SEQ ID NO: 19; (82) a targeting sequence sing amino acids 18—33 of SEQ ID NO: 19; (83) a targeting sequence comprising SEQ ID NO:19; (84) an exosporium n comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:20; (85) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 19; (86) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 19; (87) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 19; (88) a targeting ce comprising amino acids 10—33 of SEQ ID NO: 19; (89) a targeting ce comprising amino acids 15—33 of SEQ ID NO: 19; (90) a targeting sequence sing amino acids 1—33 of SEQ ID NO: 21; (91) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 21; (92) a targeting sequence comprising SEQ ID NO:21; (93) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:22; (94) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 21 ; (95) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 21; (96) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 21; (97) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 21; (98) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 21 ; (99) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 23; (100) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 23 ; (101) a targeting sequence comprising SEQ ID NO:23; (102) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:24; (103) a targeting sequence comprising amino acids 2—24 of SEQ ID NO:23; (104) a targeting ce comprising amino acids 5—24 of SEQ ID NO: 23; (105) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 23; (106) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 25; (107) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 25; (108) a targeting sequence comprising SEQ ID NO:25; (109) an rium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:26; (110) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 25; (111) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 25; (112) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 25; (113) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 27; (114) a targeting ce comprising amino acids 15—30 of SEQ ID NO: 27; (115) a targeting sequence comprising SEQ ID NO:27; (116) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:28; (117) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 27; (118) a ing sequence comprising amino acids 5—30 of SEQ ID NO: 27; (119) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 27; (120) a targeting ce comprising amino acids 10—30 of SEQ ID NO: 27; (121) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 29; (122) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 29; (123) a targeting sequence sing SEQ ID NO:29; (124) an exosporium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO:30; (125) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 29; (126) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 29; (127) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 29; (128) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 29; (129) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 29; (130) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 31 ; (131) a ing sequence comprising amino acids 9—24 of SEQ ID NO: 31 ; (132) a targeting sequence comprising SEQ ID NO:31; (133) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO:32; (134) a targeting sequence sing amino acids 2—24 of SEQ ID NO: 31; (135) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31; (136) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 31 ; (137) a targeting sequence comprising amino acids 1—15 of SEQ ID NO: 33; (138) a targeting ce comprising SEQ ID NO:33; (139) an exosporium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO:34; (140) a targeting sequence comprising amino acids 1—16 of SEQ ID NO: 35; (141) a targeting sequence comprising SEQ ID NO:35; (142) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:36; (143) a ing sequence comprising amino acids 1—29 of SEQ ID NO:43; (144) a targeting sequence comprising amino acids 14—29 of SEQ ID NO: 43; (145) a targeting sequence comprising SEQ ID NO: 43; (146) an exosporium protein comprising an amino acid sequence haVing at least 2015/050807 85% identity with SEQ ID NO: 44; (147) a targeting sequence comprising amino acids 2—29 of SEQ ID NO: 43 ; (148) a targeting sequence comprising amino acids 5—29 of SEQ ID NO: 43 ; (149) a targeting sequence comprising amino acids 8—29 of SEQ ID NO: 43; (150) a targeting sequence comprising amino acids 10—29 of SEQ ID NO: 43; (151) a targeting ce comprising amino acids 1—35 of SEQ ID NO: 45; (152) a targeting sequence comprising amino acids 20—35 of SEQ ID NO: 45; (153) a targeting sequence comprising SEQ ID NO: 45 ; (154) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 46; (155) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 45; (156) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 45; (157) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 45; (158) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45; (159) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 45 ; (160) a targeting ce comprising amino acids 1—43 of SEQ ID NO: 47; (161) a ing sequence comprising amino acids 28—43 of SEQ ID NO: 47; (162) a targeting sequence comprising SEQ ID NO: 47; (163) an exosporium protein sing an amino acid ce haVing at least 85% identity With SEQ ID NO: 48; (164) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 47; (165) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 47; (166) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 47; (167) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 47; (168) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 47; (169) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (170) a targeting sequence sing amino acids 25—43 of SEQ ID NO: 47; (171) a targeting sequence comprising amino acids 1—32 of SEQ ID NO: 49; (172) a targeting ce comprising amino acids 17—32 of SEQ ID NO: 49; (173) a targeting sequence comprising SEQ ID NO: 49; (174) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 50; (175) a targeting sequence sing amino acids 2—32 of SEQ ID NO: 49; (176) a ing ce comprising amino acids 5—32 of SEQ ID NO: 49; (177) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (178) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (179) a targeting sequence comprising amino acids 15—32 of SEQ ID NO: 49; (180) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 51; (181) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 51; (182) a targeting sequence comprising SEQ ID NO: 51; (183) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 52; (184) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 51; (185) a targeting sequence 33 8 comprising amino acids 5—33 of SEQ ID NO: 51; (186) a targeting ce comprising amino acids 8—33 of SEQ ID NO: 51; (187) a targeting ce comprising amino acids 10—33 of SEQ ID NO: 51; (188) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 51; (189) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 53; (190) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 53; (191) a targeting sequence comprising SEQ ID NO: 53; (192) an exosporium protein sing an amino acid sequence having at least 85% identity with SEQ ID NO: 54; (193) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 53; (194) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53 ; (195) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53 ; (196) a targeting ce comprising amino acids 10—33 of SEQ ID NO: 53 ; (197) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 53; (198) a targeting sequence sing amino acids 1—30 of SEQ ID NO: 55; (199) a ing sequence comprising amino acids 15—30 of SEQ ID NO: 55 ; (200) a targeting sequence comprising SEQ ID NO: 55 ; (201) an exosporium n comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 56; (202) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 55; (203) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 55; (204) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 55; (205) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 55; (206) a targeting sequence comprising amino acids 1—130 of SEQ ID NO: 57; (207) a targeting sequence comprising amino acids 115—130 of SEQ ID NO: 57; (208) a targeting sequence comprising SEQ ID NO: 57; (209) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 58; (210) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 57; (211) a targeting sequence comprising amino acids 5—130 of SEQ ID NO: 57; (212) a targeting ce comprising amino acids 10—130 of SEQ ID NO: 57; (213) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 57; (214) a targeting sequence sing amino acids 30— 130 of SEQ ID NO: 57; (215) a targeting ce comprising amino acids 40—130 of SEQ ID NO: 57; (216) a targeting sequence sing amino acids 50—130 of SEQ ID NO: 57; (217) a targeting sequence comprising amino acids 60—130 of SEQ ID NO: 57; (218) a targeting sequence comprising amino acids 70—130 of SEQ ID NO: 57; (219) a targeting sequence comprising amino acids 80—130 of SEQ ID NO: 57; (220) a targeting sequence comprising amino acids 90—130 of SEQ ID NO: 57; (221) a targeting sequence comprising amino acids 100—130 of SEQ ID NO: 57; (222) a targeting sequence sing amino acids 110—130 of SEQ ID NO: 57; (223) an exosporium protein fragment comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 95; (224) a ing sequence comprising SEQ ID NO: 96; (225) a targeting sequence sing SEQ ID NO: 97; (226) a targeting ce comprising SEQ ID NO: 98; (227) a targeting sequence comprising SEQ ID NO: 99; (228) a targeting sequence sing SEQ ID NO: 100; (229) a targeting sequence comprising SEQ ID NO: 101; (230) a targeting sequence comprising SEQ ID NO: 102; (231) a targeting sequence comprising SEQ ID NO: 103; (232) a targeting ce comprising SEQ ID NO: 104; (233) a targeting sequence comprising SEQ ID NO: 105; (234) a targeting sequence comprising SEQ ID NO: 106; (235) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (236) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 109; (237) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 110; (238) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 111; (239) an exosporium n comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 112; (240) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 113; (241) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 114; (242) an exosporium n comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 115; (243) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 116; (244) an exosporium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO: 117; (245) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 118; (246) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 119; (247) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 120; (248) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 121 ; (249) a targeting ce comprising amino acids 22—31 of SEQ ID NO: 1; (250) a targeting sequence comprising amino acids 22—33 of SEQ ID NO: 1; (251) a targeting sequence comprising amino acids 20—31 of SEQ ID NO: 1; (252) a targeting sequence comprising amino acids 14—23 of SEQ ID NO: 3; (253) a targeting sequence comprising amino acids 14—25 of SEQ ID NO: 3; or (254) a targeting sequence comprising amino acids 12—23 of SEQ ID NO: 3.

Embodiment 5 is a fusion protein comprising an n or a remediation enzyme and: (1) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 1; (2) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 1; (3) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 1; (4) a ing ce comprising amino acids 10—35 of SEQ ID NO: 1; (5) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 1; (6) a targeting sequence comprising amino acids 22—31 of SEQ ID NO: 1; (7) a targeting sequence comprising amino acids 22—33 of SEQ ID NO: 1; (8) a ing sequence comprising amino acids 20—31 of SEQ ID NO: 1; (9) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 3; (10) a ing sequence sing amino acids 5—27 of SEQ ID NO: 3; (11) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 3; (12) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 3; (13) a targeting sequence comprising amino acids 14—23 of SEQ ID NO: 3; (14) a targeting sequence comprising amino acids 14—25 of SEQ ID NO: 3; (15) a targeting sequence comprising amino acids 12—23 of SEQ ID NO: 3; (16) a ing sequence comprising amino acids 2—38 of SEQ ID NO: 5; (17) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 5; (18) a targeting sequence comprising amino acids 8—38 of SEQ ID NO: 5; (19) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 5; (20) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 5; (21) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 5; (22) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 7; (23) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 7; (24) a targeting sequence comprising amino acids 8—28 of SEQ ID NO: 7; (25) a targeting ce comprising amino acids 10—28 of SEQ ID NO: 7; (26) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 9; (27) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 9; (28) a targeting sequence comprising SEQ ID NO: 9; (29) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 10; (30) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 9; (31) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 9; (32) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 9; (33) a targeting sequence comprising amino acids 1—33 of SEQ ID NO:11; (34) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 11 ; (35) a targeting sequence comprising SEQ ID NO: 11 ; (36) an exosporium protein comprising an amino acid sequence having at least 85% ty With SEQ ID NO: 12; (37) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 11; (38) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 11; (39) a ing sequence comprising amino acids 8—33 of SEQ ID NO: 11 ; (40) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 11 ; (41) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 11; (42) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 13; (43) a targeting ce comprising amino acids 18—33 of SEQ ID NO: 13 ; (44) a WO 44661 ing sequence comprising SEQ ID NO:13; (45) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 14; (46) a ing sequence comprising amino acids 2—33 of SEQ ID NO: 13; (47) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13; (48) a targeting ce comprising amino acids 8—33 of SEQ ID NO: 13 ; (49) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 13 ; (50) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 13; (51) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 15; (52) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 15; (53) a targeting sequence comprising SEQ ID NO:15; (54) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:16; (55) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 15 ; (56) a targeting sequence sing amino acids 5—43 of SEQ ID NO: 15; (57) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 15; (58) a targeting ce comprising amino acids 10—43 of SEQ ID NO: 15 ; (59) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 15; (60) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 15; (61) a targeting sequence sing amino acids 25—43 of SEQ ID NO: 15; (62) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 17; (63) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 17; (64) a ing sequence comprising SEQ ID NO:17; (65) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:18; (66) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 17; (67) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 17; (68) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 17; (69) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 17; (70) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 19; (71) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 19; (72) a targeting sequence comprising SEQ ID NO:19; (73) an rium n comprising an amino acid sequence having at least 85% ty With SEQ ID NO:20; (74) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 19; (75) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 19; (76) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 19; (77) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 19; (78) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 19; (79) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 21; (80) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 21; (81) a ing sequence comprising SEQ ID NO:21; (82) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:22; (83) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 21; (84) a ing sequence comprising amino acids 5—33 of SEQ ID NO: 21; (85) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 21; (86) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 21; (87) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 21; (88) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 23; (89) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 23; (90) a ing ce comprising SEQ ID NO:23; (91) an rium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO:24; (92) a targeting sequence comprising amino acids 2—24 of SEQ ID NO:23; (93) a targeting ce comprising amino acids 5—24 of SEQ ID NO: 23; (94) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 23 ; (95) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 25; (96) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 25; (97) a targeting ce comprising SEQ ID NO:25; (98) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:26; (99) a targeting sequence sing amino acids 2—24 of SEQ ID NO: 25; (100) a ing sequence comprising amino acids 5—24 of SEQ ID NO: 25 ; (101) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 25 ; (102) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 27; (103) a targeting sequence comprising amino acids 15—30 of SEQ ID NO: 27; (104) a targeting sequence comprising SEQ ID NO:27; (105) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:28; (106) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 27; (107) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 27; (108) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 27; (109) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 27; (110) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 29; (111) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 29; (112) a targeting sequence comprising SEQ ID NO:29; (113) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:30; (114) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 29; (115) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 29; (116) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 29; (117) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 29; (118) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 29; (119) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 31; (120) a ing sequence comprising amino acids 9—24 of SEQ ID NO: 31; (121) a targeting sequence comprising SEQ ID NO:31; (122) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO:32; (123) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 31; (124) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31; (125) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 31; (126) a targeting sequence comprising amino acids 1—15 of SEQ ID NO: 33; (127) a targeting sequence comprising SEQ ID NO:33; (128) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:34; (129) a targeting sequence comprising amino acids 1—16 of SEQ ID NO: 35; (130) a targeting sequence comprising SEQ ID NO:35; (131) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:36; (132) a targeting sequence comprising amino acids 1—29 of SEQ ID NO:43; (133) a targeting sequence comprising amino acids 14—29 of SEQ ID NO: 43; (134) a targeting sequence comprising SEQ ID NO: 43; (135) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 44; (136) a targeting sequence comprising amino acids 2—29 of SEQ ID NO: 43; (137) a targeting sequence comprising amino acids 5—29 of SEQ ID NO: 43; (138) a targeting sequence comprising amino acids 8—29 of SEQ ID NO: 43; (139) a targeting sequence sing amino acids 10—29 of SEQ ID NO: 43 ; (140) a ing ce comprising amino acids 1—35 of SEQ ID NO: 45; (141) a targeting sequence comprising amino acids 20—35 of SEQ ID NO: 45; (142) a ing sequence comprising SEQ ID NO: 45; (143) an exosporium n comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 46; (144) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 45; (145) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 45 ; (146) a ing sequence sing amino acids 8—35 of SEQ ID NO: 45 ; (147) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45 ; (148) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 45; (149) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 47; (150) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 47; (151) a targeting sequence comprising SEQ ID NO: 47; (152) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 48; (153) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 47; (154) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 47; (155) a targeting ce sing amino acids 8—43 of SEQ ID NO: 47; (156) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 47; (157) a targeting ce comprising amino acids 15—43 of SEQ ID NO: 47; (158) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (159) a targeting ce comprising amino acids 25—43 of SEQ ID NO: 47; (160) a targeting sequence comprising amino acids 1—32 of SEQ ID NO: 49; (161) a targeting sequence comprising amino acids 17—32 of SEQ ID NO: 49; (162) a targeting WO 44661 2015/050807 sequence comprising SEQ ID NO: 49; (163) an exosporium protein sing an amino acid sequence having at least 85% identity with SEQ ID NO: 50; (164) a ing sequence comprising amino acids 2—32 of SEQ ID NO: 49; (165) a targeting sequence comprising amino acids 5—32 of SEQ ID NO: 49; (166) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (167) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (168) a targeting sequence comprising amino acids 15—32 of SEQ ID NO: 49; (169) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 51; (170) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 51; (171) a targeting sequence comprising SEQ ID NO: 51; (172) an exosporium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO: 52; (173) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 51; (174) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 51; (175) a targeting sequence sing amino acids 8—33 of SEQ ID NO: 51; (176) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 51; (177) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 51; (178) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 53 ; (179) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 53; (180) a targeting sequence comprising SEQ ID NO: 53; (181) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 54; (182) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 53; (183) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53; (184) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53; (185) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 53 ; (186) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 53; (187) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 55; (188) a targeting sequence sing amino acids 15—30 of SEQ ID NO: 55; (189) a targeting sequence comprising SEQ ID NO: 55; (190) an exosporium n comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 56; (191) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 55; (192) a ing sequence comprising amino acids 5—30 of SEQ ID NO: 55 ; (193) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 55 ; (194) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 55 ; (195) a targeting sequence sing amino acids 1—130 of SEQ ID NO: 57; (196) a targeting sequence comprising amino acids 115—130 of SEQ ID NO: 57; (197) a ing sequence comprising SEQ ID NO: 57; (198) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 58; (199) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 57; (200) a targeting sequence comprising amino acids 5—130 of SEQ ID NO: 57; (201) a targeting ce comprising amino acids 10—130 of SEQ ID NO: 57; (202) a targeting ce comprising amino acids 20—130 of SEQ ID NO: 57; (203) a targeting ce comprising amino acids 30—130 of SEQ ID NO: 57; (204) a targeting ce comprising amino acids 40—130 of SEQ ID NO: 57; (205) a targeting sequence comprising amino acids 50—130 of SEQ ID NO: 57; (206) a ing sequence comprising amino acids 60— 130 of SEQ ID NO: 57; (207) a targeting sequence comprising amino acids 70—130 of SEQ ID NO: 57; (208) a targeting sequence comprising amino acids 80—130 of SEQ ID NO: 57; (209) a targeting sequence comprising amino acids 90—130 of SEQ ID NO: 57; (210) a targeting sequence comprising amino acids 100—130 of SEQ ID NO: 57; (211) a targeting sequence comprising amino acids 110—130 of SEQ ID NO: 57; (212) a targeting sequence comprising SEQ ID NO: 97; (213) a targeting sequence comprising SEQ ID NO: 98; (214) a ing sequence comprising SEQ ID NO: 99; (215) a targeting sequence comprising SEQ ID NO: 100; (216) a targeting sequence comprising SEQ ID NO: 101 ; (217) a targeting sequence comprising SEQ ID NO: 102; (218) a targeting sequence comprising SEQ ID NO: 103; (219) a targeting sequence comprising SEQ ID NO: 104; (220) a ing sequence comprising SEQ ID NO: 105; (221) a targeting sequence comprising SEQ ID NO: 106; (222) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 108; (223) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 109; (224) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 110; (225) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 111; (226) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 112; (227) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 113; (228) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 114; (229) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 115; (230) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 116; (231) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 117; (232) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 118; (233) an exosporium n comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 119; (234) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 120; or (235) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 121.

Embodiment 6 is the fusion protein of any one of embodiments 1—3, n the protein or peptide of interest comprises an antigen or a remediation enzyme.

Embodiment 7 is the fusion protein of any one of embodiments 1—3, wherein the protein or peptide of interest comprises an enzyme suitable for breaking an emulsion or gel in a hydraulic fracturing fluid, or an antibacterial n or peptide.

Embodiment 8 is a fusion protein comprising an enzyme suitable for breaking an emulsion or gel in a hydraulic fracturing fluid or an cterial protein or peptide and: (1) a targeting sequence comprising an amino acid sequence haVing at least about 43% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 54%; (2) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 1; (3) a targeting sequence comprising amino acids 20—35 of SEQ ID NO: 1; (4) a ing sequence comprising SEQ ID NO: 1; (5) an exosporium protein comprising an amino acid ce haVing at least 85% ty with SEQ ID NO: 2; (6) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 1; (7) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 1; (8) a ing sequence comprising amino acids 8—35 of SEQ ID NO: 1; (9) a targeting ce comprising amino acids 10—35 of SEQ ID NO: 1; (10) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 1; (11) a targeting sequence sing amino acids 1—27 of SEQ ID NO: 3; (12) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 3; (13) a targeting ce comprising SEQ ID NO: 3; (14) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 4; (15) a targeting sequence sing amino acids 2—27 of SEQ ID NO: 3; (16) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 3; (17) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 3; (18) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 3; (19) a targeting sequence sing amino acids 1—38 of SEQ ID NO: 5; (20) a targeting sequence comprising amino acids 23—38 of SEQ ID NO: 5; (21) a targeting sequence comprising SEQ ID NO: 5; (22) an exosporium n comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 6; (23) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 5; (24) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 5; (25) a ing sequence comprising amino acids 8—38 of SEQ ID NO: 5; (26) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 5; (27) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 5; (28) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 5; (29) a targeting sequence comprising amino acids 1—28 of SEQ ID NO: 7; (30) a targeting sequence comprising amino acids 13—28 of SEQ ID NO: 7; (31) a targeting sequence comprising SEQ ID NO: 7; (32) an rium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 8; (33) a ing sequence comprising amino acids 2—28 of SEQ ID NO: 7; (34) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 7; (35) a ing sequence comprising amino acids 8—28 of SEQ ID NO: 7; (36) a targeting ce comprising amino acids 10—28 of SEQ ID NO: 7; (37) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 9; (38) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 9; (39) a targeting sequence sing SEQ ID NO: 9; (40) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 10; (41) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 9; (42) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 9; (43) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 9; (44) a targeting sequence comprising amino acids 1—33 of SEQ ID NO:11; (45) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 11; (46) a targeting sequence comprising SEQ ID NO: 11; (47) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 12; (48) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 11; (49) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 11; (50) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 11; (51) a ing sequence comprising amino acids 10—33 of SEQ ID NO: 11; (52) a targeting ce comprising amino acids 15—33 of SEQ ID NO: 11; (53) a targeting sequence comprising amino acids l—33 of SEQ ID NO: 13; (54) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 13; (55) a targeting sequence sing SEQ ID NO:13; (56) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 14; (57) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 13; (58) a targeting ce comprising amino acids 5—33 of SEQ ID NO: 13; (59) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 13; (60) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 13; (61) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 13; (62) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 15; (63) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 15 ; (64) a targeting ce comprising SEQ ID NO:15; (65) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:16; (66) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 15; (67) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 15; (68) a ing sequence comprising amino acids 8—43 of SEQ ID NO: 15; (69) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 15 ; (70) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 15; (71) a targeting sequence sing amino acids 20—43 of SEQ ID NO: 15; (72) a targeting sequence sing amino acids 25—43 of SEQ ID NO: 15; (73) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 17; (74) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 17; (75) a targeting sequence comprising SEQ ID NO:17; (76) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:18; (77) a ing ce comprising amino acids 2—27 of SEQ ID NO: 17; (78) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 17; (79) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 17; (80) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 17; (81) a ing ce comprising amino acids 1—33 of SEQ ID NO: 19; (82) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 19; (83) a targeting sequence sing SEQ ID NO:19; (84) an exosporium n comprising an amino acid sequence having at least 85% identity With SEQ ID NO:20; (85) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 19; (86) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 19; (87) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 19; (88) a ing sequence comprising amino acids 10—33 of SEQ ID NO: 19; (89) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 19; (90) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 21 ; (91) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 21; (92) a targeting sequence comprising SEQ ID NO:21; (93) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:22; (94) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 21; (95) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 21; (96) a ing sequence comprising amino acids 8—33 of SEQ ID NO: 21 ; (97) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 21 ; (98) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 21; (99) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 23; (100) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 23 ; (101) a targeting sequence comprising SEQ ID NO:23; (102) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:24; (103) a ing sequence comprising amino acids 2—24 of SEQ ID NO:23; (104) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 23; (105) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 23; (106) a targeting sequence sing amino acids 1—24 of SEQ ID NO: 25; (107) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 25 ; (108) a targeting sequence comprising SEQ ID NO:25; (109) an exosporium n comprising an amino acid sequence having at least 85% identity With SEQ ID NO:26; (110) a targeting ce comprising amino acids 2—24 of SEQ ID NO: 25; (111) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 25; (112) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 25; (113) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 27; (114) a ing sequence comprising amino acids 15—30 of SEQ ID NO: 27; (115) a targeting sequence comprising SEQ ID NO:27; (116) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:28; (117) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 27; (118) a targeting ce comprising amino acids 5—30 of SEQ ID NO: 27; (119) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 27; (120) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 27; (121) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 29; (122) a ing sequence comprising amino acids 18—33 of SEQ ID NO: 29; (123) a targeting ce comprising SEQ ID NO:29; (124) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:30; (125) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 29; (126) a targeting ce comprising amino acids 5—33 of SEQ ID NO: 29; (127) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 29; (128) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 29; (129) a targeting ce sing amino acids 15—33 of SEQ ID NO: 29; (130) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 31 ; (131) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 31 ; (132) a targeting sequence comprising SEQ ID NO:31; (133) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:32; (134) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 31; (135) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31; (136) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 31 ; (137) a targeting sequence sing amino acids 1—15 of SEQ ID NO: 33; (138) a targeting ce comprising SEQ ID NO:33; (139) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:34; (140) a targeting sequence comprising amino acids 1—16 of SEQ ID NO: 35; (141) a ing sequence comprising SEQ ID NO:35; (142) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:36; (143) a targeting sequence comprising amino acids 1—29 of SEQ ID NO:43; (144) a targeting sequence comprising amino acids 14—29 of SEQ ID NO: 43; (145) a targeting sequence comprising SEQ ID NO: 43; (146) an exosporium protein comprising an amino acid sequence having at least 3 50 85% identity with SEQ ID NO: 44; (147) a targeting sequence comprising amino acids 2—29 of SEQ ID NO: 43 ; (148) a targeting sequence comprising amino acids 5—29 of SEQ ID NO: 43 ; (149) a ing sequence comprising amino acids 8—29 of SEQ ID NO: 43; (150) a targeting ce comprising amino acids 10—29 of SEQ ID NO: 43; (151) a targeting sequence sing amino acids 1—35 of SEQ ID NO: 45; (152) a targeting sequence comprising amino acids 20—35 of SEQ ID NO: 45; (153) a targeting sequence comprising SEQ ID NO: 45 ; (154) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 46; (155) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 45; (156) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 45; (157) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 45; (158) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45; (159) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 45 ; (160) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 47; (161) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 47; (162) a targeting sequence comprising SEQ ID NO: 47; (163) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 48; (164) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 47; (165) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 47; (166) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 47; (167) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 47; (168) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 47; (169) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (170) a targeting sequence sing amino acids 25—43 of SEQ ID NO: 47; (171) a targeting sequence comprising amino acids 1—32 of SEQ ID NO: 49; (172) a targeting sequence sing amino acids 17—32 of SEQ ID NO: 49; (173) a targeting sequence comprising SEQ ID NO: 49; (174) an exosporium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO: 50; (175) a targeting ce comprising amino acids 2—32 of SEQ ID NO: 49; (176) a ing ce comprising amino acids 5—32 of SEQ ID NO: 49; (177) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (178) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (179) a targeting sequence comprising amino acids 15—32 of SEQ ID NO: 49; (180) a ing sequence comprising amino acids 1—33 of SEQ ID NO: 51; (181) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 51; (182) a ing sequence comprising SEQ ID NO: 51; (183) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 52; (184) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 51; (185) a ing sequence 3 5 1 comprising amino acids 5—33 of SEQ ID NO: 51; (186) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 51; (187) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 51; (188) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 51; (189) a targeting sequence sing amino acids 1—33 of SEQ ID NO: 53; (190) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 53; (191) a targeting sequence comprising SEQ ID NO: 53; (192) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 54; (193) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 53; (194) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53 ; (195) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53 ; (196) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 53 ; (197) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 53; (198) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 55; (199) a targeting sequence comprising amino acids 15—30 of SEQ ID NO: 55 ; (200) a targeting sequence comprising SEQ ID NO: 55 ; (201) an exosporium protein comprising an amino acid ce having at least 85% identity With SEQ ID NO: 56; (202) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 55; (203) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 55; (204) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 55; (205) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 55; (206) a targeting sequence sing amino acids 1—130 of SEQ ID NO: 57; (207) a targeting sequence comprising amino acids 115—130 of SEQ ID NO: 57; (208) a targeting sequence comprising SEQ ID NO: 57; (209) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 58; (210) a ing sequence comprising amino acids 2—130 of SEQ ID NO: 57; (211) a targeting sequence comprising amino acids 5—130 of SEQ ID NO: 57; (212) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (213) a targeting sequence sing amino acids 20—130 of SEQ ID NO: 57; (214) a targeting sequence comprising amino acids 30— 130 of SEQ ID NO: 57; (215) a targeting sequence comprising amino acids 40—130 of SEQ ID NO: 57; (216) a targeting sequence sing amino acids 50—130 of SEQ ID NO: 57; (217) a ing ce comprising amino acids 60—130 of SEQ ID NO: 57; (218) a targeting sequence sing amino acids 70—130 of SEQ ID NO: 57; (219) a targeting sequence comprising amino acids 80—130 of SEQ ID NO: 57; (220) a targeting sequence comprising amino acids 90—130 of SEQ ID NO: 57; (221) a ing sequence comprising amino acids 100—130 of SEQ ID NO: 57; (222) a targeting sequence comprising amino acids 110—130 of SEQ ID NO: 57; (223) an exosporium protein nt comprising an amino acid sequence 3 52 having at least 85% identity with SEQ ID NO: 95; (224) a targeting sequence comprising SEQ ID NO: 96; (225) a ing sequence comprising SEQ ID NO: 97; (226) a targeting sequence sing SEQ ID NO: 98; (227) a targeting ce comprising SEQ ID NO: 99; (228) a targeting sequence comprising SEQ ID NO: 100; (229) a targeting sequence comprising SEQ ID NO: 101; (230) a targeting sequence sing SEQ ID NO: 102; (231) a targeting ce comprising SEQ ID NO: 103; (232) a targeting sequence comprising SEQ ID NO: 104; (233) a targeting sequence comprising SEQ ID NO: 105; (234) a targeting sequence comprising SEQ ID NO: 106; (235) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (236) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 109; (237) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 110; (238) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 111; (239) an exosporium protein sing an amino acid sequence haVing at least 85% ty with SEQ ID NO: 112; (240) an rium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 113; (241) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 114; (242) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 115; (243) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 116; (244) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 117; (245) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 118; (246) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 119; (247) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 120; (248) an exosporium n comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 121 ; (249) a targeting ce comprising amino acids 22—31 of SEQ ID NO: 1; (250) a targeting sequence comprising amino acids 22—33 of SEQ ID NO: 1; (251) a targeting sequence comprising amino acids 20—31 of SEQ ID NO: 1; (252) a targeting sequence sing amino acids 14—23 of SEQ ID NO: 3; (253) a targeting ce comprising amino acids 14—25 of SEQ ID NO: 3; or (254) a targeting sequence comprising amino acids 12—23 of SEQ ID NO: 3.

Embodiment 9 is the fusion protein of any one of embodiments 5—8, wherein the targeting sequence comprises: (1) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 1; (2) a targeting ce comprising amino acids 5—35 of SEQ ID NO: 1; (3) a 3 53 targeting sequence sing amino acids 8—35 of SEQ ID NO: 1; (4) a ing sequence comprising amino acids 10—35 of SEQ ID NO: 1; (5) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 1; (6) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 3; (7) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 3; (8) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 3; (9) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 3; (10) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 5; (11) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 5; (12) a targeting sequence comprising amino acids 8—38 of SEQ ID NO: 5; (13) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 5; (14) a targeting sequence sing amino acids 15—38 of SEQ ID NO: 5; (15) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 5; (16) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 7; (17) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 7; (18) a targeting sequence comprising amino acids 8—28 of SEQ ID NO: 7; (19) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 7; (20) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 9; (21) a targeting ce comprising amino acids 5—24 of SEQ ID NO: 9; (22) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 9; (23) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 11 ; (24) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 11; (25) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 11; (26) a targeting ce comprising amino acids 10—33 of SEQ ID NO: 11; (27) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 11; (28) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 13; (29) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13 ; (30) a ing sequence comprising amino acids 8—33 of SEQ ID NO: 13 ; (31) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 13 ; (32) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 13; (33) a ing sequence comprising amino acids 2—43 of SEQ ID NO: 15; (34) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 15; (35) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 15; (36) a targeting ce comprising amino acids 10—43 of SEQ ID NO: 15; (37) a ing ce comprising amino acids 15—43 of SEQ ID NO: 15; (38) a targeting sequence sing amino acids 20—43 of SEQ ID NO: 15; (39) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 15; (40) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 17; (41) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 17; (42) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 17; (43) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 17; (44) a targeting sequence comprising amino 3 54 acids 2—33 of SEQ ID NO: 19; (45) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 19; (46) a ing sequence comprising amino acids 8—33 of SEQ ID NO: 19; (47) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 19; (48) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 19; (49) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 21 ; (50) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 21; (51) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 21; (52) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 21 ; (53) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 21 ; (54) a targeting sequence sing amino acids 2—24 of SEQ ID NO:23; (55) a targeting sequence sing amino acids 5—24 of SEQ ID NO: 23; (56) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 23; (57) a targeting ce sing amino acids 2—24 of SEQ ID NO: 25; (58) a targeting ce comprising amino acids 5—24 of SEQ ID NO: 25; (59) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 25; (60) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 27; (61) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 27; (62) a ing sequence comprising amino acids 8—30 of SEQ ID NO: 27; (63) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 27; (64) a targeting sequence sing amino acids 2—33 of SEQ ID NO: 29; (65) a targeting sequence sing amino acids 5—33 of SEQ ID NO: 29; (66) a targeting ce comprising amino acids 8—33 of SEQ ID NO: 29; (67) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 29; (68) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 29; (69) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 31; (70) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31; (71) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 31; (72) a targeting sequence comprising amino acids 2—29 of SEQ ID NO: 43; (73) a targeting sequence comprising amino acids 5—29 of SEQ ID NO: 43; (74) a targeting sequence comprising amino acids 8—29 of SEQ ID NO: 43; (75) a targeting sequence comprising amino acids 10—29 of SEQ ID NO: 43; (76) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 45; (77) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 45; (78) a targeting sequence sing amino acids 8—35 of SEQ ID NO: 45; (79) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45 ; (80) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 45 ; (81) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 47; (82) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 47; (83) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 47; (84) a targeting sequence sing amino acids 10—43 of SEQ ID NO: 47; (85) a targeting sequence comprising amino 3 5 5 acids 15—43 of SEQ ID NO: 47; (86) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (87) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 47; (88) a targeting sequence comprising amino acids 2—32 of SEQ ID NO: 49; (89) a targeting sequence comprising amino acids 5—32 of SEQ ID NO: 49; (90) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (91) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (92) a targeting ce comprising amino acids 15—32 of SEQ ID NO: 49; (93) a targeting ce comprising amino acids 2—33 of SEQ ID NO: 51; (94) a ing sequence comprising amino acids 5—33 of SEQ ID NO: 51; (95) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 51; (96) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 51; (97) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 51; (98) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 53; (99) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53; (100) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53; (101) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 53; (102) a targeting sequence sing amino acids 15—33 of SEQ ID NO: 53; (103) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 55; (104) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 55; (105) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 55; (106) a targeting ce comprising amino acids 10—30 of SEQ ID NO: 55; (107) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 57; (108) a targeting sequence sing amino acids 5—130 of SEQ ID NO: 57; (109) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (110) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 57; (111) a targeting ce comprising amino acids 30—130 of SEQ ID NO: 57; (112) a targeting sequence sing amino acids 40—130 of SEQ ID NO: 57; (113) a ing sequence comprising amino acids 50—130 of SEQ ID NO: 57; (114) a targeting sequence comprising amino acids 60—130 of SEQ ID NO: 57; (115) a targeting sequence sing amino acids 70— 130 of SEQ ID NO: 57; (116) a targeting sequence comprising amino acids 80—130 of SEQ ID NO: 57; (117) a targeting sequence comprising amino acids 90—130 of SEQ ID NO: 57; (118) a targeting sequence comprising amino acids 100—130 of SEQ ID NO: 57; or (119) a targeting sequence comprising amino acids 110—130 of SEQ ID NO: 57.

Embodiment 10 is the fusion protein of embodiment 9, wherein the targeting sequence comprises: (1) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 9; (2) a targeting ce comprising amino acids 5—24 of SEQ ID NO: 9; (3) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 9; (4) a targeting sequence comprising 3 56 amino acids 2—33 of SEQ ID NO: 11; (5) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 11; (6) a ing sequence comprising amino acids 8—33 of SEQ ID NO: 11; (7) a ing sequence comprising amino acids 10—33 of SEQ ID NO: 11; (8) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 11; (9) a targeting sequence sing amino acids 2—33 of SEQ ID NO: 13; (10) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13; (11) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 13; (12) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 13; (13) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 13; (14) a ing sequence comprising amino acids 2—43 of SEQ ID NO: 15; (15) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 15; (16) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 15; (17) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 15; (18) a ing sequence comprising amino acids 15—43 of SEQ ID NO: 15; (19) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 15; (20) a targeting sequence sing amino acids 25—43 of SEQ ID NO: 15; (21) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 17; (22) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 17; (23) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 17; (24) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 17; (25) a targeting ce comprising amino acids 2—33 of SEQ ID NO: 19; (26) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 19; (27) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 19; (28) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 19; (29) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 19; (30) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 21 ; (31) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 21; (32) a targeting sequence sing amino acids 8—33 of SEQ ID NO: 21; (33) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 21 ; (34) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 21 ; (35) a targeting ce comprising amino acids 2—24 of SEQ ID NO:23; (36) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 23; (37) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 23; (38) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 25; (39) a targeting sequence sing amino acids 5—24 of SEQ ID NO: 25; (40) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 25; (41) a targeting sequence sing amino acids 2—30 of SEQ ID NO: 27; (42) a targeting ce comprising amino acids 5—30 of SEQ ID NO: 27; (43) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 27; (44) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 27; (45) a targeting sequence comprising amino WO 44661 3 57 acids 2—33 of SEQ ID NO: 29; (46) a ing sequence comprising amino acids 5—33 of SEQ ID NO: 29; (47) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 29; (48) a ing sequence comprising amino acids 10—33 of SEQ ID NO: 29; (49) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 29; (50) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 31 ; (51) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31; (52) a targeting ce comprising amino acids 8—24 of SEQ ID NO: 31; (53) a targeting sequence sing amino acids 2—29 of SEQ ID NO: 43; (54) a ing sequence comprising amino acids 5—29 of SEQ ID NO: 43; (55) a targeting sequence comprising amino acids 8—29 of SEQ ID NO: 43; (56) a targeting sequence comprising amino acids 10—29 of SEQ ID NO: 43 ; (57) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 45 ; (58) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 45; (59) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 45; (60) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45; (61) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 45 ; (62) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 47; (63) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 47; (64) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 47; (65) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 47; (66) a ing ce comprising amino acids 15—43 of SEQ ID NO: 47; (67) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (68) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 47; (69) a targeting ce comprising amino acids 2—32 of SEQ ID NO: 49; (70) a targeting sequence comprising amino acids 5—32 of SEQ ID NO: 49; (71) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (72) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (73) a targeting sequence comprising amino acids 15—32 of SEQ ID NO: 49; (74) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 51; (75) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 51 ; (76) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 51 ; (77) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 51 ; (78) a targeting ce comprising amino acids 15—33 of SEQ ID NO: 51 ; (79) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 53; (80) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53 ; (81) a ing sequence comprising amino acids 8—33 of SEQ ID NO: 53; (82) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 53; (83) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 53; (84) a targeting sequence sing amino acids 2—30 of SEQ ID NO: 55; (85) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 55 ; (86) a 3 5 8 targeting ce comprising amino acids 8—30 of SEQ ID NO: 55; (87) a ing ce comprising amino acids 10—30 of SEQ ID NO: 55; (88) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 57; (89) a targeting sequence comprising amino acids 5—130 of SEQ ID NO: 57; (90) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (91) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 57; (92) a targeting sequence comprising amino acids 30—130 of SEQ ID NO: 57; (93) a targeting sequence comprising amino acids 40—130 of SEQ ID NO: 57; (94) a targeting sequence sing amino acids 50—130 of SEQ ID NO: 57; (95) a targeting sequence comprising amino acids 60—130 of SEQ ID NO: 57; (96) a targeting sequence comprising amino acids 70—130 of SEQ ID NO: 57; (97) a targeting sequence comprising amino acids 80—130 of SEQ ID NO: 57; (98) a targeting ce comprising amino acids 90—130 of SEQ ID NO: 57; (99) a targeting sequence comprising amino acids 100—130 of SEQ ID NO: 57; or (100) a targeting sequence comprising amino acids 110—130 of SEQ ID NO: 57.

] Embodiment 11 is the fusion protein of any one of embodiments 5—10, wherein the targeting sequence comprises: (1) a targeting sequence comprising amino acids 5— 24 of SEQ ID NO: 9; (2) a ing sequence comprising amino acids 8—24 of SEQ ID NO: 9; (3) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 11; (4) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 11; (5) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 11; (6) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 11; (7) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13; (8) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 13; (9) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 13; (10) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 13; (11) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 15; (12) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 15; (13) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 15; (14) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 15; (15) a targeting ce comprising amino acids 20—43 of SEQ ID NO: 15; (16) a targeting sequence sing amino acids 25—43 of SEQ ID NO: 15; (17) a targeting sequence sing amino acids 5—27 of SEQ ID NO: 17; (18) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 17; (19) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 17; (20) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 19; (21) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 19; (22) a ing sequence comprising amino acids 10—33 of SEQ ID NO: 19; (23) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 19; (24) a 3 59 targeting ce comprising amino acids 5—33 of SEQ ID NO: 21; (25) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 21; (26) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 21; (27) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 21; (28) a targeting sequence sing amino acids 5—24 of SEQ ID NO: 23; (29) a targeting ce sing amino acids 8—24 of SEQ ID NO: 23; (30) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 25; (31) a ing sequence comprising amino acids 8—24 of SEQ ID NO: 25; (32) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 27; (33) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 27; (34) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 27; (35) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 29; (36) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 29; (37) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 29; (38) a targeting sequence sing amino acids 15—33 of SEQ ID NO: 29; (39) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31; (40) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 31 ; (41) a targeting sequence comprising amino acids 5—29 of SEQ ID NO: 43; (42) a targeting ce comprising amino acids 8—29 of SEQ ID NO: 43; (43) a targeting sequence comprising amino acids 10—29 of SEQ ID NO: 43; (44) a targeting ce comprising amino acids 5—35 of SEQ ID NO: 45; (45) a targeting ce comprising amino acids 8—35 of SEQ ID NO: 45 ; (46) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45; (47) a targeting ce comprising amino acids 15—35 of SEQ ID NO: 45; (48) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 47; (49) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 47; (50) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 47; (51) a ing sequence comprising amino acids 15—43 of SEQ ID NO: 47; (52) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (53) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 47; (54) a targeting sequence comprising amino acids 5—32 of SEQ ID NO: 49; (55) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (56) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (57) a targeting sequence comprising amino acids 15—32 of SEQ ID NO: 49; (58) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 51; (59) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 51; (60) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 51; (61) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 51 ; (62) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53; (63) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53 ; (64) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 53; (65) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 53; (66) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 55; (67) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 55; (68) a targeting sequence sing amino acids 10—30 of SEQ ID NO: 55; (69) a targeting ce comprising amino acids 5—130 of SEQ ID NO: 57; (70) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (71) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 57; (72) a targeting sequence sing amino acids 30—130 of SEQ ID NO: 57; (73) a ing sequence comprising amino acids 40—130 of SEQ ID NO: 57; (74) a targeting sequence comprising amino acids 50—130 of SEQ ID NO: 57; (75) a targeting sequence comprising amino acids 60—130 of SEQ ID NO: 57; (76) a targeting sequence comprising amino acids 70—130 of SEQ ID NO: 57; (77) a targeting sequence comprising amino acids 80—130 of SEQ ID NO: 57; (78) a targeting sequence comprising amino acids 90—130 of SEQ ID NO: 57; (79) a targeting sequence comprising amino acids 100—130 of SEQ ID NO: 57; (80) a targeting sequence sing amino acids 110—130 of SEQ ID NO: 57; (81) a targeting sequence comprising amino acids 4— of SEQ ID NO: 59; (82) a targeting ce comprising amino acids 6—30 of SEQ ID NO: 59; (83) a targeting sequence sing amino acids 5—33 of SEQ ID NO: 61; (84) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 61; (85) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 61; (86) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 63 ; (87) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 63 ; (88) a targeting sequence sing amino acids 10—35 of SEQ ID NO: 63; (89) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 63; (90) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 65; (91) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 67; (92) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 67; (93) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 69; (94) a targeting ce comprising amino acids 10—38 of SEQ ID NO: 69; (95) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 69; (96) a targeting sequence comprising amino acids 5—42 of SEQ ID NO: 75; (97) a targeting sequence comprising amino acids 10—42 of SEQ ID NO: 75; (98) a targeting ce comprising amino acids 15—42 of SEQ ID NO: 75; (99) a targeting sequence comprising amino acids 20—42 of SEQ ID NO: 75; (100) a targeting sequence comprising amino acids 25—42 of SEQ ID NO: 75; (101) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 77; (102) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 81 ; (103) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 81; (104) a targeting ce comprising amino acids 15—38 of SEQ ID NO: 81; (105) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 81; (106) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 87; (107) a targeting ce comprising amino acids 10—28 of SEQ ID NO: 87; (108) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 89; (109) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 89; (110) a targeting sequence comprising amino acids 10—93 of SEQ ID NO: 91; (111) a targeting sequence comprising amino acids 20—93 of SEQ ID NO: 91; (112) a targeting sequence comprising amino acids 30—93 of SEQ ID NO: 91; (113) a targeting ce comprising amino acids 40—93 of SEQ ID NO: 91; (114) a targeting sequence comprising amino acids 50—93 of SEQ ID NO: 91; (115) a targeting sequence comprising amino acids 60—93 of SEQ ID NO: 91; (116) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 93; (117) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 93; or (118) a targeting sequence comprising amino acids 30—130 of SEQ ID NO: 93. 6] Embodiment 12 is the fusion protein of any one of embodiments 5 and 9—1 1, n the protein or peptide of interest comprises the antigen.

Embodiment 13 is the fusion protein of any one of embodiments 5 and 9—1 1, wherein the n or peptide of interest comprises the remediation enzyme.

Embodiment 14 is the fusion protein of any one of embodiments 7—1 1, wherein the n or peptide of interest comprises the enzyme suitable for breaking an emulsion or gel in a hydraulic fracturing fluid.

Embodiment 15 is the fusion protein of any one of embodiments 7—1 1, wherein the protein or peptide of st comprises the antibacterial protein or peptide. ment 16 is a recombinant Bacillus cereus family member that expresses a fusion protein of any one of embodiments 1—15.

Embodiment 17 is a recombinant Bacillus cereus family member that expresses: (i) a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family ; and (ii) a modulator protein, wherein the expression of the modulator protein is sed as compared to sion of the modulator protein in a ype Bacillus cereus family member under the same conditions; and wherein co-expression of the modulator protein with the fusion protein in the recombinant Bacillus cereus family member results in sed or decreased expression of the fusion protein as compared to the expression level of the fusion protein in a recombinant Bacillus cereus family member that does not express the modulator protein at an increased level under the same conditions as compared to the expression of the modulator protein in a wild-type us cereus family member.

Embodiment 18 is a recombinant Bacillus cereus family member of embodiment 17, wherein the modulator protein comprises an Est protein, an ExsFA/BxpB protein, a CotY protein, a CotO protein, an ExsFB protein, an InhAl protein, an InhA2 protein, an Est n, an Est protein, a YjcA protein, a YjcB protein, a Bch protein, an AcpC protein, an InhA3 protein, an alanine racemase 1, an alanine racemase 2, a BclA protein, a BclB protein, a Bpr protein, a BclE protein, a BetA/BAS3290 protein, a CotE protein, an Est n, an ExsK protein, an Est protein, a YabG n, a Tgl protein, a SODAl protein, a SODA2 protein, a variant of any thereof, or a combination of any thereof.

Embodiment 19 is a recombinant Bacillus cereus family member of embodiment 17 or 18, wherein the tor protein, when co-expressed in the recombinant us cereus family member with the fusion protein, results in increased expression of the fusion protein as compared to the expression level of the fusion protein in a recombinant us cereus family member that does not express the modulator protein at an increased level under the same conditions as compared to the expression of the modulator protein in a ype Bacillus cereus family member.

Embodiment 20 is a recombinant Bacillus cereus family member of embodiment 19, wherein the modulator protein comprises a BclB protein, a CotE protein, a BxpB protein, a CotO protein, a BclA protein, a variant of any thereof, or a ation of any thereof. ment 21 is a recombinant Bacillus cereus family member of embodiment 17 or 18, wherein the modulator protein, when co-expressed in the recombinant Bacillus cereus family member with the fusion protein, results in decreased expression of the fusion n as compared to the expression level of the fusion n in a recombinant Bacillus cereus family member that does not express the modulator protein at an sed level under the same conditions as compared to the sion of the modulator n in a wild-type Bacillus cereus family member. 6] Embodiment 22 is a recombinant us cereus family member of embodiment 21, wherein the modulator protein comprises a Bch protein, an Ach protein, a YjCB protein, a variant of any thereof, or a combination of any thereof.

Embodiment 23 is a recombinant Bacillus cereus family member of embodiment 17 or 18, wherein the modulator protein comprises a CotO protein, a BclB protein, an ExsFA/BxpB protein, a YjcB protein, a variant of any thereof, or a combination of any thereof.

Embodiment 24 is a recombinant Bacillus cereus family member of any one of embodiments 17—23, wherein the modulator protein comprises an amino acid sequence having at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NOs: 123—156.

Embodiment 25 is a recombinant Bacillus cereus family member of any one of embodiments 17—24, wherein the modulator protein comprises SEQ ID NO: 124, 126, 134, 135, 143, or 144.

Embodiment 26 is a recombinant Bacillus cereus family member of any one of embodiments 17—25, comprising a vector encoding the modulator n. 1] Embodiment 27 is a recombinant us cereus family member of embodiment 26, wherein the vector comprises a multicopy plasmid vector.

Embodiment 28 is a recombinant Bacillus cereus family member that expresses a fusion protein comprising at least one protein or e of interest and a targeting sequence, exosporium protein, or exosporium protein nt that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member; wherein the recombinant Bacillus cereus family member comprises a mutation that partially or completely inactivates spores of the recombinant Bacillus cereus family member or renders spores of the recombinant Bacillus cereus family more tible to al or chemical inactivation as compared to the same spores that do not comprise the mutation, and n the mutation ses a mutation in a gene ng a germination or, a mutation in a gene encoding a spore cortex lytic enzyme, a mutation in a gene encoding a small acid-soluble spore n (SASP), or a mutation in a gene encoding a spore coat or cortex protein.

Embodiment 29 is a recombinant Bacillus cereus family member that expresses a fusion protein of any one of embodiments 1—15; wherein the recombinant Bacillus cereus family member comprises a mutation that partially or completely inactivates spores of the recombinant Bacillus cereus family member or renders spores of the recombinant Bacillus cereus family more susceptible to physical or chemical vation as compared to the same spores that do not comprise the on.

Embodiment 30 is a recombinant bacterium of the genus Bacillus that expresses a protease or a nuclease, wherein the expression of the protease or se is increased as compared to the expression of the protease or the nuclease in a wild-type bacterium of the genus us under the same conditions, and wherein the increased expression of the protease or the se partially or completely inactivates spores of the recombinant bacterium of the genus Bacillus or renders spores of the recombinant bacterium of the genus Bacillus more susceptible to physical or chemical inactivation.

Embodiment 31 is a recombinant bacterium of embodiment 30, wherein the recombinant ium of the genus Bacillus comprises a recombinant Bacillus cereus family member. 6] Embodiment 32 is a recombinant bacterium of the genus Bacillus of embodiment 30 or 31, wherein the recombinant bacterium of the genus Bacillus expresses a protease and a nuclease, wherein the sion of the protease is increased as compared to the expression of the protease in a wild-type bacterium of the genus Bacillus under the same conditions and the expression of the nuclease is sed as compared to the expression of the nuclease in a wild-type bacterium of the genus Bacillus under the same conditions. ment 33 is a recombinant bacterium of the genus Bacillus of any one of ments 30—32, wherein the protease comprises a non-specific se.

Embodiment 34 is a recombinant bacterium of the genus Bacillus of any one of embodiments 30—33, wherein the protease comprises a serine protease, a threonine protease, a cysteine protease, an aspartate protease, a glutamic acid protease, an alkaline protease, a subtilisin, a histidine protease, or a metalloprotease. 9] Embodiment 35 is a recombinant ium of the genus Bacillus of any one of embodiments 30, 32, and 34, wherein the protease comprises a germination spore protease.

Embodiment 36 is a recombinant bacterium of the genus Bacillus of embodiment 35, wherein the germination spore se comprises an active form of the germination spore protease.

Embodiment 37 is a recombinant bacterium of the genus Bacillus of embodiment 35 or 36, wherein the germination spore protease comprises a Bacillus subtilis germination spore protease, a Bacillus mycoia’es germination spore protease, or a Bacillus giensis germination spore protease.

Embodiment 38 is a recombinant bacterium of the genus Bacillus of any one of ments 35—37, wherein the germination spore protease comprises an amino acid ce having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 233 or 234.

Embodiment 39 is a recombinant bacterium of the genus Bacillus of any one of ments 30—38, wherein the nuclease comprises an endonuclease or an exonuclease.

Embodiment 40 is a recombinant bacterium of the genus Bacillus of embodiment 39, wherein the nuclease comprises a non-specific endonuclease. ment 41 is a recombinant bacterium of the genus us of embodiment 40, wherein the non-specific endonuclease comprises Bacillus subtilis endonuclease 1.

Embodiment 42 is a recombinant bacterium of the genus Bacillus of embodiment 40 or 41, n the non-specific endonuclease comprises an amino acid having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 232. 7] Embodiment 43 is a recombinant bacterium of the genus Bacillus of any one of embodiments 30—42, wherein the nuclease or protease is expressed under the control of a promoter comprising a sigma G promoter sequence. 8] Embodiment 44 is a recombinant bacterium of the genus Bacillus of embodiment 43, wherein the promoter comprises a nucleic acid sequence having at least 95%, at least 98%, at least 99%, or 100% identity with a nucleic acid sequence of any of SEQ ID NOs: 23 5—246.

Embodiment 45 is a inant bacterium of the genus Bacillus of any one of embodiments 30—44, wherein spores of the recombinant bacterium of the genus us are more susceptible to inactivation by ultraviolet irradiation, gamma irradiation, or by treatment with bleach, hydrogen peroxide, form, phenol, or acetic acid as compared to the same spores that do not expresses the protease or the nuclease at an sed level as compared to expression of the protease or the nuclease in a wild-type bacterium of the genus Bacillus, treated under the same conditions.

Embodiment 46 is a recombinant Bacillus cereus family member of any one of embodiments 31—45, wherein the recombinant Bacillus cereus family member further comprises a on that partially or completely vates spores of the recombinant Bacillus cereus family member or renders spores of the recombinant Bacillus cereus family more WO 44661 susceptible to physical or chemical inactivation as compared to the same spores that do not comprise the mutation.

Embodiment 47 is a recombinant Bacillus cereus family member of embodiment 46, wherein the mutation comprises a mutation in a gene encoding a ation receptor, a mutation in a gene encoding a spore cortex lytic enzyme, a mutation in a gene encoding a small acid-soluble spore protein (SASP), or a mutation in a gene encoding a spore coat or cortex protein. 2] Embodiment 48 is a recombinant Bacillus cereus family member of embodiment 28, 29, or 47, wherein the mutation comprises a mutation in a gene encoding a germination receptor.

Embodiment 49 is a inant Bacillus cereus family member of embodiment 48, wherein the mutation in the gene encoding the germination receptor comprises a knock-out mutation of the gene encoding the germination receptor. 4] Embodiment 50 is a recombinant Bacillus cereus family member of embodiment 48 or 49, wherein the germination receptor comprises GerA, GerB, GerK, GerH, GerI, GerG, GerL, GerQ, GerR, GerS, GerN, GerU, or GerX.

Embodiment 51 is a recombinant Bacillus cereus family member of any one of embodiments 28, 29, and 47—50, n the mutation comprises a mutation in a gene encoding a spore cortex lytic enzyme. ment 52 is a recombinant Bacillus cereus family member of embodiment 5 l wherein the mutation in the gene encoding the spore cortex lytic enzyme comprises a knock-out mutation of the gene encoding the spore cortex lytic enzyme.

Embodiment 53 is a recombinant us cereus family member of embodiment 51 or 52, n the spore cortex lytic enzyme comprises SleB or Cle.

Embodiment 54 is a recombinant Bacillus cereus family member of any one of embodiments 28, 29, and 47—53, wherein the mutation ses a mutation in a gene encoding a SASP.

Embodiment 55 is a recombinant us cereus family member of embodiment 54, wherein the mutation in the gene encoding the SASP comprises a knock-out mutation of the gene encoding the SASP.

Embodiment 56 is a recombinant us cereus family member of embodiment 54 or 55, wherein the mutation in the gene encoding the SASP comprises a mutation in a SspA gene, a mutation in a SspB gene, a mutation in a SspC gene, a mutation in a SspD gene, a mutation in a SspE gene, a mutation in a SspF gene, a mutation in a SspG gene, a on in a SspH gene, a mutation in a SspI gene, a on in a SspJ gene, a mutation in a SspK gene, a mutation in a SspL gene, a mutation in a SspM gene, a mutation in a SspN gene, a mutation in a SspO gene, a mutation in a SspP gene, or a combination thereof.

Embodiment 57 is a recombinant Bacillus cereus family member of any one of embodiments 54—56, wherein the SASP comprises SASPu, SASPB, or SASPy.

Embodiment 58 is a recombinant Bacillus cereus family member of any one of ments 54—57, wherein spores of the recombinant Bacillus cereus family member are more susceptible to inactivation by ultraViolet irradiation or gamma irradiation as compared to the same spores that do not comprise the mutation in the gene encoding the SASP.

Embodiment 59 is a recombinant Bacillus cereus family member of any one of embodiments 28, 29, and 47—58, wherein the on comprises a mutation in a gene encoding a spore coat or cortex protein. 4] Embodiment 60 is a recombinant Bacillus cereus family member of embodiment 59, wherein the mutation in the gene encoding the spore coat or cortex n comprises a knock-out mutation of the gene encoding the spore coat or cortex protein. ment 61 is a recombinant Bacillus cereus family member of embodiment 59 or 60, wherein the spore coat or cortex protein comprises CotA, CotB, or CotC.

Embodiment 62 is a recombinant Bacillus cereus family member of any one of embodiments 59—61, wherein spores of the recombinant Bacillus cereus family member are more susceptible to vation by ultraViolet irradiation, gamma irradiation or by treatment with bleach, hydrogen peroxide, chloroform, , or acetic acid, as compared to the same spores that do not comprise the mutation in the spore coat or cortex protein, d under the same conditions. 7] Embodiment 63 is a recombinant Bacillus cereus family member of any one of embodiments 31—62, wherein the recombinant Bacillus cereus family member also expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member.

Embodiment 64 is a recombinant us cereus family member that expresses an exosporium n, wherein the expression of the exosporium protein is sed as compared to the expression of the exosporium protein in a wild-type Bacillus cereus family member under the same conditions, and wherein the exosporium protein comprises: an rium enzyme, wherein the exosporium enzyme comprises an enzyme involved in nutrient solubilization, an inosine-uridine hydrolase, a se, an enzyme that catalyzes the degradation of a free radical, an arginase, or an e racemase; or a BclA protein, a BclB protein, a CotE protein a CotO protein, an Est protein, an ExsFA/BxpB protein, a CotY protein, an ExsFB protein, an Est protein, an Est protein, a YjcA protein, a YjcB protein, a Bch protein, a Bpr protein, a BclE protein, a BetA/BAS329O protein, an Est protein, an ExsK protein, an Est protein, a YabG protein, or a Tgl protein. 9] Embodiment 65 is a recombinant Bacillus cereus family member of embodiment 64, wherein the exosporium protein is not part of a fusion protein.

Embodiment 66 is a recombinant us cereus family member of embodiment 64 or 65, wherein the exosporium protein comprises an exosporium enzyme, wherein the exosporium enzyme comprises an enzyme involved in nt solubilization, an e-uridine hydrolase, a protease, an enzyme that zes the degradation of a free radical, an arginase, or an alanine se.

Embodiment 67 is a recombinant Bacillus cereus family member of embodiment 66, wherein the enzyme involved in nutrient solubilization comprises an enzyme involved in phosphate solubilization.

Embodiment 68 is a recombinant us cereus family member of embodiment 67, wherein the enzyme involved in phosphate solubilization comprises an acid phosphatase.

Embodiment 69 is a recombinant Bacillus cereus family member of embodiment 68, wherein the acid phosphatase comprises AcpC. 4] Embodiment 70 is a recombinant Bacillus cereus family member of embodiment 68 or 69, wherein the acid atase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 137.

Embodiment 71 is a recombinant Bacillus cereus family member of embodiment 64 or 65, wherein the exosporium enzyme comprises an inosine-uridine hydrolase.

Embodiment 72 is a recombinant Bacillus cereus family member of embodiment 71, wherein the inosine-uridine hydrolase comprises IunHl or IunH2. 2015/050807 Embodiment 73 is a recombinant Bacillus cereus family member of embodiment 71 or 72, wherein the e-uridine hydrolase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 250 or 251. 8] Embodiment 74 is a recombinant Bacillus cereus family member of ment 64 or 65, wherein the exosporium enzyme comprises a protease.

Embodiment 75 is a recombinant Bacillus cereus family member of embodiment 74, wherein the protease comprises a metalloprotease.

Embodiment 76 is a recombinant Bacillus cereus family member of embodiment 75, wherein the metalloprotease comprises InhAl or InhA3.

, InhA2, Embodiment 77 is a recombinant Bacillus cereus family member of ment 75 or 76, wherein the metalloprotease comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

Embodiment 78 is a recombinant Bacillus cereus family member of ment 64 or 65, wherein the enzyme that catalyzes the degradation of a free radical comprises a superoxide dismutase.

Embodiment 79 is a recombinant Bacillus cereus family member of embodiment 78, wherein the superoxide dismutase comprises superoxide dismutase 1 ) or superoxide dismutase 2 (SODA2).

Embodiment 80 is a recombinant us cereus family member of embodiment 78 or 79, wherein the superoxide dismutase comprises an amino acid ce having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 155 or 156.

Embodiment 81 is a recombinant Bacillus cereus family member of embodiment 64 or 65, wherein the rium enzyme comprises an arginase.

Embodiment 82 is a recombinant Bacillus cereus family member of embodiment 81, wherein the arginase comprises a Bacillus giensis arginase.

Embodiment 83 is a recombinant Bacillus cereus family member of embodiment 81 or 82, wherein the arginase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 249. 2015/050807 Embodiment 84 is a recombinant Bacillus cereus family member of embodiment 64 or 65, n the exosporium enzyme comprises an alanine racemase.

Embodiment 85 is a recombinant Bacillus cereus family member of embodiment 84, n the alanine racemase comprises alanine racemase 1 (ALRl) or alanine racemase 2 (ALR2).

Embodiment 86 is a recombinant Bacillus cereus family member of embodiment 84 or 85, wherein the alanine racemase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 247 or 248.

Embodiment 87 is a recombinant Bacillus cereus family member of embodiment 64 or 65, wherein the exosporium protein comprises a BclA protein, a BclB protein, a CotE protein a CotO protein, an Est protein, an ExsFA/BxpB protein, a CotY protein, an ExsFB protein, an Est protein, an Est protein, a chA protein, a chB protein, a Bch protein, a Bpr n, a BclE protein, a BetA/BAS3290 protein, an Est n, an ExsK n, an Est protein, a YabG protein, or a Tgl protein.

Embodiment 88 is a recombinant Bacillus cereus family member of embodiment 87, wherein the exosporium protein comprises a BclA protein, a BclB protein, a CotE protein, or a CotO n. 3] Embodiment 89 is a recombinant Bacillus cereus family member of embodiment 87 or 88, wherein the exosporium protein ses a BclA protein. 4] Embodiment 90 is a recombinant Bacillus cereus family member of embodiment 89, wherein the BclA protein ses an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 141 or 142.

Embodiment 91 is a Bacillus cereus family member of embodiment 87 or 88, wherein the exosporium protein comprises a BclB protein.

Embodiment 92 is a Bacillus cereus family member of embodiment 91, wherein the BclB protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 143 or 144.

Embodiment 93 is a recombinant Bacillus cereus family member of embodiment 87 or 88, wherein the exosporium protein comprises a CotE protein. ment 94 is a recombinant Bacillus cereus family member of embodiment 93, wherein the CotE protein comprises an amino acid sequence having at least 2015/050807 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 149.

Embodiment 95 is a recombinant Bacillus cereus family member of embodiment 87 or 88, wherein the exosporium n comprises a CotO protein. ment 96 is a recombinant Bacillus cereus family member of embodiment 95, n the CotO protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 126.

Embodiment 97 is a recombinant Bacillus cereus family member of embodiment 87, wherein the exosporium protein comprises an Est protein, an ExsFA/BxpB protein, a CotY protein, an ExsFB protein, an Est protein, an Est protein, a YjcA protein, a YjcB protein, a Bch protein, a Bpr protein, a BclE protein, a BetA/BAS3290 protein, an Est protein, an ExsK protein, an Est protein, a YabG protein, or a Tgl protein.

Embodiment 98 is a recombinant Bacillus cereus family member of ment 97, wherein the exosporium protein comprises: an Est protein, wherein the Est protein ses an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% ty with SEQ ID NO: 123; an ExsFA/BxpB protein, wherein the ExsFA/BxpB protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 124; a CotY protein, wherein the CotY protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 125; an ExsFB protein, n the ExsFB protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 127 or 128; an Est protein, n the Ex] protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 131; an Est protein, wherein the Est protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 132; 2015/050807 a YjcA protein, wherein the chA n comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 133; a chB protein, wherein the chB protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 134 or 135; a Bch protein, wherein the Bch protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% with SEQ ID NO: 136; a Bpr n, wherein the Bpr n comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% with SEQ ID NO: 145; a BclE protein, wherein the BclE protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 146 or 147; a BetA/BAS3290 protein, wherein the BetA/BAS3290 protein comprises an amino acid ce having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 148; an Est protein, wherein the Est protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 150; an ExsK protein, wherein the ExsK protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 151 ; an Est protein, wherein the Est protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 152; a YabG protein, n the YabG protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 153; or a le protein, wherein the le protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 156.

Embodiment 99 is a recombinant Bacillus cereus family member of any one of ments 64—98, n the recombinant Bacillus cereus family member also expresses a fusion protein comprising at least one n or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion n to the exosporium of the recombinant Bacillus cereus family member.

Embodiment 100 is a recombinant Bacillus cereus family member that expresses a fusion protein sing at least one protein or peptide of interest and a targeting ce, exosporium protein, or exosporium protein nt that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member; wherein the recombinant Bacillus cereus family member comprises a mutation or expresses a protein, wherein the expression of the protein is increased as compared to the expression of the protein in a wild-type Bacillus cereus family member under the same conditions, and wherein the mutation or the increased expression of the n results in us cereus spores having an exosporium that is easier to remove from the spore as compared to the rium of a wild-type spore.

] Embodiment 101 is a recombinant Bacillus cereus family member that expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member; n the recombinant Bacillus cereus family member: (i) comprises a mutation in a CotE gene; (ii) expresses an Est protein, wherein the expression of the Est protein is increased as compared to the expression of the Est protein in a wild-type Bacillus cereus family member under the same ions, and wherein the Est protein comprises a carboxy-terminal tag comprising a ar protein; (iii) expresses a BclB protein, wherein the expression of the BclB protein is increased as compared to the expression of the BclB protein in a wild-type Bacillus cereus family member under the same conditions; (iV) expresses a YjcB protein, wherein the expression of the chB protein is sed as compared to the expression of the YjcB protein in a wild-type Bacillus cereus family member under the same conditions; (V) comprises a mutation in an Est gene; (Vi) comprises a mutation in a CotY gene; (Vii) comprises a mutation in an Est gene; or (viii) comprises a mutation in a CotO gene.

Embodiment 102 is a inant Bacillus cereus family member of embodiment 100 or 101, wherein the recombinant Bacillus cereus family member comprises a mutation in a CotE gene.

Embodiment 103 is a recombinant Bacillus cereus family member of embodiment 102, wherein the mutation in the CotE gene partially or tely inhibits the ability of CotE to attach the exosporium to the spore.

Embodiment 104 is a recombinant Bacillus cereus family member of embodiment 102 or 103, wherein the mutation in the CotE gene comprises a knock-out of the CotE gene or a nt negative form of the CotE gene. 9] Embodiment 105 is a recombinant Bacillus cereus family member of any one of embodiments 100—104, wherein the recombinant Bacillus cereus family member expresses an Est protein, wherein the expression of the Est protein is increased as compared to the expression of the Est protein in a ype us cereus family member under the same conditions, and wherein the Est protein comprises a carboxy-terminal tag comprising a globular protein.

Embodiment 106 is a recombinant Bacillus cereus family member of embodiment 105, n the globular protein has a lar weight of between 25 kDa and 100 kDa. ment 107 is a recombinant Bacillus cereus family member of embodiment 105 or 106, wherein the globular protein comprises a green fluorescent protein (GFP) or a variant thereof ment 108 is a inant Bacillus cereus family member of any one of embodiments 105—107, wherein the expression of the Est protein comprising the carboxy- terminal tag comprising a globular protein inhibits binding of the Est protein to its targets in the exosporium.

Embodiment 109 is a recombinant Bacillus cereus family member of any one of embodiments 8, wherein the recombinant Bacillus cereus family member expresses a BclB protein, wherein the expression of the BclB protein is increased as compared to the expression of the BclB protein in a wild-type Bacillus cereus family member under the same conditions.

Embodiment 110 is a recombinant Bacillus cereus family member of embodiment 109, wherein the expression of the BclB protein results in the formation of a fragile exosporium.

Embodiment 111 is a recombinant Bacillus cereus family member of any one of embodiments 100—110, wherein the recombinant Bacillus cereus family member expresses a YjcB protein, wherein the expression of the YjcB protein is increased as compared to the expression of the YjcB protein in a wild-type Bacillus cereus family member under the same conditions.

Embodiment 112 is a recombinant Bacillus cereus family member of embodiment 111, wherein the expression of the YjcB protein causes the exosporium to form in pieces rather than in a te structure.

Embodiment 113 is a recombinant Bacillus cereus family member of any one of embodiments 2, wherein the recombinant Bacillus cereus family member comprises a mutation an Est gene. 8] Embodiment 114 is a recombinant Bacillus cereus family member of embodiment 113, wherein the mutation in the Est gene partially or completely inhibits the y ofEst to complete the formation of the exosporium or attach the rium to the spore.

Embodiment 115 is a recombinant Bacillus cereus family member of ment 113 or 114, wherein the mutation in the Est gene comprises a knock-out of the Est gene.

Embodiment 116 is a recombinant Bacillus cereus family member of any one of embodiments 100—115, wherein the recombinant Bacillus cereus family member comprises a mutation in a CotY gene.

Embodiment 117 is a inant Bacillus cereus family member of embodiment 116, wherein the mutation in the CotY gene comprises a knock-out of the CotY gene. 2] Embodiment 118 is a recombinant us cereus family member of embodiment 116 or 117, n the mutation in the CotY gene results in the formation of a fragile exosporium.

Embodiment 119 is a recombinant Bacillus cereus family member of any one of embodiments 100—118, wherein the inant Bacillus cereus family member comprises a mutation in an Est gene.

Embodiment 120 is a recombinant Bacillus cereus family member of embodiment 119, wherein the mutation in the Est gene comprises a knock-out of the Est gene.

] Embodiment 121 is a recombinant Bacillus cereus family member of embodiment 119 or 120, wherein the mutation in the Est gene results in the formation of a fragile exosporium.

Embodiment 122 is a recombinant us cereus family member of any one of embodiments 100—121, wherein the recombinant Bacillus cereus family member ses a mutation in a CotO gene. 7] ment 123 is a recombinant Bacillus cereus family member of embodiment 122, wherein the mutation in the CotO gene comprises a out of the CotO gene or a nt negative form of the CotO gene. 8] Embodiment 124 is a recombinant Bacillus cereus family member of embodiment 122 or 123, wherein the mutation in the CotO gene causes the exosporium to form in strips.

Embodiment 125 is a recombinant us cereus family member that expresses a fusion n comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member, wherein the recombinant Bacillus cereus family member comprises an endophytic strain of bacteria, a strain of bacteria that is capable of degrading an herbicide or a pesticide, or a probiotic strain of bacteria.

Embodiment 126 is a recombinant Bacillus cereus family member of embodiment 125, wherein the endophytic strain comprises Bacillus cereus family member EE349 (NRRL No. B-50928), Bacillus cereus family member EE439 (NRRL B-50979), Bacillus thuringiensis EE417 (NRRL B-50979), Bacillus cereus EE444 (NRRL B-50977), Bacillus thuringiensis EE319 (NRRL B-50983), Bacillus thuringiensis EE-B00184 (NRRL B- 67122), us cereus family member EE-B00377 (NRRL 9); Bacillus pseudomycoia’es EE-B00366 (NRRL B-67120); or Bacillus mycoia’es EE-B00363 (NRRL B-67121).

Embodiment 127 is a recombinant us cereus family member of embodiment 125, wherein the endophytic strain comprises Bacillus cereus family member EE439 (NRRL B-50979), Bacillus thuringiensis EE417 (NRRL B-50979), Bacillus cereus EE444 (NRRL B-50977), or Bacillus thuringiensis EE319 (NRRL B-50983), Bacillus thuringiensis EE-B00184 (NRRL B-67122), Bacillus cereus family member EE-B00377 mRRL B-67l l9); Bacillus pseudomycoia’es EE-B00366 (NRRL B-67120); or Bacillus mycoia’es EE- B00363 (NRRL B-67121).

Embodiment 128 is a recombinant Bacillus cereus family member of embodiment 125, wherein the strain of bacteria that is capable of degrading an herbicide or a pesticide comprises Bacillus cereus family member EE349 (NRRL No. B-50928), Bacillus cereus family member EE-B00377 (NRRL B-67l l9); Bacillus pseudomycoia’es EE-B00366 (NRRL B-67120); or Bacillus mycoia’es EE-B00363 (NRRL B-67121).

Embodiment 129 is a recombinant us cereus family member of ment 125, wherein the probiotic strain of bacteria comprises Bacillus cereus family member EE349 (NRRL No. B-50928), us cereus family member EE439 (NRRL B- 50979), Bacillus thuringiensis EE4l7 (NRRL B-50979), or Bacillus cereus EE444 (NRRL B- Embodiment 130 is a recombinant Bacillus cereus family member of any one of ments 16—28, 48—63, and 99—129, wherein the targeting sequence, exosporium protein, or exosporium protein fragment comprises: (1) a targeting sequence comprising an amino acid sequence haVing at least about 43% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 54%; (2) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 1 ; (3) a targeting sequence comprising amino acids 20—35 of SEQ ID NO: 1; (4) a targeting sequence comprising SEQ ID NO: 1; (5) an exosporium protein comprising an amino acid sequence haVing at least 85% identity with SEQ ID NO: 2; (6) a targeting ce sing amino acids 2—35 of SEQ ID NO: 1; (7) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 1 ; (8) a targeting sequence sing amino acids 8—35 of SEQ ID NO: 1 ; (9) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 1; (10) a ing sequence comprising amino acids 15—35 of SEQ ID NO: 1; (l l) a targeting sequence comprising amino acids l—27 of SEQ ID NO: 3; (12) a targeting ce comprising amino acids 12—27 of SEQ ID NO: 3; (13) a targeting sequence comprising SEQ ID NO: 3; (14) an exosporium protein comprising an amino acid ce haVing at least 85% identity with SEQ ID NO: 4; (15) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 3; (16) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 3; (17) a targeting sequence sing amino acids 8—27 of SEQ ID NO: 3; (18) a targeting ce comprising amino acids 10—27 of SEQ ID NO: 3; (19) a targeting sequence comprising amino acids l—38 of SEQ ID NO: 5; (20) a targeting sequence comprising amino acids 23—38 of SEQ ID NO: 5; (21) a targeting sequence comprising SEQ ID NO: 5; (22) an exosporium n comprising an amino acid sequence having at least 85% ty with SEQ ID NO: 6; (23) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 5; (24) a targeting sequence sing amino acids 5—38 of SEQ ID NO: 5; (25) a targeting sequence comprising amino acids 8—38 of SEQ ID NO: 5; (26) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 5; (27) a targeting sequence sing amino acids 15—38 of SEQ ID NO: 5; (28) a ing sequence comprising amino acids 20—38 of SEQ ID NO: 5; (29) a targeting sequence comprising amino acids 1—28 of SEQ ID NO: 7; (30) a targeting sequence comprising amino acids l3—28 of SEQ ID NO: 7; (3 l) a targeting sequence comprising SEQ ID NO: 7; (32) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 8; (33) a ing sequence sing amino acids 2—28 of SEQ ID NO: 7; (34) a targeting ce comprising amino acids 5—28 of SEQ ID NO: 7; (35) a targeting sequence comprising amino acids 8—28 of SEQ ID NO: 7; (36) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 7; (37) a targeting sequence comprising amino acids l—24 of SEQ ID NO: 9; (38) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 9; (3 9) a targeting sequence comprising SEQ ID NO: 9; (40) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 10; (41) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 9; (42) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 9; (43) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 9; (44) a targeting sequence comprising amino acids l—33 of SEQ ID NO:ll; (45) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 11; (46) a targeting sequence comprising SEQ ID NO: 11 ; (47) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 12; (48) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 11; (49) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 11 ; (50) a targeting sequence sing amino acids 8—33 of SEQ ID NO: 11 ; (5 l) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 11; (52) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: ll; (53) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 13; (54) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 13; (55) a ing sequence comprising SEQ ID NO: 13; (5 6) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 14; (57) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 13; (58) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13; (59) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 13; (60) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 13; (61) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 13; (62) a ing sequence comprising amino acids 1—43 of SEQ ID NO: 15; (63) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 15; (64) a targeting sequence comprising SEQ ID NO:15; (65) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 16; (66) a targeting ce comprising amino acids 2—43 of SEQ ID NO: 15; (67) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 15 ; (68) a targeting sequence sing amino acids 8—43 of SEQ ID NO: 15; (69) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 15; (70) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 15; (71) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 15; (72) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 15 ; (73) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 17; (74) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 17; (75) a targeting sequence comprising SEQ ID NO:17; (76) an rium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:18; (77) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 17; (78) a targeting sequence sing amino acids 5—27 of SEQ ID NO: 17; (79) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 17; (80) a ing sequence comprising amino acids 10—27 of SEQ ID NO: 17; (81) a targeting sequence sing amino acids 1—33 of SEQ ID NO: 19; (82) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 19; (83) a targeting sequence comprising SEQ ID NO:19; (84) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:20; (85) a targeting ce sing amino acids 2—33 of SEQ ID NO: 19; (86) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 19; (87) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 19; (88) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 19; (89) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 19; (90) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 21; (91) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 21; (92) a targeting sequence comprising SEQ ID NO:21; (93) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:22; (94) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 21 ; (95) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 21; (96) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 21; (97) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 21; (98) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 21 ; (99) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 23; (100) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 23 ; (101) a targeting sequence comprising SEQ ID NO:23; (102) an 3 80 rium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:24; (103) a targeting sequence comprising amino acids 2—24 of SEQ ID NO:23; (104) a ing sequence comprising amino acids 5—24 of SEQ ID NO: 23; (105) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 23; (106) a targeting ce comprising amino acids 1—24 of SEQ ID NO: 25; (107) a targeting ce sing amino acids 9—24 of SEQ ID NO: 25; (108) a targeting sequence comprising SEQ ID NO:25; (109) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:26; (110) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 25; (111) a targeting sequence sing amino acids 5—24 of SEQ ID NO: 25; (112) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 25; (113) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 27; (114) a targeting ce comprising amino acids 15—30 of SEQ ID NO: 27; (115) a targeting sequence comprising SEQ ID NO:27; (116) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:28; (117) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 27; (118) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 27; (119) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 27; (120) a targeting ce comprising amino acids 10—30 of SEQ ID NO: 27; (121) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 29; (122) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 29; (123) a targeting sequence comprising SEQ ID NO:29; (124) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:30; (125) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 29; (126) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 29; (127) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 29; (128) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 29; (129) a targeting sequence sing amino acids 15—33 of SEQ ID NO: 29; (130) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 31 ; (131) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 31 ; (132) a targeting sequence comprising SEQ ID NO:31; (133) an exosporium n comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:32; (134) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 31; (135) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 31; (136) a targeting sequence sing amino acids 8—24 of SEQ ID NO: 31 ; (137) a targeting sequence comprising amino acids 1—15 of SEQ ID NO: 33; (138) a targeting sequence sing SEQ ID NO:33; (139) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:34; (140) a targeting ce comprising amino acids 1—16 of SEQ W0 2016/044661 3 81 ID NO: 35; (141) a targeting sequence comprising SEQ ID NO:35; (142) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:36; (143) a targeting sequence comprising amino acids 1—29 of SEQ ID NO:43; (144) a targeting sequence comprising amino acids 14—29 of SEQ ID NO: 43; (145) a targeting sequence comprising SEQ ID NO: 43; (146) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 44; (147) a targeting sequence comprising amino acids 2—29 of SEQ ID NO: 43 ; (148) a targeting sequence comprising amino acids 5—29 of SEQ ID NO: 43 ; (149) a targeting sequence comprising amino acids 8—29 of SEQ ID NO: 43; (150) a targeting sequence comprising amino acids 10—29 of SEQ ID NO: 43; (151) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 45; (152) a targeting sequence comprising amino acids 20—35 of SEQ ID NO: 45 ; (153) a ing sequence comprising SEQ ID NO: 45 ; (154) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 46; (155) a targeting ce comprising amino acids 2—35 of SEQ ID NO: 45; (156) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 45; (157) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 45; (158) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45; (159) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 45 ; (160) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 47; (161) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 47; (162) a targeting sequence sing SEQ ID NO: 47; (163) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 48; (164) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 47; (165) a ing sequence comprising amino acids 5—43 of SEQ ID NO: 47; (166) a ing ce comprising amino acids 8—43 of SEQ ID NO: 47; (167) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 47; (168) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 47; (169) a targeting sequence comprising amino acids 20—43 of SEQ ID NO: 47; (170) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 47; (171) a targeting sequence comprising amino acids 1—32 of SEQ ID NO: 49; (172) a targeting sequence comprising amino acids 17—32 of SEQ ID NO: 49; (173) a targeting sequence comprising SEQ ID NO: 49; (174) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 50; (175) a targeting sequence comprising amino acids 2—32 of SEQ ID NO: 49; (176) a targeting ce comprising amino acids 5—32 of SEQ ID NO: 49; (177) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (178) a targeting sequence sing amino acids 10—32 of SEQ ID NO: 49; (179) a targeting sequence comprising amino 3 82 acids 15—32 of SEQ ID NO: 49; (180) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 51; (181) a targeting ce comprising amino acids 18—33 of SEQ ID NO: 51; (182) a targeting sequence sing SEQ ID NO: 51; (183) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 52; (184) a ing sequence comprising amino acids 2—33 of SEQ ID NO: 51; (185) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 51; (186) a ing sequence comprising amino acids 8—33 of SEQ ID NO: 51; (187) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 51; (188) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 51; (189) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 53; (190) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 53; (191) a ing sequence comprising SEQ ID NO: 53; (192) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 54; (193) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 53; (194) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 53 ; (195) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53 ; (196) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 53 ; (197) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 53; (198) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 55; (199) a targeting sequence sing amino acids 15—30 of SEQ ID NO: 55 ; (200) a targeting sequence comprising SEQ ID NO: 55 ; (201) an exosporium protein sing an amino acid sequence having at least 85% identity With SEQ ID NO: 56; (202) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 55; (203) a targeting sequence sing amino acids 5—30 of SEQ ID NO: 55; (204) a targeting sequence sing amino acids 8—30 of SEQ ID NO: 55; (205) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 55; (206) a targeting sequence comprising amino acids 1—130 of SEQ ID NO: 57; (207) a targeting sequence comprising amino acids 115—130 of SEQ ID NO: 57; (208) a ing sequence comprising SEQ ID NO: 57; (209) an rium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 58; (210) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 57; (211) a targeting sequence comprising amino acids 5—130 of SEQ ID NO: 57; (212) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (213) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 57; (214) a targeting sequence comprising amino acids 30— 130 of SEQ ID NO: 57; (215) a targeting sequence comprising amino acids 40—130 of SEQ ID NO: 57; (216) a targeting sequence comprising amino acids 50—130 of SEQ ID NO: 57; (217) a targeting sequence comprising amino acids 60—130 of SEQ ID NO: 57; (218) a targeting 3 83 sequence comprising amino acids 70—130 of SEQ ID NO: 57; (219) a targeting ce comprising amino acids 80—130 of SEQ ID NO: 57; (220) a targeting sequence comprising amino acids 90—130 of SEQ ID NO: 57; (221) a targeting sequence comprising amino acids 100—130 of SEQ ID NO: 57; (222) a targeting sequence sing amino acids 110—130 of SEQ ID NO: 57; (223) an exosporium n fragment comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 95; (224) a targeting sequence comprising SEQ ID NO: 96; (225) a targeting ce comprising SEQ ID NO: 97; (226) a targeting sequence comprising SEQ ID NO: 98; (227) a targeting sequence comprising SEQ ID NO: 99; (228) a targeting sequence comprising SEQ ID NO: 100; (229) a targeting sequence comprising SEQ ID NO: 101; (230) a targeting sequence comprising SEQ ID NO: 102; (231) a ing sequence comprising SEQ ID NO: 103; (232) a targeting ce comprising SEQ ID NO: 104; (233) a targeting sequence comprising SEQ ID NO: 105; (234) a targeting sequence comprising SEQ ID NO: 106; (235) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (236) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 109; (237) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 110; (238) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 111; (239) an exosporium n comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 112; (240) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 113; (241) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 114; (242) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 115; (243) an rium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 116; (244) an rium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 117; (245) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 118; (246) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 119; (247) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 120; (248) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 121 ; (249) a targeting sequence comprising amino acids 22—31 of SEQ ID NO: 1; (250) a targeting sequence comprising amino acids 22—33 of SEQ ID NO: 1; (251) a targeting sequence comprising amino acids 20—31 of SEQ ID NO: 1; (252) a targeting sequence comprising amino acids 14—23 of SEQ ID NO: 3; (253) a targeting sequence 3 84 comprising amino acids 14—25 of SEQ ID NO: 3; (254) a ing sequence sing amino acids 12—23 of SEQ ID NO: 3; (255) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 59; (256) a targeting sequence comprising SEQ ID NO: 59; (257) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 60; (258) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 59; (259) a targeting sequence comprising amino acids 4—30 of SEQ ID NO: 59; (260) a targeting sequence comprising amino acids 6—30 of SEQ ID NO: 59; (261) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 61; (262) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 61; (263) a ing sequence comprising SEQ ID NO: 61 ; (264) an exosporium protein comprising an amino acid ce having at least 85% sequence identity With SEQ ID NO: 62; (265) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 61 ; (266) a targeting ce comprising amino acids 5—33 of SEQ ID NO: 61 ; (267) a targeting ce comprising amino acids 10—33 of SEQ ID NO: 61; (268) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 61; (269) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 63; (270) a targeting sequence comprising SEQ ID NO: 63; (271) an rium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 64; (272) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 63; (273) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 63; (274) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 63 ; (275) a ing sequence sing amino acids 10—35 of SEQ ID NO: 63 ; (276) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 63 ; (277) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 65; (278) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 65; (279) a targeting sequence comprising SEQ ID NO: 65; (280) an exosporium protein sing an amino acid sequence having at least 85% identity with SEQ ID NO: 66; (281) a targeting sequence comprising SEQ ID NO: 107; (282) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 65; (283) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 65; (284) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 67; (285) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 67; (286) a targeting sequence comprising SEQ ID NO: 67; (287) an exosporium protein comprising an amino acid ce having at least 85% identity With SEQ ID NO: 68; (288) an targeting sequence comprising amino acids 2—27 of SEQ ID NO: 67; (289) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 67; (290) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 67; (291) a targeting sequence comprising amino acids 1—38 of SEQ ID NO: 69; (292) a targeting sequence 2015/050807 3 85 comprising amino acids 23—38 of SEQ ID NO: 69; (293) a targeting sequence comprising SEQ ID NO: 69; (294) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 70; (295) a targeting ce comprising amino acids 2—38 of SEQ ID NO: 69; (296) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 69; (297) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 69; (298) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 69; (299) an exosporium protein comprising SEQ ID NO: 72; (300) a targeting sequence comprising SEQ ID NO: 73; (301) an exosporium n comprising an amino acid sequence having at least 95% identity With SEQ ID NO: 74; (302) a targeting sequence comprising amino acids 1—42 of SEQ ID NO: 75; (303) a targeting sequence comprising amino acids 27—42 of SEQ ID NO: 75; (304) a targeting sequence comprising SEQ ID NO: 75; (305) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 76; (306) a targeting sequence comprising amino acids 2—42 of SEQ ID NO: 75; (307) a targeting sequence comprising amino acids 5—42 of SEQ ID NO: 75; (308) a targeting sequence comprising amino acids 10—42 of SEQ ID NO: 75; (309) a targeting sequence comprising amino acids 15—42 of SEQ ID NO: 75; (310) a targeting sequence comprising amino acids 20—42 of SEQ ID NO: 75; (311) a targeting sequence comprising amino acids 25—42 of SEQ ID NO: 75; (312) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 77; (313) a targeting ce sing amino acids 9—24 of SEQ ID NO: 77; (314) a targeting sequence comprising SEQ ID NO: 77; (315) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 78; (316) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 77; (317) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 77; (318) an exosporium protein sing an amino acid sequence having at least 85% ty With SEQ ID NO: 80; (319) a targeting ce comprising amino acids 1—38 of SEQ ID NO: 81; (320) a targeting sequence comprising amino acids 23—38 of SEQ ID NO: 81; (321) a targeting sequence comprising SEQ ID NO: 81; (322) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 82; (323) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 81; (324) a ing sequence comprising amino acids 5—38 of SEQ ID NO: 81; (325) a targeting ce comprising amino acids 10—38 of SEQ ID NO: 81; (326) a targeting ce comprising amino acids 15—38 of SEQ ID NO: 81; (327) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 81; (328) a targeting sequence comprising amino acids 1—34 of SEQ ID NO: 83; (329) a targeting sequence comprising SEQ ID NO: 83; (330) an exosporium protein comprising an amino acid sequence having at least 3 86 85% ty With SEQ ID NO: 84; (33 1) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 86; (332) a targeting sequence comprising amino acids 1—28 of SEQ ID NO: 87; (333) a targeting ce comprising amino acids 13—28 of SEQ ID NO: 87; (334) a targeting sequence comprising SEQ ID NO: 87; (335) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 88; (336) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 87; (337) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 87; (338) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 87; (339) a targeting sequence comprising amino acids 1—28 of SEQ ID NO: 89; (340) a targeting sequence comprising SEQ ID NO: 89; (341) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 90; (342) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 89; (343) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 89; (344) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 89; (345) a targeting sequence comprising amino acids 1—93 of SEQ ID NO: 91 ; (346) a targeting sequence comprising SEQ ID NO: 91 ; (347) an rium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 92; (348) a targeting sequence comprising amino acids 2—93 of SEQ ID NO: 91 ; (349) a targeting sequence comprising amino acids 10—93 of SEQ ID NO: 91 ; (350) a targeting sequence comprising amino acids 20—93 of SEQ ID NO: 91 ; (35 l) a targeting sequence comprising amino acids 30—93 of SEQ ID NO: 91 ; (352) a targeting sequence comprising amino acids 40—93 of SEQ ID NO: 91 ; (353) a targeting sequence comprising amino acids 50—93 of SEQ ID NO: 91 ; (354) a targeting sequence comprising amino acids 60—93 of SEQ ID NO: 91; (355) a ing sequence comprising amino acids 1—130 of SEQ ID NO: 93; (356) a targeting sequence comprising SEQ ID NO: 93; (357) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 94; (358) a ing sequence comprising amino acids 2—130 of SEQ ID NO: 93; (359) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 93; (360) a targeting sequence sing amino acids 20—130 of SEQ ID NO: 93; (361) a targeting sequence comprising amino acids 30—130 of SEQ ID NO: 93; (362) an exosporium n sing an amino acid sequence haVing at least 85% identity With SEQ ID NO: 122; (363) a ing sequence consisting of amino acids 20—33 of SEQ ID NO: 1 ; (364) a ing sequence consisting of amino acids 21—33 of SEQ ID NO: 1 ; (365) a targeting sequence ting of amino acids 23— 31 of SEQ ID NO: 1; (366) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 96; (367) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 96; (368) a 3 87 targeting sequence consisting of amino acids 12—25 of SEQ ID NO: 3; (369) a targeting sequence consisting of amino acids 13—25 of SEQ ID NO: 3; (370) a ing sequence consisting of amino acids 15—23 of SEQ ID NO: 3; (371) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 97; (372) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 98; (373) a targeting sequence consisting of amino acids 23—36 of SEQ ID NO: ; (374) a targeting sequence consisting of amino acids 23—34 of SEQ ID NO: 5; (375) a ing sequence consisting of amino acids 24—36 of SEQ ID NO: 5; (376) a targeting sequence consisting of amino acids 26—34 of SEQ ID NO: 5; (377) a targeting sequence consisting of amino acids 13—26 of SEQ ID NO: 7; (378) a targeting sequence consisting of amino acids 13—24 of SEQ ID NO: 7; (379) a targeting sequence consisting of amino acids 14— 26 of SEQ ID NO: 7; (380) a targeting sequence consisting of amino acids 16—24 of SEQ ID NO: 7; (381) a targeting sequence ting of amino acids 9—22 of SEQ ID NO: 9; (382) a targeting sequence consisting of amino acids 9—20 of SEQ ID NO: 9; (383) a targeting ce consisting of amino acids 10—22 of SEQ ID NO: 9; (384) a ing sequence consisting of amino acids 12—20 of SEQ ID NO: 9; (385) a targeting ce consisting of amino acids 1—15 of SEQ ID NO: 105; (386) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 105; (387) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 11; (388) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 11; (389) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 11; (390) a targeting sequence ting of amino acids 1—15 of SEQ ID NO: 98 ; (391) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 98 ; (392) a targeting sequence consisting of amino acids 18— 31 of SEQ ID NO: 13; (393) a targeting sequence ting of amino acids 18—29 of SEQ ID NO: 13 ; (394) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 13 ; (395) a ing sequence consisting of amino acids 21—29 of SEQ ID NO: 13; (396) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 99; (397) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 99; (398) a targeting sequence consisting of amino acids 28—41 of SEQ ID NO: 15; (399) a targeting sequence consisting of amino acids 28— 39 of SEQ ID NO: 15; (400) a targeting sequence consisting of amino acids 29—41 of SEQ ID NO: 15 ; (401) a targeting sequence consisting of amino acids 31—39 of SEQ ID NO: 15 ; (402) a targeting ce consisting of amino acids 12—25 of SEQ ID NO: 17; (403) a targeting sequence consisting of amino acids 13—25 of SEQ ID NO: 17; (404) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 100; (405) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 19; (406) a targeting sequence consisting of amino acids 18— 2015/050807 3 88 29 of SEQ ID NO: 19; (407) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 19; (408) a targeting sequence consisting of amino acids 21—29 of SEQ ID NO: 19; (409) a targeting ce consisting of amino acids 18—31 of SEQ ID NO: 21; (410) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 21; (411) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 21; (412) a targeting sequence consisting of amino acids 21—29 of SEQ ID NO: 21; (413) a targeting sequence consisting of amino acids 1— of SEQ ID NO: 101; (414) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 101 ; (415) a targeting ce consisting of amino acids 9—22 of SEQ ID NO: 23; (416) a targeting sequence consisting of amino acids 9—20 of SEQ ID NO: 23; (417) a targeting sequence consisting of amino acids 10—22 of SEQ ID NO: 23; (418) a targeting sequence consisting of amino acids 12—20 of SEQ ID NO: 23; (419) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 102; (420) a targeting sequence consisting of amino acids 1— 13 of SEQ ID NO: 102; (421) a targeting sequence consisting of amino acids 9—22 of SEQ ID NO: 25 ; (422) a targeting sequence consisting of amino acids 9—20 of SEQ ID NO: 25 ; (423) a targeting sequence consisting of amino acids 10—22 of SEQ ID NO: 25; (424) a targeting sequence consisting of amino acids 12—20 of SEQ ID NO: 25; (425) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 103; (426) a ing ce consisting of amino acids 1—13 of SEQ ID NO: 103; (427) a targeting ce consisting of amino acids 15— 28 of SEQ ID NO: 27; (428) a targeting sequence consisting of amino acids 15—26 of SEQ ID NO: 27; (429) a targeting ce consisting of amino acids 16—28 of SEQ ID NO: 27; (430) a targeting sequence consisting of amino acids 18—26 of SEQ ID NO: 27; (431) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 104; (432) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 104; (433) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 33; (434) a targeting sequence consisting of amino acids 1—11 of SEQ ID NO: 33 ; (435) a targeting sequence consisting of amino acids 3—11 of SEQ ID NO: 33 ; (436) a targeting ce consisting of amino acids 1—14 of SEQ ID NO: 35; (437) a targeting ce consisting of amino acids 1—12 of SEQ ID NO: 35; (438) a targeting sequence consisting of amino acids 2—14 of SEQ ID NO: 35; (439) a targeting sequence consisting of amino acids 14—27 of SEQ ID NO: 43; (440) a targeting ce consisting of amino acids 14—25 of SEQ ID NO: 43; (441) a ing sequence consisting of amino acids 15— 27 of SEQ ID NO: 43; (442) a targeting sequence consisting of amino acids 20—33 of SEQ ID NO: 45; (443) a targeting sequence consisting of amino acids 20—31 of SEQ ID NO: 45 ; (444) a targeting sequence consisting of amino acids 21—33 of SEQ ID NO: 45; (445) a targeting 3 89 sequence consisting of amino acids 1—15 of SEQ ID NO: 106; (446) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 106; (447) a targeting sequence consisting of amino acids 28—41 of SEQ ID NO: 47; (448) a targeting ce consisting of amino acids 28— 39 of SEQ ID NO: 47; (449) a ing sequence consisting of amino acids 18—31 of SEQ ID NO: 53; (450) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 53; (451) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 53; (452) a targeting sequence comprising amino acids 18—31 of SEQ ID NO: 61; (453) a targeting sequence comprising amino acids 18—29 of SEQ ID NO: 61; (454) a targeting ce sing amino acids 19—31 of SEQ ID NO: 61; (455) a targeting sequence sing amino acids 9—22 of SEQ ID NO: 65; (456) a targeting sequence comprising amino acids 9—20 of SEQ ID NO: 65; (457) a targeting sequence comprising amino acids 10—22 of SEQ ID NO: 65; (458) a targeting sequence comprising amino acids 1—15 of SEQ ID NO: 107; (459) a targeting sequence comprising amino acids 1—13 of SEQ ID NO: 107; (460) a targeting sequence comprising amino acids 12—25 of SEQ ID NO: 67; (461) a targeting sequence comprising amino acids 12—23 of SEQ ID NO: 67; (462) a targeting sequence comprising amino acids 13—25 of SEQ ID NO: 67; (463) a targeting sequence comprising amino acids 15—23 of SEQ ID NO: 67; (464) a targeting sequence comprising amino acids 23—36 of SEQ ID NO: 69; (465) a targeting ce comprising amino acids 23—34 of SEQ ID NO: 69; (466) a targeting sequence comprising amino acids 24—36 of SEQ ID NO: 69; (467) a targeting ce comprising amino acids 26—34 of SEQ ID NO: 69; (468) a targeting sequence comprising amino acids 27—40 of SEQ ID NO: 75; (469) a targeting sequence comprising amino acids 27—38 of SEQ ID NO: 75; (470) a targeting sequence comprising amino acids 9—22 of SEQ ID NO: 77; (471) a targeting sequence comprising amino acids 9—20 of SEQ ID NO: 77; (472) a targeting sequence comprising amino acids 10fifi—22 of SEQ ID NO: 77; (473) a targeting ce comprising amino acids 12—20 of SEQ ID NO: 77; (474) a targeting sequence comprising amino acids 23—36 of SEQ ID NO: 81; (475) a targeting sequence comprising amino acids 23—34 of SEQ ID NO: 81; (476) a targeting sequence comprising amino acids 24—36 of SEQ ID NO: 81; (477) a targeting sequence sing amino acids 26—34 of SEQ ID NO: 81; (478) a targeting sequence comprising amino acids 13—26 of SEQ ID NO: 87; (479) a targeting sequence comprising amino acids 13—24 of SEQ ID NO: 87; or (480) a targeting sequence comprising amino acids 14—26 of SEQ ID NO: 2015/050807 ment 131 is a fusion protein of any one of embodiments 4, 8, and 12— or a recombinant Bacillus cereus family member of embodiment 16 or 130, wherein the targeting ce ses: an amino acid sequence having at least about 50% identity with amino acids 20— of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 63%; an amino acid sequence haVing at least about 50% identity with amino acids 20— of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 72%; an amino acid sequence haVing at least about 56% identity with amino acids 20— of SEQ ID NO: 1, wherein the ty with amino acids 25—35 is at least about 63%; an amino sequence haVing at least about 62% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 72%; an amino acid sequence haVing at least about 68% identity with amino acids 20— of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 8 1%; an amino sequence haVing at least about 75% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 72%; an amino sequence haVing at least about 75% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 8 1 % ; an amino acid sequence haVing at least about 81% identity with amino acids 20— of SEQ ID NO:1, wherein the identity with amino acids 25—35 is at least about 8 1 % ; or an amino acid ce haVing at least about 81% identity with amino acids 20— of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 90%.

Embodiment 132 is a fusion protein of any one of embodiments 4, 8, and 12— or a recombinant Bacillus cereus family member of embodiment 16 or 130, wherein the targeting sequence consists of: (a) an amino acid sequence consisting of 16 amino acids and having at least about 43% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 54%; (b) amino acids 1—35 of SEQ ID NO: 1; (c) amino acids 20—35 of SEQ ID NO: 1; (d) SEQ ID NO: 1; (e) SEQ ID NO: 96; or (f) SEQ ID NO: 120.

Embodiment 133 is a fusion protein or a recombinant Bacillus cereus family member of embodiment 131, wherein the targeting sequence consists of the amino acid sequence.

Embodiment 134 is a fusion protein of any one of embodiments 4, 8, and 12— or a inant Bacillus cereus family member of ment 16 or 130, wherein the fusion protein comprises an exosporium protein or an rium protein fragment comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, and 121. 9] Embodiment 135 is a fusion protein of embodiment 1 or a recombinant Bacillus cereus family member of embodiment 16 or 130, wherein the fusion protein comprises an exosporium protein comprising an amino acid sequence haVing at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 122. ment 136 is a fusion n of any one of embodiments 1—1 5 and 131—135 or a recombinant Bacillus cereus family member of any one of embodiments 16—29, 48—63, and 99—135, wherein the targeting ce, exosporium protein, or exosporium protein fragment comprises the amino acid sequence GXT at its carboxy terminus, wherein X is any amino acid.

Embodiment 137 is a fusion protein of any one of embodiments 1—1 5 and 131—136 or a recombinant Bacillus cereus family member of any one of embodiments 16—29, 48—63, and 99—136, wherein the targeting sequence, exosporium protein, or exosporium protein fragment comprises an alanine residue at the position of the targeting ce that corresponds to amino acid 20 of SEQ ID NO: 1.

Embodiment 138 is a fusion protein of any one of embodiments 1—1 5 and 131—137, or a recombinant Bacillus cereus family member of any one of ments 16—29, 48—63 and 99—137, n the ing sequence, exosporium protein, or exosporium protein fragment further comprises a methionine, serine, or threonine residue at the amino acid on immediately preceding the first amino acid of the targeting sequence, exosporium protein, or exosporium protein fragment or at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1.

Embodiment 139 is a fusion protein of any one of embodiments 1—15 and 131—138 or a recombinant Bacillus cereus family member of any one of embodiments 16—29, 48—63, and 99—138, wherein the fusion protein further comprises an amino acid linker between the targeting sequence, the exosporium protein, or the exosporium protein nt and the protein or peptide of interest.

Embodiment 140 is a fusion protein or a recombinant Bacillus cereus family member of embodiment 139, wherein the linker comprises a polyalanine linker, a polyglycine , or a linker comprising a mixture of both alanine and glycine residues.

Embodiment 141 is a fusion protein or a recombinant Bacillus cereus family member of embodiment 139 or 140, wherein the linker comprises a se recognition site. 6] Embodiment 142 is a fusion protein sing at least one protein or peptide of interest and a spore coat protein, wherein the spore coat n comprises a CotB/H protein, a spore protein X n, or a CotY protein, wherein the CotY protein comprises an amino acid sequence haVing at least 80% ty with SEQ ID NO: 258 or 259.

Embodiment 143 is a fusion protein of embodiment 142, wherein the spore coat protein comprises a CotB/H protein.

Embodiment 144 is a fusion protein of embodiment 142, wherein the spore coat protein comprises a spore protein X protein.

Embodiment 145 is a fusion protein of embodiment 142, wherein the spore coat protein comprises a CotY protein, wherein the CotY protein comprises an amino acid sequence haVing at least 80% identity with SEQ ID NO: 258 or 259.

Embodiment 146 is a fusion protein of any one of embodiments 5, wherein the spore coat protein comprises an amino acid ce haVing at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 255, 257, 258, or 259.

Embodiment 147 is a recombinant spore-forming bacterium that expresses a fusion protein of any one of embodiments 142—146.

Embodiment 148 is a recombinant spore-forming bacterium of embodiment 147, wherein the inant spore-forming bacterium ses an endophytic strain of bacteria, a plant growth-promoting strain of bacteria, or a strain of bacteria that is both endophytic and plant growth-promoting.

Embodiment 149 is a recombinant spore-forming bacterium that expresses a fusion n comprising at least one n or peptide of interest and a spore coat protein that targets the fusion protein to the surface of a spore of the bacterium, wherein the spore coat protein comprises a CotB protein, a CotC protein, a CgeA n, a CotB/H protein, a CotG protein, a spore coat protein X protein, or a CotY n; and wherein the recombinant sporeforming bacterium comprises an ytic strain of bacteria, a plant growth-promoting strain of bacteria, or a strain of bacteria that is both endophytic and plant growth-promoting.

Embodiment 150 is a plant seed coated with a recombinant spore-forming bacterium, wherein the recombinant forming bacterium expresses a fusion protein comprising at least one protein or peptide of interest and a spore coat protein that targets the fusion protein to the surface of a spore of the ium, wherein the spore coat protein comprises a cotB protein, a CotC protein, a CgeA protein, a CotB/H n, a Cot G protein, a spore protein X protein, or a cotY protein. ment 151 is a recombinant spore-forming bacterium of embodiment 147, 148, or 149 or a plant seed of embodiment 150, wherein the recombinant spore-forming bacterium comprises a bacterium of the genus Bacillus, Lysinibacillus, Virginibacillus, Clostria'ia, or acillus. 6] Embodiment 152 is a recombinant bacterium of the genus Bacillus of any one of embodiments 30—45, wherein the recombinant bacterium comprises a recombinant spore- forming bacterium and wherein the recombinant spore-forming bacterium also expresses a fusion protein comprising at least one protein or peptide of interest and a spore coat protein that targets the fusion protein to the e of a spore of the bacterium, wherein the spore coat protein comprises a CotB protein, a CotC protein, a CgeA n, a CotB/H protein, a Cot G protein, a spore coat protein X protein, or a CotY protein.

Embodiment 153 is a recombinant bacterium of the genus us of embodiment 152, wherein the recombinant bacterium comprises an endophytic strain of bacteria, a plant -promoting strain of bacteria, or a strain of bacteria that is both endophytic and plant -promoting.

Embodiment 154 is a plant seed coated with a inant spore-forming ium of embodiment 152 or 153.

Embodiment 155 is a plant seed of embodiment 150, 151, or 154, wherein the recombinant spore-forming bacterium comprises an endophytic strain of bacteria, a plant growth-promoting strain of bacteria, or a strain of bacteria that is both endophytic and plant growth-promoting.

Embodiment 156 is a recombinant spore-forming bacterium of embodiment 148, 149, or 153, or a seed of embodiment 155, wherein the endophytic strain of bacteria, the plant growth-promoting strain of bacteria, or the strain of bacteria that is both endophytic and plant growth-promoting comprises us megaterium EE3 85 (NRRL B-50980), Bacillus sp.

EE387 (NRRL B-50981), Bacillus ans EE388 (NRRL B-50982), Bacillus subtilis EE405 (NRRL B-50978), Lysinibacillusfusiformis EE442 (NRRL B-50975), Lysinibacillus sphaericus EE443 (NRRL B-50976), Bacillus pumilus EE-B00143 (NRRL B-67123), Bacillus subtilis EE148 (NRRL B-50927), Bacillus subtilis EE218 (NRRL B-50926), or Bacillus megaterium EE281 (NRRL B-50925).

Embodiment 157 is a recombinant spore-forming bacterium or seed of embodiment 156, n the endophytic strain of bacteria comprises Bacillus subtilis EE405 (NRRL B-50978) or Bacillus megaterium EE385 (NRRL B-50980). ment 158 is a inant spore-forming ium of embodiment 148, 149, or 153, or a seed of ment 155, wherein the endophytic strain of bacteria, the plant -promoting strain of bacteria, or the strain of bacteria that is both endophytic and plant growth-promoting comprises Bacillus cereus family member EE349 (NRRL No. B- 50928), Bacillus cereus family member EE439 (NRRL B-50979), Bacillus thuringiensis EE417 (NRRL B-50979), Bacillus cereus EE444 (NRRL B-50977), Bacillus thuringiensis EE319 (NRRL B-50983), Bacillus thuringiensis EE-B00184 (NRRL B-67122), Bacillus cereus family member 377 (NRRL 9), Bacillus pseudomycoia’es EE-B00366 (NRRL B-67120), Bacillus mycoia’es EE-B00363 (NRRL B-67121), Bacillus mycoia’es BT155 (NRRL No. B- 50921), Bacillus mycoia’es EE118 (NRRL No. B-50918), Bacillus mycoia’es EE141 (NRRL No.

B-50921), Bacillus mycoia’es BT46-3 (NRRL No. B-50922), Bacillus cereus family member EE128 (NRRL No. B-50917), Bacillus thuringiensis BT013A (NRRL No. B-50924), or us cereus family member EE349 (NRRL No. B-50928).

Embodiment 159 is a recombinant spore-forming bacterium of any one of embodiments 149, 151, 152, 153, and 8, or a seed of embodiment 150, 151, and 154— 158, wherein the spore coat protein ses an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NOs: 252—259. ment 160 is a recombinant spore-forming bacterium that expresses a fusion protein comprising at least one protein or peptide of interest and a protein that targets the fusion protein to the e of a spore of the bacterium, wherein the recombinant forming bacterium is not a inant Bacillus cereus family member, and the protein that targets the fusion protein to the surface of a spore of the bacterium comprises amino acids 20—35 of SEQ ID NO: 1, SEQ ID NO: 96, or an amino acid sequence having at least 85%, at least 90%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO: 114, SEQ ID NO: 120, or SEQ ID NO: 121.

Embodiment 161 is a recombinant forming bacterium of embodiment 160, wherein the protein that targets the fusion protein to a surface of a spore of the bacterium comprises amino acids 20—35 of SEQ ID NO: 1, SEQ ID NO: 96, SEQ ID NO: 108, SEQ ID NO: 120, or SEQ ID NO: 121. ment 162 is a recombinant spore-forming bacterium of ment 160 or 161, wherein the recombinant-spore forming bacterium comprises an endophytic strain of bacteria, a plant growth-promoting strain of bacteria, or a strain of bacteria that is both endophytic and plant growth-promoting.

Embodiment 163 is a recombinant spore-forming bacterium of embodiment 162, wherein the endophytic strain of bacteria, the plant growth-promoting strain of ia, or the strain of bacteria that is both endophytic and plant growth-promoting comprises Bacillus megaterium EE385 (NRRL B-50980), us sp. EE387 (NRRL B-50981), Bacillus circulans EE388 (NRRL B-50982), Bacillus subtilis EE405 (NRRL B-50978), Lysinibacillusfusiformis EE442 (NRRL B-50975), Lysinibacillus sphaericus EE443 (NRRL B-50976), Bacillus pumilus EE-B00143 (NRRL B-67123), Bacillus subtilis EE148 (NRRL B-50927), Bacillus subtilis EE218 mRRL B-50926), or Bacillus megaterium EE281 (NRRL B-50925) Embodiment 164 is a inant spore-forming bacterium of embodiment 163, n the endophytic strain of bacteria comprises Bacillus sp. EE3 87 (NRRL B-50981).

Embodiment 165 is a fusion protein of any one of embodiments 142—146, a inant spore-forming bacterium of any one of embodiments 147—149, 151—153, and 156— 164, or a seed of any one of embodiments 150, 151, and 154—159, wherein the fusion protein further ses an amino acid linker between the spore coat protein and the protein or peptide of interest.

Embodiment 166 is a fusion protein, recombinant spore-forming bacterium or seed of ment 165, wherein the linker comprises a anine linker, a polyglycine linker, or a linker comprising a mixture of both alanine and glycine residues.

Embodiment 167 is a fusion protein, recombinant spore-forming bacterium or seed of ment 165 or 166, wherein the linker comprises a protease recognition site.

Embodiment 168 is a fusion protein of any one of ments 1—3, and 142—146, a recombinant Bacillus cereus family member of any one of embodiments 16—29, 48— 63, and 99—141, a recombinant spore-forming bacterium of any one of embodiments 147—149, 151—153, and 156—167, or a plant seed of any one of embodiments 150, 151, and 154—167, wherein the protein or peptide of interest comprises a plant growth stimulating protein or peptide, a protein or peptide that protects a plant from a pathogen, a protein or peptide that enhances stress resistance in a plant, a plant binding protein or peptide, an enzyme that catalyzes the production of nitric oxide, or a nucleic acid binding protein or peptide. ment 169 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 168, wherein the protein or peptide of interest comprises a plant growth stimulating protein or e. ment 170 is a fusion protein, a recombinant Bacillus cereus family member, a inant spore-forming bacterium, or a plant seed of embodiment 169, wherein the plant growth ating protein or peptide comprises a peptide e, a non-hormone peptide, an enzyme involved in the production or activation of a plant growth stimulating compound, or an enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source. ment 171 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of ment 170, wherein the plant growth stimulating protein or peptide comprises a peptide e.

Embodiment 172 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 171, wherein the peptide e comprises a phytosulfokine, clavata 3 (CLV3), systemin, ZmlGF, or a SCIVSPl 1. 2015/050807 Embodiment 173 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 172, wherein the phytosulfokine comprises ulfokine-u.

Embodiment 174 is a fusion protein, a inant Bacillus cereus family member, a recombinant spore-forming ium, or a plant seed of embodiment 170, wherein the plant growth ating protein or peptide comprises a rmone peptide.

Embodiment 175 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 174, wherein the rmone peptide comprises a RKN 16D10, Hg-Syv46, an eNOD40 peptide, melittin, mastoparan, Mas7, RHPP, POLARIS, or kunitz trypsin inhibitor (KTI). ment 176 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 170, wherein the plant growth stimulating protein or peptide comprises an enzyme involved in the production or activation of a plant growth stimulating compound.

Embodiment 177 is a fusion n, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 176, wherein the plant growth stimulating compound ses a compound ed by bacteria or fungi in the rhizosphere.

Embodiment 178 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 177, wherein the nd produced by bacteria or fungi in the rhizosphere comprises tanediol.

Embodiment 179 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 176, wherein the plant growth stimulating compound comprises a plant growth hormone. ment 180 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 179, n the plant growth hormone comprises a cytokinin or a cytokinin derivative, ethylene, an auxin or an auxin derivative, a gibberellic acid or a gibberellic acid derivative, abscisic acid or an abscisic acid derivative, a jasmonic acid or a ic acid derivative.

Embodiment 181 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 180, wherein the plant growth hormone comprises a cytokinin or a cytokinin derivative, and the cytokinin or the cytokinin derivative comprises kinetin, cis-zeatin, trans-zeatin, 6-benzylaminopurine, WO 44661 dihydroxyzeatin, N6-(D2—isopentenyl) adenine, lzeatin, N6-(D2-isopentenyl) adenosine, 2-methylthio-cis-ribosylzeatin, cis-ribosylzeatin, trans-ribosylzeatin, 2-methylthio-transribosylzeatin , ribosylzeatin-S-monosphosphate, N6-methylaminopurine, N6- ylaminopurine, 2’-deoxyzeatin de, 4-hydroxymethyl-trans butenylaminopurine, ortho-topolin, meta-topolin, benzyladenine, methyltopolin, metamethyltopolin , or a combination thereof; or wherein the plant growth hormone comprises an auxin or an auxin derivative, and the auxin or the auxin derivative comprises an active auxin, an inactive auxin, a conjugated auxin, a naturally occurring auxin, a synthetic auxin, or a combination thereof.

Embodiment 182 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 180 or 181, wherein the plant growth hormone comprises an auxin or an auxin derivative, and the auxin or the auxin derivative comprises indoleacetic acid, indolepyruvic acid, indole acetaldoxime, indoleacetamide, indoleacetonitrile, indoleethanol, indolepyruvate, indoleacetaldoxime, indolebutyric acid, a phenylacetic acid, 4-chloroindoleacetic acid, a glucose-conjugated auxin, or a ation thereof.

Embodiment 183 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of any one of embodiments 176—182, wherein the plant growth stimulating protein or peptide comprises an enzyme involved in the production or activation of a plant growth stimulating compound, and the enzyme involved in the production or activation of a plant growth ating compound comprises an acetoin ase, an indoleacetamide hydrolase, a tryptophan ygenase, an acetolactate synthetase, an (x-acetolactate decarboxylase, a pyruvate decarboxylase, a diacetyl reductase, a butanediol dehydrogenase, an aminotransferase, a tryptophan decarboxylase, an amine oxidase, an indolepyruvate decarboxylase, an indoleacetaldehyde dehydrogenase, a tryptophan side chain oxidase, a nitrile hydrolase, a nitrilase, a peptidase, a protease, an ine ate isopentenyltransferase, a phosphatase, an adenosine kinase, an adenine phosphoribosyltransferase, CYP735A, a S’ribonucleotide phosphohydrolase, an adenosine nucleosidase, a zeatin cis-trans isomerase, a zeatin O-glucosyltransferase, a B-glucosidase, a cis- hydroxylase, a CK cis-hydroxylase, a CK N—glucosyltransferase, a bonucleotide phosphohydrolase, an adenosine nucleosidase, a purine nucleoside phosphorylase, a zeatin reductase, a hydroxylamine reductase, a lutarate dioxygenase, a gibberellic 2B/3B hydrolase, a gibberellin 3-oxidase, a ellin 20-oxidase, a chitosanase, a chitinase, a B-l,3- glucanase, a B-1,4-glucanase, a B-1,6-glucanase, an aminocyclopropane-l-carboxylic acid deaminase, or an enzyme involved in producing a nod factor.

Embodiment 184 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 183, wherein the enzyme involved in the production or activation of a plant growth stimulating compound comprises an enzyme involved in producing a nod factor and the enzyme involved in producing a nod factor ses nodA, nodB, or nodI; or n the enzyme involved in the production or activation of a plant growth stimulating compound comprises an ransferase and the aminotransferase comprises a tryptophan aminotransferase.

Embodiment 185 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 183, wherein the enzyme involved in the production or activation of a plant growth stimulating nd comprises a protease or peptidase that cleaves proteins, peptides, proproteins, or proteins to create a bioactive peptide.

Embodiment 186 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant forming ium, or a plant seed of embodiment 183, wherein the protease or ase comprises isin, an acid protease, an alkaline protease, a proteinase, an endopeptidase, an exopeptidase, thermolysin, papain, pepsin, trypsin, pronase, a carboxylase, a serine protease, a glutamic protease, an aspartate protease, a cysteine se, a threonine protease, or a oprotease.

Embodiment 187 is a fusion protein, a recombinant us cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 185 or 186, wherein the bioactive peptide comprises RKN 16D10 or RHPP.

Embodiment 188 is a fusion protein, a recombinant us cereus family member, a recombinant spore-forming bacterium, or a plant seed of any one of embodiments 185—187, wherein the protease or peptidase cleaves proteins in a protein-rich meal.

Embodiment 189 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of ment 188, wherein the protein-rich meal is soybean meal or yeast extract.

Embodiment 190 is a fusion protein, a recombinant Bacillus cereus family , a recombinant spore-forming bacterium, or a plant seed of embodiment 183, wherein the enzyme involved in the production or activation of a plant growth stimulating compound comprises a chitosanase, wherein the chitosanase comprises an amino acid sequence having at least 85%, at least 95%, at least 98%, at least 99%, or a 100% identity with SEQ ID NO: 313.

Embodiment 191 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 170, wherein the plant growth stimulating protein or e comprises an enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source.

Embodiment 192 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 191, wherein the enzyme that es or modifies a bacterial, fungal, or plant nutrient source comprises a cellulase, a lipase, a lignin oxidase, a protease, a glycoside hydrolase, a atase, a nitrogenase, a nuclease, an amidase, a nitrate reductase, a e reductase, an amylase, an ammonia oxidase, a ligninase, a glucosidase, a phospholipase, a phytase, a pectinase, a glucanase, a sulfatase, a urease, a xylanase, or a siderophore.

Embodiment 193 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming ium, or a plant seed of embodiment 192, wherein the phospholipase comprises a phospholipase A1, a phospholipase A2, a phospholipase C, a phospholipase D, or a ospholipase. 8] Embodiment 194 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 193, wherein the phospholipase comprises a phospholipase C and the olipase C comprises an amino acid sequence having at least 85%, at least 95%, at least 98%, at least 99%, or a 100% identity with SEQ ID NO: 312. 9] Embodiment 195 is a fusion protein, a inant Bacillus cereus family , a recombinant spore-forming ium, or a plant seed of embodiment 192, wherein the enzyme that degrades or s a bacterial, fungal, or plant nutrient source comprises a cellulase, and the cellulase comprises an endocellulase, an exocellulase, or a B-glucosidase.

Embodiment 196 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 195, wherein the ase comprises an endocellulase, and the endocellulase comprises an endoglucanase.

Embodiment 197 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 195 or 196, n the cellulase comprises an endocellulase, and the endocellulase comprises a Bacillus subtilis ucanase, a Bacillus thuringiensis endoglucanase, a Bacillus cereus endoglucanase, or a Bacillus clausii endoglucanase.

Embodiment 198 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 197, wherein the cellulase comprises a Bacillus subtilis endoglucanase, and the us subtilis endoglucanase comprises an amino acid sequence having at least 85%, at least 95%, at least 98%, at least 99%, or a 100% identity with SEQ ID NO: 311.

Embodiment 199 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 195, wherein the cellulase comprises an exocellulase, and the exocellulase comprises a a’erma reesei exocellulase. ment 200 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 195, wherein the cellulase comprises a B-glucosidase, and the B-glucosidase comprises a Bacillus subtilis B- glucosidase, a us thuringiensis B-glucosidase, a Bacillus cereus B-glucosidase, or a Bacillus clausii B-glucosidase.

Embodiment 201 is a fusion protein, a recombinant us cereus family member, a recombinant spore-forming ium, or a plant seed of embodiment 192, wherein the enzyme that degrades or modifies a ial, fungal, or plant nutrient source ses a lipase, and the lipase comprises a Bacillus subtilis lipase, a Bacillus thuringiensis lipase, a Bacillus cereus , or a Bacillus clausii lipase. 6] Embodiment 202 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 192, wherein the enzyme that es or modifies a bacterial, fungal, or plant nutrient source comprises a lignin oxidase, and the lignin e comprises a lignin peroxidase, a laccase, a glyoxal oxidase, a ligninase, or a manganese peroxidase.

Embodiment 203 is a fusion protein, a recombinant Bacillus cereus family , a recombinant forming bacterium, or a plant seed of embodiment 192, wherein the enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source comprises a protease, and the protease comprises a subtilisin, an acid protease, an alkaline protease, a proteinase, a peptidase, an endopeptidase, an tidase, a thermolysin, a papain, a pepsin, a trypsin, a pronase, a carboxylase, a serine protease, a glutamic se, an aspartate protease, a cysteine protease, a ine protease, or a metalloprotease.

Embodiment 204 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 192, wherein the enzyme that es or modifies a ial, fungal, or plant nt source comprises a phosphatase, and the phosphatase comprises a phosphoric ter hydrolase, a omonoesterase, a phosphoric diester hydrolase, a phosphodiesterase, a triphosphoric monoester hydrolase, a phosphoryl anhydride ase, a osphatase, a phytase, a trimetaphosphatase, or a triphosphatase.

Embodiment 205 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 204, wherein the enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source comprises a phosphomonoesterase, and the phosphomonoesterase comprises PhoA4; or wherein the enzyme that degrades or modifies a bacterial, fungal or plant nutrient source comprises a phytase, and the phytase comprises Bacillus subtilis EEl48 phytase or Bacillus thuringiensis BT013A phytase.

Embodiment 206 is a fusion protein, a recombinant Bacillus cereus family member, a inant spore-forming bacterium, or a plant seed of embodiment 192, wherein the enzyme that degrades or s a bacterial, fungal, or plant nutrient source comprises a nitrogenase, and the nitrogenase comprises a Nif family enase.

Embodiment 207 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 206, wherein the Nif family nitrogenase comprises Paenibacillus massiliensis HKNXV. 2] Embodiment 208 is a fusion protein, a recombinant Bacillus cereus family member, a inant spore-forming bacterium, or a plant seed of embodiment 168, wherein the protein or peptide of interest comprises a protein or e that protects a plant from a pathogen.

Embodiment 209 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 208, wherein the protein or peptide that protects a plant from a pathogen comprises a plant immune system enhancer protein or peptide.

Embodiment 210 is a fusion protein, a recombinant us cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 209, wherein the plant immune system enhancer protein or peptide comprises a , a harpin-like protein, an u-elastin, a B-elastin, a systemin, a phenylalanine ammonia-lyase, an elicitin, a defensin, a cryptogein, a flagellin protein, or a flagellin e.

Embodiment 211 is a fusion protein, a inant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 210, wherein the flagellin e comprises flg22.

Embodiment 212 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 208, wherein the protein or peptide that protects a plant from a pathogen has antibacterial actiVity, ngal actiVity, or both antibacterial and antifungal actiVity.

Embodiment 213 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 212, wherein the protein or peptide that protects a plant from a pathogen comprises a bacteriocin, a me, a lysozyme peptide, a siderophore, an aVidin, a streptaVidin, a non-ribosomal active e, a conalbumin, an n, a errin, a lactoferrin peptide, or TasA.

Embodiment 214 is a fusion protein, a inant Bacillus cereus family , a inant spore-forming bacterium, or a plant seed of embodiment 213, wherein the lysozyme peptide comprises LysM or wherein the lactoferrin peptide comprises LfcinB.

Embodiment 215 is a fusion n, a recombinant us cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 208, wherein the protein or peptide that protects a plant from a pathogen has insecticidal actiVity, helminthicidal actiVity, suppresses insect or worm predation, or a ation thereof.

Embodiment 216 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant forming bacterium, or a plant seed of embodiment 215, wherein the protein or peptide that protects a plant from a pathogen comprises an insecticidal bacterial toxin, an endotoxin, a Cry toxin, a protease inhibitor protein or peptide, a cysteine protease, or a chitinase.

Embodiment 217 is a fusion protein, a recombinant us cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 216, wherein the protein or peptide that protects a plant from a pathogen comprises an insecticidal bacterial toxin and the insecticidal bacterial toxin comprises a VIP insecticidal toxin; n the protein or peptide that protects a plant from a pathogen comprises a se inhibitor protein or peptide and the protease inhibitor protein or peptide comprises a trypsin inhibitor or an arrowhead 2015/050807 protease inhibitor; or wherein the protein or e that protects a plant from a en comprises a Cry toxin and the Cry toxin comprises a Cry toxin from Bacillus thuringiensis.

Embodiment 218 is a fusion protein, a recombinant Bacillus cereus family , a inant spore-forming bacterium, or a plant seed of embodiment 217, wherein the Cry toxin comprises a Cry toxin from Bacillus thuringiensis and the Cry toxin comprises a Cry toxin from Bacillus thuringiensis comprises a CrySB protein or a Cry21A protein.

Embodiment 219 is a fusion n, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 208, wherein the protein that protects a plant from a pathogen comprises an enzyme.

Embodiment 220 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 219, wherein the enzyme comprises a protease or a lactonase.

Embodiment 221 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 220, wherein the protease or lactonase is specific for a bacterial ing molecule.

Embodiment 222 is a fusion protein, a recombinant Bacillus cereus family member, a inant spore-forming bacterium, or a plant seed of embodiment 221, wherein the bacterial ing molecule comprises a bacterial lactone homoserine signaling molecule. 7] Embodiment 223 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of any one of embodiments 220—222, n the enzyme comprises a lactonase and the lactonase comprises l,4-lactonase, 2-pyrone-4,6-dicarboxylate lactonase, 3-oxoadipate enol-lactonase, actinomycin lactonase, deoxylimonate A-ring-lactonase, gluconolactonase L-rhamnono-l,4-lactonase, limonin-D-ring- lactonase, d-lactonase, triacetate-lactonase, or xylono-l,4-lactonase.

Embodiment 224 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 219, wherein the enzyme is specific for a cellular component of a bacterium or fungus.

Embodiment 225 is a fusion protein, a recombinant us cereus family , a recombinant spore-forming bacterium, or a plant seed of embodiment 224, wherein the enzyme comprises a B-l,3-glucanase, a B-l,4-glucanase, a B-l,6-glucanase, a chitosanase, a chitinase, a chitosanase-like enzyme, a se, a peptidase, a proteinase, a protease, a mutanolysin, a lysin, or a lysozyme.

Embodiment 226 is a fusion protein, a recombinant Bacillus cereus family , a recombinant spore-forming bacterium, or a plant seed of embodiment 225, n the enzyme comprises a chitosanase, wherein the chitosanase comprises an amino acid sequence having at least 85%, at least 95%, at least 98%, at least 99%, or a 100% identity with SEQ ID NO: 3 13.

Embodiment 227 is a fusion protein, a recombinant us cereus family member, a recombinant forming ium, or a plant seed of embodiment 225, wherein the protease comprises an alkaline protease, an acid protease, or a neutral protease.

Embodiment 228 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of any one of embodiments 208—227, wherein the protein or peptide that protects a plant from a pathogen protects the plant from a bacterial pathogen, a fungal pathogen, a worm pathogen, or an insect pathogen.

Embodiment 229 is a fusion protein, a recombinant us cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 228, wherein the bacterial pathogen comprises an u-class Proteobacterium, a B-class bacterium, a y- class Proteobacterium, or a combination thereof; or wherein the bacterial en comprises Agrobacterium tumefaciens, Pantoea stewartii, Erwinia car0t0v0ra, nia s0lanacearum, Pseudomonas syringae, Pseudomonas aeruginosa, monas campestris, or a combination thereof.

Embodiment 230 is a fusion protein, a recombinant Bacillus cereus family , a recombinant spore-forming bacterium, or a plant seed of embodiment 228, wherein the protein or peptide that protects a plant from a pathogen protects the plant from predation by a worm or an insect pathogen.

Embodiment 231 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of ment 228 or 230, wherein the worm or insect en comprises an army worm, a black cutworm, a European corn borer, a fall armyworm, a cutworm, a Japanese , a lesser comstalk borer, a maize billbug, a seed corn maggot, a webworm, a southern lk borer, a southern corn rm, a southern potato wireworm, a stalk borer, a sugarcane beetle, a white grub, a cabbage looper, a boll weeVil, a yellow striped armyworm, a cereal leaf beetle, a chinch bug, an aphid, a beet armyworm, a Mexican bean beetle, a soybean looper, soybean stem borer, or a combination thereof.

Embodiment 232 is a fusion protein, a inant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 168, wherein the protein or peptide of interest comprises at least one protein or peptide that es stress resistance in a plant.

Embodiment 233 is a fusion protein, a recombinant Bacillus cereus family member, recombinant spore-forming bacterium, or a plant seed of embodiment 232, n the protein or peptide that enhances stress resistance in a plant comprises an enzyme that degrades a stress-related compound.

Embodiment 234 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming ium, or a seed of embodiment 233, wherein the stress-related compound comprises aminocyclopropane-l-carboxylic acid (ACC), a ve oxygen s, nitric oxide, an oxylipin, a phenolic, or a combination f.

Embodiment 235 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant forming bacterium, or a plant seed of embodiment 233 or 234, wherein the enzyme that degrades a stress-related compound comprises a superoxide dismutase, an oxidase, a catalase, an aminocyclopropane-l-carboxylic acid deaminase, a peroxidase, an antioxidant enzyme, or an antioxidant peptide.

Embodiment 236 is a fusion protein, a inant Bacillus cereus family , a recombinant spore-forming bacterium, or a plant seed of embodiment 235, wherein the enzyme that degrades a stress-related compound comprises a superoxide dismutase.

Embodiment 237 is a fusion protein, a recombinant us cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 235 or 236, wherein the superoxide dismutase comprises superoxide dismutase l (SODAl) or superoxide dismutase 2 (SODA2). 2] Embodiment 238 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 23 7, wherein the superoxide dismutase comprises an amino acid ce having at least 85%, at least 95%, at least 98%, at least 99%, or a 100% identity with SEQ ID NO: 155 or 156. ment 239 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 232, wherein the n or peptide that enhances stress resistance in a plant comprises a protein or peptide that protects a plant from an enVironmental stress. ment 240 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant forming ium, or a plant seed of embodiment 239, wherein the environmental stress comprises drought, flood, heat, freezing, salt, heavy metals, low pH, high pH, or a combination thereof.

Embodiment 241 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 239 or 240, wherein the protein or peptide that ts a plant from an enVironmental stress comprises an ice nucleation protein, a ase, a phenylalanine amminia lyase, an isochorismate synthase, an rismate pyruvate lyase, or a choline dehydrogenase.

Embodiment 242 is a fusion protein, a recombinant Bacillus cereus family , a recombinant spore-forming bacterium, or a plant seed of embodiment 168, wherein the fusion n comprises at least one plant binding protein or peptide.

Embodiment 243 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 242, wherein the plant binding protein or peptide comprises an adhesin, a in, an omptin, a lectin, an expansin, a biofilm structural n, a pilus protein, a curlus protein, an intimin, an invasin, an agglutinin, an afimbrial protein.

Embodiment 244 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 243, n the plant binding protein comprises an adhesin and the adhesin comprises a hesin; or wherein the plant binding protein comprises a biofilm structural protein and the biofilm structural protein ses TasA or YuaB.

Embodiment 245 is a fusion protein of embodiment 4, or a fusion protein, a recombinant Bacillus cereus family member, a recombinant forming bacterium, or a plant seed of embodiment 168, wherein the protein or peptide of interest comprises an enzyme that catalyzes the production of nitric oxide.

Embodiment 246 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 245, wherein the enzyme that catalyzes the production of nitric oxide comprises a nitric oxide synthase or an arginase.

Embodiment 247 is a fusion n, a recombinant Bacillus cereus family member, a recombinant spore-forming ium, or a plant seed of embodiment 245, wherein the wherein the enzyme that catalyzes the production of nitric oxide comprises a nitric oxide synthase. 2] Embodiment 248 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant forming bacterium, or a plant seed of embodiment 247, n the nitric oxide synthase comprises a Bacillus thuringiensis nitric oxide synthase or a Bacillus subtilis nitric oxide synthase.

Embodiment 249 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 248, n the nitric oxide synthase comprises a nitric oxide synthase from Bacillus thuringiensis BT013A or Bacillus subtilis 168.

Embodiment 250 is a fusion protein, a recombinant Bacillus cereus family member, a inant spore-forming bacterium, or a plant seed of any one of embodiments 245—249, wherein the nitric oxide synthase ses an amino acid sequence haVing at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 260 or 261.

Embodiment 251 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming ium, or a plant seed of any one of embodiments 245—250, wherein the fusion protein comprises SEQ ID NO: 262 or 263.

Embodiment 252 is a fusion protein of embodiment 4, or a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 168, wherein the protein or peptide of interest comprises a nucleic acid binding protein or peptide.

Embodiment 253 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant forming bacterium, or a plant seed of ment 252, n the nucleic acid binding protein or e comprises an RNA binding protein or peptide or a DNA binding protein or peptide.

Embodiment 254 is a fusion protein, a recombinant us cereus family member, a recombinant forming bacterium, or a plant seed of embodiment 253, wherein the nucleic acid binding protein or peptide comprises an RNA binding protein or peptide.

Embodiment 255 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 254, wherein the RNA g protein or peptide comprises a non-specific RNA binding protein or peptide.

Embodiment 256 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant forming bacterium, or a plant seed of embodiment 254, wherein the RNA binding protein or peptide comprises a c RNA binding n or peptide. ment 257 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 254, wherein the RNA binding protein or peptide comprises an qu protein.

Embodiment 258 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of ment 257, n the qu protein comprises a Bacillus thuringiensis qu protein.

Embodiment 259 is a fusion n, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of ment 253, wherein the nucleic acid binding protein or peptide comprises a DNA binding protein or peptide.

Embodiment 260 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 259, wherein the DNA binding protein or peptide comprises a small acid-soluble spore protein (SASP).

Embodiment 261 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant forming bacterium, or a plant seed of embodiment 260, wherein the SASP comprises a SASP encoded by an SspA gene, an SspB gene, an SspC gene, an SspD gene, an SspE gene, an SspF gene, an SspG gene, an SspH gene, an SspI gene, an SspJ gene, an SspK gene, an SspL gene, an SspM gene, an SspN gene, an SspO gene, or an SspP gene.

Embodiment 262 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant forming bacterium, or a plant seed of ment 261, wherein the SASP comprises a SASPu, a SASPB, or a SASPy.

Embodiment 263 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of any one of embodiments 260—262, wherein the SASP comprises a Bacillus thuringiensis SASP. ment 264 is a fusion protein, a recombinant Bacillus cereus family , a recombinant spore-forming bacterium, or a plant seed of any one of embodiments 252—263, wherein the nucleic acid binding protein or peptide ses an amino acid ce haVing at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NOs: 264—266.

Embodiment 265 is a fusion protein, a recombinant Bacillus cereus family member, or recombinant spore-forming bacterium, or a plant seed of any one of embodiments 252—264, wherein the fusion protein comprises SEQ ID NO: 267, 268, or 269.

Embodiment 266 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of any one of embodiments 4 and 259, wherein the nucleic acid binding n or peptide comprises a nuclease haVing an inactivated active site.

Embodiment 267 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of any one of embodiments 6, further comprising a nucleic acid molecule bound to the nucleic acid g protein or e.

Embodiment 268 is a fusion protein, a recombinant Bacillus cereus family member, a recombinant spore-forming bacterium, or a plant seed of embodiment 267, wherein the nucleic acid molecule ses a modulating RNA molecule; an RNAi molecule; a NA; an aptamer; or a DNA molecule that encodes a modulating RNA molecule, an RNAi molecule, a microRNA, or an aptamer.

Embodiment 269 is a fusion protein of embodiment 4 or a recombinant Bacillus cereus family member, or plant seed of any one of embodiments 16—29, 48—63, 99— 141, and 168—268, wherein the ing sequence comprises SEQ ID NO: 96. ment 270 is a recombinant Bacillus cereus family member that coexpresses at least one fusion protein sing a plant growth stimulating protein or peptide, a n or peptide that ts a plant from a pathogen, a protein or peptide that enhances stress resistance in a plant, an enzyme that catalyzes the production of nitric oxide, or a nucleic acid binding protein or e of any one of embodiments 168—241 and 245—269 and at least one fusion protein comprising a plant binding protein or peptide of any one of embodiments 242—244 and 269.

Embodiment 271 is a recombinant Bacillus cereus family member of any one of embodiments 16—141 and 168—270, wherein the recombinant Bacillus cereus family member comprises Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus ’es, us pseudomycoia’es, Bacillus samanii, Bacillus gaemokensis, Bacillus weihenstephensis, Bacillus t0y0iensis, or a combination thereof. 41 1 Embodiment 272 is a recombinant Bacillus cereus family member of embodiment 271, wherein the recombinant Bacillus cereus family member comprises Bacillus thuringiensis or Bacillus mycoia’es.

Embodiment 273 is a recombinant Bacillus cereus family member of embodiment 271 or 272, wherein the recombinant Bacillus cereus family member comprises a plant-growth promoting strain of bacteria.

Embodiment 274 is a recombinant Bacillus cereus family member of embodiment 273, wherein the plant-growth promoting strain of ia produces an insecticidal toxin, produces a fungicidal compound, produces a nematocidal compound, produces a bacteriocidal compound, is resistant to one or more antibiotics, comprises one or more freely replicating plasmids, binds to plant roots, colonizes plant roots, forms biofilms, solubilizes nutrients, secretes organic acids, or combinations thereof Embodiment 275 is a recombinant Bacillus cereus family member of embodiment 274, wherein the insecticidal toxin comprises a Cry toxin; wherein the fungicidal compound comprises a B-1,3-glucanase, a chitosanase, a se, or a combination thereof; or wherein the nematocidal nd comprises a Cry toxin. 0] Embodiment 276 is a recombinant Bacillus cereus family member of any one of embodiments 273—275, wherein the plant-growth promoting strain of bacteria comprises Bacillus mycoia’es BT155 (NRRL No. B-50921), Bacillus mycoia’es EE118 (NRRL No. B- 50918), Bacillus mycoia’es EE141 (NRRL No. B-50921), Bacillus ’es BT46-3 (NRRL No.

B-50922), Bacillus cereus family member EE128 (NRRL No. B-50917), Bacillus thuringiensis BT013A (NRRL No. 4), Bacillus cereus family member EE349 (NRRL No. B-50928); Bacillus cereus family member EE-B00377 (NRRL B-67119); Bacillus pseudomycoia’es EE- B00366 (NRRL B-67120); or Bacillus mycoia’es 363 (NRRL B-67121).

Embodiment 277 is a recombinant Bacillus cereus family member of embodiment 276, wherein the plant-growth promoting strain of bacteria comprises us mycoia’es BT155 (NRRL No. B-50921), Bacillus mycoia’es EE141 (NRRL No. B-50921), or Bacillus thuringiensis BT013A (NRRL No. B-50924).

Embodiment 278 is a inant Bacillus cereus family member of any one of embodiments , and 5, wherein the recombinant Bacillus cereus family member comprises an endophytic strain of bacteria. ment 279 is a inant Bacillus cereus family member of ment 278, n the endophytic strain of bacteria comprises Bacillus cereus family 4 l 2 member EE349 (NRRL No. B-50928), Bacillus cereus family member EE439 (NRRL B- 50979), Bacillus thuringiensis EE417 (NRRL B-50979), Bacillus cereus EE444 (NRRL B- 50977), or Bacillus thuringiensis EE3 l9 (NRRL 3), Bacillus giensis EE-B00184 (NRRL B-67l22), Bacillus cereus family member EE-B00377 (NRRL B-67l l9); Bacillus pseudomycoia’es EE-B00366 (NRRL 0); or Bacillus mycoia’es EE-B00363 (NRRL B- 67121).

Embodiment 280 is a recombinant Bacillus cereus family member of embodiment 279, wherein the endophytic strain of bacteria comprises Bacillus cereus family member EE439 (NRRL 9), Bacillus giensis EE417 (NRRL B-50979), Bacillus cereus EE444 (NRRL B-50977), Bacillus thuringiensis EE3 l9 (NRRL B-50983), Bacillus thuringiensis EE-BOOl84 (NRRL B-67l22), Bacillus cereus family member 377 mRRL B-67l l9); Bacillus pseudomycoia’es EE-B00366 (NRRL B-67l20); or Bacillus mycoia’es EE- B00363 (NRRL l).

] Embodiment 281 is a recombinant Bacillus cereus family member of any one of embodiments 16—141 and 186—275, wherein the recombinant us cereus family member comprises a strain of bacteria that is capable of degrading an herbicide or a pesticide.

Embodiment 282 is a recombinant Bacillus cereus family member of embodiment 28 l wherein the strain of ia that is capable of degrading an herbicide or a pesticide comprises Bacillus cereus family member EE349 (NRRL No. B-50928), Bacillus cereus family member EE-B00377 (NRRL B-67l l9); Bacillus pseudomycoia’es EE-B00366 (NRRL 0); or Bacillus mycoia’es EE-B00363 (NRRL B-67l2l).

Embodiment 283 is a recombinant Bacillus cereus family member of any one of embodiments 125, 128, 281, and 282, wherein the strain of bacteria that is capable of degrading an herbicide or a pesticide degrades a sulfonylurea herbicide, an aryl triazine herbicide, dicamba, 2,4-D, a y herbicide, a pyrethrin, a pyretheroid, or a combination thereof.

Embodiment 284 is a recombinant Bacillus cereus family member of embodiment 283, wherein the sulfonylurea herbicide comprises sulfentrazone.

Embodiment 285 is a recombinant Bacillus cereus family member of ment 282 or 283, wherein the strain of bacteria that is capable of degrading a pesticide degrades a pyrethrin.

Embodiment 286 is a recombinant Bacillus cereus family member of any one of embodiments 16—141 and 186—275, wherein the recombinant Bacillus cereus family member comprises a probiotic strain of bacteria.

Embodiment 287 is a recombinant Bacillus cereus family member of any one of embodiment 286, wherein the probiotic strain of bacteria comprises Bacillus cereus family member EE349 NRRL No. B-50928), Bacillus cereus family member EE439 (NRRL B-50979), Bacillus giensis EE4l7 (NRRL B-50979), or Bacillus cereus EE444 (NRRL 7).

Embodiment 288 is a recombinant Bacillus cereus family member of 16—141 and 168—287, wherein the recombinant Bacillus cereus family member comprises an inactivating mutation in its BclA gene.

Embodiment 289 is a recombinant Bacillus cereus family member of embodiment 288, n the inactivating mutation in the BclA gene comprises a knock-out of the BclA gene.

Embodiment 290 is a recombinant us cereus family member of any one of embodiments 16—29, 48—63, 99—141 and 168—289, wherein the fusion protein is expressed under the control of a sporulation promoter native to the targeting sequence, exosporium protein, or exosporium protein fragment of the fusion protein or a portion thereof.

] Embodiment 291 is a recombinant Bacillus cereus family member of any one of ments 17—27, wherein the modulator protein is expressed under the control of its native promoter or a portion thereof. 6] Embodiment 292 is a recombinant Bacillus cereus family member of any one of embodiments 16—29, 48—63, 99—l4l, and 168—291, wherein the fusion protein or modulator protein is expressed under the control of a high-expression sporulation promoter.

Embodiment 293 is a recombinant Bacillus cereus family member of embodiment 292, wherein the high-expression sporulation promoter comprises a sigma-K ation-specific polymerase promoter sequence. 8] ment 294 is a recombinant Bacillus cereus family member of any one of ments 290—293, wherein the ation promoter comprises a nucleic acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity with a nucleic acid ce of any one of SEQ ID NOs: 157—23 1.

Embodiment 295 is a recombinant Bacillus cereus family member of any one of embodiments 290—293, wherein the promoter comprises a nucleic acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 157, 158, 161, 162, 189, 190, 215, 220, 221, 225, 229, or 230. ment 296 is a recombinant Bacillus cereus family member of any one of embodiments 293—295, wherein the sigma-K sporulation-specific polymerase promoter sequence or sequences have 100% identity with the corresponding nucleotides of any of SEQ ID NOs: 157—231. ment 297 is a biologically pure bacterial culture wherein the bacteria in the bacterial culture is: (a) Bacillus cereus family member EE439 (NRRL B-50979); (b) Bacillus thuringiensis EE417 (NRRL B-50979); (c) Bacillus cereus EE444 (NRRL 7); (d) Bacillus thuringiensis EE319 (NRRL B-50983); (e) Bacillus thuringiensis EE-B00184 (NRRL B-67122); (f) Bacillus cereus family member EE-B00377 (NRRL B-67119); (g) Bacillus pseudomycoides EE-B00366 (NRRL B-67120); or (h) Bacillus mycoides EE-B00363 (NRRL B-67121). 2] Embodiment 298 is a biologically pure bacterial culture of embodiment 297, wherein the bacteria in the bacterial e is Bacillus cereus family member EE439 (NRRL B- 50979) and the bacteria has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with the ce of SEQ ID NO: 277.

Embodiment 299 is a ically pure bacterial culture of embodiment 297, wherein the bacteria in the bacterial e is Bacillus giensis EE417 (NRRL B-50979) and the bacteria has a 16S ribosomal RNA ce haVing at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 278.

Embodiment 300 is a biologically pure bacterial culture of embodiment 297, wherein the bacteria in the bacterial culture is Bacillus cereus EE444(NRRL B-50977) and the bacteria has a 16S mal RNA sequence haVing at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 279.

Embodiment 301 is a biologically pure bacterial culture of embodiment 297, wherein the bacteria in the bacterial culture is Bacillus giensis EE319 (NRRL B-50983) and the bacteria has a 16S ribosomal RNA sequence haVing at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 280.

Embodiment 302 is a biologically pure bacterial culture of ment 297, wherein the bacteria in the bacterial culture is Bacillus thuringiensis 184 (NRRL B- 67122) and the bacteria has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 301.

Embodiment 303 is a biologically pure bacterial culture of embodiment 297, wherein the bacteria in the bacterial culture is Bacillus cereus family member EE-B00377 (NRRL B-67119) and the bacteria has a 16S ribosomal RNA sequence haVing at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 304.

Embodiment 304 is a biologically pure bacterial culture of embodiment 297, n the bacteria in the bacterial culture is Bacillus pseudomycoia’es EE-B00366 (NRRL B- 67120) and the ia has a 16S ribosomal RNA sequence haVing at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 303.

Embodiment 305 is a biologically pure bacterial culture of embodiment 297, wherein the bacteria in the bacterial culture is us mycoia’es EE-B00363 (NRRL B-67121) and the bacteria has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 302.

Embodiment 306 is a biologically pure bacterial culture wherein the bacteria in the bacterial culture are mutants of the bacteria of any of embodiments 297—305, comprising one or more mutations, wherein the bacteria are endophytic.

Embodiment 307 is a ically pure bacterial culture wherein the bacteria in the bacterial culture is: (a) Bacillus megaterium EE385 (NRRL 0); (b) Bacillus sp. EE387 (NRRL B-50981); (c) Bacillus circulans EE388 (NRRL B-50982); (d) Bacillus subtilis EE405 (NRRL B-50978); (e) Lysinibacillus fusiformis EE442 (NRRL B-50975); (f) Lysinibacillus sphaericus EE443 (NRRL B-50976); or (g) us pumilus 143 (NRRL 3).

Embodiment 308 is a biologically pure bacterial culture of embodiment 307, wherein the bacteria in the bacterial culture is Bacillus rium EE385 (NRRL 0) and the bacteria has a 16S mal RNA sequence having at least 98%, at least 99%, or 100% ce identity with the sequence of SEQ ID NO: 281. 4 l 6 Embodiment 309 is a biologically pure bacterial culture of embodiment 307, wherein the ia in the bacterial culture is Bacillus sp. EE3 87 (NRRL B-5098 l) and the bacteria has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 282. ment 310 is a biologically pure bacterial culture of embodiment 307, wherein the bacteria in the bacterial culture is Bacillus circulans EE388 (NRRL B-50982) and the bacteria has a 16S ribosomal RNA sequence haVing at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 283.

] Embodiment 3 ll is a biologically pure ial culture of ment 307, wherein the bacteria in the bacterial culture is Bacillus subtilis EE405(NRRL B-50978) and the bacteria has a 16S ribosomal RNA sequence haVing at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 284.

Embodiment 3 12 is a biologically pure bacterial e of embodiment 307, n the bacteria in the bacterial culture is Lysinibacillusfusiformis EE442 (NRRL B-50975) and the ia has a 16S mal RNA sequence haVing at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 285.

Embodiment 3 13 is a biologically pure bacterial culture of embodiment 307, wherein the bacteria in the bacterial culture is Lysinibacillus sphaericus EE443 (NRRL B- 50976) and the bacteria has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 286.

Embodiment 3 14 is a biologically pure ial culture of ment 307, wherein the bacteria in the bacterial culture is Bacillus pumilus EE-BOOl43 (NRRL B-67l23) and the bacteria has a 16S ribosomal RNA sequence haVing at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 305.

Embodiment 315 is a biologically pure bacterial culture wherein the bacteria in the bacterial culture are mutants of the bacteria of any one of embodiments 307—3 14 sing one or more mutations, wherein the bacteria are endophytic.

Embodiment 3 16 is a biologically pure bacterial e wherein the bacteria in the bacterial e are mutants of the bacteria of any one of embodiments 307—3 1 5 comprising one or more mutations, wherein the bacteria are probiotic.

Embodiment 3 17 is an inoculum for application to plants, plant seeds, a plant growth medium, or an area surrounding a plant or a plant seed, wherein the inoculum comprises 4 l 7 an effective amount of a biologically pure bacterial culture of any one of embodiments 297—3 16 and an agriculturally acceptable carrier. ment 318 is an inoculum of embodiment 3 17, wherein the inoculum comprises an effective amount of a mixture comprising at least two biologically pure bacterial es of any one of embodiments 297—316.

Embodiment 319 is an inoculum of embodiment 3 17 or 318, wherein the inoculum further comprises an effective amount of a rhizobacteria.

Embodiment 320 is an inoculum of embodiment 3 19, wherein the rhizobacteria is a biologically pure bacterial culture of a rhizobacteria strain.

Embodiment 321 is an inoculum of embodiment 3 19 or 320, wherein the rhizobacteria ses Bradyrhz'zobz’um genus bacteria, Rhizobz'um genus bacteria, or a combination f.

Embodiment 322 is an inoculum of embodiment 321, wherein the Bradyrhz'zobz’um genus bacteria comprises Bradyrhizobiumjaponicum.

Embodiment 323 is an inoculum of embodiment 322, wherein the Rhizobz'um genus ia comprises z'um phaseolz‘, Rhizobz'um leguminosarum, or a combination thereof.

Embodiment 324 is a plant seed coated with: (i) an enzyme that catalyzes the tion of nitric oxide; (ii) a superoxide dismutase; or (iii) a recombinant microorganism that ses an enzyme that catalyzes the production of nitric oxide or a superoxide dismutase, wherein the expression of the enzyme that catalyzes the production of nitric oxide or the superoxide dismutase is increased as compared to the expression of the enzyme that catalyzes the production of nitric oxide or the superoxide dismutase in a wild-type microorganism under the same conditions.

Embodiment 325 is a plant seed of embodiment 324, wherein the plant seed is coated with the enzyme that catalyzes the production of nitric oxide. 0] Embodiment 326 is a plant seed of embodiment 324 or 325, wherein the plant seed is coated with the recombinant microorganism that expresses an enzyme that catalyzes the production of nitric oxide. 1] Embodiment 327 is a plant seed of any one of embodiments 324—326, wherein the enzyme that zes the production of nitric oxide ses a nitric oxide synthase or an arginase.

Embodiment 328 is a plant seed of embodiment 327, n the enzyme that zes the production of nitric oxide comprises a nitric oxide synthase.

Embodiment 329 is a plant seed of embodiment 328, wherein the nitric oxide synthase comprises a nitric oxide synthase from Bacillus thuringiensis BT013A or us is 168.

Embodiment 330 is a plant seed of embodiment 328 or 329, wherein the nitric oxide synthase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 260 or 261.

Embodiment 331 is a plant seed of any one of embodiments 324—330, wherein the plant seed is coated with the superoxide dismutase.

Embodiment 332 is a plant seed of any one of ments 324—331, wherein the plant seed is coated with the recombinant microorganism that expresses a superoxide dismutase.

Embodiment 333 is a plant seed of any one of embodiments 324—332, wherein the superoxide dismutase comprises superoxide dismutase 1 (SODAl) or superoxide dismutase 2 (SODAZ).

Embodiment 334 is a plant seed of any one of embodiments 331—333, wherein the superoxide dismutase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 155 or 156.

Embodiment 335 is a plant seed of any one of embodiments 4, n the recombinant microorganism comprises a Bacillus species, ecliia c0li, an Aspergillus species, or a Saccliromyces species.

Embodiment 336 is a plant seed of embodiment 335, wherein the Bacillus species comprises a Bacillus cereus family member, Bacillus is, Bacillus licheniformis, or Bacillus rium.

Embodiment 337 is a plant seed of embodiment 335, wherein the Aspergillus species comprises Aspergillus niger or wherein the Saccliromyces s comprises Sacchromyces cerevisiae. 2] Embodiment 338 is a formulation comprising a recombinant Bacillus cereus family member of any one of embodiments 16—141 and 168—296 or a recombinant spore- forming bacterium of any one of embodiments 147—149, 152—154, and 156—268 and an agriculturally acceptable carrier. 4 l 9 ment 339 is a formulation comprising exosporium fragments derived from spores of a recombinant Bacillus cereus family member of any one of embodiments 100— 124, 130—141, and 168—296 and an lturally acceptable carrier.

Embodiment 340 is a plant seed of any one of embodiments 150, 151, and 154—268, wherein the seed is coated with a formulation comprising the recombinant spore- forming bacterium and an agriculturally acceptable carrier. ] ment 341 is a plant seed of any one of embodiments 324—337, wherein the seed is coated with a formulation comprising the enzyme or the recombinant microorganism and an agriculturally acceptable carrier.

Embodiment 342 is a formulation of embodiment 338 or 339, a plant seed of embodiment 340 or 341, or an inoculum of any one of ments 317—323, wherein the agriculturally acceptable carrier comprises a dispersant, a surfactant, an additive, water, a thickener, an anti-caking agent, residue breakdown, a composting formulation, a granular application, diatomaceous earth, an oil, a coloring agent, a stabilizer, a preservative, a polymer, a g, or a combination thereof.

Embodiment 343 is a formulation, plant seed, or inoculum of embodiment 342, wherein the agriculturally acceptable carrier ses an additive, and the additive comprises an oil, a gum, a resin, a clay, a polyoxyethylene glycol, a terpene, a viscid organic, a fatty acid ester, a sulfated alcohol, an alkyl sulfonate, a petroleum sulfonate, an alcohol sulfate, a sodium alkyl butane diamate, a polyester of sodium tane dioate, a benzene itrile derivative, a proteinaceous material, or a combination thereof; the agriculturally acceptable carrier comprises a thickener, and the ner comprises a long chain alkylsulfonate of polyethylene glycol, a polyoxyethylene oleate, or a combination thereof; the agriculturally acceptable carrier comprises a surfactant, and the surfactant comprises a heavy eum oil, a heavy petroleum distillate, a polyol fatty acid ester, a polyethoxylated fatty acid ester, an aryl alkyl polyoxyethylene glycol, an alkyl amine e, an alkyl aryl sulfonate, a polyhydric alcohol, an alkyl phosphate, or a combination thereof; or the lturally acceptable r comprises an anti-caking agent, and the anti-caking agent comprises a sodium salt, a calcium carbonate, diatomaceous earth, or a combination thereof.

Embodiment 344 is a formulation, plant seed, or inoculum of embodiment 343, wherein additive comprises a proteinaceous material, and the proteinaceous material comprises a milk product, wheat flour, n meal, blood, albumin, gelatin, alfalfa meal, yeast extract, or a combination thereof; or the aking agent comprises a sodium salt, and the sodium salt comprises a sodium salt of monomethyl naphthalene sulfonate, a sodium salt of yl naphthalene sulfonate, a sodium sulfite, a sodium sulfate, or a combination thereof. ment 345 is a formulation, plant seed, of any one of embodiment 338—344 or an inoculum of any one of ments 317—323 and 342—344, wherein the lturally able carrier ses vermiculite, charcoal, sugar factory carbonation press mud, rice husk, carboxymethyl cellulose, peat, perlite, fine sand, calcium carbonate, flour, alum, a starch, talc, polyVinyl pyrrolidone, or a combination thereof Embodiment 346 is a formulation, plant seed, of any one of embodiment 338—345 or an inoculum of any one of ments 317—323 and 342—345, wherein the formulation or inoculum ses a seed coating formulation, a liquid formulation for application to plants or to a plant growth medium, or a solid formulation for application to plants or to a plant growth medium.

Embodiment 347 is a formulation, plant seed, or um of embodiment 346, wherein the seed coating formulation comprises an aqueous or oil-based solution for application to seeds or a powder or granular formulation for application to seeds.

Embodiment 348 is a formulation, plant seed, or inoculum of embodiment 346, wherein the liquid formulation for application to plants or to a plant growth medium comprises a trated formulation or a ready-to-use formulation.

Embodiment 349 is a formulation, plant seed, or inoculum of embodiment 346, wherein the solid formulation for application to plants or to a plant growth medium comprises a granular formulation or a powder agent.

Embodiment 350 is a formulation of any one of ment 338 and 342— 349, a plant seed of embodiment 340 or 341, or an um of any one of embodiments 3 17— 323 and 342—349, wherein the formulation or inoculum further comprises an agrochemical, the agrochemical comprising a fertilizer, a micronutrient izer material, an insecticide, an herbicide, a plant growth amendment, a fungicide, an insecticide, a molluscicide, an algicide, a bacterial inoculant, a fungal inoculant, or a combination thereof.

Embodiment 351 is a formulation, plant seed, or inoculum of embodiment 350, wherein the formulation or inoculum comprises a bacterial inoculant and the bacterial inoculant comprises a growth promoting strain of bacteria. 6] Embodiment 352 is a formulation, plant seed, or inoculum of embodiment l wherein the plant-growth promoting strain of bacteria produces an icidal toxin, produces a fungicidal compound, produces a nematocidal compound, produces a bacteriocidal compound, is resistant to one or more antibiotics, comprises one or more freely replicating plasmids, binds to plant roots, colonizes plant roots, forms biofilms, solubilizes nutrients, secretes organic acids, or combinations thereof.

Embodiment 353 is a formulation, plant seed, or inoculum of embodiment 352, wherein the insecticidal toxin comprises a Cry toxin; wherein the fungicidal compound comprises a B-l,3-glucanase, a anase, a lyticase, or a combination thereof; or n the cidal nd comprises a Cry toxin.

Embodiment 354 is a formulation, plant seed, or inoculum of any one of embodiments 35 1—353, wherein the plant-growth promoting strain of bacteria comprises Bacillus aryabhattai CAP53 (NRRL No. B-508l9), Bacillus aryabhattai CAP56 (NRRL No. B- 50817), Bacillusflexus BT054 (NRRL No. B-508l6), Parac0ccus kona'ratievae NC35 (NRRL No. B-50820), Bacillus mycoia’es BTl55 (NRRL No. B-5092l), Enterobacter cl0acae CAPl2 (NRRL No. 2), Bacillus nealsonii BOBA57 (NRRL No. NRRL B-5082l), Bacillus mycoia’es EEl l8 (NRRL No. B-509l8), Bacillus subtilis EEl48 (NRRL No. B-50927), Alcaligenesfaecalis EE107 (NRRL No. B-50920), Bacillus mycoia’es EEl4l (NRRL NO. B- 50916), Bacillus mycoia’es BT46-3 (NRRL No. B-50922), us cereus family member EEl28 (NRRL No. B-509l7), Bacillus thuringiensis BT013A (NRRL No. B-50924), Paenibacillus massiliensis BT23 (NRRL No. B-50923), Bacillus cereus family member EE349 (NRRL No. B-50928), Bacillus subtilis EE2l8 (NRRL No. 6), Bacillus megaterium EE28l (NRRL No. B-50925), Bacillus cereus family member EE-B00377 (NRRL B-67l l9); Bacillus pseudomycoia’es EE-B00366 (NRRL B-67l20), Bacillus mycoia’es EE-B00363 (NRRL l), Bacillus s EE-B00143 (NRRL B-67l23), or Bacillus thuringiensis EE-BOOl84 (NRRL 2), or a combination thereof 9] Embodiment 355 is a formulation, plant seed, or inoculum of embodiment 354, wherein the plant-growth promoting strain of bacteria comprises Parac0ccus kona'ratievae NC35 (NRRL No. B-50820), Bacillus arjyabhattai CAP53 (NRRL No. B-508l9), or Bacillus megaterium EE28l (NRRL No. 5) and the ation r comprises a recombinant Bacillus cereus family member of embodiment 276 or 277.

Embodiment 356 is a plant seed coated with a recombinant Bacillus cereus family member of any one of embodiments 16—141 and 168—296, exosporium fragments derived from spores of a recombinant Bacillus cereus family member of any one of embodiments 100— 124, 130—141, and 168—296 a recombinant spore-forming ium of any one of embodiments 147—149, 15 1—153, and 156—268, a biologically pure ial culture of any one of embodiments 297—316, an inoculum of any one of embodiments 3 and 342—355, or a formulation of any one of embodiments 338, 339, and 342—355.

Embodiment 357 is a method for stimulating plant growth comprising: introducing into a plant growth medium a recombinant us cereus family member of any one of embodiments 16—141 and 168—296 or a formulation comprising a recombinant Bacillus cereus family member of any one of ments 338 and 342—355; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed a recombinant Bacillus cereus family member of any one of embodiments 16—141 and 168—296 or a formulation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 5; wherein the recombinant Bacillus cereus family member expresses a fusion protein comprising a plant growth stimulating protein or peptide.

Embodiment 358 is a method for stimulating plant growth comprising: ucing into a plant growth medium a recombinant spore-forming bacterium of any one of embodiments 9, 15 1—153, and 156—268 or a formulation comprising a recombinant spore-forming bacterium of any one of embodiments 338 and 342—355; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed a recombinant spore-forming bacterium of any one of ments 9, 15 l— l53, and 156—268 or a formulation comprising a recombinant forming bacterium of any one of embodiments 338 and 342—355; wherein the recombinant spore-forming bacterium expresses a fusion protein comprising a plant growth stimulating protein or e.

Embodiment 359 is a method for protecting a plant from a pathogen or enhancing stress resistance in a plant comprising: introducing into a plant growth medium a recombinant Bacillus cereus family member of any one of embodiments 16—141 and 168—296 or a formulation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 5 or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed a recombinant Bacillus cereus family member of any one of embodiments l6—l4l and 6 or a ation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; wherein the recombinant Bacillus cereus family member expresses a fusion protein comprising a protein or peptide that protects a plant from a pathogen or a protein or peptide that enhances stress resistance in a plant.

Embodiment 360 is a method for protecting a plant from a en or ing stress resistance in a plant sing: introducing into a plant growth medium a recombinant spore-forming bacterium of any one of embodiments 147—149, 151—153, and 8 or a formulation comprising a recombinant spore-forming bacterium of any one of embodiments 338 and 342—355; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed a recombinant spore-forming bacterium of any one of embodiments 147—149, 15 l— 153, and 8 or a ation comprising a recombinant forming bacterium of any one of embodiments 338 and 342—355; wherein the recombinant spore-forming bacterium expresses a fusion protein comprising a protein or peptide that protects a plant from a pathogen or a protein or peptide that enhances stress resistance in a plant.

Embodiment 361 is a method of embodiment 359 or 360, wherein the method is a method for protecting a plant from a pathogen and plants grown in the plant growth medium comprising the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium are less susceptible to infection with the pathogen as compared to plants grown under the same conditions in the identical plant growth medium that does not contain the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium.

Embodiment 362 is a method of embodiment 359 or 360, wherein the method is a method for enhancing stress resistance in a plant and plants grown in the plant growth medium comprising the recombinant Bacillus cereus family member or the recombinant spore- forming bacterium are less susceptible to stress as compared to plants grown under the same conditions in the identical plant growth medium that does not contain the recombinant us cereus family member or the recombinant spore-forming bacterium.

Embodiment 363 is a method for immobilizing a recombinant Bacillus cereus family member spore on a plant comprising: 2015/050807 introducing into a plant growth medium a recombinant Bacillus cereus family member of any one of embodiments 16—141 and 168—296 or a formulation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed a recombinant Bacillus cereus family member of any one of embodiments 16—141 and 168—296 or a formulation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; wherein the recombinant us cereus family member expresses a fusion protein comprising a plant binding n or peptide.

Embodiment 364 is a method for lizing a spore of a recombinant spore-forming bacterium on a plant comprising: introducing into a plant growth medium a recombinant spore-forming bacterium of any one of embodiments 147—149, 151—153, and 156—268 or a formulation comprising a recombinant spore-forming ium of any one of embodiments 338 and 342—355; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed a recombinant spore-forming bacterium of any one of embodiments 147—149, 15 l— 153, and 156—268 or a formulation comprising a recombinant forming bacterium of any one of embodiments 338 and 342—355; wherein the recombinant spore-forming ium expresses a fusion protein comprising a plant binding peptide.

Embodiment 365 is a method for ating germination of a plant seed comprising: introducing into a plant growth medium a recombinant us cereus family member of any one of embodiments 16—141 and 168—296 or a formulation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed a recombinant Bacillus cereus family member of any one embodiments 16—141 and 6 or a formulation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; wherein the recombinant Bacillus cereus family member expresses a fusion protein comprising a superoxide dismutase or an enzyme that catalyzes the production of nitric oxide.

Embodiment 366 is a method for stimulating germination of a plant seed comprising: introducing into a plant growth medium a recombinant spore-forming bacterium of any one of embodiments 147—149, 151—153, and 156—268 or a formulation comprising a recombinant spore-forming bacterium of any one of ments 338 and 342—355; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed a recombinant spore-forming ium of any one of embodiments 147—149, 15 l— 153, and 156—268 or a formulation comprising a recombinant spore-forming bacterium of any one of embodiments 338 and 5; wherein the inant spore-forming bacterium expresses a fusion protein comprising a superoxide dismutase or an enzyme that zes the production of nitric oxide.

Embodiment 367 is a method of any one of embodiments 357—360 and 363— 366, wherein the plant growth stimulating protein or peptide, the protein or peptide that protects a plant from a en, the protein or e that enhances stress resistance in a plant, the plant g n or peptide, or the superoxide dismutase or the enzyme that catalyzes the production of nitric oxide is physically attached to the exosporium of the recombinant Bacillus cereus family member or to the spore coat of the recombinant spore-forming bacterium.

Embodiment 368 is a method of any one of embodiments 365—367, wherein the method comprises applying the recombinant Bacillus cereus family member, the recombinant forming bacterium, or the formulation to a plant seed.

Embodiment 369 is a method of any one of ments embodiment 365— 371, further comprising applying L-arginine to the plant growth medium, the plant seed, the plant, or the area surrounding the plant or the plant seed.

Embodiment 370 is a method of embodiment 369, wherein the method comprises applying L-arginine to an aerial n of the plant.

Embodiment 371 is a method of embodiment 369, wherein the method comprises applying L-arginine to the plant seed.

Embodiment 372 is a method of any one of embodiments 365—371, wherein seeds in the plant growth medium comprising the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium or seeds to which the recombinant us cereus family member or the inant spore-forming bacterium has been applied have an increased germination rate or have a longer taproot after germination as compared to seeds grown under the same conditions in the identical plant growth medium that does not contain the inant Bacillus cereus family member or the recombinant spore-forming bacterium or seeds to which the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium has not been applied grown under the same conditions.

Embodiment 373 is a method for delivering nucleic acids to a plant comprising: introducing into a plant growth medium a recombinant Bacillus cereus family member of any one of embodiments 16—141 and 168—296 or a formulation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed a recombinant Bacillus cereus family member of ment 16—141 and 168—296 or the ation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; wherein the recombinant Bacillus cereus family member expresses a fusion protein sing a nucleic acid binding protein, and n the nucleic acid binding protein or peptide is bound to a nucleic acid molecule.

Embodiment 374 is a method of 373, wherein the recombinant Bacillus cereus family member comprises an endophytic strain of bacteria.

Embodiment 375 is a method of embodiment 374, wherein the endophytic strain of bacteria comprises us cereus family member EE349 (NRRL No. 8), Bacillus cereus family member EE439 (NRRL B-50979), Bacillus thuringiensis EE4l7 (NRRL B-50979), Bacillus cereus EE444 (NRRL B-50977), or Bacillus thuringiensis EE3 l9 (NRRL B- , Bacillus giensis EE-BOOl84 (NRRL B-67122), Bacillus cereus family member EE-B00377 (NRRL B-67l l9); Bacillus pseudomycoides EE-B00366 (NRRL B-67120); or Bacillus mycoides EE-B00363 (NRRL B-67121).

Embodiment 376 is a method of ment 375, wherein the endophytic strain of bacteria comprises Bacillus cereus family member EE439 (NRRL B-50979), Bacillus thuringiensis EE417 (NRRL B-50979), us cereus EE444 (NRRL B-50977), or Bacillus thuringiensis EE3 l9 (NRRL B-50983), Bacillus thuringiensis l84 (NRRL B-67122), Bacillus cereus family member EE-B00377 (NRRL B-67l l9); Bacillus pseudomycoia’es EE- B00366 (NRRL B-67120); or Bacillus mycoia’es EE-B00363 (NRRL B-67121).

Embodiment 377 is a method for delivering nucleic acids to a plant comprising: introducing into a plant growth medium a recombinant spore-forming bacterium of any one of embodiments 9, 3, and 156—268 or a formulation sing a recombinant forming bacterium of any one of embodiments 338 and 342—355; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed a recombinant spore-forming bacterium of any one of embodiments 147—149, 151— 153, and 156—268 or a formulation comprising a recombinant spore-forming bacterium of any one of embodiments 338 and 342—355; wherein the recombinant spore-forming bacterium expresses a fusion n comprising a nucleic acid binding protein, and wherein the nucleic acid binding protein or peptide is bound to a c acid molecule.

Embodiment 378 is a method of embodiment 377, wherein the recombinant spore-forming bacterium comprises an ytic strain of bacteria.

Embodiment 379 is a method of embodiment 378, wherein the endophytic strain of bacteria comprises Bacillus megaterium EE385 (NRRL 0), Bacillus sp. EE387 (NRRL B-5098l), Bacillus circulans EE388 (NRRL B-50982), Bacillus subtilis EE405 (NRRL B-50978), Lysinibacillusfusiformis EE442 (NRRL B-50975), or Lysinibacillus spliaericus EE443 (NRRL B-50976), or Bacillus pumilus EE-B00143 (NRRL 3).

Embodiment 380 is a method for delivering a nucleic acid molecule to an animal, insect, or worm comprising feeding to an animal, an insect, or a worm a plant modified to comprise a level of the nucleic acid molecule that is greater than the level of the nucleic acid molecule in the same plant that has not been modified, grown under the same conditions.

Embodiment 381 is a method for ring a nucleic acid molecule to an , insect, or worm comprising feeding to an animal, insect, or worm: a recombinant Bacillus cereus family member expressing a fusion protein comprising a protein or peptide of st and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion n to the exosporium of the recombinant Bacillus cereus family member; or a recombinant spore-forming bacterium that ses a fusion protein comprising at least one protein or peptide of st and a spore coat protein that targets the fusion protein to the surface of a spore of the bacterium; wherein the protein or peptide of interest comprises a nucleic acid binding protein or peptide and the nucleic acid molecule is bound to the nucleic acid binding protein or peptide.

Embodiment 382 is the method of embodiment 380 or 381, wherein the worm is a nematode.

Embodiment 383 is a method for delivering a nucleic acid molecule to a fungus or a protozoan, comprising contacting a fungus or a protozoan with: a recombinant Bacillus cereus family member expressing a fusion protein comprising a protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that s the fusion n to the exosporium of the recombinant Bacillus cereus family member; or a recombinant spore-forming ium that expresses a fusion protein comprising at least one protein or peptide of interest and a spore coat protein that targets the fusion protein to the surface of a spore of the bacterium; wherein the protein or peptide of interest comprises a nucleic acid binding n or peptide and the c acid molecule is bound to the c acid binding protein or peptide.

Embodiment 384 is a method of any one of embodiments 373—379, and 381— 383, wherein the c acid binding protein or peptide is physically attached to the rium of the recombinant Bacillus cereus family member or to the spore coat of the recombinant spore- forming bacterium.

Embodiment 385 is a method of any one of embodiments 373—3 84, wherein the nucleic acid molecule ses a modulating RNA molecule; an RNAi molecule; a microRNA; an aptamer; or a DNA molecule that encodes a modulating RNA molecule, an RNAi le, a microRNA, or an aptamer. ment 386 is a method of any one of embodiments 381—385, wherein the recombinant Bacillus cereus family member comprises a recombinant Bacillus cereus family member of any one of embodiments 16—29, 48—63, 100—141, and 186—296.

Embodiment 387 is a method of any one of embodiments 381—3 86, wherein the fusion protein comprises a fusion protein of any one of embodiments 6.

Embodiment 388 is a method of any one of embodiments 381—385, n the spore coat protein comprises a CotB protein, a CotC protein, a CgeA protein, a CotB/H protein, a CotG protein, a spore coat protein X protein, or a CotY protein. ment 389 is a method of embodiment 388, wherein the spore coat protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% ty with any of SEQ ID NOs: 9.

Embodiment 390 is a method for stimulating plant growth comprising: introducing into a plant growth medium a recombinant Bacillus cereus family member of any one of embodiments 64—70, 74—77, and 87—99 or a formulation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed a recombinant Bacillus cereus family member of any one of embodiments 64—70, 74—77, and 87—99 or a formulation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; wherein the recombinant Bacillus cereus family member ses an enzyme involved in nutrient solubilization, a protease, a BclA protein, a BclB protein, a CotE protein a CotO protein, an Est protein, an ExsFA/BxpB protein, a CotY protein, an ExsFB protein, an Est protein, an Est protein, a chA protein, a chB protein, a Bch protein, a Bpr protein, a BclE n, a BetA/BAS329O protein, an Est protein, an ExsK protein, an Est protein, a YabG protein, or a Tgl protein, n the expression of the enzyme involved in nutrient solubilization, the protease, a BclA protein, a BclB protein, a CotE protein a CotO protein, an Est protein, an ExsFA/BxpB n, a CotY protein, an ExsFB protein, an Est n, an Est protein, a chA protein, a chB protein, a Bch protein, a Bpr protein, a BclE n, a BetA/BAS329O n, an Est protein, an ExsK protein, an Est protein, a YabG protein, or a Tgl protein is increased as compared to the expression of the enzyme involved in nutrient solubilization, the protease, a BclA protein, a BclB protein, a CotE protein a CotO protein, an Est protein, an ExsFA/BxpB protein, a CotY n, an ExsFB protein, an Est protein, an Est n, a chA protein, a chB protein, a Bch protein, a Bpr protein, a BclE protein, a BetA/BAS329O protein, an 13st protein, an ExsK protein, an 13st protein, a YabG protein, or a Tgl protein in a wild-type Bacillus cereus family member under the same conditions.

Embodiment 391 is a method for enhancing stress resistance in a plant comprising: introducing into a plant growth medium a recombinant Bacillus cereus family member of any one of embodiments 64, 65, and 78—83 or a formulation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; or applying to a plant, a plant seed, or an area nding a plant or a plant seed a recombinant Bacillus cereus family member of any one of embodiments 64, 65, and 78—83 or a formulation comprising a recombinant Bacillus cereus family member of any one of ments 338 and 342—355; wherein the recombinant Bacillus cereus family member expresses a superoxide dismutase or an arginase, n the expression of the superoxide dismutase or the arginase is increased as compared to the expression of the superoxide dismutase or the arginase in a wild-type Bacillus cereus family member under the same conditions.

Embodiment 392 is a method for protecting a plant from a pathogen comprising: ucing into a plant growth medium a recombinant Bacillus cereus family member of any one of embodiments 64. 65, and 74—77 or a formulation sing a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; or applying to a plant, a plant seed, or an area nding a plant or a plant seed a recombinant Bacillus cereus family member of any one of embodiments 64. 65, and 74—77 or a ation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; n the inant Bacillus cereus family member expresses a protease, wherein the expression of the protease is increased as compared to the sion of the protease in a wild-type Bacillus cereus family member under the same conditions. 43 1 Embodiment 393 is a method for delaying germination of a spore of a Bacillus cereus family member comprising modifying the Bacillus cereus family member to express an inosine-uridine hydrolase or an alanine racemase, wherein the expression of the inosine-uridine hydrolase or the alanine racemase is increased as compared to the sion of the e-uridine hydrolase or the alanine racemase in a wild-type Bacillus cereus family member under the same conditions.

Embodiment 394 is a method of any one of embodiments 357—379 and 381— 393, further comprising inactivating the recombinant Bacillus cereus family member or the recombinant spore-forming bacterium prior to introduction into the plant growth medium or application to a plant, a plant seed, or an area surrounding a plant or a plant seed.

Embodiment 395 is a method of embodiment 394, n the inactivating comprises subjecting the recombinant Bacillus cereus family member or the inant spore- forming bacterium to heat treatment; gamma ation; x-ray irradiation; UV-A irradiation; UV-B irradiation; treatment with gluteraldehyde, formaldehyde, hydrogen peroxide, acetic acid, bleach, chloroform, or phenol, or a combination thereof.

Embodiment 396 is a method of embodiment 394 or 395, n the inactivating ses modifying the recombinant Bacillus cereus family member or recombinant spore-forming bacterium to express a germination spore protease or a non-specific endonuclease, wherein the expression of the germination spore protease or the non-specific endonuclease is increased as compared to the expression of the germination spore protease or the non-specific endonuclease in a wild-type Bacillus cereus family member under the same conditions, and wherein the inant spore-forming bacterium comprises a recombinant bacterium of the genus Bacillus.

Embodiment 397 is a method for removing exosporium from spores of a recombinant Bacillus cereus family member comprising: subjecting a suspension comprising spores of a inant Bacillus cereus family member of any one of embodiments 4 to centrifugation or filtration to produce fragments of exosporium that are separated from the spores; wherein the rium nts comprise the fusion protein.

Embodiment 398 is a method of embodiment 397, wherein the method ses ting the suspension comprising the spores to centrifugation, and further comprises collecting the supernatant, n the supernatant comprises the fragments of the rium and is substantially free of spores.

Embodiment 399 is a method of embodiment 397, wherein the method comprises subjecting the suspension comprising the spores to filtration, and further comprises collecting the e, wherein the filtrate comprises the fragments of the exosporium and is substantially free of spores.

Embodiment 400 is a method for stimulating plant growth comprising: introducing exosporium fragments or a formulation of embodiment 339 into a plant growth medium; or applying exosporium nts or a formulation of embodiment 339 to a plant, a plant seed, or an area surrounding a plant or a plant seed; wherein the exosporium fragments are d from spores of a recombinant Bacillus cereus family member of any one of embodiments 100—124 and comprise the fusion protein, and the fusion protein comprises a plant growth stimulating protein or peptide.

Embodiment 401 is a method for protecting a plant from a pathogen or enhancing stress resistance in a plant comprising: introducing exosporium fragments or a formulation of embodiment 339 into a plant growth ; or applying exosporium fragments of or a ation of embodiment 339 to a plant, a plant seed, or an area surrounding a plant or a plant seed; wherein the exosporium fragments are derived from spores of a recombinant Bacillus cereus family member of any one of embodiments 100—124 and comprise the fusion n, and the fusion protein comprises a protein or peptide that ts a plant from a en or a protein or peptide that enhances stress ance in a plant.

Embodiment 402 is a method of embodiment 401, wherein the method is a method for protecting a plant from a pathogen and wherein the fusion protein comprises n or peptide that protects a plant from a pathogen. 7] Embodiment 403 is a method for protecting a plant from a pathogen of embodiment 402, wherein plants grown in the plant growth medium comprising the exosporium fragments are less susceptible to infection with the pathogen as compared to plants grown under the same conditions in the identical plant growth medium that does not contain the exosporium fragments . 2015/050807 Embodiment 404 is a method of embodiment 401, wherein the method is a method for ing stress resistance in a plant and wherein the fusion protein comprises a n or peptide that enhances stress resistance in a plant.

Embodiment 405 is a method for enhancing stress resistance in a plant of ment 404, wherein plants grown in the plant growth medium comprising the exosporium fragments are less tible to stress as compared to plants grown under the same conditions in the identical plant growth medium that does not contain the exosporium fragments.

Embodiment 406 is a method for immobilizing exosporium fragments on a plant comprising: introducing exosporium nts or a formulation of embodiment 339 into a plant growth medium; or applying exosporium fragments or a formulation of embodiment 339 to a plant, a plant seed, or an area surrounding a plant or a plant seed; wherein the exosporium fragments are derived from spores of a recombinant Bacillus cereus family member of any one of embodiments 4 and comprise the fusion protein, and the fusion protein comprises a plant binding protein or peptide.

Embodiment 407 is a method of any one of embodiments 363, 364, and 406, wherein the plant binding protein or peptide ively targets and maintains the recombinant Bacillus cereus family member, the recombinant spore-forming bacterium, or the exosporium fragments on a plant.

Embodiment 408 is a method of embodiment 407, wherein the plant binding protein or peptide selectively targets and maintains the recombinant Bacillus cereus family member, the recombinant spore-forming bacterium, or the exosporium fragments on plant roots, uctures of roots, an aerial portion of a plant, or a ucture of an aerial portion of a plant.

Embodiment 409 is a method for stimulating germination of a plant seed comprising: ucing exosporium fragments or a formulation of embodiment 339 into a plant growth medium; or applying exosporium fragments or a formulation of embodiment 339 to a plant seed or an area surrounding a plant or a plant seed; wherein the exosporium fragments are derived from spores of a recombinant us cereus family member of any one of embodiments 100—124 and comprise the fusion protein, and the fusion protein comprises a superoxide dismutase or an enzyme that catalyzes the production of nitric oxide.

Embodiment 410 is a method of embodiment 409, wherein the method comprises applying the exosporium fragments to a plant seed.

Embodiment 411 is a method of ment 409 or 410, further comprising applying nine to the plant growth medium, the plant seed, or the area surrounding the plant seed.

Embodiment 412 is a method of embodiment 411, n the method comprises applying L-arginine to the plant seed.

Embodiment 413 is a method of any one of embodiments 409—412, wherein seeds in the plant grth medium sing the exosporium fragments or seeds to which the exosporium nts have been applied have an increased germination rate or have an longer taproot after ation as compared to the same seeds grown under the same conditions in the identical plant growth medium that does not contain the exosporium fragments or the same seeds grown under the same conditions to which the exosporium nts have not been applied.

Embodiment 414 is a method for delivering nucleic acids to a plant comprising: introducing exosporium fragments or a formulation of embodiment 339 into a plant growth medium; or applying exosporium fragments or a formulation of embodiment 339 to a plant, a plant seed, or an area surrounding a plant or a plant seed; wherein the rium fragments are derived from spores of a recombinant Bacillus cereus family member of any one of embodiments 100—124 and comprise the fusion protein, and the fusion protein comprises a c acid binding protein or peptide, and wherein the nucleic acid binding protein or peptide is bound to a nucleic acid molecule. ment 415 is a method for delivering a nucleic acid molecule to an animal, insect, or worm comprising feeding to an animal, insect, or worm exosporium fragments, wherein the exosporium fragments are derived from a recombinant Bacillus cereus family member of any one of embodiments 100—124 and comprise the fusion protein, wherein 43 5 the fusion protein comprises a nucleic acid binding protein or peptide, and wherein the nucleic acid binding protein or peptide is bound to a nucleic acid molecule.

Embodiment 416 is the method of embodiment 415, wherein the worm is a nematode.

Embodiment 417 is a method for delivering a nucleic acid molecule to a fungus or a protozoan, comprising contacting a fungus or a protozoan with rium fragments, wherein the exosporium fragments are derived from a recombinant Bacillus cereus family member of any one of embodiments 100—124 and comprise the fusion n, wherein the fusion protein comprises a nucleic acid binding protein or peptide, and wherein the nucleic acid binding protein or peptide is bound to a c acid molecule.

Embodiment 418 is a method of any one of embodiments 414—417, wherein the nucleic acid molecule comprises a modulating RNA molecule; an RNAi molecule; a microRNA; an aptamer; or a DNA molecule that s a ting RNA molecule, an RNAi molecule, a microRNA, or an aptamer.

Embodiment 419 is a method of any one of embodiments 414—418, wherein the fusion n comprises a fusion protein of any one of embodiments 253—266. 4] ment 420 is a method for stimulating germination of a plant seed comprising introducing into a plant growth medium, or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed: (i) an enzyme that catalyzes the production of nitric oxide; (ii) a xide dismutase; or (iii) a inant microorganism that expresses an enzyme that catalyzes the production of nitric oxide or a superoxide dismutase, wherein the expression of the enzyme that zes the production of nitric oxide or the superoxide dismutase is increased as compared to the expression of the enzyme that catalyzes the production of nitric oxide or the superoxide dismutase in a wild type microorganism under the same conditions.

Embodiment 421 is a method of embodiment 420, wherein the method comprises applying the enzyme or the microorganism to a plant seed.

Embodiment 422 is a method of embodiment 420 or 421, further comprising applying L-arginine to the plant growth medium, the plant seed, or the area nding the plant seed.

Embodiment 423 is a method of embodiment 422, n the method comprises applying L-arginine to the plant seed. 8] Embodiment 424 is a method of any one of ments 420—423, wherein seeds in the plant growth medium comprising the enzyme or the microorganism or seeds to which the enzyme or the microorganism has been applied have an increased germination rate or a longer taproot after germination as compared to seeds grown under the same conditions in the identical plant growth medium that does not contain enzyme or the microorganism or seeds to which the enzyme or the microorganism has not been d, grown under the same conditions.

Embodiment 425 is a method of any one of embodiments 420—424, wherein the enzyme that catalyzes the production of nitric oxide synthase comprises a nitric oxide synthase or an arginase.

Embodiment 426 is a method of embodiment 425, wherein the enzyme that catalyzes the production of nitric oxide ses a nitric oxide synthase. 1] ment 427 is a method of embodiment 426, wherein the nitric oxide synthase comprises a nitric oxide synthase from Bacillus thuringiensis BT013A or Bacillus subtilis 168.

Embodiment 428 is a method of embodiment 426 or 427, wherein the nitric oxide synthase comprises an amino acid sequence haVing at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% ce identity with SEQ ID NO: 260 or 261.

Embodiment 429 is a method of any one of ments 8, wherein the superoxide dismutase comprises a superoxide dismutase l (SODAl) or a superoxide dismutase 2 (SODA2).

Embodiment 430 is a method of embodiment 429, wherein the superoxide dismutase comprises an amino acid sequence haVing at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 155 or 156.

Embodiment 431 is a method of any one of embodiments 420—430, wherein the recombinant microorganism comprises a Bacillus species, Escherechia c0li, an illus species, or a Saccliromyces species.

Embodiment 432 is a method of embodiment 43 l wherein the Bacillus species comprises a Bacillus cereus family member, Bacillus subtilis, Bacillus licheniformis, or Bacillus megaterium.

Embodiment 433 is a method of ment 43 1, wherein the Aspergillus species comprises Aspergillus niger or wherein the Saccliromyces s comprises Sacchromyces cerevisiae.

Embodiment 434 is a method of any one of embodiments 420—433, wherein the enzyme or the recombinant microorganism is introduced into the plant growth medium, or applied to a plant, a plant seed, or an area surrounding a plant or a plant seed in a ation sing the enzyme or the recombinant microorganism and an agriculturally acceptable carrier.

Embodiment 435 is a method for delivering enzymes to a plant comprising: introducing into a plant growth medium a recombinant Bacillus cereus family member of embodiment 16—141 and 168—296 or the ation comprising a recombinant Bacillus cereus family member of any one of ments 338 and 342—355; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed a inant Bacillus cereus family member of embodiment 16—141 and 168—296 or the formulation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; wherein the protein or peptide of st ses an enzyme. 0] Embodiment 436 is a method for delivering enzymes to a plant comprising: introducing into a plant growth medium a recombinant forming bacterium of any one of embodiments 147—149, 151—153, and 156—268 or a formulation comprising a recombinant spore-forming bacterium of any one of embodiments 338 and 342—355; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed a recombinant spore-forming bacterium of any one of embodiments 147—149, 15 l— 153, and 156—268 or a formulation comprising a recombinant spore-forming ium of any one of embodiments 338 and 342—355; wherein the protein or peptide of interest comprises an enzyme.

Embodiment 437 is a method for delivering enzymes to a plant of embodiment 435 or 436, wherein the enzyme is physically attached to the rium of the recombinant Bacillus cereus family member or to the spore coat of the recombinant spore- forming bacterium.

Embodiment 438 is a method for delivering enzymes to a plant comprising: introducing exosporium nts or a formulation of embodiment 339 into a plant growth medium; or 43 8 applying exosporium nts or a formulation of embodiment 339 to a plant, a plant seed, or an area surrounding a plant or a plant seed; wherein the exosporium fragments are derived from spores of a recombinant Bacillus cereus family member of any one of ments 100—124 and se the fusion protein, and the protein or peptide of interest comprises an enzyme.

Embodiment 439 is a method of embodiment 438, wherein the method further comprises ng the plant with a penetrating agent, a surfactant, a detergent, an oil, or a combination thereof.

Embodiment 440 is the method of any one of embodiments 43 5—441, wherein the enzyme is suitable for degrading biomass, digesting cellulosic material, aiding digestion in a digestive system of a target animal to which the plant can be fed, or for biofuel production.

Embodiment 441 is the method of embodiment 440, wherein the enzyme comprises a nonspecific protease, a oprotease, a cellulase, a xylanase, a phosphatase, an endoglucanase, an canase, a B-glucosidase, an e, a pectinase, a xylosidase, a , a phospholipase, or a combination thereof.

Embodiment 442 is the method of any one of embodiments 434—44l, wherein the plant is a crop selected from corn, alfalfa, wheat, a pasture crop, a forage crop, n, switchgrass, jicama, sweet sorghum, sugarcane, or a combination thereof.

Embodiment 443 is a method for altering a property of a plant comprising: introducing into a plant growth medium a recombinant Bacillus cereus family member of embodiment 16—141 and 168—296 or the formulation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; or applying to a plant, a plant seed, or an area surrounding a plant or a plant seed a recombinant Bacillus cereus family member of embodiment 16—141 and 6 or the formulation comprising a recombinant Bacillus cereus family member of any one of embodiments 338 and 342—355; n the protein or peptide of interest comprises a plant signaling molecule or an enzyme that affects plant composition. 8] ment 444 is a method for altering a property of a plant comprising: introducing into a plant growth medium a recombinant spore-forming bacterium of any one of embodiments 147—149, 151—153, and 156—268 or a formulation comprising a recombinant spore-forming bacterium of any one of embodiments 338 and 342—355; or ng to a plant, a plant seed, or an area surrounding a plant or a plant seed a recombinant spore-forming bacterium of any one of embodiments 147—149, 15 l— 153, and 156—268 or a ation comprising a recombinant spore-forming bacterium of any one of embodiments 338 and 342—355; wherein the protein or peptide of interest comprises a plant signaling molecule or an enzyme that affects plant composition. 9] Embodiment 445 is a method of embodiment 443 or 444, wherein the protein or peptide of interest is physically attached to the exosporium of the recombinant Bacillus cereus family member or to the spore coat of the recombinant spore-forming bacterium.

Embodiment 446 is a method for altering a property of a plant sing: introducing exosporium fragments or a formulation of embodiment 339 into a plant growth ; or applying exosporium fragments or a formulation of embodiment 339 to a plant, a plant seed, or an area nding a plant or a plant seed; n the exosporium fragments are derived from spores of a recombinant Bacillus cereus family member of any one of embodiments 100—124 and comprise the fusion protein, and the protein or peptide of interest comprises a plant signaling molecule or an enzyme that affects plant composition.

Embodiment 447 is the method of embodiment 446, n the method further comprises treating the plant with a penetrating agent, a surfactant, a detergent, an oil, or a combination thereof.

Embodiment 448 is the method of any one of embodiments 43 5—447, wherein the inant Bacillus cereus family member, the recombinant spore-forming bacterium, or the exosporium fragments are applied to foliage of the plant prior to harvest.

Embodiment 449 is the method of any one of embodiments 443—450, wherein the enzyme comprises an endoglucanase, a protease, a phospholipase, an aminocyclopropane-l- carboxylic acid ase, a lipase, or a combination thereof.

Embodiment 450 is the method of any one of embodiments 443—448, n the plant signaling molecule comprises a flagellin peptide, a cryptogein, a harpin, a harpin-like protein, an enzyme that degrades or s a bacterial, fungal, or plant nutrient source, or a combination thereof.

] Embodiment 451 is the method of any one of embodiments 443—450, wherein the plant is selected from grape, sugar cane, switchgrass, sweet sorghum, a grain, a cereal, rapeseed, canola, soybean, sunflower, marijuana, or a combination thereof.

Embodiment 452 is the method of any one of embodiments 443—451, wherein the property of the plant which is to be altered comprises a metabolic change. 7] Embodiment 453 is the method of embodiment 452, wherein the metabolic change comprises increased macronutrient or micronutrient uptake or content of the plant tissue through enlargement of its root system, increased protein content, increased nitrogen uptake, modified oil content, modified sugar or sucrose content, increased medicinal compound content, increased cannabinoid content, or a combination thereof.

Embodiment 454 is a method of any one of embodiments 357—379, 390—396, 400—414, and 420—453, wherein the plant growth medium is mented with a substrate or a cofactor for an enzyme. 9] Embodiment 455 is a method of embodiment 454, wherein the ate or the cofactor comprises tryptophan, an adenosine monophosphate, an adenosine diphosphate, an ine triphosphate, indole, a trimetaphosphate, ferrodoxin, acetoin, diacetyl, pyruvate, acetolactate, pectin, cellulose, methylcellulose, starch, chitin, pectin, a protein meal, a ose derivative, a ate, acetoin, chitosan, an inactive derivative of acetic acid, an inactive derivative of gibberellic acid, a xylan, an oxylan, a fat, a wax, an oil, a phytic acid, a lignin, a humic acid, choline, a choline derivative, e, a polyproline, a e-rich protein, a proline-rich meal, phenylalanine, chorismate, L-arginine, NADH, NADPH, ATP, GTP, cytochrome C, cytochrome p450, or a combination thereof.

Embodiment 456 is a method of embodiment 455, wherein the adenosine triphosphate comprises inetriphosphate.

Embodiment 457 is a method of any one of embodiments 357—379, 390—396, 400—414, and 420—456, sing g seeds with the recombinant Bacillus cereus family member, the recombinant spore-forming bacterium, or the exosporium fragments or a formulation containing the recombinant Bacillus cereus family member, the recombinant spore- forming bacterium, or the or exosporium fragments prior to planting.

Embodiment 458 is a method of any one of embodiments 357—379, 390—396, 4, and 420—457, comprising applying the recombinant Bacillus cereus family , the inant spore-forming bacterium, or the exosporium fragments, or a formulation ning the recombinant Bacillus cereus family member, the inant spore-forming bacterium, or the exosporium fragments to an aerial portion of a plant.

Embodiment 459 is a method of any one of embodiments 357—379, 390—396, 400—414, and 420—458, wherein introducing the recombinant Bacillus cereus family member, the recombinant spore-forming bacterium, or the exosporium fragments into the plant growth medium comprises applying a liquid or solid formulation containing the recombinant Bacillus cereus family member, the recombinant spore-forming bacterium, or the rium fragments to the medium.

Embodiment 460 is a method of embodiment 459, wherein the method comprises applying the formulation to the plant growth medium prior to, concurrently with, or after planting of seeds, seedlings, cuttings, bulbs, or plants in the plant growth medium.

Embodiment 461 is a method of any one of ments 357—379, 390—396, 400—414, and 420—460, further comprising introducing at least one agrochemical into the plant growth medium or applying at least one agrochemical to plants or seeds.

Embodiment 462 is a method of embodiment 461, wherein the agrochemical comprises a fertilizer, a micronutrient fertilizer material, an insecticide, an herbicide, a fungicide, a cicide, an algicide, a plant growth amendment, a bacterial inoculant, a fungal inoculant, or a combination thereof. 7] Embodiment 463 is a formulation, plant seed, or inoculum of embodiment 350, or a method of embodiment 462, wherein agrochemical comprises a fertilizer, and the fertilizer ses a liquid fertilizer; wherein the agrochemical comprises a micronutrient fertilizer material and the micronutrient fertilizer material comprises boric acid, a borate, a boron frit, copper sulfate, a copper frit, a copper chelate, a sodium tetraborate decahydrate, an iron sulfate, an iron oxide, iron um sulfate, an iron frit, an iron chelate, a manganese sulfate, a manganese oxide, a manganese chelate, a manganese chloride, a manganese frit, a sodium molybdate, molybdic acid, a zinc sulfate, a zinc oxide, a zinc carbonate, a zinc frit, zinc phosphate, a zinc e, or a combination thereof; wherein the agrochemical ses an icide, and the insecticide comprises an organophosphate, a carbamate, a pyrethroid, an acaricide, an alkyl phthalate, boric acid, a , a fluoride, sulfur, a haloaromatic substituted urea, a hydrocarbon ester, a biologically-based insecticide, or a combination thereof; wherein the agrochemical comprises an herbicide, and the herbicide ses a chlorophenoxy nd, a henolic compound, a resolic compound, a dipyridyl compound, an acetamide, an aliphatic acid, an anilide, a benzamide, a benzoic acid, a benzoic acid derivative, anisic acid, an anisic acid derivative, a benzonitrile, benzothiadiazinone dioxide, a thiocarbamate, a carbamate, a carbanilate, chloropyridinyl, a cyclohexenone derivative, a dinitroaminobenzene derivative, a fluorodinitrotoluidine compound, olidinone, nicotinic acid, isopropylamine, an isopropylamine derivative, oxadiazolinone, a phosphate, a ate, a picolinic acid nd, a triazine, a triazole, a uracil, a urea derivative, endothall, sodium chlorate, or a combination thereof; wherein the agrochemical comprises a fungicide, and the fungicide comprises a substituted e, a rbamate, an ethylene bis dithiocarbamate, a thiophthalidamide, a copper nd, an organomercury compound, an tin compound, a cadmium compound, anilazine, benomyl, cyclohexamide, dodine, etridiazole, iprodione, metlaxyl, thiamimefon, triforine, or a combination f; wherein the agrochemical comprises a fungal inoculant and the fungal inoculant comprises a fungal inoculant of the family Glomeraceae, a fungal inoculant of the family Claroidoglomeraceae, a fungal inoculant of the family Gigasporaceae, a fungal inoculant of the family Acaulosporaceae, a fungal inoculant of the family Sacculosporaceae, a fungal inoculant of the family Entrophosporaceae, a fungal inoculant of the family Pacidsporaceae, a fungal inoculant of the family Diversisporaceae, a fungal inoculant of the family Paraglomeraceae, a fungal inoculant of the family Archaeosporaceae, a fungal inoculant of the family Geosiphonaceae, a fungal inoculant of the family Ambisporaceae, a fungal inoculant of the family Scutellosporaceae, a fungal inoculant of the family Dentiscultataceae, a fungal inoculant of the family traceae, a fungal inoculant of the phylum Basidiomycota, a fungal inoculant of the phylum Ascomycota, a fungal inoculant of the phylum cota, or a combination thereof; or wherein the emical comprises a ial inoculant and the bacterial inoculant comprises a bacterial inoculant of the genus Rhizobz'um, a bacterial inoculant of the genus Bradyrhz'zobz’um, a ial ant of the genus Mesorhz'zobz'um, a bacterial inoculant of the genus Azorhz'zobz'um, a bacterial inoculant of the genus Allorhz'zobz'um, a bacterial inoculant of the genus Sinorhz'zobz'um, a bacterial inoculant of the genus Kluyvera, a ial inoculant of the genus Azotobacter, a bacterial inoculant of the genus Pseudomonas, a bacterial inoculant of the genus Azospirz'llz'um, a bacterial inoculant of the genus Bacillus, a bacterial inoculant of the genus omyces, a bacterial inoculant of the genus Paenz’bacz’llus, a bacterial inoculant of the genus Paracoccus, a bacterial inoculant of the genus Enterobacter, a bacterial inoculant of the genus Alcalz'genes, a bacterial inoculant of the genus Mycobacterl'um, a bacterial inoculant of the genus derma, a bacterial inoculant of the genus Gliocladz'um, a bacterial inoculant of the genus , a bacterial inoculant of the genus Klebsz'ella, or a combination thereof.

Embodiment 464 is a formulation, plant seed, or um of embodiment 350 or a method of embodiment 462, wherein the agrochemical comprises a fungicide, and the fungicide comprises aldimorph, ampropylfos, ampropylfos potassium, andoprim, anilazine, azaconazole, azoxystrobin, benalaxyl, benodanil, benomyl, benzamacril, benzamacryl-isobutyl, bialaphos, binapacryl, biphenyl, bitertanol, blasticidin-S, boscalid, bromuconazole, bupirimate, bate, calcium polysulphide, capsimycin, captafol, captan, carbendazim, carvon, quinomethionate, chlobenthiazone, chlorfenazole, chloroneb, chloropicrin, chlorothalonil, chlozolinate, clozylacon, eb, cymoxanil, cyproconazole, inil, cyprofuram, debacarb, dichlorophen, diclobutrazole, uanid, diclomezine, dicloran, diethofencarb, dimethirimol, dimethomorph, dimoxystrobin, diniconazole, diniconazole-M, dinocap, diphenylamine, dipyrithione, ditalimfos, dithianon, dodemorph, dodine, drazoxolon, edifenphos, onazole, etaconazole, ethirimol, etridiazole, famoxadon, fenapanil, fenarimol, fenbuconazole, fenfuram, fenitropan, fenpiclonil, fenpropidin, fenpropimorph, fentin acetate, fentin hydroxide, , ferimzone, am, flumetover, fluoromide, fluquinconazole, flurprimidol, flusilazole, amide, flutolanil, flutriafol, folpet, fosetyl-aluminium, fosetyl- sodium, fthalide, fuberidazole, xyl, furametpyr, furcarbonil, furconazole, furconazole-cis, furmecyclox, guazatine, hexachlorobenzene, hexaconazole, zole, imazalil, imibenconazole, iminoctadine, tadine albesilate, iminoctadine triacetate, iodocarb, iprobenfos (IBP), iprodione, irumamycin, thiolane, isovaledione, kasugamycin, kresoxim— methyl, copper preparations, such as: copper hydroxide, copper naphthenate, copper oxychloride, copper sulphate, copper oxide, oxine-copper and Bordeaux mixture, mancopper, mancozeb, maneb, meferimzone, mepanipyrim, il, metconazole, methasulfocarb, methfuroxam, metiram, clam, fovax, mycin, myclobutanil, myclozolin, nickel dimethyldithiocarbamate, nitrothal-isopropyl, nuarimol, ofurace, oxadixyl, oxamocarb, oxolinic acid, oxycarboxim, thiin, paclobutrazole, pefurazoate, penconazole, pencycuron, phosdiphen, pimaricin, piperalin, polyoxin, polyoxorim, probenazole, prochloraz, procymidone, propamocarb, propanosine-sodium, propiconazole, propineb, prothiocinazole, pyrazophos, pyrifenox, pyrimethanil, pyroquilon, pyroxyfur, quinconazole, quintozene (PCNB), sulphur and sulphur preparations, tebuconazole, tecloftalam, tecnazene, tetcyclasis, tetraconazole, thiabendazole, thicyofen, thifluzamide, thiophanate-methyl, tioxymid, tolclofos-methyl, tolylfluanid, triadimefon, menol, triazbutil, triazoxide, trichlamide, lazole, tridemorph, trifloxystrobin, izole, triforine, uniconazole, validamycin A, Vinclozolin, nazole, zarilamide, zineb, ziram and also Dagger G, OK-8705, OK-880l, a-(l,l- dimethylethyl)-(3-(2-phenoxyethyl)- l H- l ,2,4-triazole- l -eth anol, a-(2,4-dichlorophenyl)— [3 - 3-propyl-l H-- l ,2,4-triazoleethanol, a-(2,4-dichlorophenyl)- [3-methoxy-a-methyl-l H- l riazol e-l -ethanol, a-(5 -methyl - l ,3 -dioxan-5 -yl)- [3 - [ [4-(trifluoromethyl) -phenyl] -met hylene] -l H- l ,2,4-triazole-l -ethanol, (SRS ,6RS)—6-hydroxy-2,2,7,7-tetramethyl-5 -(l H-l ,2,4- triazol- l -yl)-3 -octanone, (methoxyimino)-N-methylphenoxy-phenylacetamide, l - isopropyl {2-methyl- l -[[[ l -(4-methylphenyl)-ethyl]-amino]—carbonyl] -propyl} carbamate, - dichlorophenyl)—2-(l H- l riazol- l -yl)-ethanone-O-(phenyl methyl)-oxime, l-(2-methyl- l - alenyl)-l H-pyrrole-2,5 -dione, l-(3 , 5 -dichlorophenyl)-3 -(2-propenyl)-2,5 - pyrrolidindione, l - [(diiodomethyl)-sulphonyl] methyl-benzene, l -[[2-(2,4-dichlorophenyl)— l , 3 -dioxolanyl] -methyl] -1 H-imidazole, l-[[2-(4-chlorophenyl)—3 -phenyloxiranyl] -methyl] -1 H- l ,2,4-triazole, l-[ l -[2- [(2,4-dichlorophenyl)—methoxy] l] -ethenyl] -l H-imidazole, 1- methyl nonyl(phenylmethyl)—3 -pyrroIidinole, 2',6'-dibromomethyl-4'-trifluoromethoxy- 4'-trifluoro-methyl- l , 3 -thiazole -carboxanilide, 2,2-dichloro-N—[ l -(4-chlorophenyl)-ethyl]- l - ethylmethyl-cyclopropanecarboxamide, 2,6-dichloro(methylthio)pyrimidinylthiocyanate , 2,6-dichloro-N-(4-trifluoromethylbenzyl)—benzamide, 2,6-dichloro-N—[[4- (trifluoromethyl)-phenyl]-methyl]-benzamide, 2-(2,3 ,3 -triiodopropenyl)-2H-tetrazole, 2- [(l - methylethyl)-sul phonyl]-5 -(trichloromethyl)- l ,3 ,4-thiadiazole, 2-[[6-deoxyO-(4methyl- (3 -D-glycopyranosyl)-a-D-glucopyranos yl]-amino]—4-methoxy-l H-pyrrolo [2,3-d]pyri midinecarbonitrile, obutane, 2-bromo(bromomethyl)-pentanedinitrile, 2-chloro-N-(2,3 - dihydro-l , l ,3 -trimethyl-l H-indenyl)—3 -pyridinecarboxamide, 2-chloro-N-(2,6- dimethylphenyl)-N-(isothiocyanatomethyl)-acetamide, 2-phenylphenol (OPP), 3,4-dichloro- l - [4-(difluoromethoxy)—phenyl]-pyrrole-2,5 -dione, 3 ,5-dichloro-N-[cyano [( l -methylpropynyl)— oxy]-methyl]-benzamide, 3 -(l , l -dimethylpropyl- l -oxo- l ne-Z-carbonitrile, 3 - [2-(4- chlorophenyl)ethoxy-3 -isoxazolidinyl]-pyridine, 4-chlorocyano-N,N—dimethyl-S -(4- methylphenyl)-l H-imidazole-l-sulphonamide, 4-methyl-tetrazolo[ l ,5 -a]quinazolin-S(4H)—one, 8-(l , l hylethyl)-N-ethyl-N-propyl- l ,4-dioxaspiro [4, 5 ]decanemethanamine, 8- yquinoline te, 9H-xanthene[(phenylamino)-carbonyl]carboxylic hydrazide, bis-( 1 -methylethyl)-3 -methyl[(3 -methylbenzoyl)-oxy]—2, 5 -thiophenedicarboxylate, cis— l -(4- chlorophenyl)(l H- l ,2,4-triazol- l -yl)-cycloheptanol, cis—4-[3 - [4-(l l -dimethylpropyl)— phenylmethylpropyl]-2,6-dimethyl-morpholine hydrochloride, ethyl [(4-chlorophenyl)-azo]— cyanoacetate, potassium bicarbonate, methanetetrathiol-sodium salt, methyl 1 -(2,3 -dihydro-2,2- dimethyl-inden- l -yl)-l H-imidazole-S xylate, methyl N—(2,6-dimethylphenyl)—N—(5 - isoxazolylcarbonyl)-DL-alaninate, methyl N—(chloroacetyl)-N—(2,6-dimethylphenyl)-DL- alaninate, N—(2,3-dichlorohydroxyphenyl)-l-methyl-cyclohexanecarboxamide, N—(2,6- dimethyl phenyl)methoxy-N-(tetra hydrooxofuranyl)-acetamide, N—(2,6-dimethyl p henyl)methoxy-N-(tetrahydrooxothienyl)-acetamide, N-(2-chloronitrophenyl) nitro-benzenesulphonamide, N-(4-cyclohexylphenyl)-l ,4,5 ,6-tetrahydro pyrimidinamine, N—(4-hexylphenyl)—l,4,5,6-tetrahydropyrimidinamine, N—(S-chloro methylphenyl)methoxy-N—(2-oxooxazolidinyl)-acetamide, N—(6-methoxy)—3-pyridinyl)- cyclopropanecarboxamide, N—[2,2,2-trichloro-l-[(chloroacetyl)-amino]-ethyl]-benzamide, N—[3- chloro-4,5-bis(2-propinyloxy)-phenyl]-N'-methoxy-methanimidamide, N—formyl-N-hydroxy- nine-sodium salt, 0,0-diethyl [2-(dipropylamino)oxoethyl]- ethylphosphoramidothioate, 0-methyl yl phenylpropylphosphoramidothioate, S-methyl l,2,3-benzothiadiazolecarbothioate, and spiro[2H]-l-benzopyrane-2,l'(3'H)-isobenzofuran]— 3'-one, N-trichloromethyl)thio cyclohexane-l,2-dicarboximide, tetramethylthioperoxydicarbonic diamide, methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)- DL-alaninate, 4-(2,2-difluoro-l,3-benzodioxolyl)-l-H-pyrrolcarbonitril or a combination thereof.

Embodiment 465 is a ation, plant seed, or inoculum of embodiment 350 or a method of ment 462, wherein the agrochemical comprises a ial inoculant of the genus Bacillus, and the bacterial inoculant of the genus Bacillus comprises us argri, Bacillus aizawai, Bacillus alb0lactis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus coagulans, Bacillus endoparasiticus, Bacillus endorhythmos, Bacillus kurstaki, Bacillus lactic0la, us lactimorbus, Bacillus lactis, Bacillus laterosporus, Bacillus lentimorbus, us iformis, Bacillus megaterium, Bacillus medusa, Bacillus metiens, Bacillus natto, us nigrificans, Bacillus popillae, Bacillus pumilus, Bacillus siamensis, us sphearicus, Bacillus spp., Bacillus subtilis, Bacillus thuringiensis, us unifagellatu, or a ation thereof.

Embodiment 466 is a formulation, plant seed, or inoculum of embodiment 350 or a method of embodiment 462, wherein the agrochemical comprises an herbicide, and the herbicide comprises 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, atrazine, aminopyralid, benefin, bensulfuron, bensulide, bentazon, bromacil, bromoxynil, butylate, carfentrazone, chlorimuron, chlorsulfuron, clethodim, clomazone, alid, cloransulam, cycloate, DCPA, desmedipham, dicamba, dichlobenil, diclofop, diclosulam, diflufenzopyr, dimethenamid, , diuron, DSMA, endothall, EPTC, ethalfluralin, ethofumesate, fenoxaprop, fluazifop-P, flucarbazone, flufenacet, flumetsulam, flumiclorac, flumioxazin, uron, pyr, fomesafen, foramsulfuron, glufosinate, glyphosate, halosulfuron, hexazinone, imazamethabenz, imazamox, imazapic, imazaquin, imazethapyr, en, isoxaflutole, lactofen, linuron, MCPA, MCPB, ione, metolachlor-s, metribuzin, metsulfuron, molinate, MSMA, napropamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxyfluorfen, paraquat, pelargonic acid, pendimethalin, dipham, picloram, primisulfuron, prodiamine, prometryn, pronamide, propanil, prosulfuron, n, pyrithiobac, orac, quizalofop, rimsulfuron, sethoxydim, siduron, simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, terbacil, thiazopyr, thifensulfuron, thiobencarb, tralkoxydim, ate, lfuron, uron, triclopyr, trifluralin, triflusulfuron, or a combination thereof.

Embodiment 467 is a formulation, plant seed, or inoculum of embodiment 350 or a method of embodiment 463, wherein the formulation or inoculum comprises an herbicide and a strain of bacteria that is capable of ing the herbicide.

Embodiment 468 is a formulation, plant seed, inoculum, or method of embodiment 467, wherein the strain of bacteria that is capable of degrading an herbicide comprises Bacillus cereus family member EE349 (NRRL No. B-50928), Bacillus cereus family member EE-B00377 (NRRL B-67l l9), us pseudomycoia’es EE-B00366 (NRRL B-67120), or Bacillus mycoia’es EE-B00363 (NRRL B-67121), or a combination thereof.

Embodiment 469 is a formulation, plant seed, inoculum, or method of any one of embodiments 463—468, wherein the herbicide comprises a sulfonylurea, an aryl triazine, dicamba, a phenoxy herbicide, 2,4-D, a pyrethrin, a pyrethroid, or a combination thereof.

Embodiment 470 is a ation, plant seed, inoculum, or method of embodiment 469, wherein the sulfonylurea comprises sulfentrazone.

Embodiment 471 is a formulation, plant seed, or um of any one of embodiments 350 and 463—470 or a method of any one of embodiments 461—470, wherein the fertilizer comprises ammonium sulfate, ammonium nitrate, ammonium sulfate e, ammonium chloride, ammonium bisulfate, ammonium polysulf1de, ammonium thiosulfate, aqueous ammonia, anhydrous ammonia, ammonium polyphosphate, aluminum sulfate, m nitrate, calcium ammonium nitrate, calcium sulfate, calcined magnesite, calcitic limestone, calcium oxide, calcium nitrate, tic limestone, hydrated lime, calcium carbonate, diammonium phosphate, monoammonium ate, ium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, potassium magnesium sulfate, potassium sulfate, sodium nitrates, magnesian limestone, magnesia, urea, urea-formaldehydes, urea ammonium nitrate, sulfur-coated urea, polymer-coated urea, isobutylidene diurea, KZSO4—2MgSO4, e, sylvinite, kieserite, Epsom salts, elemental sulfur, marl, ground oyster shells, fish meal, oil cakes, fish manure, blood meal, rock phosphate, super phosphates, slag, bone meal, wood ash, manure, bat guano, peat moss, compost, green sand, cottonseed meal, feather meal, crab meal, fish on, humic acid, or a ation thereof.

Embodiment 472 is a method for ring beneficial bacteria to an animal comprising feeding to an animal a plant modified to comprise a level of an endophytic and probiotic strain of bacteria that is greater than the level of the endophytic and probiotic strain of bacteria in the same plant that has not been modified grown under the same conditions.

Embodiment 473 is a method of embodiment 472, wherein the plant fed to the animal comprises a plant grown in a plant growth medium ning the endophytic and probiotic strain of ia or a formulation comprising the endophytic and probiotic strain of bacteria, a plant to which the ytic and probiotic strain of bacteria was applied, a plant grown from a plant seed to which the endophytic and probiotic strain of ia was applied, a plant grown in an area to which the endophytic and probiotic strain of bacteria was d, or a seed grown in the area to which the endophytic and probiotic strain of bacteria was applied. ment 474 is a method of embodiment 472 or 473, wherein the endophytic and probiotic strain of bacteria comprises a Bacillus or Lysinibacillus species.

Embodiment 475 is a method of embodiment 474, wherein Bacillus species comprises Bacillus subtilis, Bacillusfirmus, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus rin, Bacillus megaterium, Bacillus pumilus, Bacillus licheniformis, or a combination thereof.

Embodiment 476 is a method of any one of embodiments 472—475, wherein the endophytic and tic strain of bacteria comprises Bacillus cereus family member EE349 (NRRL No. B-50928), Bacillus cereus family member EE439 (NRRL B-50979), Bacillus thuringiensis EE4l7 (NRRL 9), Bacillus cereus EE444 (NRRL B-50977), Bacillus megaterium EE385 (NRRL B-50980), Bacillus sp. EE387 (NRRL B-5098l), Bacillus circulans EE388 (NRRL B-50982), Bacillus subtilis EE405 (NRRL B-50978), Lysinibacillusfusiformis EE442 (NRRL B-50975), or Lysinibacillus sphaericus EE443 (NRRL B-50976), us pumilus EE-BOOl43 (NRRL B-67123), or a combination thereof. 1] Embodiment 477 is a method for ring proteins or peptides to an animal comprising feeding to an animal a recombinant Bacillus cereus family member expressing a fusion protein comprising a protein or peptide of interest and a targeting sequence, exosporium n, or rium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member, or feeding to an animal exosporium fragments derived from a recombinant Bacillus cereus family member expressing a fusion protein comprising a protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the inant Bacillus cereus family member.

Embodiment 478 is the method of ment 477, wherein the recombinant Bacillus cereus family member comprises a recombinant Bacillus cereus family member of any one of embodiments 16—29, 48—63, 125—141, and 186—296 or wherein the exosporium fragments comprise exosporium fragments derived from a Bacillus cereus family member of any one of embodiments 100—124.

Embodiment 479 is a method of embodiment 477 or 478, wherein the recombinant Bacillus cereus family member comprises an endophytic strain of bacteria.

Embodiment 480 is a method of embodiment 479, n the ytic strain of bacteria comprises Bacillus cereus family member EE349 (NRRL No. B-50928), Bacillus cereus family member EE439 (NRRL B-50979), Bacillus thuringiensis EE417 (NRRL B-50979), Bacillus cereus EE444 (NRRL B-50977), Bacillus thuringiensis EE3 l9 (NRRL B- 50983), Bacillus thuringiensis EE-B00184 (NRRL B-67122), us cereus family member EE-B00377 (NRRL B-67l l9); Bacillus pseudomycoia’es EE-B00366 (NRRL B-67120); or Bacillus mycoia’es EE-B00363 (NRRL 1).

] Embodiment 481 is a method of embodiment 480, wherein the endophytic strain of bacteria ses Bacillus cereus family member EE439 (NRRL B-50979), Bacillus thuringiensis EE417 (NRRL 9), Bacillus cereus EE444 (NRRL B-50977), or us thuringiensis EE3 l9 (NRRL B-50983), Bacillus thuringiensis EE-BOOl84 (NRRL B-67122), Bacillus cereus family member EE-B00377 (NRRL B-67l l9); Bacillus pseudomycoia’es EE- B00366 (NRRL B-67120); or Bacillus mycoia’es EE-B00363 (NRRL B-67121).

Embodiment 482 is a method of any one of embodiments 477—479, wherein the recombinant Bacillus cereus family member comprises a probiotic strain of ia.

Embodiment 483 is a method of embodiment 482, wherein the probiotic strain of bacteria comprises Bacillus cereus family member EE349 (NRRL No. B-50928), us cereus family member EE439 (NRRL B-50979), Bacillus thuringiensis EE417 (NRRL B-50979), us cereus EE444 (NRRL B-50977), Bacillus thuringiensis BT013A (NRRL No.

B-50924), or a combination thereof.

Embodiment 484 is a method of any one of embodiments 479—481, wherein the recombinant Bacillus cereus family member is sed within a plant that is fed to the animal.

Embodiment 485 is a method of embodiment 477 or 478, n the recombinant Bacillus cereus family comprises a strain of ia that is capable of colonizing the phylloplane of a plant.

Embodiment 486 is a method of ment 485, wherein the strain of bacteria that is capable of colonizing the phylloplane of a plant comprises Bacillus ’es BTl55 (NRRL No. B-50921), Bacillus mycoia’es EEl l8 (NRRL No. 8), Bacillus mycoia’es EEl4l (NRRL NO. B-509l6), us mycoia’es BT46-3 (NRRL No. B-50922), us cereus family member EE218 (NRRL No. B-50926), Bacillus giensis BT013A (NRRL No. B-50924), Bacillus cereus family member EE-B00377 (NRRL B-67l l9), Bacillus pseudomycoia’es EE-B00366 (NRRL B-67120), or Bacillus mycoia’es EE-B00363 (NRRL B- Embodiment 487 is a method of any embodiment 485 or 486, wherein the recombinant Bacillus cereus family member is present on the phylloplane of a plant that is fed to the animal.

Embodiment 488 is a vaccine comprising a pharmaceutically acceptable carrier and a recombinant Bacillus cereus family member that expresses a fusion protein of embodiment 12.

Embodiment 489 is a vaccine comprising a pharmaceutically acceptable carrier and exosporium fragments, wherein the exosporium fragments are derived from a recombinant Bacillus cereus family member of any one of embodiments 100—124 and comprise the fusion protein, and the fusion protein comprises an antigen.

Embodiment 490 is a vaccine comprising a pharmaceutically acceptable carrier and a inant Bacillus cereus family member of any one of ments 17—27, wherein the fusion protein comprises an antigen. ment 491 is the vaccine of any one of embodiments 488—490, wherein the vaccine is suitable for intravenous, intrarterial, intraperitoneal, intramuscular, aneous, intrapleural, topical, oral, intranasal, intradermal, transepithelial administration or by inhalation.

Embodiment 492 is the vaccine of any one of embodiments 488—490, wherein the vaccine is suitable for oral or transepithelial administration.

Embodiment 493 is a method of producing an immunogenic response in a subject, the method sing administering the vaccine of any one of embodiments 488—492 to the subject. 8] Embodiment 494 is a method of ng contaminants in an environment sing exposing a contaminated environment to spores of a recombinant Bacillus cereus family member that expresses a fusion n of embodiment 13.

Embodiment 495 is a method of reducing contaminants in an environment comprising exposing a contaminated environment to exosporium fragments, wherein the exosporium fragments are derived from a recombinant Bacillus cereus family member of any one of embodiments 100—124 and comprise the fusion protein, and wherein the fusion protein comprises a remediation enzyme. 0] Embodiment 496 is a method of reducing contaminants in an environment comprising exposing a contaminated environment to spores of a recombinant Bacillus cereus family member of any one of embodiments 17—27, wherein the fusion protein comprises a remediation enzyme. 1] Embodiment 497 is the method of any one of embodiments 494—496, wherein the contaminated environment comprises a gas, liquid, semi-liquid, gel, film, semi-solid, or solid. 2] Embodiment 498 is the method of embodiment 497, wherein the solid comprises surface soil, subsurface soil, compost, crop residue, leaves, mulch, cut trees, a biofilm, a slime layer, mold, sludge, sand, slag, sediment, sewage-derived solids, waste rock, nuclear waste, munitions and ordinance, hospital waste, junked auto parts, metal cuttings, insulation waste, food waste, asbestos, batteries, industrial scrap, l waste, wood waste, textile waste, glass waste, leather waste, rubber waste, plastic waste, electronic component waste, agricultural waste, photographic waste, ceramic waste, pharmaceutical waste, wax, spent sts, or a combination thereof. ment 499 is the method of embodiment 497, wherein the liquid comprises drinking water, groundwater, surface water, brine, storage tanks, lagoons, an c system, industrial wastewater, acid mine drainage, spent autofluid, spent plating baths, degreasing solutions, dry cleaning ons, machine coolants, drilling fluid waste, g fluid waste, hydraulic fracturing fluid waste, lubricant waste, paint, greywater, oily wastewater, pulp mill effluent, a water treatment , a septic system, a sewer system, a precipitation lagoon, a holding pond, a lake, a river, or combinations thereof. 45 1 Embodiment 500 is the method of embodiment 497, wherein the gas ses air, a flue gas, a process t , l gas, natural gas, propane gas, or a combination thereof.

Embodiment 501 is the method of any one of embodiments 494—500, wherein the contaminant comprises a chemical warfare agent comprising sarin (GB; o—isopropyl methylphosphonofluoridate); soman (GD; o—pinacolyl methylphosphonofl'uorida‘tc); cyclosarin (GE; o—cyclohexyl methylphosphonofluoridate); VX (CS—ethyl S—[Z— (diisopropylamino)ethyl]methylphosphonothioate); tabun (GA; N,N~dimethylethyl horoamidocyanidate), DFP (diisopropyl phophorofluoridate), or a d agent.

Embodiment 502 is the method of any one of embodiments 494—500, n the contaminant comprises an inorganic compound comprising arsenic, antimony, barium, beryllium, cadmium, chromium, copper, iron, lead, manganese, mercury, nickel, selenium, silver, tin, thallium, uranium, zinc or a combination thereof.

Embodiment 503 is the method of any one of embodiments 494—500, wherein the inant comprises an organic compound comprising a polycyclic aromatic hydrocarbon (PAH), a nated aromatic compound, a chlorinated aliphatic compound, a nitroaromatic compound (NAC), a phenolic compound, a cyano compound, dioxin, or a combination thereof Embodiment 504 is the method of any one of embodiments 0, n the contaminant comprises a crude oil, a refined oil, a fuel oil, a diesel oil, a gasoline, a hydraulic oil, and kerosene, or a volatile tuent thereof.

Embodiment 505 is the method of any one of embodiments 494—500, wherein the contaminant comprises an explosive, a fertilizer, a pesticide, an icide, or an herbicide.

Embodiment 506 is the method of any one of embodiments 494—500, n the enzyme comprises a phosphate binding protein, a protease, a carbohydrate hydrolyase, a lipase, a phospholipase, a nuclease, a nutrient binding protein, a cellulase, an oxidoreductase, a monooxygenase, a diooxygenase, a laccase, a lignin peroxidase, a manganese peroxidase, a peroxidase, a dehalogenase, a catalase, an amylase, a reductase, an oxidase, an amidase, a ligninase, a xylanase, a pectinase, a xylosidase, an endoglucanase, an exoglucanase, a glucosidase, a m inhibitory peptide, an herbicide-degrading enzyme, a pesticide-degrading enzyme, or a combination thereof Embodiment 507 is the method of embodiment 506, wherein the pesticide- degrading enzyme comprises a pyrethrinase.

Embodiment 508 is the method of embodiment 506, wherein the enzyme comprises an herbicide-degrading enzyme or a ide-degrading enzyme and the recombinant Bacillus cereus family member comprises a strain of bacteria that is capable of degrading an herbicide or a pesticide.

Embodiment 509 is the method of embodiment 508, wherein the strain of bacteria that is capable of degrading an herbicide or a pesticide comprises Bacillus cereus family member EE349 (NRRL No. B-50928), Bacillus cereus family member EE-B00377 (NRRL B- 67119); Bacillus pseudomycoia’es EE-B00366 (NRRL B-67120); or Bacillus mycoia’es EE- B00363 (NRRL B-67121).

Embodiment 510 is the method of any one of embodiments 9, wherein the recombinant Bacillus cereus family member spores or exosporium fragments contact the contaminated enVironment by incorporating the spores or exosporium fragments into a stream containing the contaminant, ting a stream containing the inant with an lization material containing the spores or exosporium fragments, incorporating the spores or exosporium fragments into granules to be mixed with the contaminated environment, ng the spores or exosporium fragments onto or into the contaminated enVironment, injecting the spores or exosporium fragments into the contaminated enVironment, or drenching the contaminated enVironment with the spores or exosporium fragments. ment 511 is a method for phytoremediation of contaminated soil, the method comprising: introducing recombinant us cereus family member spores into contaminated soil; or applying the recombinant Bacillus cereus family member spores to a plant planted in contaminated soil, or a plant seed for ng in contaminated soil, or an area of contaminated soil surrounding a plant or a plant seed; wherein the recombinant Bacillus cereus family member spores express a fusion n comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or rium n fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member spore, wherein the fusion protein comprises the fusion protein of embodiment l3 and n the recombinant Bacillus cereus family member comprises an endophytic strain of bacteria or a root colonizing strain of bacteria.

Embodiment 512 is a method for phytoremediation of contaminated soil, the method comprising expressing a remediation enzyme in a Bacillus cereus family member spore, wherein the expression of the ation enzyme in the recombinant Bacillus cereus family member spore is increased as compared to the sion of the remediation enzyme in a wild- type Bacillus cereus family member spore.

Embodiment 513 is a method for emediation of contaminated soil, the method comprising: introducing a recombinant spore-forming bacterium into contaminated soil; or applying the recombinant spore-forming bacterium to a plant planted in contaminated soil, or a plant seed to be planted in contaminated soil, or an area of contaminated soil surrounding a plant or a plant seed, wherein the recombinant spore-forming bacterium expresses a fusion protein comprising at least one protein or peptide of interest and a spore coat protein that targets the fusion protein to the surface of a spore of the bacterium, wherein the spore coat protein ses a CotB protein, a CotC protein, a CgeA protein, a CotB/H n, a Cot G protein, a spore coat protein X protein, or a CotY protein; the recombinant spore-forming bacterium comprises an endophytic strain of bacteria or a root colonizing strain of bacteria; and the protein or peptide of interest comprises a remediation enzyme.

Embodiment 514 is a method of embodiment 513, n the recombinant spore-forming bacterium comprises an endophytic strian of bacteria.

Embodiment 515 is a method for phytoremediation of contaminated soil, the method comprising introducing exosporium fragments into contaminated soil or applying exosporium fragments to a plant planted in inated soil, or a plant seed to be planted in inated soil, or an area of contaminated soil surrounding a plant or a plant seed, wherein the exosporium fragments are derived from spores of a recombinant Bacillus cereus family member of any one of embodiments 100—124 and comprise the fusion protein, wherein the fusion protein comprises a remediation enzyme. ment 516 is a method for phytoremediation of contaminated soil comprising introducing spores of a recombinant us cereus family member of any one of ments 17—27 into contaminated soil or applying spores of a recombinant us cereus family member of any one of embodiments 17—27 to a plant d in contaminated soil, or a plant seed to be planted in contaminated soil, or an area of inated soil surrounding a plant or a plant seed, wherein the fusion protein comprises a remediation enzyme.

Embodiment 517 is the method of any one of embodiments 511—516, wherein the spores or exosporium fragments are applied to the plant or the plant seed, and the plant or plant grown from the plant seed is tolerant to a target contaminant to be remediated from the contaminated soil.

Embodiment 518 is the method of any one of embodiments 494—517, wherein the recombinant Bacillus cereus family member spores comprise an endophytic strain of Embodiment 519 is the method of embodiment 518, wherein the ytic strain comprises Bacillus cereus family member EE349 (NRRL No. B-50928), Bacillus cereus family member EE439 (NRRL B-50979), Bacillus thuringiensis EE4l7 (NRRL B-50979), Bacillus cereus EE444 (NRRL B-50977), or Bacillus giensis EE3l9 (NRRL B-50983), Bacillus thuringiensis l84 mRRL 2), Bacillus cereus family member EE- B00377 (NRRL B-67l l9); Bacillus mycoia’es EE-B00366 (NRRL B-67120); or Bacillus mycoia’es EE-B00363 (NRRL B-67121).

Embodiment 520 is a method of reducing contaminants in an enVironment comprising exposing a contaminated enVironment to spores of a Bacillus cereus family member strain that is capable of degrading an herbicide or a pesticide; n the contaminants in the enVironment comprise an herbicide, a pesticide, or a combination thereof; and wherein the Bacillus cereus family member strain that is capable of ing an herbicide or a ide comprises Bacillus cereus family member EE349 (NRRL No. B-50928), Bacillus cereus family member EE-B00377 (NRRL B-67l l9); Bacillus pseudomycoia’es EE-B00366 (NRRL B-67120); Bacillus mycoia’es EE-B00363 (NRRL B-67121), or a combination thereof.

Embodiment 521 is the method of embodiment 520, wherein the Bacillus cereus family member strain that is capable of degrading an herbicide or a pesticide comprises a recombinant Bacillus cereus family member that expresses a fusion protein comprising at least one n or peptide of interest and a targeting ce, exosporium protein, or exosporium n fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member.

Embodiment 522 is the method of embodiment 521, wherein the n or peptide of interest ses an herbicide-degrading enzyme, a pesticide-degrading enzyme, or a combination thereof. 45 5 Embodiment 523 is a method of ng a hydraulic fracturing fluid to break an emulsion or gel within the fluid, the method comprising adding spores of a recombinant Bacillus cereus family member to a hydraulic fracturing fluid, wherein the recombinant us cereus family member expresses a fusion protein comprising at least one protein or peptide of interest and a ing sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the rium of the recombinant us cereus family member, and the protein or peptide of interest ses an enzyme le for ng the emulsion or gel.

Embodiment 524 is a method of embodiment 523, wherein the recombinant Bacillus cereus family member comprises a recombinant Bacillus cereus family member of any one of embodiments 16—29, 48—63, 125—141, and 168—296.

Embodiment 525 is a method of treating a hydraulic fracturing fluid to break an emulsion or gel within the fluid, the method comprising adding exosporium fragments to a hydraulic fracturing fluid, n the exosporium fragments are derived from a inant Bacillus cereus family member of any one of embodiments 100—124 and se the fusion protein, and wherein the fusion protein comprises an enzyme le for breaking the emulsion or gel.

Embodiment 526 is the method of any one of embodiments 523—525, wherein the fusion protein comprises a fusion protein of embodiment 14.

Embodiment 527 is the method of any one of embodiments 523—526, wherein the enzyme comprises a hemicellulase, an amylase, a pectinase, a carbohydrate yase, a cellulase, an agarase, a polygalacturonase, an endoglucanase, or a combination thereof Embodiment 528 is the method of any one of embodiments 523—527, wherein the emulsion or gel comprises a polymer, Arabica gum, agar, xanthan gum, cellulose, carboxymethylcellulose, carboxymethylhydroxyethyl cellulose, hydroxyethyl methylcellulose, guar, a guar derivative, or a combination thereof.

Embodiment 529 is the method of any one of embodiments 523—528, wherein the spores are injected into a well that is in contact with a subterranean hydrocarbon-containing formation.

Embodiment 530 is a method of disinfecting a surface comprising exposing a surface to a recombinant Bacillus cereus family member that expresses a fusion protein of embodiment 15. ment 531 is a method of disinfecting a surface comprising exposing a e to exosporium fragments, wherein the exosporium fragments are derived from a recombinant Bacillus cereus family member of any one of embodiments 100—124 and comprise the fusion protein, and wherein the fusion protein comprises an antibacterial n or peptide.

Embodiment 532 is a method of disinfecting a surface comprising exposing a surface to a inant Bacillus cereus family member of any one of embodiments 17—27, wherein the fusion protein comprises an cterial protein or peptide.

Embodiment 533 is the method of any one of embodiments 530—532, wherein the cterial protein or peptide minimizes or ts viral agents, bacteria, amoebas, pests, or molds from forming on or binding to the surface.

Embodiment 534 is the method of any one of embodiments 530—533, wherein the cterial protein or peptide comprises a protease, a nuclease, an crobial peptide, LysM, LfcinB, a lysostaphin, an albumin, a defensin, a bacteriocin, a lipopeptide, an innate immune system peptide, a lysozyme, a lyticase, or a combination thereof.

Embodiment 535 is Use of fusion proteins sing an enzyme as the protein or peptide of interest of any one of embodiments 13 or 15 or a recombinant Bacillus cereus family member expressing a fusion protein comprising an enzyme as the protein or peptide of interest of any one of embodiments 13 or 15 for grease, oil, or fat treatment or degumming; leather hide processing; biofuel, biodiesel, or anol ion; sugar processing or conversion; starch treatment; paper or linen processing; animal or fungal byproduct treatment or amino acid recovery; targeted digestion of facility wastes; feed or food additives; dietary supplements; animal nutrition; industrial cleaning; grain processing; cosmetic manufacturing; odor control; food or beverage processing; brewing ement or additives; detergent additives; or textile or yarn processing.

Embodiment 536 is Use of exosporium nts for grease, oil, or fat treatment or degumming; r hide processing; biofuel, biodiesel, or bioethanol formation; sugar processing or conversion; starch treatment; paper or linen processing; animal or fungal byproduct treatment or amino acid recovery; targeted ion of facility wastes; feed or food additives; dietary supplements; animal nutrition; industrial cleaning; grain processing; cosmetic manufacturing; odor control; food or beverage processing; brewing enhancement or additives; detergent additives; or textile or yarn processing; wherein the exosporium nts are derived from recombinant a recombinant Bacillus cereus family member of any one of embodiments 100—124 and se the fusion protein, and wherein the fusion protein comprises an enzyme.

Embodiment 537 is Use of a inant Bacillus cereus family member of any one of embodiments 17—27 for grease, oil, or fat treatment or degumming; leather hide processing; biofuel, biodiesel, or bioethanol formation; sugar processing or conversion; starch treatment; paper or linen processing; animal or fungal byproduct treatment or amino acid recovery; targeted digestion of facility wastes; feed or food additives; dietary supplements; animal nutrition; rial cleaning; grain processing; cosmetic manufacturing; odor control; food or beverage processing; brewing enhancement or additives; detergent additives; or textile or yarn sing.; wherein the fusion n comprises an enzyme.

Embodiment 538 is the uses of any one of embodiments 535—537, wherein the enzyme comprises a B-lactamase, a protease, a lipase, a phospholipase, a cellulase, an endoglucanase, an exogluconase, a pectinase, a ligninase, an amylase, a polygalacturonase, a glucosidase, a galactosidase, a ydrate hydrolyase, a cell wall hydrolase, a se, a llulase, a xylanase, a mannase, a laccase, a lactase, an esterase, a phytase, a phosphatase, an invertase, a glucose e, a catalase, a lyticase, an acetolactate decarboxylase, or a urease.

Embodiment 539 is the use of embodiment 538, wherein the amylase comprises an u-amylase, a ase, or a glucoamylase; or wherein the esterase comprises a pectin methyl esterase.

Embodiment 540 is a method of any one of embodiments 447—487, 493, 495— 510, 515—519, 521—529, and 4, a vaccine of any one of embodiments 489—492, or a use of any one of embodiments 536—539, n the targeting ce, exosporium protein, or exosporium protein fragment comprises: (1) a targeting sequence comprising an amino acid ce having at least about 43% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 54%; (2) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 1; (3) a ing sequence comprising amino acids 20—35 of SEQ ID NO: 1 ; (4) a targeting sequence comprising SEQ ID NO: 1 ; (5) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 2; (6) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 1 ; (7) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 1 ; (8) a targeting ce comprising amino acids 8—35 of SEQ ID NO: 1; (9) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: l; (10) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 1; (l l) a targeting sequence sing amino acids 1—27 of SEQ ID NO: 3; (12) a ing sequence comprising amino acids 12—27 of SEQ ID NO: 3; (13) a targeting sequence comprising SEQ ID NO: 3; (14) an exosporium protein comprising an amino acid sequence having at least 85% 45 8 identity with SEQ ID NO: 4; (15) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 3; (16) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 3; (17) a ing sequence comprising amino acids 8—27 of SEQ ID NO: 3; (18) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 3; (19) a targeting sequence comprising amino acids 1—38 of SEQ ID NO: 5; (20) a ing sequence comprising amino acids 23—38 of SEQ ID NO: 5; (21) a targeting sequence comprising SEQ ID NO: 5; (22) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 6; (23) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 5; (24) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 5; (25) a targeting sequence comprising amino acids 8—38 of SEQ ID NO: 5; (26) a targeting ce comprising amino acids 10—38 of SEQ ID NO: 5; (27) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 5; (28) a ing sequence comprising amino acids 20—38 of SEQ ID NO: 5; (29) a targeting sequence comprising amino acids 1—28 of SEQ ID NO: 7; (30) a targeting sequence comprising amino acids 13—28 of SEQ ID NO: 7; (31) a targeting sequence comprising SEQ ID NO: 7; (32) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 8; (33) a targeting ce comprising amino acids 2—28 of SEQ ID NO: 7; (34) a targeting ce comprising amino acids 5—28 of SEQ ID NO: 7; (35) a targeting sequence sing amino acids 8—28 of SEQ ID NO: 7; (36) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 7; (37) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 9; (38) a targeting ce comprising amino acids 9—24 of SEQ ID NO: 9; (39) a targeting ce comprising SEQ ID NO: 9; (40) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 10; (41) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 9; (42) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 9; (43) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 9; (44) a ing sequence comprising amino acids 1—33 of SEQ ID NO:11; (45) a targeting ce comprising amino acids 18—33 of SEQ ID NO: 11 ; (46) a targeting sequence comprising SEQ ID NO: 11 ; (47) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 12; (48) a targeting sequence sing amino acids 2—33 of SEQ ID NO: 11 ; (49) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 11; (50) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 11; (51) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 11 ; (52) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 11 ; (53) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 13; (54) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 13; (55) a targeting sequence comprising SEQ ID NO: 13; (56) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 14; (57) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 13; (58) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 13; (59) a targeting ce comprising amino acids 8—33 of SEQ ID NO: 13; (60) a targeting sequence sing amino acids 10—33 of SEQ ID NO: 13; (61) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 13; (62) a targeting ce comprising amino acids 1—43 of SEQ ID NO: 15; (63) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 15; (64) a targeting sequence comprising SEQ ID NO:15; (65) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 16; (66) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 15 ; (67) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 15 ; (68) a ing sequence comprising amino acids 8—43 of SEQ ID NO: 15; (69) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 15; (70) a targeting ce comprising amino acids 15—43 of SEQ ID NO: 15 ; (71) a targeting ce comprising amino acids 20—43 of SEQ ID NO: 15; (72) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 15; (73) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 17; (74) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 17; (75) a targeting sequence comprising SEQ ID NO:17; (76) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:18; (77) a targeting sequence comprising amino acids 2—27 of SEQ ID NO: 17; (78) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 17; (79) a targeting sequence comprising amino acids 8—27 of SEQ ID NO: 17; (80) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 17; (81) a targeting ce comprising amino acids 1—33 of SEQ ID NO: 19; (82) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 19; (83) a targeting sequence comprising SEQ ID NO:19; (84) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:20; (85) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 19; (86) a ing sequence comprising amino acids 5—33 of SEQ ID NO: 19; (87) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 19; (88) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 19; (89) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 19; (90) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 21; (91) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 21 ; (92) a targeting ce comprising SEQ ID NO:21; (93) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:22; (94) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 21; (95) a ing sequence comprising amino acids 5—33 of SEQ ID NO: 21; (96) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 21; (97) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 21; (98) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 21; (99) a targeting ce comprising amino acids 1—24 of SEQ ID NO: 23; (100) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 23; (101) a targeting sequence comprising SEQ ID NO:23; (102) an exosporium protein comprising an amino acid ce having at least 85% identity With SEQ ID NO:24; (103) a targeting sequence comprising amino acids 2—24 of SEQ ID NO:23; (104) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 23; (105) a targeting sequence comprising amino acids 8—24 of SEQ ID NO: 23; (106) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 25; (107) a ing sequence comprising amino acids 9—24 of SEQ ID NO: 25; (108) a targeting sequence comprising SEQ ID NO:25; (109) an rium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:26; (110) a ing sequence comprising amino acids 2—24 of SEQ ID NO: 25; (111) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 25; (112) a ing sequence comprising amino acids 8—24 of SEQ ID NO: 25; (113) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 27; (114) a targeting sequence comprising amino acids 15—30 of SEQ ID NO: 27; (115) a targeting sequence comprising SEQ ID NO:27; (116) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:28; (117) a targeting sequence comprising amino acids 2—30 of SEQ ID NO: 27; (118) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 27; (119) a targeting sequence comprising amino acids 8—30 of SEQ ID NO: 27; (120) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 27; (121) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 29; (122) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 29; (123) a targeting sequence comprising SEQ ID NO:29; (124) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO:30; (125) a targeting sequence sing amino acids 2—33 of SEQ ID NO: 29; (126) a targeting ce comprising amino acids 5—33 of SEQ ID NO: 29; (127) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 29; (128) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 29; (129) a ing sequence comprising amino acids 15—33 of SEQ ID NO: 29; (130) a targeting sequence sing amino acids 1—24 of SEQ ID NO: 31; (131) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 31; (132) a targeting sequence comprising SEQ ID NO:31; (133) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:32; (134) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 31; (135) a targeting sequence sing amino acids 5—24 of SEQ ID NO: 31; (136) a targeting ce comprising amino acids 8—24 of SEQ ID NO: 31; (137) a targeting sequence comprising amino acids 1—15 of SEQ ID NO: 33; (138) a targeting sequence comprising SEQ ID NO:33; (139) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:34; (140) a ing sequence comprising amino acids 1—16 of SEQ ID NO: 35; (141) a targeting sequence comprising SEQ ID NO:35; (142) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO:36; (143) a targeting sequence comprising amino acids 1—29 of SEQ ID NO:43; (144) a targeting sequence comprising amino acids 14—29 of SEQ ID NO: 43; (145) a targeting ce comprising SEQ ID NO: 43; (146) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 44; (147) a targeting sequence comprising amino acids 2—29 of SEQ ID NO: 43 ; (148) a targeting sequence comprising amino acids 5—29 of SEQ ID NO: 43 ; (149) a ing sequence comprising amino acids 8—29 of SEQ ID NO: 43; (150) a targeting ce sing amino acids 10—29 of SEQ ID NO: 43; (151) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 45; (152) a targeting sequence comprising amino acids 20—35 of SEQ ID NO: 45 ; (153) a targeting sequence comprising SEQ ID NO: 45 ; (154) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 46; (155) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 45; (156) a ing sequence comprising amino acids 5—35 of SEQ ID NO: 45; (157) a ing sequence sing amino acids 8—35 of SEQ ID NO: 45; (158) a targeting sequence comprising amino acids 10—35 of SEQ ID NO: 45; (159) a ing sequence comprising amino acids 15—35 of SEQ ID NO: 45 ; (160) a targeting sequence comprising amino acids 1—43 of SEQ ID NO: 47; (161) a targeting sequence comprising amino acids 28—43 of SEQ ID NO: 47; (162) a ing sequence comprising SEQ ID NO: 47; (163) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 48; (164) a targeting sequence comprising amino acids 2—43 of SEQ ID NO: 47; (165) a targeting sequence comprising amino acids 5—43 of SEQ ID NO: 47; (166) a targeting sequence comprising amino acids 8—43 of SEQ ID NO: 47; (167) a targeting sequence comprising amino acids 10—43 of SEQ ID NO: 47; (168) a targeting sequence comprising amino acids 15—43 of SEQ ID NO: 47; (169) a targeting sequence sing amino acids 20—43 of SEQ ID NO: 47; (170) a targeting sequence comprising amino acids 25—43 of SEQ ID NO: 47; (171) a targeting sequence comprising amino acids 1—32 of SEQ ID NO: 49; (172) a targeting sequence comprising amino acids 17—32 of SEQ ID NO: 49; (173) a ing sequence comprising SEQ ID NO: 49; (174) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 50; (175) a targeting sequence comprising amino acids 2—32 of SEQ ID NO: 49; (176) a targeting sequence comprising amino acids 5—32 of SEQ ID NO: 49; (177) a targeting sequence comprising amino acids 8—32 of SEQ ID NO: 49; (178) a targeting sequence comprising amino acids 10—32 of SEQ ID NO: 49; (179) a targeting sequence comprising amino acids 15—32 of SEQ ID NO: 49; (180) a targeting sequence sing amino acids 1—33 of SEQ ID NO: 51; (181) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 51; (182) a ing sequence comprising SEQ ID NO: 51; (183) an exosporium protein comprising an amino acid ce haVing at least 85% identity With SEQ ID NO: 52; (184) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 51; (185) a ing sequence comprising amino acids 5—33 of SEQ ID NO: 51; (186) a ing sequence comprising amino acids 8—33 of SEQ ID NO: 51; (187) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 51; (188) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 51; (189) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 53; (190) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 53; (191) a targeting sequence comprising SEQ ID NO: 53; (192) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 54; (193) a targeting sequence comprising amino acids 2—33 of SEQ ID NO: 53; (194) a targeting sequence sing amino acids 5—33 of SEQ ID NO: 53 ; (195) a targeting sequence comprising amino acids 8—33 of SEQ ID NO: 53 ; (196) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 53 ; (197) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 53; (198) a targeting ce comprising amino acids 1—30 of SEQ ID NO: 55; (199) a targeting sequence comprising amino acids 15—30 of SEQ ID NO: 55 ; (200) a targeting sequence comprising SEQ ID NO: 55 ; (201) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 56; (202) a targeting sequence sing amino acids 2—30 of SEQ ID NO: 55; (203) a targeting sequence comprising amino acids 5—30 of SEQ ID NO: 55; (204) a targeting sequence sing amino acids 8—30 of SEQ ID NO: 55; (205) a targeting sequence comprising amino acids 10—30 of SEQ ID NO: 55; (206) a targeting sequence comprising amino acids 1—130 of SEQ ID NO: 57; (207) a targeting sequence comprising amino acids 115—130 of SEQ ID NO: 57; (208) a targeting sequence sing SEQ ID NO: 57; (209) an exosporium protein comprising an amino acid sequence haVing at least 85% ty With SEQ ID NO: 58; (210) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 57; (211) a targeting WO 44661 sequence comprising amino acids 5—130 of SEQ ID NO: 57; (212) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 57; (213) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 57; (214) a targeting sequence comprising amino acids 30— 130 of SEQ ID NO: 57; (215) a targeting sequence sing amino acids 40—130 of SEQ ID NO: 57; (216) a targeting sequence comprising amino acids 50—130 of SEQ ID NO: 57; (217) a targeting sequence comprising amino acids 60—130 of SEQ ID NO: 57; (218) a targeting sequence sing amino acids 70—130 of SEQ ID NO: 57; (219) a targeting sequence comprising amino acids 80—130 of SEQ ID NO: 57; (220) a targeting ce comprising amino acids 90—130 of SEQ ID NO: 57; (221) a targeting sequence comprising amino acids 100—130 of SEQ ID NO: 57; (222) a targeting sequence comprising amino acids 110—130 of SEQ ID NO: 57; (223) an exosporium protein nt comprising an amino acid ce having at least 85% identity With SEQ ID NO: 95; (224) a targeting sequence comprising SEQ ID NO: 96; (225) a targeting sequence comprising SEQ ID NO: 97; (226) a targeting sequence comprising SEQ ID NO: 98; (227) a targeting sequence comprising SEQ ID NO: 99; (228) a ing sequence comprising SEQ ID NO: 100; (229) a targeting sequence comprising SEQ ID NO: 101; (230) a ing sequence comprising SEQ ID NO: 102; (231) a targeting sequence comprising SEQ ID NO: 103; (232) a targeting sequence comprising SEQ ID NO: 104; (233) a targeting sequence comprising SEQ ID NO: 105; (234) a targeting sequence sing SEQ ID NO: 106; (235) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (236) an exosporium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO: 109; (237) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 110; (238) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 111; (239) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 112; (240) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 113; (241) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 114; (242) an exosporium protein sing an amino acid sequence haVing at least 85% identity With SEQ ID NO: 115; (243) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 116; (244) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 117; (245) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 118; (246) an exosporium protein sing an amino acid ce haVing at least 85% identity With SEQ ID NO: 119; (247) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 120; (248) an exosporium protein comprising an amino acid ce having at least 85% identity With SEQ ID NO: 121; (249) a targeting sequence comprising amino acids 22—31 of SEQ ID NO: 1; (250) a targeting sequence comprising amino acids 22—33 of SEQ ID NO: 1; (251) a targeting sequence comprising amino acids 20—31 of SEQ ID NO: 1; (252) a targeting sequence comprising amino acids 14—23 of SEQ ID NO: 3; (253) a ing sequence comprising amino acids 14—25 of SEQ ID NO: 3; (254) a targeting ce comprising amino acids 12—23 of SEQ ID NO: 3; (255) a targeting sequence comprising amino acids 1—30 of SEQ ID NO: 59; (256) a targeting sequence comprising SEQ ID NO: 59; (257) an rium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 60; (258) a ing sequence sing amino acids 2—30 of SEQ ID NO: 59; (259) a targeting sequence comprising amino acids 4—30 of SEQ ID NO: 59; (260) a targeting ce comprising amino acids 6—30 of SEQ ID NO: 59; (261) a targeting sequence comprising amino acids 1—33 of SEQ ID NO: 61; (262) a targeting sequence comprising amino acids 18—33 of SEQ ID NO: 61; (263) a targeting sequence sing SEQ ID NO: 61 ; (264) an exosporium protein comprising an amino acid sequence having at least 85% sequence identity With SEQ ID NO: 62; (265) a targeting sequence sing amino acids 2—33 of SEQ ID NO: 61 ; (266) a targeting sequence comprising amino acids 5—33 of SEQ ID NO: 61 ; (267) a targeting sequence comprising amino acids 10—33 of SEQ ID NO: 61; (268) a targeting sequence comprising amino acids 15—33 of SEQ ID NO: 61; (269) a targeting sequence comprising amino acids 1—35 of SEQ ID NO: 63; (270) a targeting sequence comprising SEQ ID NO: 63; (271) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 64; (272) a targeting sequence comprising amino acids 2—35 of SEQ ID NO: 63; (273) a targeting sequence comprising amino acids 5—35 of SEQ ID NO: 63; (274) a targeting sequence comprising amino acids 8—35 of SEQ ID NO: 63 ; (275) a targeting sequence sing amino acids 10—35 of SEQ ID NO: 63 ; (276) a targeting sequence comprising amino acids 15—35 of SEQ ID NO: 63 ; (277) a ing sequence sing amino acids 1—24 of SEQ ID NO: 65; (278) a targeting ce comprising amino acids 9—24 of SEQ ID NO: 65; (279) a targeting sequence comprising SEQ ID NO: 65; (280) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 66; (281) a targeting sequence comprising SEQ ID NO: 107; (282) a targeting sequence comprising amino acids 2—24 of SEQ ID NO: 65; (283) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 65; (284) a targeting sequence comprising amino acids 1—27 of SEQ ID NO: 67; (285) a targeting sequence comprising amino acids 12—27 of SEQ ID NO: 67; (286) a targeting sequence comprising SEQ ID NO: 67; (287) an exosporium protein comprising an amino acid ce having at least 85% identity with SEQ ID NO: 68; (288) an targeting sequence comprising amino acids 2—27 of SEQ ID NO: 67; (289) a targeting sequence comprising amino acids 5—27 of SEQ ID NO: 67; (290) a targeting sequence comprising amino acids 10—27 of SEQ ID NO: 67; (291) a targeting sequence comprising amino acids 1—38 of SEQ ID NO: 69; (292) a targeting ce comprising amino acids 23—38 of SEQ ID NO: 69; (293) a targeting sequence comprising SEQ ID NO: 69; (294) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 70; (295) a targeting sequence comprising amino acids 2—38 of SEQ ID NO: 69; (296) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 69; (297) a ing sequence comprising amino acids 10—38 of SEQ ID NO: 69; (298) a targeting sequence comprising amino acids 15—38 of SEQ ID NO: 69; (299) an exosporium protein comprising SEQ ID NO: 72; (300) a targeting ce sing SEQ ID NO: 73; (301) an exosporium protein comprising an amino acid sequence having at least 95% identity With SEQ ID NO: 74; (302) a targeting sequence comprising amino acids 1—42 of SEQ ID NO: 75; (303) a targeting sequence comprising amino acids 27—42 of SEQ ID NO: 75; (304) a targeting sequence comprising SEQ ID NO: 75; (305) an exosporium protein comprising an amino acid sequence having at least 85% ty With SEQ ID NO: 76; (306) a targeting sequence comprising amino acids 2—42 of SEQ ID NO: 75; (307) a targeting sequence comprising amino acids 5—42 of SEQ ID NO: 75; (308) a targeting sequence comprising amino acids 10—42 of SEQ ID NO: 75; (309) a ing sequence comprising amino acids 15—42 of SEQ ID NO: 75; (310) a targeting sequence comprising amino acids 20—42 of SEQ ID NO: 75; (311) a targeting sequence comprising amino acids 25—42 of SEQ ID NO: 75; (312) a targeting sequence comprising amino acids 1—24 of SEQ ID NO: 77; (313) a targeting sequence comprising amino acids 9—24 of SEQ ID NO: 77; (314) a targeting sequence sing SEQ ID NO: 77; (315) an rium protein sing an amino acid sequence having at least 85% identity With SEQ ID NO: 78; (316) a ing sequence comprising amino acids 2—24 of SEQ ID NO: 77; (317) a targeting sequence comprising amino acids 5—24 of SEQ ID NO: 77; (318) an exosporium protein comprising an amino acid sequence having at least 85% identity With SEQ ID NO: 80; (319) a targeting sequence comprising amino acids 1—38 of SEQ ID NO: 81; (320) a targeting sequence comprising amino acids 23—38 of SEQ ID NO: 81; (321) a targeting sequence comprising SEQ ID NO: 81; (322) an exosporium protein comprising an amino acid sequence having at least 85% ty With SEQ ID NO: 82; (323) a targeting sequence comprising amino WO 44661 acids 2—38 of SEQ ID NO: 81; (324) a targeting sequence comprising amino acids 5—38 of SEQ ID NO: 81; (325) a targeting sequence comprising amino acids 10—38 of SEQ ID NO: 81; (326) a ing sequence comprising amino acids 15—38 of SEQ ID NO: 81; (327) a targeting sequence comprising amino acids 20—38 of SEQ ID NO: 81; (328) a targeting sequence comprising amino acids 1—34 of SEQ ID NO: 83; (329) a targeting sequence comprising SEQ ID NO: 83; (330) an rium protein comprising an amino acid sequence having at least 85% ty With SEQ ID NO: 84; (331) an rium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 86; (332) a ing sequence comprising amino acids 1—28 of SEQ ID NO: 87; (333) a targeting sequence comprising amino acids 13—28 of SEQ ID NO: 87; (334) a targeting sequence comprising SEQ ID NO: 87; (335) an exosporium protein comprising an amino acid ce haVing at least 85% ty With SEQ ID NO: 88; (336) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 87; (337) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 87; (338) a targeting sequence comprising amino acids 10—28 of SEQ ID NO: 87; (339) a targeting sequence comprising amino acids 1—28 of SEQ ID NO: 89; (340) a targeting sequence comprising SEQ ID NO: 89; (341) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 90; (342) a targeting sequence comprising amino acids 2—28 of SEQ ID NO: 89; (343) a targeting sequence comprising amino acids 5—28 of SEQ ID NO: 89; (344) a targeting ce comprising amino acids 10—28 of SEQ ID NO: 89; (345) a targeting sequence comprising amino acids 1—93 of SEQ ID NO: 91 ; (346) a targeting sequence comprising SEQ ID NO: 91 ; (347) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 92; (348) a targeting sequence comprising amino acids 2—93 of SEQ ID NO: 91 ; (349) a targeting sequence comprising amino acids 10—93 of SEQ ID NO: 91 ; (350) a targeting sequence comprising amino acids 20—93 of SEQ ID NO: 91 ; (351) a targeting sequence comprising amino acids 30—93 of SEQ ID NO: 91 ; (352) a ing sequence comprising amino acids 40—93 of SEQ ID NO: 91 ; (353) a targeting sequence comprising amino acids 50—93 of SEQ ID NO: 91 ; (354) a targeting sequence comprising amino acids 60—93 of SEQ ID NO: 91; (355) a ing ce comprising amino acids 1—130 of SEQ ID NO: 93; (356) a targeting sequence comprising SEQ ID NO: 93; (357) an exosporium protein comprising an amino acid sequence haVing at least 85% identity With SEQ ID NO: 94; (358) a targeting sequence comprising amino acids 2—130 of SEQ ID NO: 93; (359) a targeting sequence comprising amino acids 10—130 of SEQ ID NO: 93; (360) a targeting sequence comprising amino acids 20—130 of SEQ ID NO: 93; (361) a targeting sequence comprising amino acids 30—130 of SEQ ID NO: 93; (362) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 122; (363) a targeting sequence consisting of amino acids 20—33 of SEQ ID NO: 1 ; (364) a targeting sequence consisting of amino acids 21—33 of SEQ ID NO: 1; (365) a targeting sequence consisting of amino acids 23— 31 of SEQ ID NO: 1; (366) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 96; (367) a targeting ce consisting of amino acids 1—13 of SEQ ID NO: 96; (368) a targeting sequence consisting of amino acids 12—25 of SEQ ID NO: 3; (369) a targeting sequence consisting of amino acids 13—25 of SEQ ID NO: 3; (370) a targeting sequence consisting of amino acids 15—23 of SEQ ID NO: 3; (371) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 97; (372) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 98; (373) a targeting sequence consisting of amino acids 23—36 of SEQ ID NO: ; (374) a targeting sequence consisting of amino acids 23—34 of SEQ ID NO: 5; (375) a targeting sequence consisting of amino acids 24—36 of SEQ ID NO: 5; (376) a targeting sequence consisting of amino acids 26—34 of SEQ ID NO: 5; (377) a targeting sequence consisting of amino acids 13—26 of SEQ ID NO: 7; (378) a targeting sequence consisting of amino acids 13—24 of SEQ ID NO: 7; (379) a targeting sequence consisting of amino acids 14— 26 of SEQ ID NO: 7; (380) a targeting sequence consisting of amino acids 16—24 of SEQ ID NO: 7; (381) a ing sequence consisting of amino acids 9—22 of SEQ ID NO: 9; (382) a ing ce consisting of amino acids 9—20 of SEQ ID NO: 9; (383) a ing ce consisting of amino acids 10—22 of SEQ ID NO: 9; (384) a targeting sequence consisting of amino acids 12—20 of SEQ ID NO: 9; (385) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 105; (386) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 105; (387) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 11; (388) a ing sequence consisting of amino acids 18—29 of SEQ ID NO: 11; (389) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 11; (390) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 98 ; (391) a targeting ce consisting of amino acids 1—13 of SEQ ID NO: 98 ; (392) a targeting ce consisting of amino acids 18— 31 of SEQ ID NO: 13; (393) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 13 ; (394) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 13 ; (395) a targeting sequence consisting of amino acids 21—29 of SEQ ID NO: 13; (396) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 99; (397) a targeting ce consisting of amino acids 1—13 of SEQ ID NO: 99; (398) a targeting sequence ting of amino acids 28—41 of SEQ ID NO: 15; (399) a targeting sequence consisting of amino acids 28— 39 of SEQ ID NO: 15; (400) a targeting sequence consisting of amino acids 29—41 of SEQ ID NO: 15; (401) a targeting sequence consisting of amino acids 31—39 of SEQ ID NO: 15; (402) a targeting sequence consisting of amino acids 12—25 of SEQ ID NO: 17; (403) a targeting sequence consisting of amino acids 13—25 of SEQ ID NO: 17; (404) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 100; (405) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 19; (406) a targeting ce consisting of amino acids 18— 29 of SEQ ID NO: 19; (407) a targeting ce consisting of amino acids 19—31 of SEQ ID NO: 19; (408) a targeting sequence consisting of amino acids 21—29 of SEQ ID NO: 19; (409) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 21; (410) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 21; (411) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 21; (412) a ing sequence consisting of amino acids 21—29 of SEQ ID NO: 21; (413) a targeting sequence consisting of amino acids 1— of SEQ ID NO: 101; (414) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 101 ; (415) a targeting sequence ting of amino acids 9—22 of SEQ ID NO: 23; (416) a targeting ce consisting of amino acids 9—20 of SEQ ID NO: 23; (417) a targeting sequence consisting of amino acids 10—22 of SEQ ID NO: 23; (418) a targeting sequence consisting of amino acids 12—20 of SEQ ID NO: 23; (419) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 102; (420) a targeting sequence consisting of amino acids 1— 13 of SEQ ID NO: 102; (421) a targeting sequence consisting of amino acids 9—22 of SEQ ID NO: 25; (422) a targeting sequence consisting of amino acids 9—20 of SEQ ID NO: 25 ; (423) a targeting sequence ting of amino acids 10—22 of SEQ ID NO: 25; (424) a targeting sequence consisting of amino acids 12—20 of SEQ ID NO: 25; (425) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 103; (426) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 103; (427) a targeting sequence consisting of amino acids 15— 28 of SEQ ID NO: 27; (428) a targeting sequence consisting of amino acids 15—26 of SEQ ID NO: 27; (429) a targeting sequence ting of amino acids 16—28 of SEQ ID NO: 27; (430) a targeting sequence consisting of amino acids 18—26 of SEQ ID NO: 27; (431) a targeting sequence consisting of amino acids 1—15 of SEQ ID NO: 104; (432) a targeting ce consisting of amino acids 1—13 of SEQ ID NO: 104; (433) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 33; (434) a targeting sequence consisting of amino acids 1—11 of SEQ ID NO: 33 ; (435) a ing ce consisting of amino acids 3—11 of SEQ ID NO: 33 ; (436) a targeting sequence consisting of amino acids 1—14 of SEQ ID NO: 35; (437) a targeting sequence consisting of amino acids 1—12 of SEQ ID NO: 35; (438) a targeting 2015/050807 sequence consisting of amino acids 2—14 of SEQ ID NO: 35; (439) a targeting ce consisting of amino acids 14—27 of SEQ ID NO: 43; (440) a targeting sequence consisting of amino acids 14—25 of SEQ ID NO: 43; (441) a targeting sequence consisting of amino acids 15— 27 of SEQ ID NO: 43; (442) a ing sequence ting of amino acids 20—33 of SEQ ID NO: 45 ; (443) a targeting sequence consisting of amino acids 20—31 of SEQ ID NO: 45 ; (444) a targeting sequence consisting of amino acids 21—33 of SEQ ID NO: 45; (445) a ing sequence consisting of amino acids 1—15 of SEQ ID NO: 106; (446) a targeting sequence consisting of amino acids 1—13 of SEQ ID NO: 106; (447) a targeting sequence consisting of amino acids 28—41 of SEQ ID NO: 47; (448) a targeting sequence consisting of amino acids 28— 39 of SEQ ID NO: 47; (449) a targeting sequence consisting of amino acids 18—31 of SEQ ID NO: 53; (450) a targeting sequence consisting of amino acids 18—29 of SEQ ID NO: 53 ; (451) a targeting sequence consisting of amino acids 19—31 of SEQ ID NO: 53; (452) a targeting sequence comprising amino acids 18—31 of SEQ ID NO: 61; (453) a targeting sequence comprising amino acids 18—29 of SEQ ID NO: 61; (454) a targeting sequence comprising amino acids 19—31 of SEQ ID NO: 61; (455) a targeting sequence comprising amino acids 9—22 of SEQ ID NO: 65; (456) a targeting sequence comprising amino acids 9—20 of SEQ ID NO: 65; (457) a targeting ce comprising amino acids 10—22 of SEQ ID NO: 65; (458) a targeting sequence comprising amino acids 1—15 of SEQ ID NO: 107; (459) a targeting sequence comprising amino acids 1—13 of SEQ ID NO: 107; (460) a targeting sequence comprising amino acids 12—25 of SEQ ID NO: 67; (461) a targeting sequence comprising amino acids 12—23 of SEQ ID NO: 67; (462) a ing sequence comprising amino acids 13—25 of SEQ ID NO: 67; (463) a targeting sequence comprising amino acids 15—23 of SEQ ID NO: 67; (464) a targeting sequence comprising amino acids 23—36 of SEQ ID NO: 69; (465) a targeting sequence comprising amino acids 23—34 of SEQ ID NO: 69; (466) a targeting sequence comprising amino acids 24—36 of SEQ ID NO: 69; (467) a targeting ce comprising amino acids 26—34 of SEQ ID NO: 69; (468) a targeting sequence comprising amino acids 27—40 of SEQ ID NO: 75; (469) a ing sequence comprising amino acids 27—38 of SEQ ID NO: 75; (470) a targeting sequence comprising amino acids 9—22 of SEQ ID NO: 77; (471) a targeting sequence comprising amino acids 9—20 of SEQ ID NO: 77; (472) a targeting sequence comprising amino acids 10—22 of SEQ ID NO: 77; (473) a targeting sequence sing amino acids 12—20 of SEQ ID NO: 77; (474) a targeting sequence comprising amino acids 23—36 of SEQ ID NO: 81; (475) a targeting sequence comprising amino acids 23—34 of SEQ ID NO: 81; (476) a targeting sequence comprising amino acids 24—36 of SEQ ID NO: 81; (477) a targeting sequence comprising amino acids 26—34 of SEQ ID NO: 81 ; (478) a targeting ce comprising amino acids 13—26 of SEQ ID NO: 87; (479) a targeting sequence comprising amino acids 13—24 of SEQ ID NO: 87; or (480) a targeting sequence comprising amino acids 14—26 of SEQ ID NO: Embodiment 541 is a method of embodiment 540, wherein the targeting sequence comprises: an amino acid sequence haVing at least about 50% identity with amino acids 20— of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 63%; an amino acid sequence haVing at least about 50% identity with amino acids 20— of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 72%; an amino acid sequence haVing at least about 56% identity with amino acids 20— of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 63%; an amino sequence haVing at least about 62% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 72%; an amino acid ce haVing at least about 68% identity with amino acids 20— of SEQ ID NO: 1, wherein the ty with amino acids 25—35 is at least about 8 l% ; an amino sequence haVing at least about 75% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the ty with amino acids 25—35 is at least about 72%; an amino sequence haVing at least about 75% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 8 l % ; an amino acid sequence haVing at least about 81% identity with amino acids 20— of SEQ ID NO:l, wherein the identity with amino acids 25—35 is at least about 8 l % ; or an amino acid sequence haVing at least about 81% identity with amino acids 20— of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 90%. 2015/050807 Embodiment 542 is a method of embodiment 540, wherein the targeting sequence consists of: (a) an amino acid sequence consisting of 16 amino acids and haVing at least about 43% identity with amino acids 20—35 of SEQ ID NO: 1, wherein the identity with amino acids 25—35 is at least about 54%; (b) amino acids 1—35 of SEQ ID NO: 1; (c) amino acids 20—35 of SEQ ID NO: 1; (d) SEQ ID NO: 1; (e) SEQ ID NO: 96; or (f) SEQ ID NO: 120.

Embodiment 543 is a method of ment 541, wherein the targeting sequence consists of the amino acid sequence.

Embodiment 544 is a method of embodiment 540, wherein the exosporium protein or rium protein fragment comprises an amino acid sequence haVing at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, and 122.

Embodiment 545 is a method of any one of embodiments 477—544, wherein the targeting sequence, rium protein, or exosporium protein fragment comprises the amino acid ce GXT at its carboxy terminus, wherein X is any amino acid.

Embodiment 546 is a method of any one of embodiments 477—545, wherein the targeting sequence, exosporium protein, or rium protein fragment comprises an alanine residue at the position of the targeting ce that corresponds to amino acid 20 of SEQ ID NO: 1.

Embodiment 547 is a method of any one of embodiments 477—546, wherein the targeting sequence, exosporium protein, or exosporium protein fragment r comprises a nine, serine, or threonine residue at the amino acid position immediately preceding the first amino acid of the targeting sequence, rium protein, or exosporium protein fragment or at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1. ment 548 is a method of any one of embodiments 477—547, wherein the fusion protein further comprises an amino acid linker between the targeting sequence, the rium protein, or the exosporium protein fragment and the protein or peptide of interest.

Embodiment 549 is a method of embodiment 548, wherein the linker comprises a polyalanine linker, a ycine linker, or a linker comprising a e of both alanine and glycine residues.

Embodiment 550 is a method of embodiment 548 or 549, wherein the linker comprises a se recognition site.

] Embodiment 551 is a method of any one of embodiments 447—550, wherein the fusion protein is expressed under the l of a sporulation er native to the targeting sequence, exosporium protein, or exosporium protein fragment of the fusion protein or a portion thereof.

Embodiment 552 is a method of any one of embodiments 447—551, n the fusion protein is expressed under the control of a high-expression sporulation promoter.

Embodiment 553 is a method of any embodiment 552, wherein the high- expression sporulation promoter comprises a sigma-K sporulation-specific polymerase promoter sequence. 8] Embodiment 554 is a method of any one of embodiments 551—553, wherein the sporulation promoter comprises a nucleic acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with a nucleic acid sequence of any one of SEQ ID NOs: 1.

Embodiment 555 is a method of embodiment 553 or 554, wherein the sigma- K sporulation-specific polymerase promoter sequence or sequences have 100% identity with the corresponding nucleotides of any of SEQ ID NOs: 157—23 1.

Embodiment 556 is a method for delivering proteins or peptides to an animal comprising feeding to an animal a recombinant spore-forming bacterium of any one of embodiments 147—149, 151—153, adn 156—167.

Embodiment 557 is a method of ment 556, wherein the recombinant forming ium comprises an endophytic and probiotic strain of bacteria.

Embodiment 558 is a method of embodiment 557, wherein the endophytic and probiotic strain of bacteria comprises Bacillus megaterium EE385 (NRRL B-50980), Bacillus sp. EE387 (NRRL B-50981), Bacillus circulans EE388 (NRRL 2), Bacillus subtilis EE405 (NRRL B-50978), Lysinibacillusfusiformis EE442 (NRRL B-50975), or Lysinibacillus sphaericus EE443 (NRRL B-50976), Bacillus pumilus 143 (NRRL B- 67123), or a combination thereof. 2015/050807 Embodiment 559 is a method of any one of embodiments 556—558, wherein the inant forming bacterium is comprised within a plant that is fed to the animal.

Embodiment 560 is a method of any one of embodiments 472—487 and 540— 559, wherein the plant has been processed prior to feeding to the animal.

Embodiment 561 is a method of embodiment 472—487 and 540—560, wherein the method further comprises: introducing the ytic strain of bacteria or a formulation comprising the endophytic strain of bacteria into a plant growth medium; or applying the endophytic strain of bacteria or a formulation comprising the ytic strain of bacteria to a plant, a plant seed, or an area surrounding a plant or a plant seed; n the plant fed to the animal comprises a plant grown in a plant growth medium containing the endophytic and probiotic strain of bacteria or a formulation comprising the endophytic and probiotic strain of bacteria, a plant to which the endophytic and probiotic strain of bacteria was d, a plant grown from a plant seed to which the endophytic and probiotic strain of bacteria was applied, a plant grown in an area to which the endophytic and probiotic strain of ia was applied, or a seed grown in the area to which the endophytic and probiotic strain of bacteria was applied.

Embodiment 562 is a method of any one of ments 7 and 540— 561, wherein the protein or peptide of interest comprises an enzyme. ment 563 is a method of embodiment 562, wherein the enzyme comprises a xylanase, a xylosidase, a phytase, a phosphatase, a se, a cellulase, an endoglucanase, an exogluconase, a glucanase, an amylase, a lipase, a phospholipase, a glycosylase, a galactanase, an (x-galactosidase, a B-glucosidase, an amylase, a pectinase, a biotinase, a polygalacturonase, a ligninase, or a combination thereof 8] Embodiment 564 is a method of embodiment 563, wherein the lipase comprises a phospholipase A1, a phospholipase A2, a phospholipase C, a phospholipase D, a lysophospholipase, or a combination thereof Embodiment 565 is a method of embodiment 563, wherein the amylase comprises an u-amylase or a B-amylase.

Embodiment 566 is a method of embodiment 563 wherein the enzyme comprises a xylanase or a phytase.

Embodiment 567 is a method of any one of embodiments 7 and 540— 566, wherein the animal comprises a mammal, a bird, a fish, or a crustacean.

Embodiment 568 is a method of embodiment 567, wherein the mammal comprises a sheep, goat, cow, pig, deer, alpaca, bison, camel, donkey, horse, mule, llama, , dog, or cat; wherein the bird comprises a chicken, turkey, duck, goose, quail, or pheasant; wherein the fish comprises salmon, trout, tilapia, tuna, , or a carp; or wherein the crustacean comprises a shrimp, prawn, lobster, crab, or crayfish. 3] In view of the above, it will be seen that the l objects of the invention are achieved and other advantageous results attained.

As various changes could be made in the above products, compositions, and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

Claims (10)

1. A recombinant Bacillus cereus family member that expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member; wherein the protein or peptide of interest comprises an enzyme, a plant growth ating protein or peptide, a protein or peptide that protects a plant from a pathogen, a protein or peptide that es stress resistance in a plant, a plant g protein or peptide, or a nucleic acid binding protein or peptide; and wherein the recombinant Bacillus cereus family member: (i) comprises a mutation in an ExsY gene; (ii) comprises a on in a CotE gene; (iii) expresses an ExsY protein, wherein the expression of the ExsY protein is increased as compared to the expression of the ExsY protein in a wild-type Bacillus cereus family member under the same conditions, and wherein the ExsY protein comprises a carboxy-terminal tag comprising a globular n; (iv) expresses a BclB protein, wherein the expression of the BclB protein is increased as compared to the sion of the BclB protein in a wild-type Bacillus cereus family member under the same conditions; (v) expresses a YjcB protein, wherein the expression of the YjcB protein is increased as compared to the expression of the YjcB protein in a wild-type Bacillus cereus family member under the same conditions; (vi) comprises a mutation in a CotY gene; (vii) ses a mutation in an ExsA gene; (viii) ses a mutation in a CotO gene; or (ix) a combination of any of (i) through ; and n the on in the ExsY gene, the mutation in the CotE gene, the expression of the ExsY protein, the expression of the BclB protein, the expression of the YjcB protein, the mutation in the CotY gene, the mutation in the ExsA gene, or the mutation in the CotO gene results in Bacillus cereus family member spores having an rium that is easier to remove from the spore as compared to the exosporium of a wild-type spore.

2. A recombinant Bacillus cereus family member of claim 1, wherein the recombinant Bacillus cereus family member comprises a mutation in a CotE gene, and wherein the mutation in the CotE gene comprises a knock-out of the CotE gene or a dominant negative form of the CotE gene.

3. A recombinant us cereus family member of claim 1 or 2, wherein: the recombinant Bacillus cereus family member expresses an ExsY protein, wherein the sion of the ExsY protein is increased as compared to the expression of the ExsY protein in a wild-type Bacillus cereus family member under the same conditions, and wherein the ExsY n comprises a y-terminal tag comprising a globular protein; wherein optionally: the globular protein has a molecular weight of between 25 kDa and 100 kDa; and/or the globular protein comprises a green fluorescent protein (GFP) or a variant thereof; the recombinant Bacillus cereus family member expresses a BclB protein, wherein the expression of the BclB protein is increased as compared to the expression of the BclB protein in a wild-type Bacillus cereus family member under the same conditions, and wherein the expression of the BclB protein optionally results in the ion of a fragile exosporium; the inant Bacillus cereus family member expresses a YjcB protein, wherein the expression of the YjcB protein is increased as ed to the expression of the YjcB n in a wild-type Bacillus cereus family member under the same conditions, and wherein the expression of the YjcB protein optionally causes the exosporium to form in pieces rather than in a complete structure; the inant Bacillus cereus family member comprises a mutation in a CotY gene, wherein the mutation in the CotY gene optionally comprises a knock-out of the CotY gene; the inant Bacillus cereus family member comprises a mutation in an ExsA gene, n the mutation in the ExsA gene optionally comprises a knock-out of the ExsA gene; the recombinant Bacillus cereus family member comprises a on in a CotO gene, wherein the mutation in the CotO gene comprises a knock-out of the CotO gene or a dominant ve form of the CotO gene.

4. A recombinant Bacillus cereus family member of any one of claims 1–3, wherein the recombinant Bacillus cereus family member comprises a mutation an ExsY gene, and wherein the mutation in the ExsY gene comprises a out of the ExsY gene.

5. A recombinant Bacillus cereus family member of any one of claims 1–4, wherein the targeting sequence, exosporium protein, or exosporium protein fragment comprises: (1) a targeting sequence comprising an amino acid sequence having at least about 43% identity with amino acids 20–35 of SEQ ID NO: 1, wherein the identity with amino acids 25–35 is at least about 54%; (2) a targeting sequence comprising amino acids 1–35 of SEQ ID NO: 1; (3) a targeting sequence comprising amino acids 20–35 of SEQ ID NO: 1; (4) a targeting sequence comprising SEQ ID NO: 1; (5) an exosporium protein comprising an amino acid ce having at least 85% ty with SEQ ID NO: 2; (6) a targeting sequence comprising amino acids 2–35 of SEQ ID NO: 1; (7) a targeting sequence comprising amino acids 5–35 of SEQ ID NO: 1; (8) a targeting sequence comprising amino acids 8–35 of SEQ ID NO: 1; (9) a targeting sequence comprising amino acids 10–35 of SEQ ID NO: 1; (10) a targeting sequence comprising amino acids 15–35 of SEQ ID NO: 1; (11) a targeting sequence sing amino acids 1–27 of SEQ ID NO: 3; (12) a targeting sequence comprising amino acids 12–27 of SEQ ID NO: 3; (13) a targeting sequence sing SEQ ID NO: 3; (14) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 4; (15) a targeting sequence comprising amino acids 2–27 of SEQ ID NO: 3; (16) a targeting ce comprising amino acids 5–27 of SEQ ID NO: 3; (17) a targeting sequence comprising amino acids 8–27 of SEQ ID NO: 3; (18) a targeting sequence comprising amino acids 10–27 of SEQ ID NO: 3; (19) a ing sequence comprising amino acids 1–38 of SEQ ID NO: 5; (20) a targeting sequence comprising amino acids 23–38 of SEQ ID NO: 5; (21) a targeting sequence comprising SEQ ID NO: 5; (22) an exosporium n comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 6; (23) a targeting sequence sing amino acids 2–38 of SEQ ID NO: 5; (24) a targeting sequence comprising amino acids 5–38 of SEQ ID NO: 5; (25) a targeting sequence comprising amino acids 8–38 of SEQ ID NO: 5; (26) a targeting sequence comprising amino acids 10–38 of SEQ ID NO: 5; (27) a targeting sequence comprising amino acids 15–38 of SEQ ID NO: 5; (28) a targeting sequence comprising amino acids 20–38 of SEQ ID NO: 5; (29) a targeting ce comprising amino acids 1–28 of SEQ ID NO: 7; (30) a targeting sequence comprising amino acids 13–28 of SEQ ID NO: 7; (31) a targeting ce comprising SEQ ID NO: 7; (32) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 8; (33) a targeting sequence comprising amino acids 2–28 of SEQ ID NO: 7; (34) a targeting sequence comprising amino acids 5–28 of SEQ ID NO: 7; (35) a targeting sequence comprising amino acids 8–28 of SEQ ID NO: 7; (36) a targeting sequence comprising amino acids 10–28 of SEQ ID NO: 7; (37) a targeting sequence comprising amino acids 1–24 of SEQ ID NO: 9; (38) a targeting sequence sing amino acids 9–24 of SEQ ID NO: 9; (39) a ing sequence comprising SEQ ID NO: 9; (40) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 10; (41) a targeting sequence comprising amino acids 2–24 of SEQ ID NO: 9; (42) a targeting ce comprising amino acids 5–24 of SEQ ID NO: 9; (43) a targeting sequence comprising amino acids 8–24 of SEQ ID NO: 9; (44) a targeting sequence comprising amino acids 1–33 of SEQ ID NO:11; (45) a ing sequence comprising amino acids 18–33 of SEQ ID NO: 11; (46) a targeting sequence comprising SEQ ID NO: 11; (47) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 12; (48) a targeting sequence sing amino acids 2–33 of SEQ ID NO: 11; (49) a targeting sequence comprising amino acids 5–33 of SEQ ID NO: 11; (50) a targeting sequence comprising amino acids 8–33 of SEQ ID NO: 11; (51) a ing sequence comprising amino acids 10–33 of SEQ ID NO: 11; (52) a targeting sequence comprising amino acids 15–33 of SEQ ID NO: 11; (53) a targeting sequence comprising amino acids 1–33 of SEQ ID NO: 13; (54) a targeting sequence comprising amino acids 18–33 of SEQ ID NO: 13; (55) a targeting sequence comprising SEQ ID NO:13; (56) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:14; (57) a targeting sequence sing amino acids 2–33 of SEQ ID NO: 13; (58) a targeting sequence comprising amino acids 5–33 of SEQ ID NO: 13; (59) a targeting sequence comprising amino acids 8–33 of SEQ ID NO: 13; (60) a ing sequence sing amino acids 10–33 of SEQ ID NO: 13; (61) a targeting sequence comprising amino acids 15–33 of SEQ ID NO: 13; (62) a targeting sequence comprising amino acids 1–43 of SEQ ID NO: 15; (63) a targeting sequence comprising amino acids 28–43 of SEQ ID NO: 15; (64) a targeting sequence comprising SEQ ID NO:15; (65) an exosporium protein sing an amino acid sequence having at least 85% identity with SEQ ID NO:16; (66) a ing sequence comprising amino acids 2–43 of SEQ ID NO: 15; (67) a targeting sequence comprising amino acids 5–43 of SEQ ID NO: 15; (68) a targeting sequence comprising amino acids 8–43 of SEQ ID NO: 15; (69) a targeting sequence sing amino acids 10–43 of SEQ ID NO: 15; (70) a targeting sequence comprising amino acids 15–43 of SEQ ID NO: 15; (71) a targeting sequence comprising amino acids 20–43 of SEQ ID NO: 15; (72) a targeting ce comprising amino acids 25–43 of SEQ ID NO: 15; (73) a targeting sequence comprising amino acids 1–27 of SEQ ID NO: 17; (74) a targeting sequence comprising amino acids 12–27 of SEQ ID NO: 17; (75) a targeting sequence comprising SEQ ID NO:17; (76) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:18; (77) a targeting sequence comprising amino acids 2–27 of SEQ ID NO: 17; (78) a targeting sequence comprising amino acids 5–27 of SEQ ID NO: 17; (79) a targeting ce sing amino acids 8–27 of SEQ ID NO: 17; (80) a targeting sequence sing amino acids 10–27 of SEQ ID NO: 17; (81) a targeting sequence comprising amino acids 1–33 of SEQ ID NO: 19; (82) a targeting sequence sing amino acids 18–33 of SEQ ID NO: 19; (83) a targeting sequence comprising SEQ ID NO:19; (84) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:20; (85) a targeting sequence comprising amino acids 2–33 of SEQ ID NO: 19; (86) a targeting sequence comprising amino acids 5–33 of SEQ ID NO: 19; (87) a targeting sequence comprising amino acids 8–33 of SEQ ID NO: 19; (88) a targeting sequence sing amino acids 10–33 of SEQ ID NO: 19; (89) a targeting sequence comprising amino acids 15–33 of SEQ ID NO: 19; (90) a targeting sequence sing amino acids 1–33 of SEQ ID NO: 21; (91) a targeting sequence comprising amino acids 18–33 of SEQ ID NO: 21; (92) a targeting sequence sing SEQ ID NO:21; (93) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:22; (94) a targeting sequence comprising amino acids 2–33 of SEQ ID NO: 21; (95) a targeting sequence comprising amino acids 5–33 of SEQ ID NO: 21; (96) a targeting sequence comprising amino acids 8–33 of SEQ ID NO: 21; (97) a targeting sequence sing amino acids 10–33 of SEQ ID NO: 21; (98) a targeting sequence comprising amino acids 15–33 of SEQ ID NO: 21; (99) a targeting sequence comprising amino acids 1–24 of SEQ ID NO: 23; (100) a targeting sequence sing amino acids 9–24 of SEQ ID NO: 23; (101) a targeting ce comprising SEQ ID NO:23; (102) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:24; (103) a targeting sequence comprising amino acids 2–24 of SEQ ID NO:23; (104) a targeting sequence comprising amino acids 5–24 of SEQ ID NO: 23; (105) a targeting sequence comprising amino acids 8–24 of SEQ ID NO: 23; (106) a targeting sequence comprising amino acids 1–24 of SEQ ID NO: 25; (107) a targeting sequence comprising amino acids 9–24 of SEQ ID NO: 25; (108) a targeting sequence comprising SEQ ID NO:25; (109) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:26; (110) a targeting sequence comprising amino acids 2–24 of SEQ ID NO: 25; (111) a targeting sequence comprising amino acids 5–24 of SEQ ID NO: 25; (112) a targeting sequence sing amino acids 8–24 of SEQ ID NO: 25; (113) a targeting sequence comprising amino acids 1–30 of SEQ ID NO: 27; (114) a ing sequence comprising amino acids 15–30 of SEQ ID NO: 27; (115) a ing sequence comprising SEQ ID NO:27; (116) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:28; (117) a targeting ce comprising amino acids 2–30 of SEQ ID NO: 27; (118) a targeting sequence comprising amino acids 5–30 of SEQ ID NO: 27; (119) a targeting sequence comprising amino acids 8–30 of SEQ ID NO: 27; (120) a targeting sequence comprising amino acids 10–30 of SEQ ID NO: 27; (121) a targeting sequence comprising amino acids 1–33 of SEQ ID NO: 29; (122) a targeting sequence comprising amino acids 18–33 of SEQ ID NO: 29; (123) a targeting sequence sing SEQ ID NO:29; (124) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:30; (125) a targeting sequence comprising amino acids 2–33 of SEQ ID NO: 29; (126) a ing sequence sing amino acids 5–33 of SEQ ID NO: 29; (127) a targeting sequence comprising amino acids 8–33 of SEQ ID NO: 29; (128) a ing sequence comprising amino acids 10–33 of SEQ ID NO: 29; (129) a targeting sequence comprising amino acids 15–33 of SEQ ID NO: 29; (130) a targeting sequence comprising amino acids 1–24 of SEQ ID NO: 31; (131) a targeting sequence comprising amino acids 9–24 of SEQ ID NO: 31; (132) a targeting sequence comprising SEQ ID NO:31; (133) an exosporium n comprising an amino acid sequence having at least 85% identity with SEQ ID NO:32; (134) a targeting sequence comprising amino acids 2–24 of SEQ ID NO: 31; (135) a targeting sequence comprising amino acids 5–24 of SEQ ID NO: 31; (136) a targeting sequence comprising amino acids 8–24 of SEQ ID NO: 31; (137) a targeting sequence comprising amino acids 1–15 of SEQ ID NO: 33; (138) a targeting sequence comprising SEQ ID NO:33; (139) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:34; (140) a targeting sequence comprising amino acids 1–16 of SEQ ID NO: 35; (141) a targeting sequence comprising SEQ ID NO:35; (142) an exosporium protein comprising an amino acid sequence having at least 85% ty with SEQ ID NO:36; (143) a targeting sequence comprising amino acids 1–29 of SEQ ID NO:43; (144) a targeting sequence comprising amino acids 14–29 of SEQ ID NO: 43; (145) a targeting sequence comprising SEQ ID NO: 43; (146) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 44; (147) a targeting sequence comprising amino acids 2–29 of SEQ ID NO: 43; (148) a targeting ce comprising amino acids 5–29 of SEQ ID NO: 43; (149) a targeting sequence comprising amino acids 8–29 of SEQ ID NO: 43; (150) a targeting ce comprising amino acids 10–29 of SEQ ID NO: 43; (151) a targeting ce comprising amino acids 1–35 of SEQ ID NO: 45; (152) a targeting sequence comprising amino acids 20–35 of SEQ ID NO: 45; (153) a targeting sequence comprising SEQ ID NO: 45; (154) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 46; (155) a targeting sequence comprising amino acids 2–35 of SEQ ID NO: 45; (156) a targeting sequence sing amino acids 5–35 of SEQ ID NO: 45; (157) a targeting sequence comprising amino acids 8–35 of SEQ ID NO: 45; (158) a targeting sequence comprising amino acids 10–35 of SEQ ID NO: 45; (159) a targeting sequence sing amino acids 15–35 of SEQ ID NO: 45; (160) a targeting ce comprising amino acids 1–43 of SEQ ID NO: 47; (161) a targeting sequence comprising amino acids 28–43 of SEQ ID NO: 47; (162) a targeting sequence comprising SEQ ID NO: 47; (163) an rium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 48; (164) a targeting sequence sing amino acids 2–43 of SEQ ID NO: 47; (165) a targeting sequence comprising amino acids 5–43 of SEQ ID NO: 47; (166) a targeting sequence comprising amino acids 8–43 of SEQ ID NO: 47; (167) a targeting sequence comprising amino acids 10–43 of SEQ ID NO: 47; (168) a targeting sequence comprising amino acids 15–43 of SEQ ID NO: 47; (169) a targeting sequence comprising amino acids 20–43 of SEQ ID NO: 47; (170) a targeting sequence comprising amino acids 25–43 of SEQ ID NO: 47; (171) a targeting sequence comprising amino acids 1–32 of SEQ ID NO: 49; (172) a targeting sequence comprising amino acids 17–32 of SEQ ID NO: 49; (173) a targeting ce comprising SEQ ID NO: 49; (174) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 50; (175) a ing sequence comprising amino acids 2–32 of SEQ ID NO: 49; (176) a targeting sequence comprising amino acids 5–32 of SEQ ID NO: 49; (177) a targeting sequence comprising amino acids 8–32 of SEQ ID NO: 49; (178) a targeting sequence comprising amino acids 10–32 of SEQ ID NO: 49; (179) a targeting ce comprising amino acids 15–32 of SEQ ID NO: 49; (180) a targeting sequence comprising amino acids 1–33 of SEQ ID NO: 51; (181) a targeting sequence comprising amino acids 18–33 of SEQ ID NO: 51; (182) a targeting sequence comprising SEQ ID NO: 51; (183) an rium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 52; (184) a targeting sequence comprising amino acids 2–33 of SEQ ID NO: 51; (185) a targeting sequence comprising amino acids 5–33 of SEQ ID NO: 51; (186) a targeting sequence comprising amino acids 8–33 of SEQ ID NO: 51; (187) a targeting sequence sing amino acids 10–33 of SEQ ID NO: 51; (188) a ing sequence comprising amino acids 15–33 of SEQ ID NO: 51; (189) a ing sequence comprising amino acids 1–33 of SEQ ID NO: 53; (190) a ing sequence comprising amino acids 18–33 of SEQ ID NO: 53; (191) a targeting sequence comprising SEQ ID NO: 53; (192) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 54; (193) a targeting sequence comprising amino acids 2–33 of SEQ ID NO: 53; (194) a targeting sequence comprising amino acids 5–33 of SEQ ID NO: 53; (195) a targeting sequence comprising amino acids 8–33 of SEQ ID NO: 53; (196) a targeting sequence comprising amino acids 10–33 of SEQ ID NO: 53; (197) a targeting sequence comprising amino acids 15–33 of SEQ ID NO: 53; (198) a targeting sequence comprising amino acids 1–30 of SEQ ID NO: 55; (199) a targeting sequence comprising amino acids 15–30 of SEQ ID NO: 55; (200) a targeting sequence comprising SEQ ID NO: 55; (201) an exosporium protein sing an amino acid sequence having at least 85% identity with SEQ ID NO: 56; (202) a targeting sequence sing amino acids 2–30 of SEQ ID NO: 55; (203) a targeting sequence comprising amino acids 5–30 of SEQ ID NO: 55; (204) a targeting sequence comprising amino acids 8–30 of SEQ ID NO: 55; (205) a targeting sequence sing amino acids 10–30 of SEQ ID NO: 55; (206) a targeting sequence comprising amino acids 1–130 of SEQ ID NO: 57; (207) a targeting sequence sing amino acids 115–130 of SEQ ID NO: 57; (208) a targeting sequence comprising SEQ ID NO: 57; (209) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 58; (210) a targeting ce comprising amino acids 2–130 of SEQ ID NO: 57; (211) a targeting sequence comprising amino acids 5–130 of SEQ ID NO: 57; (212) a targeting sequence comprising amino acids 10–130 of SEQ ID NO: 57; (213) a targeting sequence comprising amino acids 20–130 of SEQ ID NO: 57; (214) a targeting ce comprising amino acids 30–130 of SEQ ID NO: 57; (215) a targeting sequence comprising amino acids 40–130 of SEQ ID NO: 57; (216) a ing sequence comprising amino acids 50–130 of SEQ ID NO: 57; (217) a targeting sequence comprising amino acids 60–130 of SEQ ID NO: 57; (218) a targeting sequence comprising amino acids 70–130 of SEQ ID NO: 57; (219) a targeting sequence comprising amino acids 80–130 of SEQ ID NO: 57; (220) a targeting sequence comprising amino acids 90–130 of SEQ ID NO: 57; (221) a targeting sequence comprising amino acids 100–130 of SEQ ID NO: 57; (222) a targeting sequence comprising amino acids 110–130 of SEQ ID NO: 57; (223) an exosporium protein fragment comprising an amino acid ce having at least 85% identity with SEQ ID NO: 95; (224) a targeting sequence comprising SEQ ID NO: 96; (225) a targeting ce comprising SEQ ID NO: 97; (226) a targeting sequence sing SEQ ID NO: 98; (227) a targeting sequence comprising SEQ ID NO: 99; (228) a targeting sequence comprising SEQ ID NO: 100; (229) a targeting sequence comprising SEQ ID NO: 101; (230) a targeting sequence comprising SEQ ID NO: 102; (231) a targeting sequence comprising SEQ ID NO: 103; (232) a targeting sequence comprising SEQ ID NO: 104; (233) a targeting sequence comprising SEQ ID NO: 105; (234) a ing sequence comprising SEQ ID NO: 106; (235) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (236) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 109; (237) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 110; (238) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 111; (239) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 112; (240) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 113; (241) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 114; (242) an exosporium protein comprising an amino acid sequence having at least 85% ty with SEQ ID NO: 115; (243) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 116; (244) an exosporium protein comprising an amino acid sequence having at least 85% ty with SEQ ID NO: 117; (245) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 118; (246) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 119; (247) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 120; (248) an exosporium n comprising an amino acid ce having at least 85% identity with SEQ ID NO: 121; (249) a targeting ce comprising amino acids 22–31 of SEQ ID NO: 1; (250) a targeting sequence comprising amino acids 22–33 of SEQ ID NO: 1; (251) a ing ce comprising amino acids 20–31 of SEQ ID NO: 1; (252) a targeting sequence comprising amino acids 14–23 of SEQ ID NO: 3; (253) a targeting sequence comprising amino acids 14–25 of SEQ ID NO: 3; (254) a targeting sequence comprising amino acids 12–23 of SEQ ID NO: 3; (255) a targeting sequence comprising amino acids 1–30 of SEQ ID NO: 59; (256) a targeting ce comprising SEQ ID NO: 59; (257) an exosporium protein comprising an amino acid sequence having at least 85% ty with SEQ ID NO: 60; (258) a targeting sequence comprising amino acids 2–30 of SEQ ID NO: 59; (259) a targeting sequence comprising amino acids 4–30 of SEQ ID NO: 59; (260) a targeting sequence comprising amino acids 6–30 of SEQ ID NO: 59; (261) a targeting sequence comprising amino acids 1–33 of SEQ ID NO: 61; (262) a ing sequence sing amino acids 18–33 of SEQ ID NO: 61; (263) a targeting sequence comprising SEQ ID NO: 61; (264) an exosporium protein comprising an amino acid sequence having at least 85% ce identity with SEQ ID NO: 62; (265) a targeting sequence comprising amino acids 2–33 of SEQ ID NO: 61; (266) a targeting sequence comprising amino acids 5–33 of SEQ ID NO: 61; (267) a targeting sequence comprising amino acids 10–33 of SEQ ID NO: 61; (268) a targeting sequence comprising amino acids 15–33 of SEQ ID NO: 61; (269) a targeting sequence comprising amino acids 1–35 of SEQ ID NO: 63; (270) a targeting sequence sing SEQ ID NO: 63; (271) an rium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 64; (272) a targeting sequence comprising amino acids 2–35 of SEQ ID NO: 63; (273) a targeting sequence comprising amino acids 5–35 of SEQ ID NO: 63; (274) a targeting sequence comprising amino acids 8–35 of SEQ ID NO: 63; (275) a targeting sequence comprising amino acids 10–35 of SEQ ID NO: 63; (276) a targeting sequence comprising amino acids 15–35 of SEQ ID NO: 63; (277) a targeting sequence sing amino acids 1–24 of SEQ ID NO: 65; (278) a targeting sequence sing amino acids 9–24 of SEQ ID NO: 65; (279) a targeting sequence comprising SEQ ID NO: 65; (280) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 66; (281) a targeting sequence comprising SEQ ID NO: 107; (282) a ing sequence comprising amino acids 2–24 of SEQ ID NO: 65; (283) a targeting sequence comprising amino acids 5–24 of SEQ ID NO: 65; (284) a targeting sequence comprising amino acids 1–27 of SEQ ID NO: 67; (285) a targeting ce comprising amino acids 12–27 of SEQ ID NO: 67; (286) a ing sequence comprising SEQ ID NO: 67; (287) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 68; (288) an targeting sequence comprising amino acids 2–27 of SEQ ID NO: 67; (289) a targeting sequence comprising amino acids 5–27 of SEQ ID NO: 67; (290) a targeting sequence comprising amino acids 10–27 of SEQ ID NO: 67; (291) a targeting sequence comprising amino acids 1–38 of SEQ ID NO: 69; (292) a targeting ce comprising amino acids 23–38 of SEQ ID NO: 69; (293) a targeting sequence comprising SEQ ID NO: 69; (294) an exosporium protein comprising an amino acid ce having at least 85% identity with SEQ ID NO: 70; (295) a targeting sequence comprising amino acids 2–38 of SEQ ID NO: 69; (296) a targeting ce comprising amino acids 5–38 of SEQ ID NO: 69; (297) a targeting sequence comprising amino acids 10–38 of SEQ ID NO: 69; (298) a targeting sequence comprising amino acids 15–38 of SEQ ID NO: 69; (299) an exosporium protein comprising SEQ ID NO: 72; (300) a targeting sequence sing SEQ ID NO: 73; (301) an exosporium protein comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 74; (302) a targeting ce comprising amino acids 1–42 of SEQ ID NO: 75; (303) a targeting sequence comprising amino acids 27–42 of SEQ ID NO: 75; (304) a targeting sequence comprising SEQ ID NO: 75; (305) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 76; (306) a targeting sequence comprising amino acids 2–42 of SEQ ID NO: 75; (307) a targeting sequence comprising amino acids 5–42 of SEQ ID NO: 75; (308) a targeting sequence comprising amino acids 10–42 of SEQ ID NO: 75; (309) a targeting sequence comprising amino acids 15–42 of SEQ ID NO: 75; (310) a targeting sequence comprising amino acids 20–42 of SEQ ID NO: 75; (311) a targeting sequence comprising amino acids 25–42 of SEQ ID NO: 75; (312) a targeting sequence comprising amino acids 1–24 of SEQ ID NO: 77; (313) a targeting sequence sing amino acids 9–24 of SEQ ID NO: 77; (314) a targeting sequence comprising SEQ ID NO: 77; (315) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 78; (316) a targeting sequence comprising amino acids 2–24 of SEQ ID NO: 77; (317) a targeting sequence comprising amino acids 5–24 of SEQ ID NO: 77; (318) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 80; (319) a targeting sequence comprising amino acids 1–38 of SEQ ID NO: 81; (320) a targeting sequence comprising amino acids 23–38 of SEQ ID NO: 81; (321) a ing sequence sing SEQ ID NO: 81; (322) an exosporium protein sing an amino acid sequence having at least 85% identity with SEQ ID NO: 82; (323) a targeting sequence comprising amino acids 2–38 of SEQ ID NO: 81; (324) a targeting sequence comprising amino acids 5–38 of SEQ ID NO: 81; (325) a targeting sequence comprising amino acids 10–38 of SEQ ID NO: 81; (326) a targeting sequence sing amino acids 15–38 of SEQ ID NO: 81; (327) a targeting sequence comprising amino acids 20–38 of SEQ ID NO: 81; (328) a ing sequence comprising amino acids 1–34 of SEQ ID NO: 83; (329) a targeting sequence comprising SEQ ID NO: 83; (330) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 84; (331) an rium n comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 86; (332) a targeting sequence comprising amino acids 1–28 of SEQ ID NO: 87; (333) a targeting sequence comprising amino acids 13–28 of SEQ ID NO: 87; (334) a targeting sequence comprising SEQ ID NO: 87; (335) an exosporium protein sing an amino acid ce having at least 85% identity with SEQ ID NO: 88; (336) a targeting sequence comprising amino acids 2–28 of SEQ ID NO: 87; (337) a targeting sequence comprising amino acids 5–28 of SEQ ID NO: 87; (338) a targeting sequence comprising amino acids 10–28 of SEQ ID NO: 87; (339) a targeting ce comprising amino acids 1–28 of SEQ ID NO: 89; (340) a targeting sequence comprising SEQ ID NO: 89; (341) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 90; (342) a targeting sequence comprising amino acids 2–28 of SEQ ID NO: 89; (343) a targeting sequence sing amino acids 5–28 of SEQ ID NO: 89; (344) a targeting sequence sing amino acids 10–28 of SEQ ID NO: 89; (345) a targeting sequence comprising amino acids 1–93 of SEQ ID NO: 91; (346) a targeting sequence sing SEQ ID NO: 91; (347) an exosporium protein sing an amino acid sequence having at least 85% identity with SEQ ID NO: 92; (348) a targeting sequence sing amino acids 2–93 of SEQ ID NO: 91; (349) a targeting sequence comprising amino acids 10–93 of SEQ ID NO: 91; (350) a targeting sequence comprising amino acids 20–93 of SEQ ID NO: 91; (351) a targeting sequence comprising amino acids 30–93 of SEQ ID NO: 91; (352) a targeting sequence comprising amino acids 40–93 of SEQ ID NO: 91; (353) a targeting sequence comprising amino acids 50–93 of SEQ ID NO: 91; (354) a targeting sequence comprising amino acids 60–93 of SEQ ID NO: 91; (355) a targeting sequence comprising amino acids 1–130 of SEQ ID NO: 93; (356) a targeting sequence comprising SEQ ID NO: 93; (357) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 94; (358) a targeting sequence comprising amino acids 2–130 of SEQ ID NO: 93; (359) a targeting sequence comprising amino acids 10–130 of SEQ ID NO: 93; (360) a targeting sequence comprising amino acids 20–130 of SEQ ID NO: 93; (361) a targeting sequence comprising amino acids 30–130 of SEQ ID NO: 93; (362) an exosporium protein sing an amino acid sequence having at least 85% identity with SEQ ID NO: 122; (363) a targeting sequence consisting of amino acids 20–33 of SEQ ID NO: 1; (364) a targeting sequence ting of amino acids 21–33 of SEQ ID NO: 1; (365) a targeting sequence consisting of amino acids 23–31 of SEQ ID NO: 1; (366) a targeting ce consisting of amino acids 1–15 of SEQ ID NO: 96; (367) a targeting sequence ting of amino acids 1–13 of SEQ ID NO: 96; (368) a ing sequence consisting of amino acids 12–25 of SEQ ID NO: 3; (369) a targeting sequence consisting of amino acids 13–25 of SEQ ID NO: 3; (370) a targeting sequence consisting of amino acids 15–23 of SEQ ID NO: 3; (371) a targeting sequence ting of amino acids 1–15 of SEQ ID NO: 97; (372) a targeting sequence ting of amino acids 1–13 of SEQ ID NO: 98; (373) a targeting sequence consisting of amino acids 23–36 of SEQ ID NO: 5; (374) a targeting sequence consisting of amino acids 23–34 of SEQ ID NO: 5; (375) a targeting sequence consisting of amino acids 24–36 of SEQ ID NO: 5; (376) a targeting sequence consisting of amino acids 26–34 of SEQ ID NO: 5; (377) a targeting sequence consisting of amino acids 13–26 of SEQ ID NO: 7; (378) a targeting sequence consisting of amino acids 13–24 of SEQ ID NO: 7; (379) a targeting sequence consisting of amino acids 14–26 of SEQ ID NO: 7; (380) a targeting sequence consisting of amino acids 16–24 of SEQ ID NO: 7; (381) a targeting sequence consisting of amino acids 9–22 of SEQ ID NO: 9; (382) a targeting sequence consisting of amino acids 9–20 of SEQ ID NO: 9; (383) a targeting sequence consisting of amino acids 10–22 of SEQ ID NO: 9; (384) a targeting sequence consisting of amino acids 12–20 of SEQ ID NO: 9; (385) a targeting sequence consisting of amino acids 1–15 of SEQ ID NO: 105; (386) a targeting sequence consisting of amino acids 1–13 of SEQ ID NO: 105; (387) a targeting ce consisting of amino acids 18–31 of SEQ ID NO: 11; (388) a targeting ce consisting of amino acids 18–29 of SEQ ID NO: 11; (389) a targeting sequence consisting of amino acids 19–31 of SEQ ID NO: 11; (390) a targeting sequence consisting of amino acids 1–15 of SEQ ID NO: 98; (391) a targeting sequence consisting of amino acids 1–13 of SEQ ID NO: 98; (392) a targeting ce ting of amino acids 18–31 of SEQ ID NO: 13; (393) a targeting ce ting of amino acids 18–29 of SEQ ID NO: 13; (394) a targeting sequence consisting of amino acids 19–31 of SEQ ID NO: 13; (395) a targeting sequence consisting of amino acids 21–29 of SEQ ID NO: 13; (396) a targeting sequence consisting of amino acids 1–15 of SEQ ID NO: 99; (397) a targeting sequence consisting of amino acids 1–13 of SEQ ID NO: 99; (398) a targeting sequence consisting of amino acids 28–41 of SEQ ID NO: 15; (399) a targeting sequence consisting of amino acids 28–39 of SEQ ID NO: 15; (400) a targeting sequence consisting of amino acids 29–41 of SEQ ID NO: 15; (401) a targeting sequence consisting of amino acids 31–39 of SEQ ID NO: 15; (402) a targeting sequence consisting of amino acids 12–25 of SEQ ID NO: 17; (403) a targeting sequence consisting of amino acids 13–25 of SEQ ID NO: 17; (404) a targeting sequence consisting of amino acids 1–15 of SEQ ID NO: 100; (405) a targeting sequence consisting of amino acids 18–31 of SEQ ID NO: 19; (406) a targeting ce consisting of amino acids 18–29 of SEQ ID NO: 19; (407) a targeting sequence ting of amino acids 19–31 of SEQ ID NO: 19; (408) a targeting sequence consisting of amino acids 21–29 of SEQ ID NO: 19; (409) a targeting sequence consisting of amino acids 18–31 of SEQ ID NO: 21; (410) a targeting sequence consisting of amino acids 18–29 of SEQ ID NO: 21; (411) a targeting sequence consisting of amino acids 19–31 of SEQ ID NO: 21; (412) a targeting sequence consisting of amino acids 21–29 of SEQ ID NO: 21; (413) a targeting sequence consisting of amino acids 1–15 of SEQ ID NO: 101; (414) a targeting sequence consisting of amino acids 1–13 of SEQ ID NO: 101; (415) a ing sequence consisting of amino acids 9–22 of SEQ ID NO: 23; (416) a targeting sequence ting of amino acids 9–20 of SEQ ID NO: 23; (417) a targeting sequence consisting of amino acids 10–22 of SEQ ID NO: 23; (418) a targeting sequence ting of amino acids 12–20 of SEQ ID NO: 23; (419) a targeting sequence consisting of amino acids 1–15 of SEQ ID NO: 102; (420) a targeting sequence consisting of amino acids 1–13 of SEQ ID NO: 102; (421) a targeting sequence consisting of amino acids 9–22 of SEQ ID NO: 25; (422) a targeting sequence consisting of amino acids 9–20 of SEQ ID NO: 25; (423) a targeting sequence consisting of amino acids 10–22 of SEQ ID NO: 25; (424) a targeting sequence consisting of amino acids 12–20 of SEQ ID NO: 25; (425) a targeting sequence consisting of amino acids 1–15 of SEQ ID NO: 103; (426) a targeting sequence consisting of amino acids 1–13 of SEQ ID NO: 103; (427) a targeting sequence consisting of amino acids 15–28 of SEQ ID NO: 27; (428) a targeting sequence consisting of amino acids 15–26 of SEQ ID NO: 27; (429) a targeting sequence consisting of amino acids 16–28 of SEQ ID NO: 27; (430) a targeting sequence consisting of amino acids 18–26 of SEQ ID NO: 27; (431) a targeting sequence consisting of amino acids 1–15 of SEQ ID NO: 104; (432) a targeting sequence consisting of amino acids 1–13 of SEQ ID NO: 104; (433) a targeting sequence consisting of amino acids 1–13 of SEQ ID NO: 33; (434) a ing sequence consisting of amino acids 1–11 of SEQ ID NO: 33; (435) a targeting ce consisting of amino acids 3–11 of SEQ ID NO: 33; (436) a targeting sequence consisting of amino acids 1–14 of SEQ ID NO: 35; (437) a targeting sequence consisting of amino acids 1–12 of SEQ ID NO: 35; (438) a targeting sequence consisting of amino acids 2–14 of SEQ ID NO: 35; (439) a targeting sequence consisting of amino acids 14–27 of SEQ ID NO: 43; (440) a targeting ce consisting of amino acids 14–25 of SEQ ID NO: 43; (441) a targeting sequence consisting of amino acids 15–27 of SEQ ID NO: 43; (442) a targeting sequence consisting of amino acids 20–33 of SEQ ID NO: 45; (443) a targeting ce consisting of amino acids 20–31 of SEQ ID NO: 45; (444) a targeting sequence ting of amino acids 21–33 of SEQ ID NO: 45; (445) a targeting sequence consisting of amino acids 1–15 of SEQ ID NO: 106; (446) a targeting ce consisting of amino acids 1–13 of SEQ ID NO: 106; (447) a targeting sequence consisting of amino acids 28–41 of SEQ ID NO: 47; (448) a targeting sequence ting of amino acids 28–39 of SEQ ID NO: 47; (449) a targeting sequence consisting of amino acids 18–31 of SEQ ID NO: 53; (450) a targeting sequence consisting of amino acids 18–29 of SEQ ID NO: 53; (451) a targeting ce consisting of amino acids 19–31 of SEQ ID NO: 53; (452) a ing sequence comprising amino acids 18–31 of SEQ ID NO: 61; (453) a targeting sequence comprising amino acids 18–29 of SEQ ID NO: 61; (454) a targeting sequence comprising amino acids 19–31 of SEQ ID NO: 61; (455) a targeting sequence comprising amino acids 9–22 of SEQ ID NO: 65; (456) a ing sequence comprising amino acids 9–20 of SEQ ID NO: 65; (457) a targeting sequence comprising amino acids 10–22 of SEQ ID NO: 65; (458) a targeting ce comprising amino acids 1–15 of SEQ ID NO: 107; (459) a targeting sequence comprising amino acids 1–13 of SEQ ID NO: 107; (460) a targeting sequence comprising amino acids 12–25 of SEQ ID NO: 67; (461) a targeting ce comprising amino acids 12–23 of SEQ ID NO: 67; (462) a targeting sequence comprising amino acids 13–25 of SEQ ID NO: 67; (463) a targeting sequence comprising amino acids 15–23 of SEQ ID NO: 67; (464) a targeting sequence comprising amino acids 23–36 of SEQ ID NO: 69; (465) a targeting ce comprising amino acids 23–34 of SEQ ID NO: 69; (466) a ing ce comprising amino acids 24–36 of SEQ ID NO: 69; (467) a targeting sequence comprising amino acids 26–34 of SEQ ID NO: 69; (468) a targeting sequence comprising amino acids 27–40 of SEQ ID NO: 75; (469) a targeting sequence comprising amino acids 27–38 of SEQ ID NO: 75; (470) a targeting sequence sing amino acids 9–22 of SEQ ID NO: 77; (471) a targeting sequence comprising amino acids 9–20 of SEQ ID NO: 77; (472) a targeting sequence comprising amino acids 10–22 of SEQ ID NO: 77; (473) a targeting sequence comprising amino acids 12–20 of SEQ ID NO: 77; (474) a targeting sequence comprising amino acids 23–36 of SEQ ID NO: 81; (475) a targeting sequence comprising amino acids 23–34 of SEQ ID NO: 81; (476) a targeting sequence comprising amino acids 24–36 of SEQ ID NO: 81; (477) a targeting sequence comprising amino acids 26–34 of SEQ ID NO: 81; (478) a targeting sequence comprising amino acids 13–26 of SEQ ID NO: 87; (479) a targeting sequence comprising amino acids 13–24 of SEQ ID NO: 87; or (480) a targeting sequence comprising amino acids 14–26 of SEQ ID NO: 87.

6. A inant Bacillus cereus family member of claim 5, wherein the ing sequence comprises an amino acid sequence having at least about 50% identity with amino acids 20–35 of SEQ ID NO: 1, wherein the identity with amino acids 25–35 is at least about 63%.

7. A recombinant Bacillus cereus family member of claim 5, wherein the targeting sequence comprises an amino acid sequence having at least about 81% identity with amino acids 20–35 of SEQ ID NO: 1, wherein the identity with amino acids 25–35 is at least about 90%.

8. A recombinant Bacillus cereus family member of claim 5, wherein the ing sequence or exosporium protein comprises: (a) an amino acid sequence ting of 16 amino acids and having at least about 43% identity with amino acids 20–35 of SEQ ID NO: 1, wherein the identity with amino acids 25–35 is at least about 54%; (b) amino acids 1–35 of SEQ ID NO: 1; (c) amino acids 20–35 of SEQ ID NO: 1; (d) SEQ ID NO: 1; (e) SEQ ID NO: 96; or (f) SEQ ID NO: 120.

9. A recombinant Bacillus cereus family member of claim 5, wherein the exosporium protein or an exosporium protein fragment comprises an amino acid sequence having at least 95% ty with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, or 122.

10. A recombinant Bacillus cereus family member of any one of claims 1–9, wherein the targeting sequence, exosporium protein, or exosporium protein fragment: ses the amino acid sequence GXT at its carboxy terminus, wherein X is any amino acid; and/or further comprises a methionine, serine, or threonine residue at the amino acid position immediately preceding the first amino acid of the targeting sequence, exosporium protein, or