RU2771828C2 - Fused proteins, recombinant bacteria and methods for using recombinant bacteria - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: recombinant representative of Bacillus cereus family is described that expresses fused protein containing: (A) protein or peptide of interest; and (B) targeting sequence, exosporium protein or fragment of exosporium protein that targets fused protein in exosporium of the recombinant representative of Bacillus cereus family. A composition is also described containing exosporium fragments originating from spores of the recombinant representative of Bacillus cereus family and carrier acceptable from agricultural point of view, wherein the composition stimulates plant growth, protects a plant from pathogen and/or increases plant resistance to stressful effects. Besides, a plant seed is presented coated with exosporium fragments originating from spores of the recombinant representative of Bacillus cereus family. Methods for the stimulation of plant growth, protection of a plant from pathogen or increase in plant resistance to stressful effects, immobilization of exosporium fragments on a plant, stimulation of germination of a plant seed, delivery of nucleic acids to a plant, protection of a plant from pathogen or increase in plant resistance to stressful effects, including the use of exosporium fragments on a plant, plant seed or area surrounding the plant or plant seed, are described, where exosporium fragments originate from spores of the recombinant representative of Bacillus cereus family according to the above-described and contain fused protein, and fused protein contains protein or peptide.

EFFECT: invention expands the arsenal of means for plant protection means and increase in seed germination.

121 cl, 15 dwg, 75 tbl, 69 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US provisional application No. 62/051885, filed September 17, 2014, the contents of which are incorporated herein by reference in their entirety.

LINK TO THE SEQUENCE LIST PROVIDED IN ELECTRONIC FORM

[0002] An official copy of the sequence listing is provided in electronic form via EFS-Web as an ASCII sequence listing in a file called "3005.WO Gene Sequence Listing.txt", created on September 10, 2015 and having a size of 488 kilobytes, and it filed along with the description. The sequence listing contained in this document in ASCII format is part of the description and is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates primarily to fusion proteins containing a targeting sequence, exospore protein or exospore protein fragment, which target the fusion protein to the exospore of a member of the Bacillus cereus family. The invention also relates to recombinant members of the Bacillus cereus family expressing such fusion proteins, to compositions containing recombinant members of the Bacillus cereus family, to seeds coated with recombinant members of the Bacillus cereus family, and to methods of using recombinant members of the Bacillus cereus family (for example, to stimulate growth plants, protecting plants from pathogens, increasing plant resistance to stress, immobilizing spores of a recombinant member of the Bacillus cereus family on a plant, stimulating plant seed germination, and delivering nucleic acids to plants). In addition, the invention relates to recombinant members of the Bacillus cereus family that overexpress a protease or nuclease, where overexpression of the protease or nuclease partially or completely inactivates spores of a member of the Bacillus cereus family or increases the sensitivity of spores to physical or chemical inactivation. In addition, the present invention relates to recombinant members of the Bacillus cereus family that overexpress exosporium proteins, to seeds coated with such recombinant members of the Bacillus cereus family, and to methods of using such recombinant members of the Bacillus cereus family (for example, to stimulate plant growth, increase plant resistance stress and protect plants from pathogens).

[0004] In addition, the invention relates to various modifications of recombinant members of the Bacillus cereus family that express fusion proteins, including: (i) overexpression of modulator proteins that modulate the expression of a fusion protein in recombinant members of Bacillus cereus ; (ii) genetic inactivation of recombinant members of the Bacillus cereus family; and (iii) mutations or other genetic changes in recombinant members of the Bacillus cereus family that allow the collection of exospore fragments containing the fusion protein. The invention also relates to various methods of using exosporium fragments.

[0005] In addition, the invention relates to fusion proteins comprising a spore coat protein and a protein or peptide of interest, to recombinant bacteria that express such fusion proteins, to seeds coated with such recombinant bacteria, and to methods of using such recombinant bacteria (for example , to stimulate plant growth, protect plants from pathogens, increase plant resistance to stress, immobilize recombinant bacterial spores on the plant, stimulate plant seed germination and deliver nucleic acids to plants).

[0006] In addition, the present invention relates to biologically pure bacterial cultures of new strains of bacteria.

[0007] In addition, the present invention relates to plant seeds coated with an enzyme that catalyzes the formation of nitric oxide, or superoxide dismutase, or a recombinant spore-forming bacterium that overexpresses an enzyme that catalyzes the formation of nitric oxide, or superoxide dismutase.

[0008] The invention also relates to methods for delivering beneficial bacteria and enzymes or vaccines to animals and other uses.

STATE OF THE ART TO which THE INVENTION RELATES

[0009] In the zone surrounding the roots of a plant, there is an area called the rhizosphere. In the rhizosphere, bacteria, fungi, and other organisms compete for nutrients and for binding to the plant's root structures. The rhizosphere can be occupied by both harmful and beneficial bacteria and fungi. Bacteria, fungi, and plant root systems can be affected by the action of peptides, enzymes, and other proteins in the rhizosphere. Supplementing soil or treating plants with some of these peptides, enzymes, or other proteins may have beneficial effects on the overall population of beneficial soil bacteria and fungi, create an overall healthier soil environment for plant growth, improve plant growth, and provide plant protection 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 hampered by the short lifespan of enzymes, proteins, and peptides in soil. In addition, the prevalence of proteases naturally present in the soil leads to the degradation of proteins in the soil. The environment around the roots of a plant (rhizosphere) is a unique mixture of bacteria, fungi, nutrients and roots that has qualities that are different from native soil. The symbiotic relationship between these organisms is unique and can be changed for the better by incorporating exogenous proteins. The high concentration of fungi and bacteria in the rhizosphere causes even more degradation of proteins due to abnormally high levels of proteases and other elements harmful to proteins in the soil. In addition, enzymes and other proteins introduced into the soil can quickly leach out of plant roots.

[0010] Thus, there is a need in the art for a method for efficiently delivering peptides, enzymes, and other proteins to plants (eg, plant root systems) and for extending the time period during which such molecules remain active. Moreover, there is a need in the art for a method for selectively targeting such peptides, enzymes and proteins to the rhizosphere, and in particular plant leaves and plant roots.

SUMMARY OF THE INVENTION

[0011] The features of the present invention are defined in the appended claims and in the list of embodiments provided below in the section titled "EMBODIMENTS". Other objectives and features will become apparent in part and are described in part below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGS. 1A and 1B show an amino acid sequence alignment of the N-terminal portion of the BclA of Bacillus anthracis strain Sterne with the corresponding region from various exospore proteins of the Bacillus cereus family.

[0013] Figure 2 shows exemplary fluorescence microscopy results for the expression of fusion proteins containing various exospore proteins associated with the mCherry reporter on exospores of a recombinant member of the Bacillus cereus family.

[0014] Figure 3 shows data showing recombinant Bacillus thuringiensis BT013A spores expressing a fusion protein containing a DNA binding protein.

[0015] Figure 4 is a transmission electron microscopy photograph showing exospore fragments and spores of a member of the Bacillus cereus family that have lost exospores obtained using a recombinant member of the Bacillus cereus family having its CotE gene knockout.

[0016] Figure 5 is an SDS-PAGE gel photograph showing a standard protein marker (lane 1) and exospore fragment proteins generated using a recombinant member of the Bacillus cereus family having a knockout mutation in its CotE gene (lane 2).

[0017] Figure 6 presents data illustrating acid phosphatase enzymatic activity in exospore fragments derived from a member of the Bacillus cereus family having a knockout mutation of its CotE gene.

[0018] Figure 7 presents data illustrating that a member of the family Bacillus cereus EE349 reduces herbicide inhibitory effects on lentil root length.

[0019] Figure 8 presents data illustrating the increased phosphatase activity of a member of the Bacillus cereus family modified to overexpress acid phosphatase (AcpC).

[0020] Figure 9 shows data demonstrating endoglucanase activity in recombinant Bacillus thuringiensis spores expressing the CotC-endoglucanase fusion protein.

[0021] Figure 10 shows bright field and fluorescence microscopy images demonstrating RNA detection on the surface of recombinant B. thuringiensis spores expressing a fusion protein containing amino acids 20-35 of SEQ ID NO: 1 and SspC associated with either single-stranded RNA (ssRNA) , or with double-stranded RNA (dsRNA).

[0022] FIG. 11 is a photograph demonstrating the effects of MIR319 microRNA on soybean root height and development after being delivered to soybean plants using recombinant B. thuringiensis spores expressing a fusion protein containing amino acids 20-35 of SEQ ID NO: 1 and SspC associated with MIR319 .

[0023] Figure 12 shows bright-field and fluorescence microscopy images demonstrating the detection of GFP and mCherry in the gut of nematodes fed normal E. coli OP50 bacterial diet (two right panels) or nematodes fed B. thuringiensis spores expressing the fusion a protein containing amino acids 20-35 of SEQ ID NO: 1 and either GFP or or mCherry (left three panels).

[0024] Figure 13 is a fluorescence microscopy image showing detection of endophytic bacteria isolated from corn plants treated with Bacillus thuringiensis EE-B00184 expressing a fusion protein containing amino acids 20-35 of SEQ ID NO: 1 and GFP. Arrows indicate individual disputes.

[0025] Figure 14 is a photograph showing the fluorescence of bacterial colonies containing recombinant members of the Bacillus cereus family expressing a fusion protein containing amino acids 20-35 of SEQ ID NO: 1 and GFP isolated from corn plants grown from seeds coated with recombinant bacteria.

[0026] FIG. 15 is a transmission electron microscopy photograph showing: (A) intact spores of Bacillus thuringiensis BT013A surrounded by attached exospores; (B) CotE knockout strain Bacillus thuringiensis BT013A spores with detached exospores; and (C) a purified preparation of exospore fragments derived from the CotE knockout strain of Bacillus thuringiensis BT013A.

DEFINITIONS

[0027] When the singular form, "one" and "specified" are used herein, they mean "at least one" or "one or more" unless otherwise indicated.

[0028] The terms "agriculturally acceptable carrier" and "carrier" are used interchangeably herein.

[0029] The term "animal" encompasses any non-human animal as well as humans. For example, when the term "animal" is used herein, the animal may be a mammal (for example, a human, a sheep, a goat, a cow, a pig, a deer, an alpaca, a bison, a camel, a donkey, a horse, a mule, a llama, a rabbit, a dog, or cat), poultry (such as chicken, turkey, duck, goose, quail, or pheasant), fish (such as salmon, trout, tilapia, tuna, catfish, or carp), or shellfish (such as small shrimp, large shrimp, lobster, crab or cancer).

[0030] "Biologically pure bacterial culture" refers to a culture of bacteria that does not contain other types of bacteria in quantities sufficient to prevent the replication of the culture or to be detected by conventional bacteriological methods. In other words, it is a culture where almost all bacterial cells present belong to a particular strain.

[0031] The terms "comprising", "including" and "having" are inclusive and mean that additional elements other than the listed elements may be present.

[0032] The term "bioactive peptide" refers to any peptide that exhibits biological activity. "Biologically active peptides" can be obtained, for example, by cleaving a protein, peptide, proprotein or preproprotein with a protease or peptidase.

[0033] The term "effective amount" refers to an amount that is sufficient to significantly increase the growth and/or protein yield and/or grain yield of a plant compared to the growth, protein yield and grain yield of a control treated plant.

[0034] "An enzyme involved in the production or activation of a plant growth promoting compound" includes any enzyme that catalyzes any step in the biological pathway for the synthesis of a compound that stimulates plant growth or alters plant structure, or any enzyme that catalyzes the conversion of an inactive or less an active derivative of a compound that stimulates the growth of a plant or changes the structure of a plant into an active or more active form of the compound. Such compounds include, for example, but are not limited to, small molecule plant hormones such as auxins and cytokinins, biologically active peptides, and small molecule plant growth promoting compounds synthesized by bacteria or fungi in the rhizosphere (eg, 2,3-butanediol).

[0035] The term "fusion protein", as used herein, refers to a protein having a polypeptide sequence that contains sequences derived from two or more separate proteins. A fusion protein can be produced by linking a nucleic acid molecule that encodes all or part of a first polypeptide to a nucleic acid molecule that encodes all or part of a second polypeptide to produce a nucleic acid sequence that, when expressed, provides a single polypeptide having functional properties, derived from each of the original proteins.

[0036] The term "germination rate", as used herein, refers to the number of seeds that germinate over a particular period of time. For example, a germination rate of 85% indicates that 85 out of 100 seeds germinated during a given time period.

[0037] The term "inactivate" or "inactivation" as used herein in relation to the inactivation of spores of a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium means that the spores are unable to germinate, or that the spores can germinate but are damaged so that germination does not result in live bacteria. The terms "partially inactivate" or "partially inactivate" mean that a certain percentage of spores are inactivated, but some spores retain the ability to germinate and return to a living replicating state. The term "genetic inactivation" refers to the inactivation of spores of a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium by mutating the spore DNA, which results in complete or partial inactivation of the spore. The terms "physical inactivation" and "chemical inactivation" refer to the inactivation of spores by any physical or chemical means, for example, heat treatment, gamma irradiation, X-ray irradiation, UV-A irradiation, UV-B irradiation, or treatment with a solvent such as as glutaraldehyde, formaldehyde, hydrogen peroxide, acetic acid, bleach, chloroform or phenol, or any combination thereof.

[0038] The terms "immobilization of a spore of a recombinant member of the Bacillus cereus family on a plant" and "immobilization of a spore of a recombinant member of the Bacillus cereus family on a plant" refers to the binding of a spore of a recombinant member of the Bacillus cereus family or a spore of a recombinant spore-forming bacterium to a plant, for example, a plant root or an aerial part of a plant, such as a leaf, stem, flower or fruit, so that the spore remains in the root structure of the plant or the above-ground part, and does not pass into the growth medium of the plant or into the environment surrounding the above-ground parts of the plant.

[0039] The term "inoculum", as described herein, is defined in several federal or state regulations as (1) "soil or plant inoculums include any carrier or culture of a particular microorganism or mixture of microorganisms provided to improve soil or growth, quality or plant yield, and includes any seed or fertilizer provided for inoculation with such crop" (New York State 10-A Consolidated Law); (2) "substances, other than fertilizers, manufactured, sold or provided for use to improve the physical condition of the soil or to promote plant growth or crop yield" (Canada Fertilizers Act); (3) "composition containing pure or specified mixtures of live bacteria, fungi or viral particles, for the treatment of seeds, seedlings or other plant propagation material to increase the ability to grow or disease resistance or otherwise modify the properties of the resulting plant or crop" (Ad hoc European Working Group, 1997) or (4) "any chemical or biological substance, mixture of substances, or device distributed in this state for application to soil, plants or seeds, for soil amendment purposes; or which is intended to improve germination, growth, quality , yield, product quality, reproduction, taste, or other desired plant characteristics, or which are intended to provide any chemical, biochemical, biological, or physical soil modification" (Section 14513, California Food and Agriculture Code).

[0040] A "modulator protein" includes any protein that, when overexpressed in a member of the Bacillus cereus family expressing any of the fusion proteins described herein, modulates the expression of the fusion protein such that the expression of the fusion protein is increased or decreased relative to the expression of the fusion protein. a protein in a member of the Bacillus cereus family that does not overexpress the modulator protein.

[0041] "Plant growth media" includes any material that is capable of supporting plant growth.

[0042] "Protein or peptide that enhances the plant immune system" as used herein includes any protein or peptide that has a beneficial effect on the plant immune system.

[0043] The term "plant growth promoting protein or peptide" as used herein includes any protein or peptide that enhances the growth of a plant exposed to the protein or peptide.

[0044] The term "probiotic" as used herein refers to microorganisms (eg, bacteria) that provide a health benefit when consumed or administered to an animal.

[0045] The terms "plant growth promotion" and "plant growth promotion" are used interchangeably herein and refer to the ability to enhance or increase at least one of the height, weight, leaf size, root size, or stem size of a plant, increase yield protein from the plant or increase grain yield from the plant.

[0046] A "protein or peptide that protects a plant from a pathogen" as used herein includes any protein or peptide that renders a plant exposed to the protein or peptide less susceptible to pathogen infection.

[0047] A "protein or peptide that enhances plant stress tolerance" as used herein includes any protein or peptide that renders a plant exposed to the protein or peptide more stress tolerant.

[0048] The term "plant-binding protein or peptide" refers to any peptide or protein capable of binding specifically or non-specifically to any part of a plant (e.g. roots or aerial parts of a plant such as leaves, stems, flowers or fruits) or material plants.

[0049] The term "pyrethrinase" refers to any enzyme that degrades pyrethrin or pyrethroid.

[0050] The term "rhizosphere" is used interchangeably with the term "root zone" to refer to the segment of soil that surrounds and is affected by the roots of a plant.

[0051] The term "targeting sequence" as used herein refers to a polypeptide sequence which, when present as part of a longer polypeptide or protein, results in the localization of the longer polypeptide or protein in a specific subcellular region. The targeting sequences described herein result in the localization of proteins in the exospore of a member of the Bacillus cereus family.

DESCRIPTION OF THE INVENTION

I. Fusion proteins for expression in family members Bacillus cereus and recombinant members of the family Bacillus cereus expressing such fusion proteins

[0052] The present invention provides fusion proteins comprising a targeting sequence, an exospore protein or an exospore protein fragment that targets the fusion protein to the exospore of a member of the Bacillus cereus family, and at least one protein or peptide of interest. When expressed in bacteria members of the Bacillus cereus family, these fusion proteins target the exospore layer of the spore and physically orient themselves so that the protein or peptide of interest is exposed outside the spore.

[0053] This Bacillus exospore display (BEMD) system can be used to deliver peptides, enzymes, and other proteins to plants (e.g., leaves, fruits, flowers, stems, or roots of a plant) or to a plant growth medium, such as soil. Peptides, enzymes and proteins delivered to the soil or other plant growth medium in this manner are retained and exhibit activity in the soil for extended periods of time. Introduction of recombinant bacteria members of the Bacillus cereus family expressing the fusion proteins described herein into the soil or rhizosphere of a plant results in beneficial enhancement of plant growth under many different soil conditions. The use of BEMD to produce these enzymes allows them to continue to show their beneficial effects on the plant and rhizosphere during the first months of plant life.

A. Targeting sequences, exospore proteins and exospore protein fragments for targeting proteins or peptides of interest to exospores of a member of the Bacillus cereus family

[0054] For ease of reference, descriptions of the amino acid sequences for targeting sequences, exospore proteins, and exospore protein fragments that can be used to target proteins or peptides of interest to exospores of members of the Bacillus cereus family are presented in Table 1 along with their SEQ ID NO.

Table 1. Peptide and protein sequences used to target proteins or peptides of interest to exospores of members of the family Bacillus cereus

Protein, protein fragment, or targeting sequence SEQID NO: A.k. 1-41 BclA ( B. anthracis Sterne) one* Full-length BclA ( B. anthracis Sterne) 2* A.k. 1-33 BetA/BAS3290 ( B. anthracis Sterne) 3 Full size BetA/BAS3290 ( B. anthracis Sterne) 4 Met+a.k. 2-43 BAS4623 ( B. anthracis Sterne) 5 Full size BAS4623( B. anthracis Sterne) 6 A.k. 1-34 BclB ( B. anthracis Sterne) 7 Full-length BclB ( B. anthracis Sterne) eight A.k. 1-30 BAS1882 ( B. anthracis Sterne) nine Full-length BAS1882 ( B. anthracis Sterne) ten A.k. 1-39 gene product 2280 ( B. weihenstephensis KBAB4) eleven Full-length product of the 2280 KBAB4 gene ( B. weihenstephensis KBAB4) 12 A.k. 1-39 gene product 3572 ( B. weihenstephensis KBAB4) thirteen Full-length product of the 3572 KBAB4 gene ( B. weihenstephensis KBAB4) fourteen A.k. 1-49 exospore leader peptide ( B. cereus VD200) fifteen Exospore ( B. cereus VD200) full-length leader peptide sixteen A.k. 1-33 exospore leader peptide ( B. cereus VD166) 17 Exosporium full-length leader peptide ( B. cereus VD166) eighteen A.k. 1-39 of the hypothetical protein IKG_04663 ( B. cereus VD200) nineteen Hypothetical protein IKG_04663, incomplete ( B. cereus VD200) 20 A.k. 1-39 β-propeller protein YVTN ( B. weihenstephensis KBAB4) 21 Full-length YVTN β-propeller protein ( B. weihenstephensis KBAB4) 22 A.k. 1-30 of the hypothetical protein bcerkbab4_2363 ( B. weihenstephensis KBAB4) 23 Full-length hypothetical protein bcerkbab4_2363 ( B. weihenstephensis KBAB4) 24 A.k. 1-30 hypothetical protein bcerkbab4_2131 ( B. weihenstephensis KBAB4) 25 Full-length hypothetical protein bcerkbab4_2131 ( B. weihenstephensis KBAB4) 26 A.k. 1-36 triple helix repeat containing collagen ( B. weihenstephensis KBAB4) 27 Triple-helix repeat-containing full-length collagen, KBAB4 ( B. weihenstephensis KBAB4) 28 A.k. 1-39 hypothetical protein bmyco0001_21660 ( B. mycoides 2048) 29 Full-length hypothetical protein bmyco0001_21660 ( B. mycoides 2048) thirty A.k. 1-30 hypothetical protein bmyc0001_22540 ( B. mycoides 2048) 31 Full length hypothetical protein bmyc0001_22540 ( B. mycoides 2048) 32 A.k. 1-21 hypothetical protein bmyc0001_21510 ( B. mycoides 2048) 33 Full length hypothetical protein bmyc0001_21510 ( B. mycoides 2048) 34 A.k. 1-22 collagen triple helix repeat protein ( B. thuringiensis 35646) 35 Full length collagen triple helix repeat protein ( B. thuringiensis 35646) 36 A.k. 1-35 of the hypothetical protein WP_69652 ( B. cereus ) 43 Full-length hypothetical protein WP_69652 ( B. cereus ) 44 A.k. 1-41 exospore leader sequence WP016117717 ( B. cereus ) 45 Full-length exospore leader sequence WP016117717 ( B. cereus ) 46 A.k. WP002105192 ( B. cereus ) exospore peptide 1-49 47 Full length peptide of exosporium WP002105192 ( B. cereus ) 48 A.k. 1-38 of the hypothetical protein WP87353 ( B. cereus ) 49 Full length hypothetical protein WP87353 ( B. cereus ) fifty A.k. 1-39 peptide exospore 02112369 ( B. cereus ) 51 Exosporium full-length peptide 02112369 ( B. cereus ) 52 A.k. 1-39 protein exospore WP016099770 ( B. cereus ) 53 Full-length exospore protein WP016099770 ( B. cereus ) 54 A.k. 1-36 hypothetical protein YP006612525 ( B. thuringiensis ) 55 Full length hypothetical protein YP006612525 ( B. thuringiensis ) 56 A.k. 1-136 of the hypothetical protein TIGR03720 ( B. mycoides ) 57** Full-length hypothetical TIGR03720 protein ( B. mycoides ) 58** A.k. 1-36 collagen protein with triple helical repeat domain ( B. cereus ATCC 10987) 59 Full-length collagen protein with a three-strand repeat domain ( B. cereus ATCC 10987) 60 A.k. 1-39 collagen-like protein ( B. cereus E33L) 61 Full-length collagen-like protein ( B. cereus E33L) 62 A.k. 1-41 triple-stranded repeat-containing collagen ( B. weihenstephanensis KBAB4) 63 Full-length collagen containing a three-stranded repeat ( B. weihenstephanensis KBAB4) 64 A.k. 1-30 hypothetical protein BALH_2230
( B. thuringiensis str. Al Hakam) 65 Full-length hypothetical protein BALH_2230 ( B. thuringiensis str. Al Hakam) 66 A.k. 1-33 trihelix repeat-containing collagen ( B. cereus ATCC 14579) 67 Full-length collagen containing a three-stranded repeat ( B. cereus ATCC 14579) 68 A.k. 1-44 collagen triple helix repeat ( B. cereus ) 69 Full-length three-stranded collagen repeat ( B. cereus ) 70 A.k. 1-38 triple helix repeat containing collagen ( B. cereus ATCC 14579) 71 Full-length collagen containing a three-stranded repeat ( B. cereus ATCC 14579) 72 A.k. 1-30 of the hypothetical protein BCZK1835 ( B. cereus E33L) 73 Full length hypothetical protein BCZK1835 ( B. cereus E33L) 74 A.k. 1-48 triple helix repeat containing collagen ( B. weihenstephanensis KBAB4) 75 Full-length collagen containing a three-stranded repeat ( B. weihenstephanensis KBAB4) 76 A.k. 1-30 triple helix repeat containing collagen ( B. cereus ATCC 14579) 77 Full-length collagen containing a three-stranded repeat ( B. cereus ATCC 14579) 78 A.k. 1-39 of the hypothetical BC4725 protein ( B. cereus ATCC 14579) 79 Full-length hypothetical BC4725 protein ( B. cereus ATCC 14579) 80 A.k. 1-44 of the hypothetical protein BCZK4476 ( B. cereus E33L) 81 Full length hypothetical protein BCZK4476 ( B. cereus E33L) 82 A.k. 1-40 collagen containing a three-stranded repeat ( B. anthracis strain "Ames Ancestor") 83 Full-length collagen containing a three-stranded repeat ( B. anthracis strain "Ames Ancestor") 84 A.k. 1-34 BclA protein ( B. thuringiensis serovar konkukian strain 97-27) 85 Full length BclA protein ( B. thuringiensis serovar konkukian strain 97-27) 86 A.k. 1-34 conserved hypothetical protein ( B. cereus ATCC 10987) 87 Full-length conserved hypothetical protein ( B. cereus ATCC 10987) 88 A.k. 1-34 triple helix repeat containing collagen ( B. cereus ATCC 14579) 89 Full-length collagen containing a three-stranded repeat ( B. cereus ATCC 14579) 90 A.k. 1-99 partial sequence of exosporium ( B. cereus ) leader peptide 91 Partial sequence of exosporium ( B. cereus ) leader peptide 92 A.k. 1-136 hypothetical ER45_27600 protein, partial sequence ( B. weihenstephanensis ) 93 Hypothetical protein ER45_27600, partial sequence ( B. weihenstephanensis ) 94 A.k. 1-196 BclA ( B. anthracis Sterne) 95* Met+a.k. 20-35 BclA ( B. anthracis Sterne) 96 Met+a.k. 12-27 BetA/BAS3290 ( B. anthracis Sterne) 97 Met+a.k. 18-33 gene product 2280 ( B. weihenstephensis KBAB4) 98 Met+a.k. 18-33 gene product 3572 ( B. weihenstephensis KBAB4) 99 Met+a.k. 12-27 exospore leader peptide ( B. cereus VD166) 100 Met+a.k. 18-33 β-propeller protein YVTN ( B. weihenstephensis KBAB4) 101 Met+a.k. 9-24 of the hypothetical protein bcerkbab4_2363 ( B. weihenstephensis KBAB4) 102 Met+a.k. 9-24 of the hypothetical protein bcerkbab4_2131 ( B. weihenstephensis KBAB4) 103 Met+a.k. 9-24 hypothetical protein bmyc0001_22540 ( B. mycoides 2048) 104 Met+a.k. 9-24 BAS1882 ( B. anthracis Sterne) 105 Met+a.k. 20-35 exospore leader sequence WP016117717 ( B. cereus ) 106 Met+a.k. 9-24 of the hypothetical protein BALH_2230 ( B. thuringiensis str. Al Hakam) 107 Full-length InhA ( B. mycoides ) 108 Full-length BAS1141 (ExsY) ( B. anthracis Sterne) 109 Full size BAS1144 (BxpB/ExsFA) ( B. anthracis Sterne) 110 Full-length BAS1145 (CotY) ( B. anthracis Sterne) 111 Full-length BAS1140 ( B. anthracis Sterne) 112 Full-length ExsFB ( B. anthracis H9401) 113 Full-length InhA1 ( B. thuringiensis HD74) 114 Full-length ExsJ ( B. cereus ATCC 10876) 115 Full-length ExsH ( B. cereus ) 116 Full-length YjcA ( B. anthracis Ames) 117 Full-length YjcB ( B. anthracis ) 118 Full-length BclC ( B. anthracis Sterne) 119 Full length acid phosphatase ( Bacillus thuringiensis serovar konkukian strain 97-27) 120 Full-length InhA2 ( B. thuringiensis HD74) 121 Full length InhA3 ( B. mycoides ) 122

a.k.=amino acids

* BclA of B. anthracis Sterne has 100% sequence identity with BclA of B. thuringiensis . Thus, SEQ ID NOs: 1, 2, and 95 also correspond to B. thuringiensis BclA amino acids 1-41, B. thuringiensis full-length BclA, and B. thuringiensis BclA amino acids 1-196, respectively. Similarly, SEQ ID NO: 96 also corresponds to the methionine residue plus amino acids 20-35 of BclA B. thuringiensis .

** The hypothetical B. mycoides TIGR03720 protein has 100% sequence identity with the hypothetical B. mycoides protein WP003189234. Thus, SEQ ID NOs: 57 and 58 also correspond to amino acids 1-136 of the B. mycoides hypothetical protein WP003189234 and the full-length hypothetical B. mycoides protein WP003189234, respectively.

[0055] Bacillus is a genus of rod-shaped bacteria. The Bacillus cereus family of bacteria includes any Bacillus species that is capable of producing exospores. The bacterial family Bacillus cereus thus includes the species Bacillus anthracis , Bacillus cereus , Bacillus thuringiensis , Bacillus mycoides , Bacillus pseudomycoides , Bacillus samanii, Bacillus gaemokensis , Bacillus weihenstephensis , and Bacillus toyoiensis . Under stressful environmental conditions, bacteria of the Bacillus cereus family undergo sporulation and form oval endospores that can remain dormant for long periods of time. The outer layer of the endospore is known as the exospore and contains a basal layer surrounded by an outer fluff of hair-like projections. The filaments on the hair-like cannon are mainly formed by the collagen-like glycoprotein BclA, while the basal layer is composed of a number of different proteins. Another collagen-related protein, BclB, is also present in the exospore and exhibits on endospores of members of the Bacillus cereus family. It has been shown that BclA, the main component of the surface fluff, is associated with the exospore with its amino-terminus (N-terminus) located in the stratum basale and its carboxy-terminus (C-terminus) protruding outward from the spore.

[0056] It has previously been discovered that certain sequences of the N-terminal regions of BclA and BclB can be used to target a peptide or protein to the exospores of Bacillus cereus endospores (see U.S. Patent Application Publications Nos. 2010/0233124 and 2011/0281316, and Thompson et al. , Targeting of the BclA and BclB proteins to the Bacillus anthracis spore surface , Molecular Microbiology 70(2):421-34 (2008)). The BetA/BAS3290 protein of Bacillus anthracis was also found to localize in the exospore.

[0057] In particular, amino acids 20-35 of BclA from the strain Bacillus anthracis Sterne were found to be sufficient to target exospores. Alignment of BclA amino acid sequences 1-41 (SEQ ID NO: 1) with the corresponding N-terminal regions of several other Bacillus cereus family exospore proteins and Bacillus cereus family proteins having related sequences is shown in FIGS. 1A and 1B. As can be seen from figa and 1B, there is a region of high homology among all proteins in the region corresponding to amino acids 20-41 BclA. However, in these sequences, the amino acids corresponding to amino acids 36-41 of BclA contain a secondary structure and are not necessary for the localization of the fusion protein in the exospore. The conserved region of the BclA targeting sequence (amino acids 20-35 of SEQ ID NO: 1) is shown in bold in Figures 1A and 1B and corresponds to the minimum targeting sequence required for exospore localization. A more highly conserved region, spanning amino acids 25-35 of BclA in the targeting sequence, is underlined in the sequences of Figures 1A and 1B, and is the recognition sequence for ExsFA/BxpB/ExsFB and homologues that targets and assembles the described proteins on the exospore surface. Amino acid sequences SEQ ID NO. 3, 5, and 7 in FIG. 1A are amino acids 1-33 of BetA/BAS3290 of Bacillus anthracis Sterne, methionine followed by amino acids 2-43 of BAS4623 of Bacillus anthracis Sterne, and amino acids 1-34 of BclB of Bacillus anthracis Sterne, respectively (for BAS4623 it was found that the replacement of valine, located in position 1 of the native protein, with methionine leads to 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 conserved region corresponding to amino acids 20-35 of BclA (underlined).

[0058] Additional proteins of members of the Bacillus cereus family also contain a conserved targeting region. In particular, in FIGS. 1A and 1B, SEQ ID NO: 9 is amino acids 1-30 of the Bacillus anthracis Sterne strain BAS1882, SEQ ID NO: 11 is amino acids 1-39 of the Bacillus weihenstephensis KBAB4 gene 2280 product, SEQ ID NO: 13 is is amino acids 1-39 of Bacillus weihenstephensis KBAB4 gene 3572, SEQ ID NO: 15 is amino acids 1-49 of Bacillus cereus VD200 exospore leader peptide, SEQ ID NO: 17 is amino acids 1-33 of Bacillus cereus VD166 exospore leader, 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 of Bacillus weihenstephensis KBAB4 β-propeller protein YVTN, SEQ ID NO: 23 is amino acids 1-30 of bcerkbab4_2363 hypothetical protein Bacillus weihenstephensis KBAB4, SEQ ID NO: 25 is amino acids 1-30 of the hypothetical protein bcerkbab4_2131 Bacillus weihenstephensis KBAB4, SEQ ID NO : 27 is amino acids 1-36 of collagen containing triple helical repeats, Bacillus weihenstephensis KBAB4, SEQ ID NO: 29 is amino acids 1-39 of the hypothetical protein bmyco0001_21660 Bacillus mycoides 2048, SEQ ID NO: 31 is amino acids 1-30 of the hypothetical protein bmyc0001_22540 Bacillus mycoides 2048, SEQ ID NO: 33 is amino acids 1-21 of the hypothetical protein bmyc0001_21510 Bacillus mycoides 2048, SEQ ID NO: 35 is amino acids 1-22 of the triple helical repeat collagen protein Bacillus thuringiensis 35646, SEQ ID NO: 43 is amino acids 1-35 of Bacillus cereus hypothetical protein WP_69652, SEQ ID NO: 45 is amino acids 1-41 of Bacillus cereus exospore WP016117717 leader sequence, SEQ ID NO: 47 is amino acids 1-49 of Bacillus cereus exospore WP002105192 peptide, SEQ ID NO: 49 is is amino acids 1-38 of the hypothetical protein WP87353 Bacillus cereus , SEQ ID NO: 51 is amino acids 1-39 of Bacillus cereus exosporium 02112369 peptide, SEQ ID NO: 53 is amino acids 1-39 of Bacillus cereus exosporium WP016099770 protein, SEQ ID NO: 55 is amino acids 1-36 of hypothetical Bacillus protein YP006612525 thuringiensis , SEQ ID NO: 57 is amino acids 1-136 of the hypothetical Bacillus mycoides TIGR03720 protein, SEQ ID NO: 59 is amino acids 1-36 of B. cereus triple-stranded repeat domain collagen protein ATCC 10987, SEQ ID NO: 61 is amino acids 1-39 of B. cereus E33L collagen-like protein, SEQ ID NO: 63 are amino acids 1-41 of collagen containing triple helical repeats, B. weihenstephanensis KBAB4, SEQ ID NO: 65 are amino acids 1-30 of the hypothetical BALH_2230 B protein. thuringiensis strain Al Hakam, SEQ ID NO: 67 is amino acids 1-33 of collagen containing triple helical repeats, B. cereus ATCC 14579, SEQ ID NO : 69 is amino acids 1-44 of B. cereus collagen triple helical repeat, SEQ ID NO: 71 is amino acids 1-38 of collagen containing B. cereus triple helical repeats ATCC 14579, SEQ ID NO: 73 is amino acids 1-30 of the putative protein BCZK1835 B. cereus E33L, SEQ ID NO: 75 is amino acids 1-48 of collagen containing triple helical repeats, B. weihenstephanensis KBAB4, SEQ ID NO: 77 is amino acids 1-30 of collagen containing triple helical repeats, B. cereus ATCC 14579 , SEQ ID NO: 79 is amino acids 1-39 of B. cereus ATCC 14579 hypothetical BC4725 protein, SEQ ID NO: 81 is amino acids 1-44 of B. cereus E33L hypothetical BCZK4476 protein, SEQ ID NO: 83 is amino acids 1- 40 collagen containing triple helical repeats, B. anthracis strain "Ames Ancestor", SEQ ID NO: 85 is amino acids 1-34 of the BclA protein of B. thuringiensis serovar konkukian strain 97-27, S EQ ID NO: 87 is amino acids 1-34 of the conserved hypothetical protein B. cereus ATCC 10987, SEQ ID NO: 89 is amino acids 1-34 of collagen containing triple helical repeats, B. cereus ATCC 14579, SEQ ID NO: 91 is amino acids 1-99 of the partial B. cereus exospore leader peptide sequence, and SEQ ID NO: 93 are amino acids 1-136 of the hypothetical B. weihenstephanensis ER45_27600 protein . As shown in Figures 1A and 1B, each of the N-terminal regions of these proteins contains a region that is conserved at amino acids 20-35 of BclA (SEQ ID NO: 1) and a more conserved region at amino acids 25-35 of BclA.

[0059] Any portion of BclA that includes amino acids 20-35 can be used to target the fusion protein to exospores. In addition, full-length exospore proteins or fragments of exospore proteins can be used to target fusion proteins to exospores. Thus, full-length BclA or a BclA fragment that includes amino acids 20-35 can be used to target exospores. For example, full-length BclA (SEQ ID NO: 2) or a medium-sized fragment of BclA that lacks the C-terminus, such as SEQ ID NO: 95 (BclA amino acids 1-196), can be used to target fusion proteins to exospores. Fragments of medium size, such as the fragment of SEQ ID NO: 95, have a smaller secondary structure than full-length BclA, and were found to be suitable for use as a targeting sequence. The targeting sequence may also contain significantly shorter portions of BclA that include amino acids 20-35 such as SEQ ID NO: 1 (BclA amino acids 1-41), amino acids 1-35 of SEQ ID NO: 1, amino acids 20-35 of SEQ ID NO : 1 or SEQ ID NO: 96 (methionine residue linked to amino acids 20-35 of BclA). Even shorter BclA fragments, which include only some of amino acids 20-35, also show the ability to target fusion proteins into exospores. For example, the targeting sequence may comprise 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.

[0060] Alternatively, any portion of BetA/BAS3290, BAS4623, BclB, BAS1882, KBAB4 gene product 2280, KBAB4 gene product 3572, exospore leader peptideB. cereus VD200, exospore leader peptideB. cereus VD166, hypothetical protein IKG_04663B. cereus VD200, β-propeller protein YVTNB. weihenstephensis KBAB4, the hypothetical protein bcerkbab4_2363B. weihenstephensis KBAB4, the hypothetical protein bcerkbab4_2131B. weihenstephensis KBAB4, collagen containing triple helical repeats,B. weihenstephensis KBAB4, hypothetical protein bmyco0001_21660B. mycoides 2048, hypothetical protein bmyc0001_22540B. mycoides 2048, hypothetical protein bmyc0001_21510B. mycoides 2048, triple helical repeat collagen proteinB. thuringiensis 35646, hypothetical protein WP_69652B. cereus, exospore leader sequence WP016117717B. cereus, exospore peptide WP002105192 peptideB. cereus, the hypothetical protein WP87353B. cereus, exospore peptide 02112369B. cereus, exospore squirrel WP016099770B. cereus, hypothetical protein YP006612525B. thuringiensis, the hypothetical protein TIGR03720B. mycoides, a collagen protein with a triple helical repeat domainB. cereus ATCC 10987, a collagen-like proteinB. cereus E33L, collagen containing triple helical repeats,B. weihenstephanensisKBAB4, hypothetical BALH_2230 proteinB. thuringiensis strain Al Hakam, collagen containing triple helix repeats,B. cereus ATCC 14579, collagen triple helix repeatsB. cereus, collagen containing triple helix repeats,B. cereus ATCC 14579, hypothetical protein BCZK1835B. cereus E33L, collagen containing triple helical repeats,B. weihenstephanensis KBAB4, collagen containing triple helical repeats,B. cereus ATCC 14579, hypothetical protein BC4725B. cereus ATCC 14579, hypothetical protein BCZK4476B. cereus E33L, collagen containing triple helical repeats,B. anthracis strain "Ames Ancestor", protein BclAB. thuringiensisserovar konkukian strain 97-27, a conserved hypothetical proteinB. cereus ATCC 10987, collagen containing triple helical repeats,B. cereus ATCC 14579, incomplete exospore leader peptide sequenceB. cereus or hypothetical protein ER45_27600B. weihenstephanensis, which includes amino acids corresponding to amino acids 20-35 of BclA can serve as a targeting sequence.

[0061] As can be seen from Figure 1A, BetA/BAS3290 amino acids 12-27, BAS4623 amino acids 23-38, BclB amino acids 13-28, BAS1882 amino acids 9-24, KBAB4 gene product 2280 amino acids 18-33, amino acids 18-33 KBAB4 gene product 3572, amino acids 28-43 of the B. cereus VD200 exosporium leader peptide, amino acids 12-27 of the B. cereus VD166 exosporium leader peptide, amino acids 18-33 of the hypothetical B. cereus VD200 IKG_04663 protein, amino acids 18-33 of the YVTN β-propeller protein B. weihenstephensis KBAB4, amino acids 9-24 of hypothetical protein bcerkbab4_2363 B. weihenstephensis KBAB4, amino acids 9-24 of hypothetical protein bcerkbab4_2131 B. weihenstephensis KBAB4, amino acids 15-30 of triple-stranded repeat collagen B. weihenstephensis KBAB4, amino acids 18-33 of hypothetical protein bmyco0001_21660 B. mycoides 2048, amino acids 9-24 of the hypothetical protein bmyc0001_22540 B. mycoides 2048, amino acids 1-15 of the hypothetical protein bmyc0001_21510 B. mycoides 2048, amino acids 1-16 B. thuringiensis triple-stranded repeat collagen protein 35646, amino acids 14-29 of the hypothetical B. cereus WP_69652 protein, amino acids 20-35 of the B. cereus exospore leader sequence WP016117717, amino acids 28-43 of the WP002105192 B. cereus exospore peptide, amino acids 17- 32 of the hypothetical B. cereus protein WP87353, amino acids 18-33 of the B. cereus exosporium 02112369 peptide, amino acids 18-33 of the B. cereus exosporium protein WP016099770, amino acids 15-30 of the hypothetical YP006612525 B. thuringiensis hypothetical protein, and amino acids 115-130 B03 of the hypothetical B. thuringiensis protein B030303 mycoides correspond to amino acids 20-35 of BclA. As can be seen from FIG. 1B, B. cereus ATCC 10987 triple strand repeat domain collagen protein amino acids 15-30, B. cereus E33L collagen-like protein amino acids 18-33, B. weihenstephanensis tri strand repeat collagen containing amino acids 20-35 KBAB4, amino acids 9-24 of the hypothetical protein BALH_2230 of B. thuringiensis strain Al Hakam, amino acids 12-27 of collagen containing three-stranded repeats, B. cereus ATCC 14579, amino acids 23-38 of three-stranded repeats of B. cereus collagen, amino acids 17-32 of collagen containing triple helix repeats, B. cereus ATCC 14579, amino acids 9-24 of hypothetical protein BCZK1835 B. cereus E33L, amino acids 27-42 of collagen containing triple helical repeats, B. weihenstephanensis KBAB4, amino acids 9-24 of collagen containing three helical repeats, B. cereus ATCC 14579, amino acids 18-33 of B. cereus ATCC hypothetical BC4725 protein 14579, amino acids 23-38 of B. cereus E33L hypothetical protein BCZK4476, amino acids 19-34 collagen containing triple helical repeats, B. anthracis strain "Ames Ancestor", amino acids 13-28 of the BclA protein of B. thuringiensis serovar konkukian strain 97-27, amino acids 13-28 of the conservative hypothetical protein B. cereus ATCC 10987, amino acids 13-28 of collagen, containing triple helical repeats, B. cereus ATCC 14579, amino acids 78-93 of the partial sequence of the B. cereus exospore leader peptide, and amino acids 115-130 of the hypothetical B. weihenstephanensis ER45_27600 protein correspond to amino acids 20-35 of BclA. Thus, any portion of these proteins that includes the appropriate amino acids listed above can serve as a targeting sequence.

[0062] Moreover, any amino acid sequence containing amino acids 20-35 of BclA or any of the above corresponding amino acids can serve as a targeting sequence.

[0063] Thus, the targeting sequence may 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 targeting sequence may 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 exospore protein may comprise a full-length BclA (SEQ ID NO: 2) or an exosporium protein fragment may contain a medium-sized BclA fragment that lacks a C-terminus, such as SEQ ID NO: 59 (BclA amino acids 1-196). Alternatively, the exospore protein fragment may consist of SEQ ID NO: 59.

[0064] The targeting sequence may comprise amino acids 2-35 of SEQ ID NO: 1; amino acids 5-35 SEQ ID NO: 1; amino acids 8-35 SEQ ID NO: 1; amino acids 10-35 of SEQ ID NO: 1 or amino acids 15-35 of SEQ ID NO: 1.

[0065] The targeting sequence may also comprise 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 may contain full-length BetA/BAS3290 (SEQ ID NO: 4) . It was also found that the methionine residue associated with amino acids 12-27 of BetA/BAS3290 can be used as a targeting sequence. Thus, the targeting sequence may comprise SEQ ID NO: 97. The targeting sequence may 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.

[0066] The targeting sequence may comprise amino acids 2-27 of SEQ ID NO: 3; amino acids 5-27 SEQ ID NO: 3; amino acids 8-27 of SEQ ID NO: 3 or amino acids 10-27 of SEQ ID NO: 3.

[0067] The targeting sequence may 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 may comprise the full length BAS4623 (SEQ ID NO: 6).

[0068] The targeting sequence may comprise amino acids 2-38 of SEQ ID NO: 5; amino acids 5-38 SEQ ID NO: 5; amino acids 8-38 SEQ ID NO: 5; amino acids 10-38 SEQ ID NO: 5; amino acids 15-38 of SEQ ID NO: 5 or amino acids 20-38 of SEQ ID NO: 5.

[0069] Alternatively, the targeting sequence may 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 may contain full-length BclB (SEQ ID NO: 8).

[0070] The targeting sequence may comprise amino acids 2-28 of SEQ ID NO: 7; amino acids 5-28 SEQ ID NO: 7; amino acids 8-28 of SEQ ID NO: 7 or amino acids 10-28 of SEQ ID NO: 7.

[0071] The targeting sequence may 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 may contain the full length BAS1882 (SEQ ID NO: 10). Also, a methionine residue linked to amino acids 9-24 of BAS1882 can be used as a targeting sequence. Thus, the targeting sequence may comprise SEQ ID NO: 105.

[0072] The targeting sequence may 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.

[0073] The targeting sequence may 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 may comprise the full length B. weihenstephensis KBAB4 gene product 2280 (SEQ ID NO: 12). The methionine residue linked to amino acids 18-33 of the B. weihenstephensis KBAB4 gene product 2280 can also be used as a targeting sequence. Thus, the targeting sequence may comprise SEQ ID NO: 98.

[0074] The targeting sequence may comprise amino acids 2-33 of SEQ ID NO: 11; amino acids 5-33 SEQ ID NO: 11; amino acids 8-33 SEQ ID NO: 11; amino acids 10-33 of SEQ ID NO: 11 or amino acids 15-33 of SEQ ID NO: 11.

[0075] The targeting sequence may 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 may comprise the full length B. weihenstephensis KBAB4 gene product 3572 (SEQ ID NO: 14). The methionine residue linked to amino acids 18-33 of the B. weihenstephensis KBAB4 gene product 3572 can also be used as a targeting sequence. Thus, the targeting sequence may comprise SEQ ID NO: 99.

[0076] The targeting sequence may comprise amino acids 2-33 of SEQ ID NO: 13; amino acids 5-33 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.

[0077] Alternatively, the targeting sequence may 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 may comprise the full length B. cereus VD200 exosporium leader peptide (SEQ ID NO: 16).

[0078] The targeting sequence may comprise amino acids 2-43 of SEQ ID NO: 15; amino acids 5-43 SEQ ID NO: 15; amino acids 8-43 SEQ ID NO: 15; amino acids 10-43 SEQ ID NO: 15; amino acids 15-43 SEQ ID NO: 15; amino acids 20-43 of SEQ ID NO: 15 or amino acids 25-43 of SEQ ID NO: 15.

[0079] The targeting sequence may 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 protein may comprise the full-length exosporium leader peptide of B. cereus VD166 (SEQ ID NO: 18). Also, a methionine residue linked to amino acids 12-27 of B. cereus VD166 can be used as a targeting sequence. Thus, the targeting sequence may comprise SEQ ID NO: 100.

[0080] The targeting sequence may comprise amino acids 2-27 of SEQ ID NO: 17; amino acids 5-27 SEQ ID NO: 17; amino acids 8-27 of SEQ ID NO: 17 or amino acids 10-27 of SEQ ID NO: 17.

[0081] The targeting sequence may 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 may comprise the full length hypothetical B. cereus VD200 protein IKG_04663 (SEQ ID NO: 20).

[0082] The targeting sequence may 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.

[0083] 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 may comprise the full-length B. weihenstephensis KBAB4 YVTN β-propeller protein (SEQ ID NO: 22). Also, the methionine residue linked to amino acids 18-33 of the β-propeller protein KBAB4 YVTN B. weihenstephensis can be used as a targeting sequence. Thus, the targeting sequence may comprise SEQ ID NO: 101.

[0084] The targeting sequence may comprise amino acids 2-33 of SEQ ID NO: 21; amino acids 5-33 SEQ ID NO: 21; amino acids 8-33 SEQ ID NO: 21; amino acids 10-33 of SEQ ID NO: 21 or amino acids 15-33 of SEQ ID NO: 21.

[0085] The targeting sequence may 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 exosporium protein may comprise the full-length hypothetical B. weihenstephensis KBAB4 protein bcerkbab4_2363 (SEQ ID NO: 24). Also, a methionine residue linked to amino acids 9-24 of the putative B. weihenstephensis KBAB4 protein bcerkbab4_2363 can be used as a targeting sequence. Thus, the targeting sequence may comprise SEQ ID NO: 102.

[0086] The targeting sequence may 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.

[0087] The targeting sequence comprises 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 may comprise the full-length hypothetical bcerkbab4_2131 B. weihenstephensis KBAB4 protein (SEQ ID NO: 26). Also, a methionine residue linked to amino acids 9-24 of the hypothetical B. weihenstephensis KBAB4 protein bcerkbab4_2131 can be used as a targeting sequence. Thus, the targeting sequence may comprise SEQ ID NO: 103.

[0088] The targeting sequence may 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.

[0089] 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 may comprise a full-length collagen containing a three-stranded repeat, B. weihenstephensis KBAB4 ( SEQ ID NO: 28).

[0090] The targeting sequence may comprise amino acids 2-30 of SEQ ID NO: 27; amino acids 5-30 SEQ ID NO: 27; amino acids 8-30 of SEQ ID NO: 27 or amino acids 10-30 of SEQ ID NO: 27.

[0091] The targeting sequence may 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 may comprise the full-length hypothetical bmyco0001_21660 B. mycoides 2048 protein (SEQ ID NO: 30).

[0092] The targeting sequence may comprise amino acids 2-33 of SEQ ID NO: 29; amino acids 5-33 SEQ ID NO: 29; amino acids 8-33 SEQ ID NO: 29; amino acids 10-33 of SEQ ID NO: 29 or amino acids 15-33 of SEQ ID NO: 29.

[0093] The targeting sequence may 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 may comprise the full-length hypothetical bmyc0001_22540 B. mycoides 2048 protein (SEQ ID NO: 32). Also, a methionine residue linked to amino acids 9-24 of the hypothetical bmyc0001_22540 B. mycoides 2048 protein can be used as a targeting sequence. Thus, a targeting sequence can contain SEQ ID NO: 104.

[0094] The targeting sequence may 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.

[0095] Alternatively, the targeting sequence contains amino acids 1-15 of SEQ ID NO: 33, SEQ ID NO: 33, or the exosporium protein contains the full-length hypothetical bmyc0001_21510 B. mycoides 2048 protein (SEQ ID NO: 34).

[0096] The targeting sequence may also comprise amino acids 1-16 of SEQ ID NO: 35, SEQ ID NO: 35, or the exosporium protein may comprise full length B. thuringiensis triple helical repeat collagen protein 35646 (SEQ ID NO: 36).

[0097] The targeting sequence may comprise 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 may comprise the full-length hypothetical B. cereus protein WP_69652 (SEQ ID NO: 44).

[0098] The targeting sequence may comprise amino acids 2-29 of SEQ ID NO: 43; amino acids 5-29 SEQ ID NO: 43; amino acids 8-29 of SEQ ID NO: 43 or amino acids 10-29 of SEQ ID NO: 43.

[0099] Alternatively, the targeting sequence may 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 may comprise the full length B. cereus exosporium leader sequence WP016117717 (SEQ ID NO: 46). Also, a methionine residue linked to amino acids 20-35 of the B. cereus exosporium leader sequence WP016117717 can be used as a targeting sequence. Thus, the targeting sequence may comprise SEQ ID NO: 106.

[00100] The targeting sequence may comprise amino acids 2-35 of SEQ ID NO: 45; amino acids 5-35 SEQ ID NO: 45; amino acids 8-35 SEQ ID NO: 45; amino acids 10-35 of SEQ ID NO: 45 or amino acids 15-35 of SEQ ID NO: 45.

[00101] The targeting sequence may comprise 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 may comprise the full-length B. cereus exosporium WP002105192 peptide (SEQ ID NO: 48).

[00102] The targeting sequence may comprise amino acids 2-43 of SEQ ID NO: 47; amino acids 5-43 SEQ ID NO: 47; amino acids 8-43 SEQ ID NO: 47; amino acids 10-43 SEQ ID NO: 47; amino acids 15-43 SEQ ID NO: 47; amino acids 20-43 of SEQ ID NO: 47 or amino acids 25-43 of SEQ ID NO: 47.

[00103] The targeting sequence may 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 may comprise the full-length hypothetical B. cereus WP87353 protein (SEQ ID NO: fifty).

[00104] The targeting sequence may comprise amino acids 2-32 of SEQ ID NO: 49; amino acids 5-32 SEQ ID NO: 49; amino acids 8-32 SEQ ID NO: 49; amino acids 10-32 of SEQ ID NO: 49 or amino acids 15-32 of SEQ ID NO: 49.

[00105] Alternatively, the targeting sequence may 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 exosporium protein may comprise the full length B. cereus exosporium 02112369 peptide (SEQ ID NO : 52).

[00106] The targeting sequence may comprise amino acids 2-33 of SEQ ID NO: 51; amino acids 5-33 SEQ ID NO: 51; amino acids 8-33 SEQ ID NO: 51; amino acids 10-33 of SEQ ID NO: 51 or amino acids 15-33 of SEQ ID NO: 51.

[00107] The targeting sequence may 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 may comprise the full-length B. cereus exosporium protein WP016099770 (SEQ ID NO: 54).

[00108] The targeting sequence may comprise amino acids 2-33 of SEQ ID NO: 53; amino acids 5-33 SEQ ID NO: 53; amino acids 8-33 SEQ ID NO: 53; amino acids 10-33 of SEQ ID NO: 53 or amino acids 15-33 of SEQ ID NO: 53.

[00109] Alternatively, the targeting sequence may 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 may comprise the full length hypothetical B. thuringiensis protein YP006612525 (SEQ ID NO : 56).

[00110] The targeting sequence may comprise amino acids 2-30 of SEQ ID NO: 55; amino acids 5-30 SEQ ID NO: 55; amino acids 8-30 of SEQ ID NO: 55 or amino acids 10-30 of SEQ ID NO: 55.

[00111] The targeting sequence may also comprise amino acids 1-130 of SEQ ID NO: 57, amino acids 115-130 of SEQ ID NO: 57 or SEQ ID NO: 57, or the exosporium protein may comprise the full length hypothetical B. mycoides TIGR03720 protein (SEQ ID NO : 58).

[00112] The targeting sequence may comprise amino acids 2-130 of SEQ ID NO: 57; amino acids 5-130 SEQ ID NO: 57; amino acids 10-130 SEQ ID NO: 57; amino acids 20-130 SEQ ID NO: 57; amino acids 30-130 SEQ ID NO: 57; amino acids 40-130 SEQ ID NO: 57; amino acids 50-130 SEQ ID NO: 57; amino acids 60-130 SEQ ID NO: 57; amino acids 70-130 SEQ ID NO: 57; amino acids 80-130 SEQ ID NO: 57; amino acids 90-130 SEQ ID NO: 57; amino acids 100-130 of SEQ ID NO: 57 or amino acids 110-130 of SEQ ID NO: 57.

[00113] The targeting sequence may comprise amino acids 1-30 of SEQ ID NO: 59 or SEQ ID NO: 59; or the exosporium protein may comprise a full-length B. cereus triple helical repeat domain collagen protein ATCC 10987 (SEQ ID NO: 60).

[00114] The targeting sequence may 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.

[00115] The targeting sequence may 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 may comprise a full-length B. cereus E33L collagen-like protein (SEQ ID NO: 62).

[00116] The targeting sequence may comprise amino acids 2-33 of SEQ ID NO: 61; amino acids 5-33 SEQ ID NO: 61; amino acids 10-33 of SEQ ID NO: 61 or amino acids 15-33 of SEQ ID NO: 61.

[00117] The targeting sequence may comprise amino acids 1-35 of SEQ ID NO: 63 or SEQ ID NO: 63; or the exosporium protein may comprise a full-length collagen containing triple helical repeats, B. weihenstephanensis KBAB4 (SEQ ID NO: 64).

[00118] The targeting sequence may comprise amino acids 2-35 of SEQ ID NO: 63; amino acids 5-35 SEQ ID NO: 63; amino acids 8-35 SEQ ID NO: 63; amino acids 10-35 of SEQ ID NO: 63 or amino acids 15-35 of SEQ ID NO: 63.

[00119] The targeting sequence may comprise amino acids 1-24 of SEQ ID NO: 65; amino acids 9-24 SEQ ID NO: 65; SEQ ID NO: 65 or SEQ ID NO: 107; or the exosporium protein may comprise the full length hypothetical BALH_2230 protein of B. thuringiensis strain Al Hakam (SEQ ID NO: 66).

[00120] The targeting sequence may comprise amino acids 2-24 of SEQ ID NO: 65 or amino acids 5-24 of SEQ ID NO: 65.

[00121] The targeting sequence may comprise amino acids 1-27 of SEQ ID NO: 67; amino acids 12-27 of SEQ ID NO: 67 or SEQ ID NO: 67; or the exospore protein may comprise a full-length collagen containing triple helical repeats, B. cereus ATCC 14579 (SEQ ID NO: 68).

[00122] The targeting sequence may comprise amino acids 2-27 of SEQ ID NO: 67; amino acids 5-27 of SEQ ID NO: 67 or amino acids 10-27 of SEQ ID NO: 67.

[00123] The targeting sequence may comprise amino acids 1-38 of SEQ ID NO: 69; amino acids 23-38 of SEQ ID NO: 69 or SEQ ID NO: 69; or the exosporium protein may comprise a full length B. cereus collagen triple helix repeat (SEQ ID NO: 70).

[00124] The targeting sequence may comprise amino acids 2-38 of SEQ ID NO: 69; amino acids 5-38 SEQ ID NO: 69; amino acids 10-38 of SEQ ID NO: 69 or amino acids 15-38 of SEQ ID NO: 69.

[00125] The exosporium protein may comprise a full-length collagen containing triple helical repeats, B. cereus ATCC 14579 (SEQ ID NO: 72).

[00126] The targeting sequence may comprise SEQ ID NO: 73, or the exosporium protein may comprise the full-length putative B. cereus E33L protein BCZK1835 (SEQ ID NO: 74).

[00127] The targeting sequence may comprise amino acids 1-42 of SEQ ID NO: 75; amino acids 27-42 of SEQ ID NO: 75 or SEQ ID NO: 75; or the exospore protein may comprise a full-length collagen containing triple helical repeats, B. weihenstephanensis KBAB4 (SEQ ID NO: 76).

[00128] The targeting sequence may comprise amino acids 2-42 of SEQ ID NO: 75; amino acids 5-42 SEQ ID NO: 75; amino acids 10-42 SEQ ID NO: 75; amino acids 15-42 SEQ ID NO: 75; amino acids 20-42 of SEQ ID NO: 75 or amino acids 25-42 of SEQ ID NO: 75.

[00129] The targeting sequence may 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 exospore protein may comprise a full-length collagen containing triple helical repeats, B. cereus ATCC 14579 (SEQ ID NO: 78).

[00130] The targeting sequence may comprise amino acids 2-24 of SEQ ID NO: 77 or amino acids 5-24 of SEQ ID NO: 77.

[00131] The exosporium protein may comprise the full length hypothetical B. cereus ATCC 14579 BC4725 protein (SEQ ID NO: 80).

[00132] The targeting sequence may 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 protein may contain the full-length hypothetical B. cereus E33L protein BCZK4476 (SEQ ID NO: 82).

[00133] The targeting sequence may comprise amino acids 2-38 of SEQ ID NO: 81; amino acids 5-38 SEQ ID NO: 81; amino acids 10-38 SEQ ID NO: 81; amino acids 15-38 of SEQ ID NO: 81 or amino acids 20-38 of SEQ ID NO: 81.

[00134] The targeting sequence may comprise amino acids 1-34 of SEQ ID NO: 83 or SEQ ID NO: 83; or the exosporium protein may comprise full-length collagen containing triple helical repeats, B. anthracis strain "Ames Ancestor" (SEQ ID NO: 84).

[00135] The exosporium protein may comprise the full-length BclA protein of B. thuringiensis serovar konkukian strain 97-27 (SEQ ID NO: 86).

[00136] The targeting sequence may 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 exospore protein may comprise the full-length conserved B. cereus ATCC 10987 hypothetical protein (SEQ ID NO: 88).

[00137] The targeting sequence may 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.

[00138] The targeting sequence may comprise amino acids 1-28 of SEQ ID NO: 89 or SEQ ID NO: 89; or the exospore protein may comprise a full-length collagen containing triple helical repeats, B. cereus ATCC 14579 (SEQ ID NO: 90).

[00139] The targeting sequence may 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.

[00140] The targeting sequence may comprise amino acids 1-93 of SEQ ID NO: 91 or SEQ ID NO: 91; or the exosporium protein may contain a partial sequence of the B. cereus exospore leader peptide (SEQ ID NO: 92).

[00141] The targeting sequence may comprise amino acids 2-93 of SEQ ID NO: 91; amino acids 10-93 SEQ ID NO: 91; amino acids 20-93 SEQ ID NO: 91; amino acids 30-93 SEQ ID NO: 91; amino acids 40-93 SEQ ID NO: 91; amino acids 50-93 of SEQ ID NO: 91 or amino acids 60-93 of SEQ ID NO: 91.

[00142] The targeting sequence may comprise amino acids 1-130 of SEQ ID NO: 93 or SEQ ID NO: 93; or the exosporium protein may contain a partial sequence of the hypothetical B. weihenstephanensis ER45_27600 protein (SEQ ID NO: 94).

[00143] The targeting sequence may comprise amino acids 2-130 of SEQ ID NO: 93; amino acids 10-130 SEQ ID NO: 93; amino acids 20-130 of SEQ ID NO: 93 or amino acids 30-130 of SEQ ID NO: 93.

[00144] Moreover, as illustrated in the examples provided herein below, it has been found that sequences shorter than amino acids 20-35 of BclA can be used to target the fusion protein into the exospores of a recombinant member of the Bacillus cereus family. In particular, BclA amino acids 20-33, BclA amino acids 20-31, BclA amino acids 21-33, or BclA amino acids 23-31 can be used to target the fusion protein to the exospore of a recombinant member of the Bacillus cereus family. Thus, the targeting sequence may 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. Also for targeting the fusion protein in the exospore of a recombinant member of the Bacillus cereus family, the appropriate regions of any of the SEQ ID NOs shown in FIGS. 1A and 1B can be used. By "relevant regions" is meant that when sequences are aligned with SEQ ID NO: 1 as shown in Figures 1A and 1B, the regions of other amino acid sequences that align with the amino acids of SEQ ID NO: 1 are the "relevant regions" 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 the fusion protein into exospores of recombinant Bacillus cereus as these regions align with amino acids 20-33 of SEQ ID NO: 1 as shown in FIG. 1A.

[00145] Even shorter regions within amino acids 20-35 of BclA can also be used to target the fusion protein into the exospores of a recombinant member of the Bacillus cereus family. In particular, you can use any amino acid sequence that includes amino acids 25-30 of SEQ ID NO: 1 or the corresponding amino acids from any of the sequences shown in figa and 1B. The skilled artisan will appreciate that starting at amino acids 25-30 of SEQ ID NO: 1 or the corresponding region of any of the sequences shown in FIGS. 1A and 1B, additional amino acids can be added at the N-terminus, C-terminus, and both at the N -terminus and C-terminus to obtain a targeting sequence that will be effective for targeting the fusion protein into the exospores of the recombinant member of the Bacillus cereus family.

[00146] In addition, from the alignment of the sequences in Figures 1A and 1B, it can be readily seen that while BclA amino acids 20-35 are conserved and amino acids 25-35 are more conserved, some degree of variation can occur in this region without being influenced by on the ability of the targeting sequence to target the protein into exospores. Figures 1A and 1B show the percent identity of each of the respective amino acids of each sequence with BclA amino acids 20-35 ("% identity 20-35") and with BclA amino acids 25-35 ("% identity 25-35"). Thus, for example, compared to BclA amino acids 20-35, the corresponding BetA/BAS3290 amino acids are approximately 81.3% identical, the corresponding BAS4623 amino acids are approximately 50.0% identical, the corresponding BclB amino acids are approximately 43.8% identical. identical, the corresponding amino acids of BAS1882 are approximately 62.5% identical, the corresponding amino acids of the 2280 KBAB4 gene product are approximately 81.3% identical, and the corresponding amino acids of the 3572 KBAB4 gene product are approximately 81.3% identical. The identity of the sequences throughout this region for the remaining sequences is shown in figa and 1B.

[00147] For BclA amino acids 25-35, the corresponding BetA/BAS3290 amino acids are approximately 90.9% identical, the corresponding BAS4623 amino acids are approximately 72.7% identical, the corresponding BclB amino acids are approximately 54.5% identical, the corresponding the amino acids of BAS1882 are approximately 72.7% identical, the corresponding amino acids of the 2280 KBAB4 gene product are approximately 90.9% identical, and the corresponding amino acids of the 3572 KBAB4 gene product are approximately 81.8% identical. The identity of the sequences throughout this region for the remaining sequences is shown in figa and 1B.

[00148] Thus, the targeting sequence may comprise an amino acid sequence having at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 54%. Alternatively, the targeting sequence consists of a 16 amino acid sequence having at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1, where amino acids 25-35 have at least about 54% identity.

[00149] The targeting sequence may also comprise an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 63%. Alternatively, the targeting sequence consists of a 16 amino acid sequence with at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 63%.

[00150] The targeting sequence may also comprise an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 72%. Alternatively, the targeting sequence consists of a 16 amino acid sequence with at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 72%.

[00151] The targeting sequence may also comprise an amino acid sequence having at least about 56% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 63%. Alternatively, the targeting sequence consists of a 16 amino acid sequence with at least about 56% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 63%.

[00152] Alternatively, the targeting sequence may comprise an amino acid sequence having at least about 62% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 72%. The targeting sequence may also consist of a 16 amino acid sequence having at least about 62% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 of SEQ ID NO: 1 is at least about 72%.

[00153] The targeting sequence may comprise an amino acid sequence having at least 68% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%. Alternatively, the targeting sequence consists of a 16 amino acid sequence with at least 68% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%.

[00154] The targeting sequence may also comprise an amino acid sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 72%. Alternatively, the targeting sequence consists of a 16 amino acid sequence with at least about 75% identity to amino acids 20-35 of SEQ ID NO: 1, where the identity to amino acids 25-35 of SEQ ID NO: 1 is at least about 72 %.

[00155] The targeting sequence may also comprise an amino acid sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%. Alternatively, the targeting sequence consists of a 16 amino acid sequence with at least about 75% identity to amino acids 20-35 of SEQ ID NO: 1, where the identity to amino acids 25-35 of SEQ ID NO: 1 is at least about 81 %.

[00156] The targeting sequence may also comprise an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%. Alternatively, the targeting sequence consists of a 16 amino acid sequence with at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%.

[00157] The targeting sequence may comprise an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 90%. Alternatively, the targeting sequence consists of a 16 amino acid sequence with at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 90%.

[00158] The skilled artisan will appreciate that variants of the sequences described above can also be used as targeting sequences, provided that the targeting sequence contains amino acids 20-35 of BclA, the corresponding amino acids of BetA/BAS3290, BAS4263, BclB, BAS1882, KBAB4 gene product 2280, or a 3572 KBAB gene product, or a sequence having sequence identity as described above with amino acids 20-35 and 25-35 of BclA is present.

[00159] Certain exospore proteins of the Bacillus cereus family that lack regions of homology to amino acids 25-35 of BclA can also be used to target a peptide or protein to the exospore of a member of the Bacillus cereus family. In particular, fusion proteins may include an exosporium protein containing SEQ ID NO: 108 (InhA B. mycoides ), an exosporium protein containing SEQ ID NO: 109 (BAS1141 B. anthracis Sterne (ExsY)), an exosporium protein containing SEQ ID NO : 110 (BAS1144 B. anthracis Sterne (BxpB/ExsFA)), exosporium protein containing SEQ ID NO: 111 (BAS1145 B. anthracis Sterne (CotY)), exosporium protein containing SEQ ID NO: 112 (BAS1140 B. anthracis Sterne ), exosporium protein containing SEQ ID NO: 113 (ExsFB B. anthracis H9401), exosporium protein containing SEQ ID NO: 114 (InhA1 B. thuringiensis HD74), exosporium protein containing SEQ ID NO: 115 (ExsJ B. cereus ATCC 10876), exosporium protein containing SEQ ID NO: 116 (ExsH B. cereus ), exosporium protein containing SEQ ID NO: 117 (YjcA B. anthracis Ames), exosporium protein containing SEQ ID NO: 118 (YjcB B. anthracis ), exosporium protein containing SEQ ID NO: 119 (BclC B. anthracis Sterne), exosporium protein containing SEQ ID NO: 120 ( Bacillus thuri acid phosphatase ngiensis serovar konkukian strain 97-27), an exosporium protein containing SEQ ID NO: 121 (InhA2 B. thuringiensis HD74), or an exosporium protein containing SEQ ID NO: 122 (InhA3 B. mycoides ). Incorporation of an exospore protein comprising any of SEQ ID NOs: 108-122 into the fusion proteins described herein results in the targeting of a member of the B. cereus family into the exospore.

[00160] Moreover, exospore proteins having a high degree of sequence identity to any of the full-length exospore proteins or exospore protein fragments described above can also be used to target a peptide or protein to exospores of a member of the Bacillus cereus family. Thus, the fusion protein may comprise an exosporium protein or an exosporium protein fragment containing an amino acid sequence having at least 85% identity with any 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 may comprise an exosporium protein having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with any 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.

[00161] During sporulation of a recombinant member of the Bacillus cereus family expressing any of the fusion proteins described herein, the exosporium targeting protein or exosporium protein fragment is recognized by the exospore spore assembly apparatus and targeted to the exospore, resulting in the exposure of the protein or peptide of interest , which is part of the fusion protein, outside the spore.

[00162] As further illustrated by the examples provided herein below, the use of different targeting sequences allows for control of the expression level of the fusion protein on the spore surface of a member of the Bacillus cereus family. The use of certain targeting sequences described herein will result in higher levels of fusion protein expression, while the use of other targeting sequences will result in lower levels of fusion protein expression on the spore surface.

[00163] In any of the fusion proteins described herein, the targeting sequence, exosporium protein, or exosporium protein fragment may contain the amino acid sequence GXT at their C-terminus, where X is any amino acid.

[00164] In any of the fusion proteins described herein, the targeting sequence, exosporium protein, or exosporium protein fragment may contain an alanine residue at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1.

[00165] In any of the fusion proteins described herein, the targeting sequence, exosporium protein, or exosporium protein fragment may 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 fragment. exospore protein, or at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1.

b. Fusion proteins for expression in recombinant members of the Bacillus cereus family

[00166] The present invention provides fusion proteins comprising at least one protein or peptide of interest and an exospore targeting sequence or protein. When the protein or peptide of interest is any protein or peptide of interest, the fusion protein may comprise: (1) a targeting sequence comprising amino acids 1-30 of SEQ ID NO: 59; (2) a targeting sequence containing SEQ ID NO: 59; (3) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 60; (4) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 59; (5) a targeting sequence containing amino acids 4-30 of SEQ ID NO: 59; (6) a targeting sequence containing amino acids 6-30 of SEQ ID NO: 59; (7) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 61; (8) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 61; (9) a targeting sequence containing SEQ ID NO: 61; (10) an exosporium protein containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 62; (11) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 61; (12) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 61; (13) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 61; (14) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 61; (15) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 63; (16) a targeting sequence containing SEQ ID NO: 63; (17) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 64; (18) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 63; (19) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 63; (20) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 63; (21) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 63; (22) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 63; (23) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 65; (24) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 65; (25) a targeting sequence containing SEQ ID NO: 65; (26) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 66; (27) a targeting sequence containing SEQ ID NO: 107; (28) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 65; (29) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 65; (30) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 67; (31) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 67; (32) a targeting sequence containing SEQ ID NO: 67; (33) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 68; (34) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 67; (35) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 67; (36) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 67; (37) a targeting sequence containing amino acids 1-38 of SEQ ID NO: 69; (38) a targeting sequence containing amino acids 23-38 of SEQ ID NO: 69; (39) a targeting sequence containing SEQ ID NO: 69; (40) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 70; (41) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 69; (42) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 69; (43) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 69; (44) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 69; (45) exosporium protein containing SEQ ID NO: 72; (46) a targeting sequence containing SEQ ID NO: 73; (47) exospore protein containing an amino acid sequence having at least 95% identity with SEQ ID NO: 74; (48) a targeting sequence containing amino acids 1-42 of SEQ ID NO: 75; (49) a targeting sequence containing amino acids 27-42 of SEQ ID NO: 75; (50) a targeting sequence containing SEQ ID NO: 75; (51) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 76; (52) a targeting sequence containing amino acids 2-42 of SEQ ID NO: 75; (53) a targeting sequence containing amino acids 5-42 of SEQ ID NO: 75; (54) a targeting sequence containing amino acids 10-42 of SEQ ID NO: 75; (55) a targeting sequence containing amino acids 15-42 of SEQ ID NO: 75; (56) a targeting sequence containing amino acids 20-42 of SEQ ID NO: 75; (57) a targeting sequence containing amino acids 25-42 of SEQ ID NO: 75; (58) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 77; (59) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 77; (60) a targeting sequence containing SEQ ID NO: 77; (61) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 78; (62) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 77; (63) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 77; (64) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 80; (65) a targeting sequence containing amino acids 1-38 of SEQ ID NO: 81; (66) a targeting sequence containing amino acids 23-38 of SEQ ID NO: 81; (67) a targeting sequence containing SEQ ID NO: 81; (68) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 82; (69) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 81; (70) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 81; (71) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 81; (72) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 81; (73) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 81; (74) a targeting sequence containing amino acids 1-34 of SEQ ID NO: 83; (75) a targeting sequence containing SEQ ID NO: 83; (76) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 84; (77) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 86; (78) a targeting sequence containing amino acids 1-28 of SEQ ID NO: 87; (79) a targeting sequence containing amino acids 13-28 of SEQ ID NO: 87; (80) a targeting sequence containing SEQ ID NO: 87; (81) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 88; (82) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 87; (83) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 87; (84) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 87; (85) a targeting sequence containing amino acids 1-28 of SEQ ID NO: 89; (86) a targeting sequence containing SEQ ID NO: 89; (87) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 90; (88) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 89; (89) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 89; (90) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 89; (91) a targeting sequence containing amino acids 1-93 of SEQ ID NO: 91; (92) a targeting sequence comprising SEQ ID NO: 91; (93) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 92; (94) a targeting sequence containing amino acids 2-93 of SEQ ID NO: 91; (95) a targeting sequence containing amino acids 10-93 of SEQ ID NO: 91; (96) a targeting sequence containing amino acids 20-93 of SEQ ID NO: 91; (97) a targeting sequence containing amino acids 30-93 of SEQ ID NO: 91; (98) a targeting sequence containing amino acids 40-93 of SEQ ID NO: 91; (99) a targeting sequence containing amino acids 50-93 of SEQ ID NO: 91; (100) a targeting sequence containing amino acids 60-93 of SEQ ID NO: 91; (101) a targeting sequence containing amino acids 1-130 of SEQ ID NO: 93; (102) a targeting sequence comprising SEQ ID NO: 93; (103) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 94; (104) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 93; (105) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 93; (106) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 93; (107) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 93; or (108) an exospore protein containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 122.

[00167] For example, when the protein or peptide of interest is any protein or peptide of interest, the fusion protein may comprise: (1) a targeting sequence comprising amino acids 2-30 of SEQ ID NO: 59; (2) a targeting sequence containing amino acids 4-30 of SEQ ID NO: 59; (3) a targeting sequence containing amino acids 6-30 of SEQ ID NO: 59; (4) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 61; (5) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 61; (6) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 61; (7) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 61; (8) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 63; (9) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 63; (10) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 63; (11) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 63; (12) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 63; (13) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 65; (14) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 65; (15) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 67; (16) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 67; (17) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 67; (18) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 69; (19) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 69; (20) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 69; (21) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 69; (22) a targeting sequence containing amino acids 2-42 of SEQ ID NO: 75; (23) a targeting sequence containing amino acids 5-42 of SEQ ID NO: 75; (24) a targeting sequence containing amino acids 10-42 of SEQ ID NO: 75; (25) a targeting sequence containing amino acids 15-42 of SEQ ID NO: 75; (26) a targeting sequence containing amino acids 20-42 of SEQ ID NO: 75; (27) a targeting sequence containing amino acids 25-42 of SEQ ID NO: 75; (28) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 77; (29) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 77; (30) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 81; (31) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 81; (32) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 81; (33) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 81; (34) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 81; (35) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 87; (36) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 87; (37) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 87; (38) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 89; (39) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 89; (40) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 89; (41) a targeting sequence containing amino acids 2-93 of SEQ ID NO: 91; (42) a targeting sequence containing amino acids 10-93 of SEQ ID NO: 91; (43) a targeting sequence containing amino acids 20-93 of SEQ ID NO: 91; (44) a targeting sequence containing amino acids 30-93 of SEQ ID NO: 91; (45) a targeting sequence containing amino acids 40-93 of SEQ ID NO: 91; (46) a targeting sequence containing amino acids 50-93 of SEQ ID NO: 91; (47) a targeting sequence containing amino acids 60-93 of SEQ ID NO: 91; (48) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 93; (49) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 93; (50) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 93; or (51) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 93.

[00168] Alternatively, when the protein or peptide of interest is any protein or peptide of interest, the fusion protein may comprise: (1) a targeting sequence consisting of amino acids 20-33 of SEQ ID NO: 1; (2) a targeting sequence 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 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 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 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 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 sequence 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 consisting of amino acids 12-25 of SEQ ID NO: 17; (41) a targeting 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 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 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 consisting 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 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 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: 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 targeting 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 targeting 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 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: 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 sequence 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 sequence consisting of amino acids 18-31 of SEQ ID NO: 53; (88) a targeting sequence 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 containing amino acids 18-31 of SEQ ID NO: 61; (91) a targeting sequence containing amino acids 18-29 of SEQ ID NO: 61; (92) a targeting sequence containing amino acids 19-31 of SEQ ID NO: 61; (93) a targeting sequence containing amino acids 9-22 of SEQ ID NO: 65; (94) a targeting sequence containing amino acids 9-20 of SEQ ID NO: 65; (95) a targeting sequence containing amino acids 10-22 of SEQ ID NO: 65; (96) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 107; (97) a targeting sequence containing amino acids 1-13 of SEQ ID NO: 107; (98) a targeting sequence containing amino acids 12-25 of SEQ ID NO: 67; (99) a targeting sequence containing amino acids 12-23 of SEQ ID NO: 67; (100) a targeting sequence containing amino acids 13-25 of SEQ ID NO: 67; (101) a targeting sequence containing amino acids 15-23 of SEQ ID NO: 67; (102) a targeting sequence containing amino acids 23-36 of SEQ ID NO: 69; (103) a targeting sequence containing amino acids 23-34 of SEQ ID NO: 69; (104) a targeting sequence containing amino acids 24-36 of SEQ ID NO: 69; (105) a targeting sequence containing amino acids 26-34 of SEQ ID NO: 69; (106) a targeting sequence containing amino acids 27-40 of SEQ ID NO: 75; (107) a targeting sequence containing amino acids 27-38 of SEQ ID NO: 75; (108) a targeting sequence containing amino acids 9-22 of SEQ ID NO: 77; (109) a targeting sequence containing amino acids 9-20 of SEQ ID NO: 77; (110) a targeting sequence containing amino acids 10-22 of SEQ ID NO: 77; (111) a targeting sequence containing amino acids 12-20 of SEQ ID NO: 77; (112) a targeting sequence containing amino acids 23-36 of SEQ ID NO: 81; (113) a targeting sequence containing amino acids 23-34 of SEQ ID NO: 81; (114) a targeting sequence containing amino acids 24-36 of SEQ ID NO: 81; (115) a targeting sequence containing amino acids 26-34 of SEQ ID NO: 81; (116) a targeting sequence containing amino acids 13-26 of SEQ ID NO: 87; (117) a targeting sequence containing amino acids 13-24 of SEQ ID NO: 87; or (118) a targeting sequence containing amino acids 14-26 of SEQ ID NO: 87. The targeting sequence may also consist of any of these sequences.

[00169] The present invention provides fusion proteins comprising at least one protein or peptide of interest and a targeting sequence, an exosporium protein or an exosporium protein fragment. The protein or peptide of interest may be an enzyme that catalyses the production of nitric oxide, or a nucleic acid binding protein or peptide. When the protein or peptide of interest comprises an enzyme that catalyzes the production of nitric oxide or a nucleic acid-binding protein or peptide, the targeting sequence, exosporium protein or exosporium protein fragment can be any targeting sequence, exosporium protein or exosporium protein fragment that targets the fusion protein. into exospores of a recombinant member of the Bacillus cereus family. For example, the targeting sequence, exosporium protein, or exosporium protein fragment may be any of the targeting sequences, exosporium proteins, or exosporium protein fragments set forth in paragraphs [00166]-[00168] above for use with any protein or peptide of interest, or: ( 1) a targeting sequence containing an amino acid sequence having at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 54%; (2) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 1; (3) a targeting sequence containing amino acids 20-35 of SEQ ID NO: 1; (4) a targeting sequence containing SEQ ID NO: 1; (5) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 2; (6) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 1; (7) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 1; (8) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 1; (9) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 1; (10) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 1; (11) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 3; (12) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 3; (13) a targeting sequence containing SEQ ID NO: 3; (14) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 4; (15) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 3; (16) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 3; (17) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 3; (18) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 3; (19) a targeting sequence containing amino acids 1-38 of SEQ ID NO: 5; (20) a targeting sequence containing amino acids 23-38 of SEQ ID NO: 5; (21) a targeting sequence containing SEQ ID NO: 5; (22) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 6; (23) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 5; (24) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 5; (25) a targeting sequence containing amino acids 8-38 of SEQ ID NO: 5; (26) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 5; (27) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 5; (28) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 5; (29) a targeting sequence containing amino acids 1-28 of SEQ ID NO: 7; (30) a targeting sequence containing amino acids 13-28 of SEQ ID NO: 7; (31) a targeting sequence containing SEQ ID NO: 7; (32) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 8; (33) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 7; (34) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 7; (35) a targeting sequence containing amino acids 8-28 of SEQ ID NO: 7; (36) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 7; (37) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 9; (38) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 9; (39) a targeting sequence containing SEQ ID NO: 9; (40) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 10; (41) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 9; (42) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 9; (43) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 9; (44) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 11; (45) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 11; (46) a targeting sequence containing SEQ ID NO: 11; (47) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 12; (48) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 11; (49) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 11; (50) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 11; (51) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 11; (52) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 11; (53) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 13; (54) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 13; (55) a targeting sequence containing SEQ ID NO: 13; (56) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 14; (57) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 13; (58) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 13; (59) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 13; (60) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 13; (61) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 13; (62) a targeting sequence containing amino acids 1-43 of SEQ ID NO: 15; (63) a targeting sequence containing amino acids 28-43 of SEQ ID NO: 15; (64) a targeting sequence containing SEQ ID NO: 15; (65) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 16; (66) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 15; (67) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 15; (68) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 15; (69) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 15; (70) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 15; (71) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 15; (72) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 15; (73) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 17; (74) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 17; (75) a targeting sequence containing SEQ ID NO: 17; (76) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 18; (77) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 17; (78) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 17; (79) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 17; (80) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 17; (81) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 19; (82) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 19; (83) a targeting sequence containing SEQ ID NO: 19; (84) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 20; (85) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 19; (86) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 19; (87) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 19; (88) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 19; (89) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 19; (90) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 21; (91) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 21; (92) a targeting sequence containing SEQ ID NO: 21; (93) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 22; (94) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 21; (95) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 21; (96) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 21; (97) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 21; (98) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 21; (99) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 23; (100) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 23; (101) a targeting sequence comprising SEQ ID NO: 23; (102) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 24; (103) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 23; (104) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 23; (105) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 23; (106) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 25; (107) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 25; (108) a targeting sequence comprising SEQ ID NO: 25; (109) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 26; (110) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 25; (111) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 25; (112) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 25; (113) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 27; (114) a targeting sequence containing amino acids 15-30 of SEQ ID NO: 27; (115) a targeting sequence comprising SEQ ID NO: 27; (116) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 28; (117) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 27; (118) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 27; (119) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 27; (120) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 27; (121) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 29; (122) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 29; (123) a targeting sequence comprising SEQ ID NO: 29; (124) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 30; (125) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 29; (126) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 29; (127) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 29; (128) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 29; (129) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 29; (130) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 31; (131) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 31; (132) a targeting sequence containing SEQ ID NO: 31; (133) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 32; (134) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 31; (135) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 31; (136) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 31; (137) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 33; (138) a targeting sequence comprising SEQ ID NO: 33; (139) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 34; (140) a targeting sequence containing amino acids 1-16 of SEQ ID NO: 35; (141) a targeting sequence containing SEQ ID NO: 35; (142) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 36; (143) a targeting sequence containing amino acids 1-29 of SEQ ID NO: 43; (144) a targeting sequence containing amino acids 14-29 of SEQ ID NO: 43; (145) a targeting sequence containing SEQ ID NO: 43; (146) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 44; (147) a targeting sequence containing amino acids 2-29 of SEQ ID NO: 43; (148) a targeting sequence containing amino acids 5-29 of SEQ ID NO: 43; (149) a targeting sequence containing amino acids 8-29 of SEQ ID NO: 43; (150) a targeting sequence containing amino acids 10-29 of SEQ ID NO: 43; (151) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 45; (152) a targeting sequence containing amino acids 20-35 of SEQ ID NO: 45; (153) a targeting sequence containing SEQ ID NO: 45; (154) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 46; (155) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 45; (156) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 45; (157) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 45; (158) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 45; (159) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 45; (160) a targeting sequence containing amino acids 1-43 of SEQ ID NO: 47; (161) a targeting sequence containing amino acids 28-43 of SEQ ID NO: 47; (162) a targeting sequence containing SEQ ID NO: 47; (163) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 48; (164) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 47; (165) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 47; (166) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 47; (167) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 47; (168) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 47; (169) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 47; (170) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 47; (171) a targeting sequence containing amino acids 1-32 of SEQ ID NO: 49; (172) a targeting sequence containing amino acids 17-32 of SEQ ID NO: 49; (173) a targeting sequence containing SEQ ID NO: 49; (174) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 50; (175) a targeting sequence containing amino acids 2-32 of SEQ ID NO: 49; (176) a targeting sequence containing amino acids 5-32 of SEQ ID NO: 49; (177) a targeting sequence containing amino acids 8-32 of SEQ ID NO: 49; (178) a targeting sequence containing amino acids 10-32 of SEQ ID NO: 49; (179) a targeting sequence containing amino acids 15-32 of SEQ ID NO: 49; (180) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 51; (181) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 51; (182) a targeting sequence comprising SEQ ID NO: 51; (183) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 52; (184) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 51; (185) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 51; (186) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 51; (187) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 51; (188) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 51; (189) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 53; (190) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 53; (191) a targeting sequence containing SEQ ID NO: 53; (192) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 54; (193) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 53; (194) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 53; (195) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 53; (196) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 53; (197) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 53; (198) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 55; (199) a targeting sequence containing amino acids 15-30 of SEQ ID NO: 55; (200) a targeting sequence comprising SEQ ID NO: 55; (201) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 56; (202) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 55; (203) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 55; (204) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 55; (205) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 55; (206) a targeting sequence containing amino acids 1-130 of SEQ ID NO: 57; (207) a targeting sequence containing amino acids 115-130 of SEQ ID NO: 57; (208) a targeting sequence containing SEQ ID NO: 57; (209) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 58; (210) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 57; (211) a targeting sequence containing amino acids 5-130 of SEQ ID NO: 57; (212) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 57; (213) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 57; (214) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 57; (215) a targeting sequence containing amino acids 40-130 of SEQ ID NO: 57; (216) a targeting sequence containing amino acids 50-130 of SEQ ID NO: 57; (217) a targeting sequence containing amino acids 60-130 of SEQ ID NO: 57; (218) a targeting sequence containing amino acids 70-130 of SEQ ID NO: 57; (219) a targeting sequence containing amino acids 80-130 of SEQ ID NO: 57; (220) a targeting sequence containing amino acids 90-130 of SEQ ID NO: 57; (221) a targeting sequence containing amino acids 100-130 of SEQ ID NO: 57; (222) a targeting sequence containing amino acids 110-130 of SEQ ID NO: 57; (223) an exospore protein fragment containing 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 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 containing SEQ ID NO: 103; (232) a targeting sequence comprising SEQ ID NO: 104; (233) a targeting sequence containing SEQ ID NO: 105; (234) a targeting sequence comprising SEQ ID NO: 106; (235) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (236) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 109; (237) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 110; (238) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 111; (239) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 112; (240) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 113; (241) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 114; (242) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 115; (243) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 116; (244) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 117; (245) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 118; (246) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 119; (247) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 120; (248) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 121; (249) a targeting sequence containing amino acids 22-31 of SEQ ID NO: 1; (250) a targeting sequence containing amino acids 22-33 of SEQ ID NO: 1; (251) a targeting sequence containing amino acids 20-31 of SEQ ID NO: 1; (252) a targeting sequence containing amino acids 14-23 of SEQ ID NO: 3; (253) a targeting sequence containing amino acids 14-25 of SEQ ID NO: 3; or (254) a targeting sequence containing amino acids 12-23 of SEQ ID NO: 3.

[00170] For example, when the protein or peptide of interest includes an enzyme that catalyzes the production of nitric oxide, or a nucleic acid binding protein or peptide, the targeting sequence, exosporium protein or exosporium protein fragment can be: (1) a targeting sequence containing amino acids 2 -35 SEQ ID NO: 1; (2) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 1; (3) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 1; (4) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 1; (5) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 1; (6) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 3; (7) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 3; (8) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 3; (9) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 3; (10) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 5; (11) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 5; (12) a targeting sequence containing amino acids 8-38 of SEQ ID NO: 5; (13) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 5; (14) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 5; (15) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 5; (16) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 7; (17) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 7; (18) a targeting sequence containing amino acids 8-28 of SEQ ID NO: 7; (19) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 7; (20) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 9; (21) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 9; (22) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 9; (23) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 11; (24) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 11; (25) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 11; (26) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 11; (27) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 11; (28) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 13; (29) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 13; (30) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 13; (31) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 13; (32) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 13; (33) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 15; (34) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 15; (35) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 15; (36) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 15; (37) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 15; (38) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 15; (39) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 15; (40) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 17; (41) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 17; (42) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 17; (43) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 17; (44) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 19; (45) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 19; (46) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 19; (47) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 19; (48) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 19; (49) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 21; (50) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 21; (51) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 21; (52) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 21; (53) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 21; (54) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 23; (55) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 23; (56) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 23; (57) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 25; (58) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 25; (59) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 25; (60) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 27; (61) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 27; (62) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 27; (63) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 27; (64) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 29; (65) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 29; (66) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 29; (67) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 29; (68) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 29; (69) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 31; (70) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 31; (71) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 31; (72) a targeting sequence containing amino acids 2-29 of SEQ ID NO: 43; (73) a targeting sequence containing amino acids 5-29 of SEQ ID NO: 43; (74) a targeting sequence containing amino acids 8-29 of SEQ ID NO: 43; (75) a targeting sequence containing amino acids 10-29 of SEQ ID NO: 43; (76) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 45; (77) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 45; (78) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 45; (79) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 45; (80) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 45; (81) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 47; (82) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 47; (83) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 47; (84) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 47; (85) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 47; (86) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 47; (87) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 47; (88) a targeting sequence containing amino acids 2-32 of SEQ ID NO: 49; (89) a targeting sequence containing amino acids 5-32 of SEQ ID NO: 49; (90) a targeting sequence containing amino acids 8-32 of SEQ ID NO: 49; (91) a targeting sequence containing amino acids 10-32 of SEQ ID NO: 49; (92) a targeting sequence containing amino acids 15-32 of SEQ ID NO: 49; (93) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 51; (94) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 51; (95) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 51; (96) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 51; (97) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 51; (98) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 53; (99) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 53; (100) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 53; (101) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 53; (102) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 53; (103) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 55; (104) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 55; (105) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 55; (106) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 55; (107) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 57; (108) a targeting sequence containing amino acids 5-130 of SEQ ID NO: 57; (109) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 57; (110) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 57; (111) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 57; (112) a targeting sequence containing amino acids 40-130 of SEQ ID NO: 57; (113) a targeting sequence containing amino acids 50-130 of SEQ ID NO: 57; (114) a targeting sequence containing amino acids 60-130 of SEQ ID NO: 57; (115) a targeting sequence containing amino acids 70-130 of SEQ ID NO: 57; (116) a targeting sequence containing amino acids 80-130 of SEQ ID NO: 57; (117) a targeting sequence containing amino acids 90-130 of SEQ ID NO: 57; (118) a targeting sequence containing amino acids 100-130 of SEQ ID NO: 57; or (119) a targeting sequence containing amino acids 110-130 of SEQ ID NO: 57.

[00171] A fusion protein is provided that contains a reclamation antigen or enzyme and an exospore targeting sequence or protein. The exosporium targeting sequence or protein may comprise any of the exosporium targeting sequences or proteins set forth 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 containing amino acids 5-35 of SEQ ID NO: 1; (3) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 1; (4) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 1; (5) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 1; (6) a targeting sequence containing amino acids 22-31 of SEQ ID NO: 1; (7) a targeting sequence containing amino acids 22-33 of SEQ ID NO: 1; (8) a targeting sequence containing amino acids 20-31 of SEQ ID NO: 1; (9) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 3; (10) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 3; (11) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 3; (12) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 3; (13) a targeting sequence containing amino acids 14-23 of SEQ ID NO: 3; (14) a targeting sequence containing amino acids 14-25 of SEQ ID NO: 3; (15) a targeting sequence containing amino acids 12-23 of SEQ ID NO: 3; (16) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 5; (17) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 5; (18) a targeting sequence containing amino acids 8-38 of SEQ ID NO: 5; (19) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 5; (20) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 5; (21) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 5; (22) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 7; (23) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 7; (24) a targeting sequence containing amino acids 8-28 of SEQ ID NO: 7; (25) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 7; (26) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 9; (27) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 9; (28) a targeting sequence containing SEQ ID NO: 9; (29) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 10; (30) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 9; (31) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 9; (32) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 9; (33) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 11; (34) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 11; (35) a targeting sequence containing SEQ ID NO: 11; (36) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 12; (37) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 11; (38) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 11; (39) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 11; (40) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 11; (41) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 11; (42) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 13; (43) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 13; (44) a targeting sequence containing SEQ ID NO: 13; (45) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 14; (46) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 13; (47) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 13; (48) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 13; (49) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 13; (50) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 13; (51) a targeting sequence containing amino acids 1-43 of SEQ ID NO: 15; (52) a targeting sequence containing amino acids 28-43 of SEQ ID NO: 15; (53) a targeting sequence containing SEQ ID NO: 15; (54) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 16; (55) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 15; (56) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 15; (57) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 15; (58) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 15; (59) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 15; (60) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 15; (61) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 15; (62) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 17; (63) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 17; (64) a targeting sequence containing SEQ ID NO: 17; (65) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 18; (66) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 17; (67) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 17; (68) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 17; (69) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 17; (70) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 19; (71) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 19; (72) a targeting sequence containing SEQ ID NO: 19; (73) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 20; (74) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 19; (75) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 19; (76) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 19; (77) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 19; (78) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 19; (79) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 21; (80) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 21; (81) a targeting sequence containing SEQ ID NO: 21; (82) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 22; (83) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 21; (84) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 21; (85) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 21; (86) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 21; (87) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 21; (88) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 23; (89) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 23; (90) a targeting sequence containing SEQ ID NO: 23; (91) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 24; (92) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 23; (93) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 23; (94) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 23; (95) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 25; (96) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 25; (97) a targeting sequence containing SEQ ID NO: 25; (98) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 26; (99) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 25; (100) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 25; (101) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 25; (102) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 27; (103) a targeting sequence containing amino acids 15-30 of SEQ ID NO: 27; (104) a targeting sequence containing SEQ ID NO: 27; (105) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 28; (106) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 27; (107) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 27; (108) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 27; (109) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 27; (110) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 29; (111) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 29; (112) a targeting sequence comprising SEQ ID NO: 29; (113) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 30; (114) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 29; (115) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 29; (116) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 29; (117) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 29; (118) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 29; (119) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 31; (120) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 31; (121) a targeting sequence comprising SEQ ID NO: 31; (122) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 32; (123) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 31; (124) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 31; (125) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 31; (126) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 33; (127) a targeting sequence comprising SEQ ID NO: 33; (128) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 34; (129) a targeting sequence containing amino acids 1-16 of SEQ ID NO: 35; (130) a targeting sequence comprising SEQ ID NO: 35; (131) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 36; (132) a targeting sequence containing amino acids 1-29 of SEQ ID NO: 43; (133) a targeting sequence containing amino acids 14-29 of SEQ ID NO: 43; (134) a targeting sequence comprising SEQ ID NO: 43; (135) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 44; (136) a targeting sequence containing amino acids 2-29 of SEQ ID NO: 43; (137) a targeting sequence containing amino acids 5-29 of SEQ ID NO: 43; (138) a targeting sequence containing amino acids 8-29 of SEQ ID NO: 43; (139) a targeting sequence containing amino acids 10-29 of SEQ ID NO: 43; (140) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 45; (141) a targeting sequence containing amino acids 20-35 of SEQ ID NO: 45; (142) a targeting sequence containing SEQ ID NO: 45; (143) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 46; (144) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 45; (145) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 45; (146) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 45; (147) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 45; (148) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 45; (149) a targeting sequence containing amino acids 1-43 of SEQ ID NO: 47; (150) a targeting sequence containing amino acids 28-43 of SEQ ID NO: 47; (151) a targeting sequence comprising SEQ ID NO: 47; (152) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 48; (153) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 47; (154) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 47; (155) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 47; (156) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 47; (157) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 47; (158) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 47; (159) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 47; (160) a targeting sequence containing amino acids 1-32 of SEQ ID NO: 49; (161) a targeting sequence containing amino acids 17-32 of SEQ ID NO: 49; (162) a targeting sequence containing SEQ ID NO: 49; (163) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 50; (164) a targeting sequence containing amino acids 2-32 of SEQ ID NO: 49; (165) a targeting sequence containing amino acids 5-32 of SEQ ID NO: 49; (166) a targeting sequence containing amino acids 8-32 of SEQ ID NO: 49; (167) a targeting sequence containing amino acids 10-32 of SEQ ID NO: 49; (168) a targeting sequence containing amino acids 15-32 of SEQ ID NO: 49; (169) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 51; (170) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 51; (171) a targeting sequence containing SEQ ID NO: 51; (172) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 52; (173) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 51; (174) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 51; (175) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 51; (176) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 51; (177) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 51; (178) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 53; (179) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 53; (180) a targeting sequence comprising SEQ ID NO: 53; (181) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 54; (182) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 53; (183) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 53; (184) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 53; (185) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 53; (186) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 53; (187) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 55; (188) a targeting sequence containing amino acids 15-30 of SEQ ID NO: 55; (189) a targeting sequence comprising SEQ ID NO: 55; (190) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 56; (191) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 55; (192) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 55; (193) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 55; (194) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 55; (195) a targeting sequence containing amino acids 1-130 of SEQ ID NO: 57; (196) a targeting sequence containing amino acids 115-130 of SEQ ID NO: 57; (197) a targeting sequence comprising SEQ ID NO: 57; (198) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 58; (199) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 57; (200) a targeting sequence containing amino acids 5-130 of SEQ ID NO: 57; (201) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 57; (202) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 57; (203) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 57; (204) a targeting sequence containing amino acids 40-130 of SEQ ID NO: 57; (205) a targeting sequence containing amino acids 50-130 of SEQ ID NO: 57; (206) a targeting sequence containing amino acids 60-130 of SEQ ID NO: 57; (207) a targeting sequence containing amino acids 70-130 of SEQ ID NO: 57; (208) a targeting sequence containing amino acids 80-130 of SEQ ID NO: 57; (209) a targeting sequence containing amino acids 90-130 of SEQ ID NO: 57; (210) a targeting sequence containing amino acids 100-130 of SEQ ID NO: 57; (211) a targeting sequence containing amino acids 110-130 of SEQ ID NO: 57; (212) a targeting sequence comprising SEQ ID NO: 97; (213) a targeting sequence containing SEQ ID NO: 98; (214) a targeting sequence comprising SEQ ID NO: 99; (215) a targeting sequence comprising SEQ ID NO: 100; (216) a targeting sequence containing 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 targeting sequence comprising SEQ ID NO: 105; (221) a targeting sequence comprising SEQ ID NO: 106; (222) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (223) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 109; (224) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 110; (225) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 111; (226) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 112; (227) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 113; (228) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 114; (229) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 115; (230) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 116; (231) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 117; (232) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 118; (233) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 119; (234) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 120; or (235) an exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 121.

[00172] A fusion protein is provided that contains an enzyme suitable for breaking an emulsion or gel in a fracturing fluid, or an antibacterial protein or peptide and a targeting sequence, an exospore protein, or an exospore protein fragment. The targeting sequence, exosporium protein, or exosporium protein fragment may comprise any of the targeting sequences or exosporium proteins set forth in paragraphs [00166]-[00168] above for use with any protein or peptide of interest, or: (1) a targeting sequence containing an amino acid a sequence having at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 54%; (2) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 1; (3) a targeting sequence containing amino acids 20-35 of SEQ ID NO: 1; (4) a targeting sequence containing SEQ ID NO: 1; (5) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 2; (6) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 1; (7) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 1; (8) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 1; (9) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 1; (10) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 1; (11) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 3; (12) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 3; (13) a targeting sequence containing SEQ ID NO: 3; (14) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 4; (15) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 3; (16) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 3; (17) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 3; (18) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 3; (19) a targeting sequence containing amino acids 1-38 of SEQ ID NO: 5; (20) a targeting sequence containing amino acids 23-38 of SEQ ID NO: 5; (21) a targeting sequence containing SEQ ID NO: 5; (22) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 6; (23) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 5; (24) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 5; (25) a targeting sequence containing amino acids 8-38 of SEQ ID NO: 5; (26) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 5; (27) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 5; (28) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 5; (29) a targeting sequence containing amino acids 1-28 of SEQ ID NO: 7; (30) a targeting sequence containing amino acids 13-28 of SEQ ID NO: 7; (31) a targeting sequence containing SEQ ID NO: 7; (32) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 8; (33) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 7; (34) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 7; (35) a targeting sequence containing amino acids 8-28 of SEQ ID NO: 7; (36) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 7; (37) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 9; (38) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 9; (39) a targeting sequence containing SEQ ID NO: 9; (40) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 10; (41) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 9; (42) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 9; (43) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 9; (44) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 11; (45) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 11; (46) a targeting sequence containing SEQ ID NO: 11; (47) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 12; (48) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 11; (49) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 11; (50) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 11; (51) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 11; (52) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 11; (53) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 13; (54) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 13; (55) a targeting sequence containing SEQ ID NO: 13; (56) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 14; (57) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 13; (58) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 13; (59) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 13; (60) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 13; (61) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 13; (62) a targeting sequence containing amino acids 1-43 of SEQ ID NO: 15; (63) a targeting sequence containing amino acids 28-43 of SEQ ID NO: 15; (64) a targeting sequence containing SEQ ID NO: 15; (65) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 16; (66) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 15; (67) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 15; (68) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 15; (69) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 15; (70) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 15; (71) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 15; (72) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 15; (73) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 17; (74) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 17; (75) a targeting sequence containing SEQ ID NO: 17; (76) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 18; (77) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 17; (78) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 17; (79) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 17; (80) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 17; (81) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 19; (82) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 19; (83) a targeting sequence containing SEQ ID NO: 19; (84) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 20; (85) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 19; (86) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 19; (87) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 19; (88) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 19; (89) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 19; (90) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 21; (91) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 21; (92) a targeting sequence containing SEQ ID NO: 21; (93) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 22; (94) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 21; (95) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 21; (96) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 21; (97) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 21; (98) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 21; (99) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 23; (100) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 23; (101) a targeting sequence comprising SEQ ID NO: 23; (102) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 24; (103) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 23; (104) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 23; (105) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 23; (106) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 25; (107) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 25; (108) a targeting sequence comprising SEQ ID NO: 25; (109) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 26; (110) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 25; (111) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 25; (112) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 25; (113) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 27; (114) a targeting sequence containing amino acids 15-30 of SEQ ID NO: 27; (115) a targeting sequence comprising SEQ ID NO: 27; (116) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 28; (117) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 27; (118) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 27; (119) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 27; (120) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 27; (121) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 29; (122) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 29; (123) a targeting sequence comprising SEQ ID NO: 29; (124) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 30; (125) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 29; (126) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 29; (127) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 29; (128) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 29; (129) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 29; (130) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 31; (131) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 31; (132) a targeting sequence containing SEQ ID NO: 31; (133) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 32; (134) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 31; (135) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 31; (136) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 31; (137) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 33; (138) a targeting sequence comprising SEQ ID NO: 33; (139) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 34; (140) a targeting sequence containing amino acids 1-16 of SEQ ID NO: 35; (141) a targeting sequence containing SEQ ID NO: 35; (142) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 36; (143) a targeting sequence containing amino acids 1-29 of SEQ ID NO: 43; (144) a targeting sequence containing amino acids 14-29 of SEQ ID NO: 43; (145) a targeting sequence containing SEQ ID NO: 43; (146) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 44; (147) a targeting sequence containing amino acids 2-29 of SEQ ID NO: 43; (148) a targeting sequence containing amino acids 5-29 of SEQ ID NO: 43; (149) a targeting sequence containing amino acids 8-29 of SEQ ID NO: 43; (150) a targeting sequence containing amino acids 10-29 of SEQ ID NO: 43; (151) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 45; (152) a targeting sequence containing amino acids 20-35 of SEQ ID NO: 45; (153) a targeting sequence containing SEQ ID NO: 45; (154) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 46; (155) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 45; (156) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 45; (157) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 45; (158) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 45; (159) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 45; (160) a targeting sequence containing amino acids 1-43 of SEQ ID NO: 47; (161) a targeting sequence containing amino acids 28-43 of SEQ ID NO: 47; (162) a targeting sequence containing SEQ ID NO: 47; (163) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 48; (164) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 47; (165) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 47; (166) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 47; (167) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 47; (168) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 47; (169) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 47; (170) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 47; (171) a targeting sequence containing amino acids 1-32 of SEQ ID NO: 49; (172) a targeting sequence containing amino acids 17-32 of SEQ ID NO: 49; (173) a targeting sequence containing SEQ ID NO: 49; (174) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 50; (175) a targeting sequence containing amino acids 2-32 of SEQ ID NO: 49; (176) a targeting sequence containing amino acids 5-32 of SEQ ID NO: 49; (177) a targeting sequence containing amino acids 8-32 of SEQ ID NO: 49; (178) a targeting sequence containing amino acids 10-32 of SEQ ID NO: 49; (179) a targeting sequence containing amino acids 15-32 of SEQ ID NO: 49; (180) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 51; (181) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 51; (182) a targeting sequence comprising SEQ ID NO: 51; (183) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 52; (184) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 51; (185) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 51; (186) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 51; (187) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 51; (188) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 51; (189) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 53; (190) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 53; (191) a targeting sequence containing SEQ ID NO: 53; (192) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 54; (193) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 53; (194) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 53; (195) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 53; (196) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 53; (197) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 53; (198) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 55; (199) a targeting sequence containing amino acids 15-30 of SEQ ID NO: 55; (200) a targeting sequence comprising SEQ ID NO: 55; (201) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 56; (202) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 55; (203) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 55; (204) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 55; (205) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 55; (206) a targeting sequence containing amino acids 1-130 of SEQ ID NO: 57; (207) a targeting sequence containing amino acids 115-130 of SEQ ID NO: 57; (208) a targeting sequence containing SEQ ID NO: 57; (209) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 58; (210) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 57; (211) a targeting sequence containing amino acids 5-130 of SEQ ID NO: 57; (212) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 57; (213) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 57; (214) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 57; (215) a targeting sequence containing amino acids 40-130 of SEQ ID NO: 57; (216) a targeting sequence containing amino acids 50-130 of SEQ ID NO: 57; (217) a targeting sequence containing amino acids 60-130 of SEQ ID NO: 57; (218) a targeting sequence containing amino acids 70-130 of SEQ ID NO: 57; (219) a targeting sequence containing amino acids 80-130 of SEQ ID NO: 57; (220) a targeting sequence containing amino acids 90-130 of SEQ ID NO: 57; (221) a targeting sequence containing amino acids 100-130 of SEQ ID NO: 57; (222) a targeting sequence containing amino acids 110-130 of SEQ ID NO: 57; (223) an exospore protein fragment containing 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 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 containing 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) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (236) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 109; (237) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 110; (238) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 111; (239) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 112; (240) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 113; (241) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 114; (242) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 115; (243) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 116; (244) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 117; (245) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 118; (246) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 119; (247) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 120; (248) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 121; (249) a targeting sequence containing amino acids 22-31 of SEQ ID NO: 1; (250) a targeting sequence containing amino acids 22-33 of SEQ ID NO: 1; (251) a targeting sequence containing amino acids 20-31 of SEQ ID NO: 1; (252) a targeting sequence containing amino acids 14-23 of SEQ ID NO: 3; (253) a targeting sequence containing amino acids 14-25 of SEQ ID NO: 3; or (254) a targeting sequence containing amino acids 12-23 of SEQ ID NO: 3.

[00173] When the protein or peptide of interest comprises an antigen, a remediation enzyme, an enzyme suitable for breaking an emulsion or gel in a fracturing fluid, or an antibacterial protein or peptide, preferably the exosporium targeting sequence or protein comprises any of the exosporium targeting sequences or proteins, above in paragraph [00167], for use with any protein or peptide of interest, or: (1) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 1; (2) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 1; (3) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 1; (4) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 1; (5) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 1; (6) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 3; (7) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 3; (8) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 3; (9) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 3; (10) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 5; (11) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 5; (12) a targeting sequence containing amino acids 8-38 of SEQ ID NO: 5; (13) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 5; (14) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 5; (15) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 5; (16) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 7; (17) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 7; (18) a targeting sequence containing amino acids 8-28 of SEQ ID NO: 7; (19) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 7; (20) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 9; (21) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 9; (22) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 9; (23) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 11; (24) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 11; (25) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 11; (26) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 11; (27) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 11; (28) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 13; (29) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 13; (30) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 13; (31) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 13; (32) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 13; (33) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 15; (34) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 15; (35) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 15; (36) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 15; (37) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 15; (38) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 15; (39) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 15; (40) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 17; (41) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 17; (42) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 17; (43) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 17; (44) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 19; (45) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 19; (46) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 19; (47) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 19; (48) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 19; (49) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 21; (50) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 21; (51) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 21; (52) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 21; (53) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 21; (54) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 23; (55) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 23; (56) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 23; (57) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 25; (58) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 25; (59) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 25; (60) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 27; (61) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 27; (62) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 27; (63) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 27; (64) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 29; (65) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 29; (66) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 29; (67) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 29; (68) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 29; (69) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 31; (70) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 31; (71) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 31; (72) a targeting sequence containing amino acids 2-29 of SEQ ID NO: 43; (73) a targeting sequence containing amino acids 5-29 of SEQ ID NO: 43; (74) a targeting sequence containing amino acids 8-29 of SEQ ID NO: 43; (75) a targeting sequence containing amino acids 10-29 of SEQ ID NO: 43; (76) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 45; (77) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 45; (78) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 45; (79) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 45; (80) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 45; (81) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 47; (82) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 47; (83) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 47; (84) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 47; (85) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 47; (86) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 47; (87) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 47; (88) a targeting sequence containing amino acids 2-32 of SEQ ID NO: 49; (89) a targeting sequence containing amino acids 5-32 of SEQ ID NO: 49; (90) a targeting sequence containing amino acids 8-32 of SEQ ID NO: 49; (91) a targeting sequence containing amino acids 10-32 of SEQ ID NO: 49; (92) a targeting sequence containing amino acids 15-32 of SEQ ID NO: 49; (93) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 51; (94) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 51; (95) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 51; (96) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 51; (97) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 51; (98) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 53; (99) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 53; (100) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 53; (101) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 53; (102) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 53; (103) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 55; (104) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 55; (105) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 55; (106) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 55; (107) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 57; (108) a targeting sequence containing amino acids 5-130 of SEQ ID NO: 57; (109) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 57; (110) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 57; (111) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 57; (112) a targeting sequence containing amino acids 40-130 of SEQ ID NO: 57; (113) a targeting sequence containing amino acids 50-130 of SEQ ID NO: 57; (114) a targeting sequence containing amino acids 60-130 of SEQ ID NO: 57; (115) a targeting sequence containing amino acids 70-130 of SEQ ID NO: 57; (116) a targeting sequence containing amino acids 80-130 of SEQ ID NO: 57; (117) a targeting sequence containing amino acids 90-130 of SEQ ID NO: 57; (118) a targeting sequence containing amino acids 100-130 of SEQ ID NO: 57; (119) a targeting sequence containing amino acids 110-130 of SEQ ID NO: 57.

[00174] When the protein or peptide of interest comprises an antigen, a remediation enzyme, an enzyme suitable for breaking an emulsion or gel in a fracturing fluid, or an antibacterial protein or peptide, more preferably, the exosporium targeting sequence or protein comprises any of the exosporium targeting sequences or proteins above in paragraph [00167], for use with any protein or peptide of interest, or: (1) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 9; (2) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 9; (3) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 9; (4) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 11; (5) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 11; (6) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 11; (7) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 11; (8) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 11; (9) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 13; (10) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 13; (11) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 13; (12) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 13; (13) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 13; (14) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 15; (15) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 15; (16) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 15; (17) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 15; (18) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 15; (19) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 15; (20) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 15; (21) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 17; (22) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 17; (23) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 17; (24) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 17; (25) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 19; (26) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 19; (27) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 19; (28) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 19; (29) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 19; (30) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 21; (31) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 21; (32) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 21; (33) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 21; (34) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 21; (35) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 23; (36) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 23; (37) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 23; (38) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 25; (39) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 25; (40) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 25; (41) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 27; (42) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 27; (43) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 27; (44) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 27; (45) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 29; (46) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 29; (47) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 29; (48) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 29; (49) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 29; (50) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 31; (51) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 31; (52) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 31; (53) a targeting sequence containing amino acids 2-29 of SEQ ID NO: 43; (54) a targeting sequence containing amino acids 5-29 of SEQ ID NO: 43; (55) a targeting sequence containing amino acids 8-29 of SEQ ID NO: 43; (56) a targeting sequence containing amino acids 10-29 of SEQ ID NO: 43; (57) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 45; (58) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 45; (59) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 45; (60) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 45; (61) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 45; (62) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 47; (63) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 47; (64) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 47; (65) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 47; (66) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 47; (67) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 47; (68) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 47; (69) a targeting sequence containing amino acids 2-32 of SEQ ID NO: 49; (70) a targeting sequence containing amino acids 5-32 of SEQ ID NO: 49; (71) a targeting sequence containing amino acids 8-32 of SEQ ID NO: 49; (72) a targeting sequence containing amino acids 10-32 of SEQ ID NO: 49; (73) a targeting sequence containing amino acids 15-32 of SEQ ID NO: 49; (74) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 51; (75) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 51; (76) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 51; (77) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 51; (78) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 51; (79) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 53; (80) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 53; (81) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 53; (82) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 53; (83) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 53; (84) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 55; (85) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 55; (86) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 55; (87) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 55; (88) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 57; (89) a targeting sequence containing amino acids 5-130 of SEQ ID NO: 57; (90) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 57; (91) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 57; (92) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 57; (93) a targeting sequence containing amino acids 40-130 of SEQ ID NO: 57; (94) a targeting sequence containing amino acids 50-130 of SEQ ID NO: 57; (95) a targeting sequence containing amino acids 60-130 of SEQ ID NO: 57; (96) a targeting sequence containing amino acids 70-130 of SEQ ID NO: 57; (97) a targeting sequence containing amino acids 80-130 of SEQ ID NO: 57; (98) a targeting sequence containing amino acids 90-130 of SEQ ID NO: 57; (99) a targeting sequence containing amino acids 100-130 of SEQ ID NO: 57; (100) a targeting sequence containing amino acids 110-130 of SEQ ID NO: 57.

[00175] When the protein or peptide of interest comprises an antigen, a remediation enzyme, an enzyme suitable for breaking an emulsion or gel in a fracturing fluid, or an antibacterial protein or peptide, even more preferably the targeting sequence or exospore protein comprises: (1) a targeting sequence, containing amino acids 5-24 SEQ ID NO: 9; (2) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 9; (3) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 11; (4) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 11; (5) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 11; (6) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 11; (7) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 13; (8) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 13; (9) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 13; (10) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 13; (11) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 15; (12) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 15; (13) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 15; (14) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 15; (15) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 15; (16) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 15; (17) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 17; (18) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 17; (19) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 17; (20) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 19; (21) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 19; (22) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 19; (23) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 19; (24) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 21; (25) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 21; (26) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 21; (27) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 21; (28) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 23; (29) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 23; (30) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 25; (31) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 25; (32) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 27; (33) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 27; (34) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 27; (35) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 29; (36) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 29; (37) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 29; (38) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 29; (39) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 31; (40) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 31; (41) a targeting sequence containing amino acids 5-29 of SEQ ID NO: 43; (42) a targeting sequence containing amino acids 8-29 of SEQ ID NO: 43; (43) a targeting sequence containing amino acids 10-29 of SEQ ID NO: 43; (44) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 45; (45) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 45; (46) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 45; (47) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 45; (48) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 47; (49) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 47; (50) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 47; (51) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 47; (52) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 47; (53) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 47; (54) a targeting sequence containing amino acids 5-32 of SEQ ID NO: 49; (55) a targeting sequence containing amino acids 8-32 of SEQ ID NO: 49; (56) a targeting sequence containing amino acids 10-32 of SEQ ID NO: 49; (57) a targeting sequence containing amino acids 15-32 of SEQ ID NO: 49; (58) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 51; (59) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 51; (60) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 51; (61) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 51; (62) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 53; (63) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 53; (64) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 53; (65) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 53; (66) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 55; (67) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 55; (68) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 55; (69) a targeting sequence containing amino acids 5-130 of SEQ ID NO: 57; (70) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 57; (71) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 57; (72) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 57; (73) a targeting sequence containing amino acids 40-130 of SEQ ID NO: 57; (74) a targeting sequence containing amino acids 50-130 of SEQ ID NO: 57; (75) a targeting sequence containing amino acids 60-130 of SEQ ID NO: 57; (76) a targeting sequence containing amino acids 70-130 of SEQ ID NO: 57; (77) a targeting sequence containing amino acids 80-130 of SEQ ID NO: 57; (78) a targeting sequence containing amino acids 90-130 of SEQ ID NO: 57; (79) a targeting sequence containing amino acids 100-130 of SEQ ID NO: 57; (80) a targeting sequence containing amino acids 110-130 of SEQ ID NO: 57; (81) a targeting sequence containing amino acids 4-30 of SEQ ID NO: 59; (82) a targeting sequence containing amino acids 6-30 of SEQ ID NO: 59; (83) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 61; (84) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 61; (85) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 61; (86) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 63; (87) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 63; (88) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 63; (89) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 63; (90) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 65; (91) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 67; (92) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 67; (93) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 69; (94) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 69; (95) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 69; (96) a targeting sequence containing amino acids 5-42 of SEQ ID NO: 75; (97) a targeting sequence containing amino acids 10-42 of SEQ ID NO: 75; (98) a targeting sequence containing amino acids 15-42 of SEQ ID NO: 75; (99) a targeting sequence containing amino acids 20-42 of SEQ ID NO: 75; (100) a targeting sequence containing amino acids 25-42 of SEQ ID NO: 75; (101) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 77; (102) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 81; (103) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 81; (104) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 81; (105) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 81; (106) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 87; (107) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 87; (108) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 89; (109) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 89; (110) a targeting sequence containing amino acids 10-93 of SEQ ID NO: 91; (111) a targeting sequence containing amino acids 20-93 of SEQ ID NO: 91; (112) a targeting sequence containing amino acids 30-93 of SEQ ID NO: 91; (113) a targeting sequence containing amino acids 40-93 of SEQ ID NO: 91; (114) a targeting sequence containing amino acids 50-93 of SEQ ID NO: 91; (115) a targeting sequence containing amino acids 60-93 of SEQ ID NO: 91; (116) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 93; (117) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 93; or (118) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 93.

[00176] The fusion protein or peptide of interest described above may include an antigen.

[00177] The fusion protein or peptide of interest described above may include a reclamation enzyme.

[00178] The fusion protein or peptide of interest described above may include an enzyme suitable for breaking an emulsion or gel in a fracturing fluid.

[00179] The fusion protein or peptide of interest described above may include an antibacterial protein or peptide.

C. Recombinant members of the Bacillus cereus family that express fusion proteins

[00180] In addition, the present invention provides recombinant members of the Bacillus cereus family that express a fusion protein. The fusion protein may be any of the fusion proteins described in section IB above.

II. Modulation of Fusion Protein Expression in Recombinant Family Members Bacillus cereus that express the fusion protein via co-overexpression with modulator proteins

[00181] Recombinant members of the Bacillus cereus family that express the fusion proteins described herein display the protein or peptide of interest that is part 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 member of the Bacillus cereus family that also expresses the fusion protein allows modulation (i.e., increase or decrease) in the level of expression of the fusion protein, thereby thereby increasing or decreasing the amount of protein or peptide of interest that is exposed to the outside of the spore. The ability to control the amount of protein or peptide of interest that is exposed outside the spore is useful because in some cases it is desirable to increase the amount of protein or peptide of interest that is exposed. For example, when the protein of interest is an enzyme that degrades a plant nutrient source, it may be desirable to increase the amount of enzyme exhibited on the spore so that higher enzymatic activity and higher stimulation of plant growth can be achieved when the spores are introduced into the plant growth medium. or applying the spores to the plant or plant seed or the area surrounding the plant or plant seed. In other cases, it is desirable to reduce the amount of protein or peptide of interest that is exposed. For example, when the protein or peptide of interest includes a plant immune enhancing protein or peptide, it may be desirable to reduce the amount of protein or peptide exhibited on the spore, since overstimulation of the plant's immune system can lead to undesirable effects.

[00182] As further described herein below, recombinant members of the Bacillus cereus family that express the modulator protein can be used in any of the various fields and methods described herein and for any of the uses described herein. For example, recombinant members of the Bacillus cereus family that express a modulator protein can be used in methods for promoting plant growth; ways to protect plants from pathogens; ways to increase plant resistance to stress; methods for immobilizing spores of a recombinant member of the Bacillus cereus family on plants; methods of stimulating the germination of plant seeds; methods for delivering nucleic acids to a plant; methods for delivering nucleic acids to animals, insects, worms (eg, nematodes), fungi, or protozoa; methods for delivering enzymes to a plant; ways to change the properties of plants; methods for delivering proteins or peptides to an animal; vaccines and methods for achieving an immunogenic response in an individual; ways to reduce the content of contaminants in the environment; methods of phytorecultivation of contaminated soil; methods of treating the fracturing fluid to break the emulsion or gel in the fluid; surface disinfection methods; and for applications such as the processing or degumming of lubricants, oils or fats; processing hides into leather; production of biofuels, biodiesel or bioethanol; processing or conversion of sugars; starch processing; paper or linen processing; treatment of animal or fungal by-products or isolation of amino acids; directed splitting of technical waste; food and feed additives; dietary supplements; pet food; industrial cleaning; grain processing; cosmetic production; deodorization; food or beverage processing; brewing optimization or brewing additives; detergent additives or fabric or yarn processing.

[00183] For many uses of proteins (eg, enzymes), there is a biological response curve where an optimal protein or enzyme concentration results in a desired effect, and too much protein or too little protein results in undesirable or reduced effects. One example of this biological curve is the demonstration that a biological drug, such as the protein drug insulin for the treatment of diabetes, requires an optimal dose to lower blood sugar levels in diabetic patients. Too little insulin leads to a poor response and maintenance of unwanted elevated blood sugar levels and potential hyperkalemia. Too much insulin leads to low blood sugar levels and potential hypokalemia and associated morbidity.

[00184] Similar biological response curves exist for many of the proteins and peptides of interest contained in the fusion proteins described herein. Thus, for the various uses and methods for the recombinant members of the Bacillus cereus family described herein, it may be desirable to modulate the expression level of a protein or peptide of interest on the exospore. By increasing or decreasing the expression levels of the protein or peptide of interest on exospores of a recombinant member of the Bacillus cereus family, expression levels can be optimized to maintain the overall level of expression of the protein or peptide of interest at the most effective concentration.

[00185] For example, it would be desirable to modulate expression levels of a fusion protein in cases where the protein or peptide of interest includes a protein or peptide involved in direct signaling in plants, such as the flagellin flg22 peptide, and a recombinant member of the Bacillus cereus family expressing the fusion protein, applied to the plant to provide a beneficial effect on the plant. Such modulation would be useful to avoid a signal response that is high enough to result in detrimental plant responses (for example, too high a response to flg22 can result in necrosis), or a signal response that is low enough to provide low or insufficient peptide response.

[00186] The biological response curve may also be relevant for recombinant members of the Bacillus cereus family expressing a fusion protein wherein the protein or peptide of interest includes an antigen. In such cases, it would be desirable to modulate the level of expression of the antigen-containing fusion protein to achieve an optimal range for providing an appropriate immune response in the animal. Too high a dose may result in swelling of the injection site and unwanted inflammation, while too low a dose may result in insufficient vaccination or immune response.

[00187] Modulating the expression level of the fusion protein on exospores of a recombinant member of the Bacillus cereus family also provides a benefit, for example, when a recombinant member of the Bacillus cereus family is used to break an emulsion or gel in a fracturing fluid. Polysaccharide gels are often used in hydraulic fracturing gels. These gels require destruction. When the gel solution is ready to be broken down, it is desirable for the operator that the breaking down, which is an enzymatic reaction, occurs at a particular optimized rate. Too rapid degradation of the gel can lead to undesirable side effects such as accumulation of uncleaved gel fragments. On the other hand, breaking down too slowly leads to long waiting times and increased costs. Using the methods described herein below, enzyme levels on the exospore of a recombinant member of the Bacillus cereus family expressing a fusion protein containing an enzyme suitable for breaking an emulsion or gel in a fracturing fluid can be modulated to provide an optimized level of enzyme for breaking gels, which leads to preferred results when used in the field.

[00188] Provided is a recombinant member of the Bacillus cereus family 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 into the exospore of a recombinant member of the Bacillus family cereus ; and (ii) a modulator protein, wherein the expression of the modulator protein is increased compared to the expression of the modulator protein in a wild-type member of the Bacillus cereus family under the same conditions. The modulator protein, when co-expressed with a fusion protein in a recombinant member of the Bacillus cereus family, results in increased or reduced expression of the fusion protein compared to the expression level of the fusion protein in a recombinant member of the Bacillus cereus family that does not express the modulator protein at an increased level, compared with the expression of the modulator protein in a wild-type member of the Bacillus cereus family under the same conditions.

[00189] The modulator protein may include ExsY protein, ExsFA/BxpB protein, CotY protein, CotO protein, ExsFB protein, InhA1 protein, InhA2 protein, ExsJ protein, ExsH protein, YjcA protein, YjcB protein, BclC protein, AcpC protein, InhA3, alanine racemase 1, alanine racemase 2, BclA protein, BclB protein, BxpA protein, BclE protein, BetA/BAS3290 protein, CotE protein, ExsA protein, ExsK protein, ExsB protein, YabG protein, Tgl protein, SODA1 protein, SODA2 protein, variant any of them or a combination of any of them.

[00190] For example, a modulator protein, when coexpressed in a recombinant member of the Bacillus cereus family with a fusion protein, results in increased expression of the fusion protein relative to the expression level of the fusion protein in a recombinant member of the Bacillus cereus family that does not express the modulator protein at an increased level , compared with the expression of the modulator protein in a wild-type member of the Bacillus cereus family under the same conditions. When the modulator protein, when co-expressed in a recombinant member of the Bacillus cereus family with a fusion protein, results in increased expression of the fusion protein, the modulator protein may include BclB protein, CotE protein, BxpB protein, CotO protein, BclA protein, a variant of any of these. or a combination of any of them.

[00191] Alternatively, the modulator protein, when co-expressed in a recombinant member of the Bacillus cereus family with a fusion protein, results in reduced expression of the fusion protein relative to the level of expression of the fusion protein in a recombinant member of the Bacillus cereus family that does not express the modulator protein at an increased level, compared with the expression of the modulator protein in a wild-type Bacillus cereus under the same conditions. When the modulator protein, when co-expressed in a recombinant member of the Bacillus cereus family with a fusion protein, results in such reduced expression of the fusion protein, the modulator protein may include a BclC protein, an ApcC protein, a YjcB protein, a variant of any of these, or a combination of any of them.

[00192] For example, a modulator protein may include a CotO protein, a BclB protein, an ExsFA/BxpB protein, a YjcB protein, any variant thereof, or any combination thereof.

[00193] For ease of reference, descriptions of modulator proteins and their SEQ ID NOs are provided in Table 2 below.

Table 2. Amino acid sequences of modulator proteins

Modulator protein SEQID NO. ExsY, Bacillus thuringiensis 123 ExsFA/BxpB, Bacillus thuringiensis 124 CotY, Bacillus cereus 125 CotO, Bacillus anthracis 126 ExsFB variant 1, Bacillus cereus 127 ExsFB variant 2, Bacillus cereus 128 InhA1, Bacillus cereus 129 InhA3, Bacillus mycoides 130 ExsJ, Bacillus cereus ATCC 10876 131 ExsH, Bacillus cereus 132 YjcA, Bacillus cereus 133 YjcB, variant 1, Bacillus cereus 134 YjcB, variant 2, Bacillus cereus 135 BclC, Bacillus anthracis 136 AcpC, Bacillus cereus 137 InhA2, Bacillus cereus 138 Alanine racemase 1, Bacillus cereus 139 Alanine racemase 2, Bacillus 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 BxpA, 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 group 149 ExsA, Bacillus cereus 150 ExsK, Bacillus cereus AH187 151 ExsB, Bacillus cereus 152 YabG, Bacillus cereus 153 Tgl, Bacillus cereus group 154 SODA1, Bacillus cereus 155 SODA2, Bacillus thuringiensis 156

[00194] Many of the modulator proteins have homologs, paralogs, or genetic rearrangements. Thus, many proteins that share at least 70% homology with any of the modulating sequences listed above in Table 2 retain the ability to act as modulator proteins when overexpressed in a recombinant member of the Bacillus cereus family that also expresses any of the fusion proteins. described in the present description. In addition, many of the modulator proteins (eg, BclA, BclB, and BclE) have internal repeat regions that can vary significantly between strains. Additions or reductions in the number of repeats in the internal repeat region can affect the overall sequence homology, but it is expected that if the homology of the N- and C-terminal regions of the protein retains at least 75% sequence identity with any of the amino acid sequences of the modulator proteins listed in the table above, the homologues will retain the ability to act as modulator proteins.

[00195] Thus, for example, a modulator protein may comprise an amino acid sequence having at least 70% sequence identity with any of SEQ ID NOs: 123-156.

[00196] The modulator protein may comprise an amino acid sequence having at least 75% sequence identity with any of SEQ ID NOs: 123-156.

[00197] The modulator protein may comprise an amino acid sequence having at least 85% sequence identity with any of SEQ ID NOs: 123-156.

[00198] The modulator protein may comprise an amino acid sequence having at least 90% sequence identity with any of SEQ ID NOs: 123-156.

[00199] The modulator protein may comprise an amino acid sequence having at least 95% sequence identity with any of SEQ ID NOs: 123-156.

[00200] The modulator protein may comprise an amino acid sequence having at least 98% sequence identity with any of SEQ ID NOs: 123-156.

[00201] The modulator protein may comprise an amino acid sequence having at least 99% sequence identity with any of SEQ ID NOs: 123-156.

[00202] The modulator protein may comprise an amino acid sequence having 100% sequence identity with any of SEQ ID NOs: 123-156.

[00203] For example, the modulator protein may include SEQ ID NO: 124, 126, 134, 135, 143, or 144.

[00204] Recombinant members of the Bacillus cereus family that express the modulator protein may contain a vector encoding the modulator protein. For example, the vector may include a multicopy plasmid. Multicopy plasmids allow high levels of expression of the modulator protein.

III. Promoters for expression of fusion proteins and/or modulator proteins in recombinant members of the family Bacillus cereus

[00205] When the fusion protein contains a targeting sequence, an exospore protein or an exospore protein fragment that targets the fusion protein to the exospore of a member of the Bacillus cereus family, the DNA encoding the fusion protein is suitably under the control of a sporulation promoter that causes the fusion protein to be expressed on the exospore endospores of a member of the B. cereus family (eg, native bclA promoter from a member of the B. cereus family).

[00206] Thus, any of the fusion proteins described in section 1.B above can be expressed in a recombinant member of the Bacillus cereus family under the control of a sporulation promoter that is native to the targeting sequence, exosporium protein, or exosporium protein fragment of the fusion protein, or portion such a promoter.

[00207] Similarly, any of the modulator proteins described in section II above can be expressed under the control of their native promoter, or a portion thereof.

[00208] Any of the fusion or modulator proteins can be expressed under the control of a highly expressed sporulation promoter.

[00209] A highly expressed sporulation promoter includes a sporulation-specific sigma-K polymerase promoter sequence.

[00210] For ease of reference, exemplary promoter nucleotide sequences that can be used to express any of the fusion proteins or any of the modulator proteins in a recombinant member of the Bacillus cereus family are shown in Table 3 below along with their SEQ ID NO. Table 3 also provides illustrative minimum promoter sequences for many promoters. In Table 3, sporulation-specific polymerase-K promoter sequences in promoters are indicated in bold and underlined text. Some of the sequences have multiple sigma-K sequences that overlap with each other. Overlaps are indicated by double underlining in the table. The promoter sequences are immediately upstream of the start codon for each of these genes. In other words, in the sequences shown in Table 3 below, the last nucleotide of the promoter sequence immediately precedes the first nucleotide of the start codon for the coding region of the gene encoding the specified protein.

Table 3. Promoter sequences for the expression of fusion proteins and modulator proteins in recombinant members of the family Bacillus cereus

Promoter (SEQ ID NO) Promoter sequence ExsY promoter ( B. cereus F837/76) (SEQ ID NO: 157) TTTCTTAATCCTTTACCCTTTACTTTTGTAAAAGTTGATACACTTCCATCCGGCTCTGTAATTTCTAATTCATCAATAAATGGTCTTCGCAAAAAGCCTGTAATTTTATCATAAACAATTAAACGAGTGAGCCTAAAAGCAGCTAACGCGAAAATAAAAAATAAAAGCCAGCTTGTAAACAGCATAATTCCACCTTCCCTTATCCTCTTTCGCCTATTTAAAAAAAGGTCTTGAGATTGTGACCAAATCTCCTCAACTCC AATATCTTA TTAATGTAAATACAAACAAGAAGATAAGGA Minimal promoter ExsY ( B. cereus F837/76) (SEQ ID NO: 158) ACCAAATCTCCTCAACTCC AATATCTTA TTAATGTAAATACAAAACAAGAAGATAAGGA ExsFA/BxpB promoter ( B. anthracis Sterne) (SEQ ID NO: 159) ACCACCTACCGACGATCCAATCTGTACATTCCTAGCTGTACCAAATGCAAGATTAATATCGACTAACACTTGTCTTACTGTTGATTTAAGTTGCTTCTGTGCGATTCAATGCTTGCGTGATGTTACGATTTAAAACTAAATAATGAGCTAAG CATGGATTG GGTGGCAGAATTATCTGCCACCCAATCCATGCTTAACGAGTATTATTATGTAAATTTCTTAAAATTGGGAACTTGTCTAGAACATAGAACCTGTCCTTTT CATTAACTG AAAGTAGAAACAGATAAAGGAGTGAAAAAC Minimal promoter ExsFA/BxpB ( B. anthracis Sterne) (SEQ ID NO: 160) ACATAGAACCTGTCCTTTTT CATTAACTG AAAGTAGAAACAGATAAAGGAGTGAAAAAC CotY/CotZ promoter ( B. anthracis Sterne) (SEQ ID NO: 161) TAGAAGAAGAACGCCGACTACTTTATGTCGCAATTACACGGGCGAAAGAAGAACTTTACATTTCCTCTCCGCAATTTTTTAGAGGAAAAAAATTAGATATATCTCGTTTTTTATACACTGTGCGAAAAGATTTACCTGAAAAGACATCCACTAAATAAGGATGTCTTTTTTTATATTGTATTATGTACATCCCTACTATATAAATTCCCTGCTTTTATCGTAAGAATTAACGTAATATCAACCATATCCCGTT CATATTGTA GTAGTGTATGTCAGAACTCACGAGAAGGAGTGAACATA Minimal promoter CotY/CotZ ( B. anthracis Sterne) (SEQ ID NO: 162) TCAACCATATCCCGTT CATATTGTA GTAGTGTATGTCAGAACTCACGAGAAGGAGTGAACATA CotO ( B. cereus ) promoter (SEQ ID NO: 163) TAACTCAATCTTAAGAGAAATTGAGGAGCGCGCACCACTTCGTCGTACAACAACGCAAGAAGAAGTTGGGGATACAGCAGTATTCTTATTCAGTGATTTAGCACGCGGCGTAACAGGAGAAAACATTCACGTTGATTCAGGGTAT CATATCTTA GGATA AATATAATA TTAATTTTAAAGGACAATCTCTACATGTTGAGATTGTCCTTTTTATTTGTTCTTAGAAAGAACGATTTTTAACGAAAGTTCTTACCACGTTATGAATATAAGTATAATAGTACACGATTTATTCAGCTACGT Minimal promoter CotO ( B. cereus ) (SEQ ID NO: 164) ACGTTGATTCAGGGTAT CATATCTTA GGATA AATAATAATA TTAATTTTAAAGGACAATCTCTACATGTTGAGATTGTCCTTTTTATTTGTTCTTAGAAAGAACGATTTTTAACGAAAGTTCTTACCACGTTATGAATATAAGTATAATAGTACACGATTTATTCAGCTACGT ExsFB promoter ( B. cereus F837/76) (SEQ ID NO: 165) CATAAAAATCTACTTTTCTTGTCAAAGAGTATGCTTATATGCGTGCTCTTTTTATTTGGTTTTCTTTCATTTCTAAATAACATTTTCAACTCTATTCATACTATTCTTTCAACTTTAGGTTACAAACTATTTCTGTAAGCGTAGTGTTTCTTTTGTACTATAGGCAGTTAGTTTTATCCATAACAGTACACCTCTGCACTATTCACTATAAATTTT CATATATTA TATTGTGCTTGTCCAAAACATGTGGTTATTACTCACGCGATCTAAATGAAAGAAAGGAGTGAAAAT Minimal ExsFB promoter ( B. cereus F837/76) (SEQ ID NO: 166) ACTATTCACTATAAATTTT CATATATTA TATTGTGCTTGTCCAAAAACATGTGGTTATTACTCACGCGATCTAAATGAAAGAAAGGAGTGAAAAT InhA1 promoter ( B. thuringiensis serovar kurstaki strain HD-1) (SEQ ID NO: 167) AATACATGATAATGAAATCCGATTTTGTGTTTTATATAGTGAATTATCAAATATTGTGTAGATGAAACAAAGATAAAATCCCCATTAAACTCCCTCTATGGAAATTATAAATTGTTCGATAAAAACTTTCAATATTTTCAGAAAACATTGTTGAATTGTGATATATTCGTATGCTAACTATGAAATTTTTACAAATATATTAAAAACATTA CATAATATG ACTAAATATTGAAAAAATATTGAATTTTTAATAAAATTTAATTTGTAATA CATATTATT TATTAGGGGAGGAAATAAGGG InhA1 minimal promoter ( B. thuringiensis serovar kurstaki strain HD-1) (SEQ ID NO: 168) AAAATTTAATTTGTAATA CATATTATT TATTAGGGGAGGAAATAAGGG InhA2 promoter ( B. mycoides strain 219298) (SEQ ID NO: 169) AATTGTGCATATTGTCTTTTAAATTTTCTATCTAAGTTATTTAATATATAATAAATAACTCTTTTTTGTGAGTTTTTTTGATACGAGGTAAATAATCAGTACAGGGTCTGACCAGAGGACTGGAGGGCATGATTCTATAAGGGAATATTTACTATTCCATGATTATAGAACTATGTCTTTTTTATTGTATATAGAAGGGGGGATAGGTC TATATTATA GAACTTATATATATTGTGCATTC CATATTATC AATTATCTAAATTTTAAGTCTTGTTACAATTAATAAGGGAGGAAATAGTA InhA2 minimal promoter ( B. mycoides strain 219298) (SEQ ID NO: 170) ACTTATATATATTGTGCATTC CATATTATC AATTATCTAAATTTTAAGTCTTGTTACAATTAATAAGGGAGGAAATAGTA ExsJ promoter ( B. thuringiensis serovar kurstaki) (SEQ ID NO: 171) AATGACGTTTTCAAGTTTGATTATCATTCATGTTTCCTATTTTAAGAGAAACATATAACTCAACTACTTTTTTCAATGG CATCTTTTA TAGTACTTAGAATAGGAAAACACTCAACTATAAGAAAAAGTAAGGAGGAAATAA Minimal promoter ExsJ ( B. thuringiensis serovar kurstaki) (SEQ ID NO: 172) ACTACTTTTTTCAATGG CATCTTTTA TAGTACTTAGAATAGGAAAACACTCAACTATAAGAAAAAGTAAGGAGGAAATAA ExsH promoter ( B. cereus F837/76) (SEQ ID NO: 173) ATATGCTAATGCTTAGTTTTTATACTCAAGTTAAAATGTGCTTTTGGACCTAAGAGATAAACGTGGAAA AATAAAATA AACTCTTAAGTTTAGGTGTTTAATCTAAGCAGTCAATTATTAAAAA CATATAATT AATATGTGAGTCATGAA CATAATTAA ATAATGTTTTCAAGTTTAATTATCGTTCATGTTTCCTATTTTAAGCAGAACAAATAACTCAATTACTTTTTTCGATTGGATCTTTTTTAACTCTTATAATAGGAAAACACTCAACTATAAAAATAAGTAAGGAGGAAATAA Minimal promoter ExsH ( B. cereus F837/76) (SEQ ID NO: 174) AATATGTGAGTCATGAA CATAATTA AATAATGTTTTCAAGTTTAATTATCGTTCATGTTTCCTATTTTAAGCAGAACAAATAACTCAATTACTTTTTTCGATTGGATCTTTTTTAACTCTTATAATAGGAAAACACTCAACTATAAAAATAAGTAAGGAGGAAATAA YjcA promoter ( B. thuringiensis serovar kurstaki strain HD73) (SEQ ID NO: 175) TATAAAATAAAAGGGCGTGTATTTGCTACTGATGCAGTATTGTGTGCGCCTAAAAATGGAATTTCACAACCAGATCCACATGTTGTTGTAGAACAATCTTGTAATTCATTGATGAATTTTACAACGTCAACTACACAATGAGAAGAGCCATGGTGTTTATTTTCGTTACAACTCATTAATGTCACTCCTTATCTTCTTGTTTGTATTTACATT AATAAGATA TTGGAGTTGAGGAGATTTGGTCACAATCTCAAGACCTTTTTTTTAAATAGGCGAAAGAGGATAAGGGAAGGTGGAATT YjcA minimal promoter ( B. thuringiensis serovar kurstaki strain HD73) (SEQ ID NO: 176) TCTTGTTTGTATTTACATT AATAAGATA TTGGAGTTGAGGAGATTTGGTCACAATCTCAAGACCTTTTTTAAATAGGCGAAAAGGATAAGGGAAGGTGGAATT YjcB promoter ( B. thuringiensis serovar kurstaki strain HD73) (SEQ ID NO: 177) ATCAACTTTTACAAAAGTAAAGGGTAAAGGATTAAGAAAGTGGATTGGCGAATTATTAAGCTGTTATTGGTGTACAGGTGTATGGGTTAGTGCTTTTTTATTAGTTTTATATAATTGGATTCCGATCGTTGCAGAGCCGTTACTTGCATTATTAGCTATTGCAGGAGCAGCAGCAATCATTGAAACGATTACAG GATATTTTA TGGGAG AATAATATA TTTTCATAATACGAGAAAAAGCGGAGTTTAAAAGAATGAGGGAACGGAAATAAAGAGTTGTT CATATAGTA AATAGACAGAA Minimal promoter YjcB ( B. thuringiensis serovar kurstaki strain HD73) (SEQ ID NO: 178) ACGGAAATAAAGAGTTGTT CATATAGTA AATAGACAGAA BclC promoter ( B. anthracis Sterne) (SEQ ID NO: 179) TGAAGTATCTAGAGCTAATTTACGCAAAGGAATCTCAGGACAACACTTTCGCAACACC TATATTTTA AATTTAATAAAAAAAGAGACTCCGGAGTCAGAAATTATAAAGCTAGCTGGGTTCAAATCAAAAATTTCACTAAAACGATATTATCAATACGCAGAAAATGGAAAAAACGCCTTATCATAAGGCGTTTTTTCCATTTTTTCTTCAAACAAACGATTTTACTATGACCATTTAACTAATTTTTG CATCTACTA TGATGAGTTTCATTCACATTCTCATTAGAAAGGAGAGATTTA BclC minimal promoter ( B. anthracis Sterne) (SEQ ID NO: 180) ACCATTTAACTAATTTTTG CATCTACTA TGATGAGTTTCATTCACATTCTCATTAGAAAGGAGAGATTTA AcpC promoter ( B. cereus F837/76) (SEQ ID NO: 181) GACTATGTTTTATTCAG GATAAAATA TAGCACTACACTCTCTCCTCTTATTATGTAGCATCTCTCTAATCCATCATTTGTTTCATTTAGTTAAAATTGTAAATAAAATCACATGATTTGTCAATTATAATTGTCATTTCGACAATTAAACTTGTCAAAATAATTCTCATCATTTTTTCTCATCTTCTAATATAGGACATGACTATATATACAAAAAGAATATGCAAATGTT CATACAAGAAATACATCATTATTTTCTGA AcpC ( B. cereus F837/76) minimal promoter (SEQ ID NO: 182) AGACAATATGCAAATGTT CATACAAAA AATATTATTTTTCGA TATATAATA TTAACTGATTTTCTAACATCAAGGAGGGTACAT InhA3 promoter (B. thuringiensisserovar kurstaki strain HD73) (SEQ ID NO: 183) ATAGTGAGTAATATGGTAATC CATAGATTAAATAGTATA GAAAATATTTAATTCTTATTTTTATTAAAAAAGCATGAATCCCAGATTTACTGGGTTTTGATTGTAACTAAGAA CATATAAAA GTTCACTGTTATTTATAGGAGAGTCTGTTTGTTTT TATATCTTA TGTATTTCACCCTG CATAAAAAA ATATTTCTCAACATTTTATTTGTTGAAAAATATTGAATATTCGTATTATAACGAATATTATGTTGTTATCGGCAAAAAACGATAATTTGCAGACACTGGGGAGGAAATACA InhA3 minimal promoter (B. thuringiensisserovar kurstaki strain HD73) (SEQ ID NO: 184) TCTTTGTATTTCACCCTG CATAAAAAA ATATTTCTCAACATTTTATTTGTTGAAAAATATTGAATATTCGTATTATAACGAATATTATGTTGTTATCGGCAAAAAACGATAATTTGCAGACACTGGGGAGGAAATACA Alanine racemase 1 promoter ( B. cereus F837/76) (SEQ ID NO: 185) CTTCGTCAGCAATAAGTGTGAGCGGAGAATTGGTTGATCTTGGCTTTACAATTGGAGCATTGACGAAAGACTCTTTAACGTGGTCG CATAACGGA GTAGAATATATGCTCGTGTCTAAAGGTTTAGAGCCGAAGGAGCTATTAATGGTTGCTCGTTCAGTTACAGAGAAGCAAGTGAAGTAAACTTCTTAGACGTGGTGATATATGTGCACCACGTCTTTTCTTAGTTTGAAGGGTGGATTT CATAAAAGA AG CATATAAAA GAATAAGCTTCG CATATCGTG TATAAGGAAGTGTATTT Alanine racemase 1 minimal promoter ( B. cereus F837/76) (SEQ ID NO: 186) ATAAAAAGAATAAGCTTCG CATATCGTG TATAAGGAAGTGTATTT Alanine racemase 2 promoter ( B. thuringiensis serovar kurstaki strain HD73) (SEQ ID NO: 187) CATTTCAAATAATGAACGCTTCGATTGAATCGGAGCTATTTTCAAATCAATTTCAGTATATTGATCCAGCATTTGAATAGAAGTATCAACAGCAACTTTAAGTTGATGCAATGCAGATTGTACAAACATTGTAATTCTCCTCTTCTCCG TATATAATA GTTTCTTGAGGGTATTATATCATGCTCAAAATTCCGAAAATTCTAGTAGTTTGACTAG CATATTGAA AAGTAT TATATTGTA AAAGGT CATATGAAA CGTGAAATAGAATGGAATGCAATTATTGAGTTAGGAGTTAGACCA Alanine racemase 2 minimal promoter ( B. thuringiensis serovar kurstaki strain HD73) (SEQ ID NO: 188) T TATATTGTA AAAGGT CATATGAAA CGTGAAATAGAATGGAATGCAATTATTGAGTTAGGAGTTAGACCA BclA promoter ( B. cereus F837/76) (SEQ ID NO: 189) ATCGATGGAACCTGTATCAACCACTATAATTTCATCCACAATTTTTTCAACTGAGTCTAAACAACGGGCTATTGTCTTCTCCTCATCTCGAACAAT CATACATAAACTA ATTGTAATTCCTTGCTTGTTCAACATAATCACCCTCTTCCAAATCAAT CATATGTTA TA CATATACTA AACTTTCCATTTTTTTAAATTGTTCAAGTAGTTTAAGATTTCTTTTCAATAATTCAAATGTCCGTGTCATTTTCTTTCGGTTTTG CATCTACTA TATAATGAACGCTTTATGGAGGTGAATTT BclA minimal promoter ( B. cereus F837/76) (SEQ ID NO: 190) AATCAAT CATATGTTA TA CATATACTA AACTTTCCATTTTTTTAAATTGTTCAAGTAGTTTAAGATTTCTTTTCAATAATTCAAATGTCCGTGTCATTTTCTTTCGGTTTTG CATCTACTA TATAATGAACGCTTTATGGAGGTGAATTT BclB promoter ( B. thuringiensis serovar konkukian strain 97-27) (SEQ ID NO: 191) GACCTGTAAGTCTGTAGGGAAGAATAATTTCAAGAGCCAGTGATAATAGATTTTTTTGTTTTTTCATTCTTATCTTGAATATAAATCACCT CATCTTTTA ATTAGAACGTAACCAATTTAGTATTTTGAAATAGAGCTAT CATTTTATA ATATGAATACTACTAGTTATAGAAACGGCAAAAAGTTTAATATATGTAAAAATCATTTGGATATGAAAAAAGTAGC CATAGATTT TTTCGAAATGATAAATGTTTTATTTTGTTAATTAGGAAACAAAAATGTGGAATGAGGGGGATTTAA Minimal BclB promoter ( B. thuringiensis serovar konkukian strain 97-27) (SEQ ID NO: 192) ATATGAAAAAAGTAGC CATAGATTT TTTCGAAATGATAAATGTTTTATTTTGTTAATTAGGAAACAAAAATGTGGAATGAGGGGGATTTAA BxpA promoter ( B. anthracis strain Sterne) (SEQ ID NO: 193) TTTT CATCTGCTA CATCGTGAAGTAATGCTGCCATTTCAATTATAAAACGATTTCCTCCTTCTTGCTCGGATAAAGAAATCGCCAGTTTATGTACACGCTC AATATGATA CCAATCATGCCCACTGGCATCTTTTTCTAAAATATGTTTTACAAAAGTAATTGTTTTTTCTATCTTTTCTTGTTTTGTCATTTTATCTTCACCCAGTTACTTATTGTAACACGCCCGCATTTTTT CATCACATATTTTC TTGTCCGCC CATACACTA GGTGGTAGGCATCATCATGAAGGAGGAATAGAT BxpA minimal promoter ( B. anthracis strain Sterne) (SEQ ID NO: 194) ACATATTTTC TTGTCCGCC CATACACTA GGTGGTAGGCATCATCATGAAGGAGGAATAGAT BclE promoter ( B. anthracis ΔSterne) (SEQ ID NO: 195) GGTGACGACAA CATATACAA GAGGCACTCCTGCTGGTACTGTAACAGGAACAAATATGGGGCAAAGTGTAAATA CATCGGGTA TAGCACAAGCTGTCCCGAATACA GATAATATG GATTCAACGGCGGGACTCCCTTAAGAAATTAGGGGAGTCTTTATTTGGAAAAAGAGCTTATGTTACATAAAAACAGGAGTAATTGTTTTAAAAGTAGTATTGGTGACGTTGTTAGAAAATACAATTTAAGTAGAAGGTGCGTTTTTATATGA AATATATTT TATAGCTGTACTTTACCTTTCAAG BclE minimal promoter ( B. anthracis ΔSterne) (SEQ ID NO: 196) ACAAGCTGTCCCGAATACA GATAATATG GATTCAACGGCGGGACTCCCTTAAGAAATTAGGGGAGTCTTTATTTGGAAAAAGAGCTTATGTTACATAAAACAGGAGTAATTGTTTTAAAAGTAGTATTGGTGACGTTGTTAGAAAATACAATTTAAGTAGAAGGTGCGTTTTATATGA AATATATTT TATAGCTGTACTTTACCTTTCAAG BetA promoter ( B. anthracis Sterne) (SEQ ID NO: 197) ATTTATTTCATTCAATTTTTCCTATTTAGTACCTACCGCACTCACAAAAAGCACCTCTCATTAATTTATATTATAGTCATTGAAATCTAATTTAATGAAATCAT CATACTATA TGTTTTATAAGAAGTAAAGGTAC CATACTTAA TTAATACATATCTATACACTTCAATATCACAGCATGCAGTTGAATTATATCCAACTTTCATTTCAAATTAAATAAGTGCCTCCGCTATTGTGAATGTCATTTACTCTCCCTACTA CATTTAATA ATTATGACAAGCAATCATAGGAGGTTACTAC Minimal BetA promoter ( B. anthracis Sterne) (SEQ ID NO: 198) TAAGAAGTAAAGGTAC CATACTTAA TTAATACATATCTATACACTTCAATATCACAGCATGCAGTTGAATTATATCCAACTTTCATTTCAAATTAAATAAGTGCCTCCGCTATTGTGAATGTCATTTACTCTCCCTACTA CATTTAATA ATTATGACAAGCAATCATAGGAGGTTACTAC CotE promoter ( B. cereus AH820) (SEQ ID NO: 199) AGTTGTACAAGAATTTAAATCTTCACAAA CATATGTAA ATGACTTACTACAGCTAGTTGCAAGTACGATTTCTAACAACGTAACAGATGAAATATTAATTTCAACTAATGGCGATGTATTGAAGGGTGAAACGGGCGCAGCGGTAGAAAGTAAAAAAGGAAATTGTGGTTGTTAAAGAGATGTCGAAATGACATCTCTTTTTTTAGTGGATTAAACGTAAGTTCTTCTCAAAAAAAGAATGACACATTCCGCTATTGTCACG CATATGATT AAGTGAATAGTGATTGAGGAGGGTTACGA Minimal CotE promoter ( B. cereus AH820) (SEQ ID NO: 200) ACATTCCGCTATTGTCACG CATATGATT AAGTGAATAGTGATTGAGGAGGGTTACGA ExsA promoter ( B. cereus strain ATCC 10876) (SEQ ID NO: 201) AACGTTATTAGCGTAGACAAACAAGTAACGGCAGAAGCAGTTCTTG CATTAAATC GTATGTTAGAGCGTGTGTAAAGCAACGGTATTCCCGTTGCTTTTTTTCATA CATATAATC ATAACGAGAACGAAATGGG CATACATTG TTTTGAAGAAATCATTGTGGTTCTTTATGCTTATTCCACTTCGAATGATATTGAAAATCGAAGAAGTGATAAAAGTAAAAAGAAGTTAATGTTATTTAGAAAGAGTTACTTCATGAGATTTGTTACTTATA GATAAGTTA TACAGGAGGGGGAAAAT Minimal promoter ExsA ( B. cereus strain ATCC 10876) (SEQ ID NO: 202) TCATGAGATTTGTTACTTATA GATAAGTTA TACAGGAGGGGGAAAAT ExsK promoter ( B. thuringiensis serovar konkukian strain 97-27) (SEQ ID NO: 203) AAGCCGCGGTCAATGCTGTATATGCA AATAAGATT GCAGCTTTACCTGAAGAAGAGCGT GATAGCTTC ATTGCTGAAAAACGAGAAGAGTATAAGAAAGATATTGATATTTACCATTTAGCATCAGAGATGGTCATTGATGGTATTGTTCATCCAAACAATTTAAGAGAAGAGTTAAAAGGACGATTCGAAATGTATATGAGTAAATATCAAGTATTTACGGATCGTAAA CATCCTGTT TATCCAGTTTAAAAGCCCTATTTAGGGCTTTCTTGCTCAAAAAGTTAAGGAGGGGAAAACA Minimal promoter ExsK ( B. thuringiensis serovar konkukian strain 97-27) (SEQ ID NO: 204) TCAAGTATTTACGGATCGTAAA CATCCTGTT TATCCAGTTTAAAAGCCCTATTTAGGGCTTTCTTGCTCAAAAAGTTAAGGAGGGGAAAAACA ExsB promoter ( B. cereus F837/76) (SEQ ID NO: 205) AGGATTTCAGTGGGACGCCTCCTCTCTTCTTACATTAAATTAAT CATACTATA AAATGAAAGAAATGAAATGAAAAATAGCGGAAAAATCAGAAATTTTTTCTGGTAG TATACAATATGTTA CAATAAGCTTTGTCAATGAAAGAAGGAATTCCGTGCAATGCACGGGAGAGGTTCGCGAACTCCCTCTATAAAAAACTATGGAAACAAC AATATCTTT AGGTATTGTTTTGTTTTTTTATTGTGACAGTTCAAGAACGTTCTTTCTTCTTATTCGTAGTAGAGAAGGAGAATGAGTGAA Minimal ExsB promoter ( B. cereus F837/76) (SEQ ID NO: 206) ACTATGGAAACAAC AATATCTTT AGGTATTGTTTTGTTTTTTTATTGTGACAGTTCAAGAACGTTCTTTCTTCTTATTCGTAGTAGAGAAGGAGAATGAGTGAA YabG promoter ( B. cereus AH820) (SEQ ID NO: 207) TTTTGCACAACGCCGTAAAACTTTAATG AATAATTTA TCA AATAATTTA AATGGTTTCCCGAAAGATAAAGAGCTGTTGGATCGAATTTTAACAGAAGTAGGAATTGATCCAAAACGAAGAGGCGAAACGCTATCTATCGAAGAGTTTGCGACATTAAGTAATGCATTAGTTCTT CATAAGTTA TCATAAGAATACAAAAGGGACAGTTCAATTTGAACTGTCCCTTTTGTCACCTTTCTCCTCCTAAATT CATACTTTA AAAACAGGTAAGATGGCCTAACGAGTTTGGAGGTAGGAGA YabG minimal promoter ( B. cereus AH820) (SEQ ID NO: 208) TCTCCTCCTAAATT CATACTTTA AAAACAGGTAAGATGGCCTAACGAGTTTGGAGGTAGGAGA Tgl promoter ( B. thuringiensis serovar konkukian strain 97-27) (SEQ ID NO: 209) GGAAACAGAAGTCATCCCATTTGAAAATGCAGCAGGTCGTATTATAGCTGATTTCGTTATGGTTTATCCGCCAGGGATTCCAATCTTTACTCCGGGGGAAATTATTACACAAGACAACTTAGAGTATATTCGTAAAAACTTAGAAGCAGGTTTACCTGTACAAGGTCCTGAAGATATGACATTACAAACATTACGCGTGATCAAAGAGTACAAGCCTATCAGTTGATAGGCTTTTTTTCACCCTTTTTCCCTTTTCT CATACGATA TTATGTAATGTAACGTATAGGTGGGGATACTACT Minimal Tgl promoter ( B. thuringiensis serovar konkukian strain 97-27) (SEQ ID NO: 210) ACCCTTTTTCCCTTTTCT CATACGATA TTATGTAATGTAACGTATAGGTGGGGATACTACT Superoxide dismutase promoter (SODA1) ( B. cereus F837/76) (SEQ ID NO: 211) ATTGTGGACCCTTAGCTCAGCTGGTTAGAGCAGACGGCTCATAACCGTCCGGTCGTAGGTTCGAGTCCTACAGGGTCCATATCCATTTCACATGTTTATTATGTCGGCAGGAAGCTTCCTTGTAGAAGGGAGCTTTTTTTATGAAATATATGAGCATTTTAATTGAAATGAAGTGGGAATTTTGCTACTTTAATGATAGCAAGACAATGTGATTTATTTGTTTGCACCCTATGGCAATTAGGGTAGAATGAAGTTGTATGTCACTTAAGTGGCAATA CATAAACTG GGAGGAATATAACA Minimal Superoxide Dismutase (SODA1) Promoter ( B. cereus F837/76) (SEQ ID NO: 212) ACTTAAGTGGCAATA CATAAACTG GGAGGAATATAACA Superoxide dismutase (SODA2) promoter ( B. cereus AH820) (SEQ ID NO: 213) AATATAACAGAAAATTCTGATGTTTTTTCAAATCCTATAATAAGGAGTGTTCCGTATGATGCCTT TATATTTTC CGGAAGATAAAACAG AATATATTA TTCCAGGGATTGTTTGTGTTCTATTTATCATCGGTGCGATTGCTACGTGGCGTATGTTCATTCGTGTATCAAAACGAGAAGCAGAGCGATTACAGAAAGTTGAAGAAAAGCTGTTAGCTGAAAAGAAACAGTAACTCATTTTTGTATGTTTCCCTCTATGCTCGGACAATCTAAGGGCAGAATGTATTTTGGAGGGAATGAA Minimal superoxide dismutase (SODA2) promoter ( B. cereus AH820) (SEQ ID NO: 214) TCCGGAAGATAAAACAG AATATTA TTCCAGGGATTGTTTGTGTTCTATTTATCATCGGTGCGATTGCTACGTGGCGTATGTTCATTCGTGTATCAAAACGAGAAGCAGAGCGATTACAGAAAGTTGAAGAAAAGCTGTTAGCTGAAAAGAAACAGTAACTCATTTTTGTATGTTTCCCTCTATGCTCGGACAATCTAAGGGCAGAATGTATTTTGGAGGAATGAA BclA promoter ( B. anthracis Sterne) (SEQ ID NO: 215) TAATCACCCTCTTCCAAATCAAT CATATGTTA TA CATATACTA AACTTTCCATTTTTTTAAATTGTTCAAGTAGTTTAAGATTTCTTTTCAATAATTCAAATGTCCGTGTCATTTTCTTTCGGTTTTG CATCTACTA TATAATGAACGCTTTATGGAGGTGAATTT BAS1882 promoter ( B. anthracis Sterne) (SEQ ID NO: 216) aattacataacaagaactacattagggagcaagcagtctagcgaaagctaactgcttttttattaaataactattttattaaatttcatatatacaatcgcttgtccatttcatttggctctacccg catttacta ttagtaatatgaatttttcagaggtggattttatt Gene promoter 3572 ( B. weihenstephensis KBAB 4) (SEQ ID NO: 217) Ctatgatttaagatacacaatagcaaaagagaaa catattata taacgataaatgaaacttatgtatatgtatggtaactgtatatattactacaatacagtatactcataggaggtaggt YVTN β-propeller protein promoter ( B. weihenstephensis KBAB 4) (SEQ ID NO: 218) ggtaggtagatttgaaatatgatgaagaaaaggaataactaaaaggagtcgatatccgactccttttagttataaataatgtggaattagagtataattttatataggtatattgtattagatgaacgctttatcctttaattgtgattaatgatggattgtaagagaaggggcttacagtcctttttttatggtgttctataagcctttttaaaaggggtaccaccccacacccaaaaacagggggggttataactacatattggatgttttgtaacgtacaagaatcggtattaattaccctgtaaataagttatgtgtatataaggtaactt tatatattc tcctacaataaaataaaggaggtaataaa Cry1A promoter ( B. thuringiensis HD-73) (SEQ ID NO: 219) aacccttaatgcattggttaaacattgtaaagtctaaagcatggataatgggcgagaagtaagtagattgtgttaacaccctgggtcaaaaattgatatttagtaaaattagttgcactttgtgcattttt cataagatg agt catatgttt taaattgtagtaatgaaaaacagtat tatatcataatgaa ttggtagatcttaataaaggta ExsY promoter ( B. thuringiensis serovar konkukian strain 97-27) (SEQ ID NO: 220) Taattccaccttcccttatcctctttcgcctatttaaaaaaaggtcttgagattgtgaccaaatctcctcaactcc aatatctta ttaatgtaaatacaaaaagaagaagataagga CotY promoter ( B. thuringiensis Al Hakam) (SEQ ID NO: 221) Aggatgtcttttttttattgtattatgtacatccctactatataaattccctgcttttatcgtaagaattaacgtaatatcaaccatatcccgtt catattgta gtagtgtatgtcagaactcacgagaaggagtgaacataa YjcA promoter ( B. thuringiensis serovar kurstaki strain HD73) (SEQ ID NO: 222) Ttaatgtcactccttatcttcttgtttgtatttacatt aataagata ttggagttgaggagattggtcacaatctcaagacctttttttaaataggcgaaagaggataagggaaggtggaatt YjcB promoter ( B. thuringiensis serovar kurstaki strain HD73) (SEQ ID NO: 223) Atatattttcataatacgagaaaaagcggagtttaaaagaatgagggaacggaaataaagagttgtt catatagta aatagacagaa ExsFA/BxpB promoter ( B. thuringiensis Al Hakam) (SEQ ID NO: 224) Aaactaaataatgagctaagcatggattgggtggcagaattatctgccacccaatc catgcttaa cgagtattattatgtaaatttcttaaaattgggaacttgtctagaacatagaacctgtcctttt cattaactg aaagtagaaacagataaaggagtgaaaaac Rhamnose promoter ( B. thuringiensis Al Hakam) (SEQ ID NO: 225) Attcactacaacggggatgagtttgatgcggata catatgaga agtaccggaaagtgtttgtagaa cattacaa agatatattatctccatcataaaggagagatgcaaag CotO promoter ( B. anthracis Sterne) (SEQ ID NO: 226) Cgcgcaccacttcgtcgtacaacaacgcaagaagaagttggggatacagcagtattcttattcagtgatttagcacgcggcgtaacaggagaaaacattcacgttgattcagggtat catatctta ggataaatataatattaattttaaaggacaatctctacatgttgagattgtcctttttatttgttcttagaaagaacgatttttaacgaaagttcttaccacgttatgaatataagtataatagtacacgatttattcagctacgta Sigma-K promoter ( B. anthracis Sterne) (SEQ ID NO: 227) tatatcatatgtaaaattagttcttattccca catatcata tagaatcgc catattata catgcagaaaactaagtatggtattattcttaaattgtttagcaccttctaatattacagatagaatccgtcattttcaacagtgaacatggatttcttctgaacacaactctttttctttccttatttccaaaagaagaaaagcagcccattttaaaatacggctgcttgtaatgcttg InhA1 promoter ( B. thuringiensis Al Hakam) (SEQ ID NO: 228) TATCACATAACTCTTTATTTTTTAATATTTCGA CATAAAGTG AAACTTTAATCAGTGGGGGCTTTGTTCCCCCCACTGATTATTAATTGAACCAAGGGATAAAAAGATAGAGGGTCTGACCAGAAAACTGGAGGCATGATTCTATAACAAAAAGCTTAATGTTTATAGAATTATGTCTTTTATATAGGGAGGGTAGTAAACAGAGATTTGGACAAAAAATGCATTGATTTAGGATTGATTGATTTGGACAAAAAATGCATTGATTTAGGA BclA glycosyl transferase cluster operon 1 ( B. thuringiensis serovar konkukian strain 97-27) (SEQ ID NO: 229) Attttttacttagcagtaaaactgatatcagttttactgctttttcatttttaaattcaatcattaaatcttccttttctacatagt cataatgtt gtatgacattccgtaggaggcacttata BclA glycosyl transferase cluster operon 2 ( B. thuringiensis serovar kurstaki strain HD73) (SEQ ID NO: 230) acataaattcacctccataaagcgttcattatatagtagatgcaaaaccgaaagaaaatgaccggacatttgaattattgaaaagaaatcttaaactacttgaacaatttaaaaaaatggaaagtttagtatatgtataa catatgatt gatttggaagagggtgatta Glycosyl transferase promoter ( B. thuringiensis Al Hakam) (SEQ ID NO: 231) ttctattttccaa cataacatg ctacgattaaatggttttttgcaaatgccttcttgggaagaaggattagagcgttttttttagaaaccaaaagtcattaacaattttaagttaatgactttttgtttgcctttaagaggttttatgttactataattatagtatcaggtactaataacaagtataagtattctctgggaggatatatca

[00211] Sigma-K sporulation-specific polymerase promoter sequences among the promoter sequences shown in Table 3 result in high levels of fusion protein or modulator protein expression during late sporulation. The consensus sequence for the sporulation-specific sigma-K polymerase promoter sequence is CATANNNTN; however, this sequence can contain up to two mutations and still be functional. The promoter sequence of the sporulation-specific polymerase sigma-K is usually located upstream of the ribosome binding site (RBS).

[00212] Promoters having a high degree of sequence identity with any of the sequences shown in Table 3 above can also be used to express fusion or modulator proteins.

[00213] For example, a fusion or modulator protein can be expressed under the control of a promoter containing a nucleic acid sequence having at least 80% identity with any of the nucleic acid sequences of SEQ ID NOs: 157-231.

[00214] The fusion protein or modulator protein can be expressed under the control of a promoter containing a nucleic acid sequence having at least 90% identity with any of the nucleic acid sequences of SEQ ID NO: 157-231.

[00215] The fusion protein or modulator protein can be expressed under the control of a promoter containing a nucleic acid sequence having at least 95% identity with any of the nucleic acid sequences of SEQ ID NO: 157-231.

[00216] The fusion protein or modulator protein can be expressed under the control of a promoter containing a nucleic acid sequence having at least 98% identity with any of the nucleic acid sequences of SEQ ID NO: 157-231.

[00217] A fusion or modulator protein can be expressed under the control of a promoter containing a nucleic acid sequence having at least 99% identity with any of the nucleic acid sequences of SEQ ID NOs: 157-231.

[00218] A fusion or modulator protein can be expressed under the control of a promoter containing a nucleic acid sequence that has 100% identity with any of the nucleic acid sequences of SEQ ID NO: 157-231.

[00219] For example, a modulator protein or fusion protein can be expressed under the control of a BclA promoter (e.g., SEQ ID NO: 189, 190, 215, 229, or 230), a CotY promoter (e.g., SEQ ID NO: 161, 162, or 221) , an ExsY promoter (eg SEQ ID NO: 157, 158 or 220) or a rhamnose inducible promoter (eg SEQ ID NO: 225). For example, a fusion protein can be expressed under the control of a promoter containing a nucleic acid sequence having at least 80% identity with any of the nucleic acid sequences of SEQ ID NOs: 157, 158, 161, 162, 189, 190, 215, 220, 221, 225, 229 or 230.

[00220] The fusion protein can be expressed under the control of a promoter containing a nucleic acid sequence having at least 85% identity with any of the nucleic acid sequences of SEQ ID NOs: 157, 158, 161, 162, 189, 190, 215, 220, 221 , 225, 229 or 230.

[00221] The fusion protein can be expressed under the control of a promoter containing a nucleic acid sequence having at least 90% identity with any of the nucleic acid sequences of SEQ ID NO: 157, 158, 161, 162, 189, 190, 215, 220, 221 , 225, 229 or 230.

[00222] The fusion protein can be expressed under the control of a promoter containing a nucleic acid sequence having at least 95% identity with any of the nucleic acid sequences SEQ ID NOs: 157, 158, 161, 162, 189, 190, 215, 220, 221 , 225, 229 or 230.

[00223] The fusion protein can be expressed under the control of a promoter containing a nucleic acid sequence having at least 98% identity with any of the nucleic acid sequences of SEQ ID NOs: 157, 158, 161, 162, 189, 190, 215, 220, 221 , 225, 229 or 230.

[00224] The fusion protein can be expressed under the control of a promoter containing a nucleic acid sequence having at least 99% identity with any of the nucleic acid sequences of SEQ ID NOs: 157, 158, 161, 162, 189, 190, 215, 220, 221 , 225, 229 or 230.

[00225] The fusion protein can be expressed under the control of a promoter containing a nucleic acid sequence having 100% identity with any of the nucleic acid sequences of SEQ ID NOs: 157, 158, 161, 162, 189, 190, 215, 220, 221, 225, 229 or 230.

[00226] The fusion or modulator protein can be expressed under the control of a promoter comprising a sporulation-specific sigma-K polymerase promoter sequence, wherein the sporulation-specific sigma-K polymerase promoter sequence or sequences have 100% identity with the corresponding nucleotides of any of SEQ ID NO: 157-231.

[00227] Fusion proteins can be expressed under the control of a promoter that is native to the targeting sequence, exospore protein, or exospore protein fragment of the fusion protein. Thus, for example, when the targeting sequence is from BclA, the fusion protein can be expressed under the control of a native BclA promoter (eg, SEQ ID NO: 189, 190, 215, 229, or 230).

[00228] Modulator proteins can be expressed under the control of their native promoters. Thus, for example, when the modulator protein contains CotO, CotO can be expressed under the control of a native CotO promoter (eg, SEQ ID NO: 163 or 226). Native promoter sequences for each of the modulator proteins are shown in Table 3 above.

[00229] Table 3 also provides illustrative minimum promoter sequences for each modulator protein. Modulator proteins and fusion proteins can be expressed under the control of these minimal promoter sequences. For example, a modulator protein can be expressed under the control of a minimal promoter that contains a portion of the native promoter sequence. For example, when the modulator protein contains CotO, CotO can be expressed under the control of a minimal CotO promoter (SEQ ID NO: 164).

[00230] Alternatively, modulator proteins can be expressed under the control of any promoter containing a sporulation-specific sigma-K polymerase promoter sequence, regardless of whether the promoter is a native promoter for the modulator protein. As can be seen from Table 3, each of the native promoters and minimal promoters for modulator proteins contains at least one sorgma-K polymerase specific sporulation promoter sequence. Thus, for example, when the modulator protein is BxpB, BxpB can be expressed under the control of the BclA promoter (e.g., SEQ ID NO: 189, 190, 215, 229, or 230) or any of the other promoters shown in Table 3.

[00231] Moreover, the modulator protein or fusion protein can be expressed under the control of a portion of any of the promoters listed above in Table 3, provided that the promoter portion includes a sigma-K polymerase specific sporulation promoter sequence. For example, a modulator protein can be expressed under the control of a promoter region that contains the first 25, 50, 100, 150, 200, 250, or 300 nucleotides upstream of the initiation codon, provided that this region contains the sorgma-K polymerase specific sporulation promoter sequence. .

IV. Mutations and other genetic changes in recombinant members of the family Bacillus cereus that allow the collection of free exospore

[00232] As further described in the example below, recombinant members of the Bacillus cereus family that express fusion proteins comprising a protein or peptide of interest and a targeting sequence, exosporium protein or exosporium protein fragment, that target the fusion protein into the exospores of the recombinant member of the Bacillus cereus family, can be used to deliver proteins or peptides of interest to plants, seeds, plant growth media, or the area surrounding the seed or plant (eg, via soil irrigation, leaf application, or seed treatment). In addition, recombinant members of the Bacillus cereus family can be used to deliver nucleic acid molecules to animals, insects, worms (eg, nematodes), fungi, and protozoa; for delivering proteins or peptides to an animal; in vaccines and to achieve an immunogenic response; for reclamation; for treating the fracturing fluid to break the emulsion or gel in the fluid; for disinfection and for various other uses described herein below. However, in some cases the presence of live microorganisms may be undesirable, and instead it is desirable to separate the live spore from the fusion proteins in the exospore on the outer surface of the spore. For example, in some applications it is desirable to increase enzymatic activity without compromising the integrity of the spores. In such situations, exospore fragments may be preferred over live microorganisms having the enzyme in their exospore.

[00233] In addition, for some applications it may be desirable to reduce the density of the product. In such cases, it is desirable to separate the dense spores from the exospore (containing the fusion proteins). In the field of vaccines, it may be desirable to separate the spore from the exospore (containing the fusion proteins that contain the antigen) to remove potential antigens present on the spore itself from the vaccine preparation. Moreover, in some cases, the presence of live spores can lead to potential bacterial growth in the product, which may be undesirable for some applications (eg supplementation of animal feed and processing hides into leather).

[00234] Recombinant members of the Bacillus cereus family can be mutated and other genetic changes (eg, protein overexpression) that allow separation of free exospore from spores of the recombinant member of the Bacillus cereus family. The separation process provides exospore fragments that contain fusion proteins that are substantially free of the spores themselves. By "substantially free of spores" is meant that after separation of the exospore from the spores, a preparation is obtained which contains less than 5% spores by volume, preferably less than 3% spores by volume, even more preferably less than 1% spores by volume, and most preferably not contains a spore or, if spores are present, they are not detectable. These exospore fragments themselves can be used in place of recombinant members of the Bacillus cereus family, and can be used to deliver proteins or peptides of interest to plants, seeds, plant growth media, or the area surrounding a seed or plant, and for other purposes described in this description.

[00235] Thus, a recombinant member of the Bacillus cereus family is provided that expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, an exosporium protein or an exosporium protein fragment, that targets the fusion protein into the exospore of the recombinant member of the Bacillus cereus family . The recombinant member of the Bacillus cereus family contains a mutation or expresses a protein, wherein the expression of the protein is increased compared to the expression of the protein in a wild-type member of the Bacillus cereus family under the same conditions. The mutation and increased expression of the protein results in Bacillus cereus spores having an exospore that is easier to remove from the spore compared to the wild-type spore exospore.

[00236] In addition, a recombinant member of the Bacillus cereus family is provided that expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, an exosporium protein or exosporium protein fragment, that targets the fusion protein into the exospore of the recombinant member of the Bacillus cereus family . A recombinant member of the Bacillus cereus family: (i) contains a mutation in the CotE gene; (ii) expresses an ExsY protein, wherein the expression of the ExsY protein is increased compared to the expression of the ExsY protein in a wild-type member of the Bacillus cereus family under the same conditions, and where the ExsY protein contains a C-terminal tag containing a globular protein; (iii) expresses a BclB protein, wherein the expression of the BclB protein is increased compared to the expression of the BclB protein in a wild-type member of the Bacillus cereus family under the same conditions; (iv) expresses a YjcB protein, wherein the expression of the YjcB protein is increased compared to the expression of the YjcB protein in a wild-type member of the Bacillus cereus family under the same conditions; (v) contains a mutation in the ExsY gene; (vi) contains a mutation in the CotY gene; (vii) contains a mutation in the ExsA gene; or (viii) contains a mutation in the CotO gene.

[00237] A recombinant member of the Bacillus cereus family may contain a mutation in the CotE gene, such as a knockout of the CotE gene or a dominant negative form of the CotE gene. A mutation in the CotE gene can partially or completely inhibit the ability of CotE to bind exospores to spores.

[00238] A recombinant member of the Bacillus cereus family can express the ExsY protein. The ExsY protein contains a C-terminal tag containing a globular protein (e.g., green fluorescent protein (GFP) or a variant thereof) and the expression of the ExsY protein is increased compared to the expression of the ExsY protein in a wild-type member of the Bacillus cereus family under the same conditions. A globular protein may have a molecular weight of 25 kDa to 100 kDa. Expression of an ExsY protein containing a C-terminal tag containing a globular protein can also inhibit the binding of the ExsY protein to its targets in the exospore.

[00239] A recombinant member of the Bacillus cereus family can express the BclB protein, which can lead to the formation of a fragile exospore. BclB protein expression can be increased compared to BclB protein expression in a wild-type member of the Bacillus cereus family under the same conditions.

[00240] A recombinant member of the Bacillus cereus family can express the YjcB protein, which can lead to fragmented exospore formation rather than a complete structure. YjcB protein expression can be increased compared to YjcB protein expression in a wild-type member of the Bacillus cereus family under the same conditions.

[00241] A recombinant member of the Bacillus cereus family may contain a mutation in the ExsY gene, such as a knockout of the ExsY gene. A mutation in the ExsY gene can partially or completely inhibit the ability of ExsY to complete exospore formation or bind exospore to spore.

[00242] A recombinant member of the Bacillus cereus family may contain a mutation in the CotY gene, such as a knockout of the CotY gene. A mutation in the CotY gene can lead to the formation of fragile exospores.

[00243] A recombinant member of the Bacillus cereus family may contain a mutation in the ExsA gene, such as a knockout of the ExsA gene. A mutation in the ExsA gene can lead to the formation of fragile exospores.

[00244] A recombinant member of the Bacillus cereus family may contain a mutation in the CotO gene, such as a knockout of the CotO gene or a dominant negative form of the CotO gene. A mutation in the CotO gene can lead to the formation of exospores in stripes.

[00245] Exosporium fragments can be obtained from any of these recombinant members of the Bacillus cereus family and used for a variety of purposes, as further described herein below. Fragments of exosporium contain fused proteins. Purification of exospore fragments that contain fusion proteins from spores results in a cell-free protein preparation in which the fusion proteins are stabilized and maintained through covalent bonds with the exospore fragments.

[00246] Due to the strong covalent bonds between the fusion proteins and the exospore fragments, the fusion proteins become heat resistant. The heat resistance of fusion proteins associated with exosporium fragments allows their use for applications that require heat tolerant proteins or enzymes (eg, in food supplements).

V. Inactivation of spores of bacteria of the genus bacillus , including spores of recombinant members of the family Bacillus cereus

[00247] Bacillus spores can be genetically inactivated. Genetic inactivation of spores may be advantageous, for example, because it allows the delivery of spores to a plant or plant growth medium while eliminating any detrimental effects that the live bacteria may have on the plant. In addition, the use of inactivated spores can provide many of the same beneficial effects (eg prevention of bacterial growth in the product) discussed in Section IV above with respect to the use of exospore fragments.

A. Genetic inactivation via protease or nuclease overexpression

[00248] Provided is a recombinant bacterium of the genus Bacillus that expresses a protease or nuclease. The expression of the protease or nuclease is increased compared to the expression of the protease or nuclease in wild-type bacteria of the genus Bacillus under the same conditions. Increased expression of the protease or nuclease partially or completely inactivates the spores of the recombinant bacterium of the genus Bacillus or renders the spores of the recombinant bacterium of the genus Bacillus more susceptible to physical or chemical inactivation.

[00249] The recombinant Bacillus bacterium is preferably a recombinant member of the Bacillus cereus family.

[00250] A recombinant member of the Bacillus cereus family can also express 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 into the exospore of the recombinant member of the Bacillus cereus family.

[00251] A recombinant bacterium of the genus Bacillus can express both a protease and a nuclease, wherein the expression of the protease is increased compared to the expression of the protease in the wild-type bacterium of the genus Bacillus under the same conditions, and the expression of the nuclease is increased compared to the expression of the nuclease in the wild-type bacterium of the genus Bacillus under the same conditions.

[00252] The recombinant bacterial protease may include a non-specific protease.

[00253] A recombinant bacterial protease may include 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 metalloproteinase.

[00254] The recombinant bacterial protease may comprise a spore germinating protease, such as a Bacillus subtilis spore germinating protease, a Bacillus mycoides spore germinating protease, or a Bacillus thuringiensis spore germinating protease.

[00255] The spore germinating protease may include an active form of the spore germinating protease. This protease is naturally inactive in the spore. During germination, this protease is activated due to the cleavage of the protease to the proprotein active form. Thus, a recombinant bacterium may contain an active protease rather than a naturally inactive form. The active protease can cleave the protective SASP proteins in the spore before germination.

[00256] The nuclease of a recombinant bacterium may contain an endonuclease or an exonuclease. The nuclease may comprise a non-specific endonuclease such as Bacillus subtilis endonuclease 1. For example, spore germinating protease and endonuclease 1 may have the amino acid sequences shown in Table 4 below.

Table 4. Amino acid sequences of spore germinating protease and endonuclease 1

Protein SEQID NO. Endonuclease 1, B. subtilis 168 232 Protease GPR, B. subtilis 168 233 GPR protease, B. cereus 234

[00257] You can also use a protease or nuclease that has a high degree of amino acid identity with the sequences shown in Table 4 above.

[00258] Thus, for example, a spore germinating protease may comprise an amino acid sequence that shares at least 85% identity with SEQ ID NO: 233 or 234.

[00259] The spore sprouting protease may comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 233 or 234.

[00260] The spore sprouting protease may comprise an amino acid sequence having at least 95% identity with SEQ ID NO: 233 or 234.

[00261] The spore sprouting protease may comprise an amino acid sequence having at least 98% identity with SEQ ID NO: 233 or 234.

[00262] The spore sprouting protease may comprise an amino acid sequence having at least 99% identity with SEQ ID NO: 233 or 234.

[00263] The spore sprouting protease may contain an amino acid sequence that has 100% identity with SEQ ID NO: 233 or 234.

[00264] Similarly, a non-specific endonuclease may contain an amino acid sequence that shares at least 85% identity with SEQ ID NO: 232.

[00265] The non-specific endonuclease may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 232.

[00266] The non-specific endonuclease may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 232.

[00267] The non-specific endonuclease may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 232.

[00268] The non-specific endonuclease may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 232.

[00269] The non-specific endonuclease may contain an amino acid sequence that has 100% identity with SEQ ID NO: 232.

[00270] The protease or nuclease can be expressed under the control of a promoter containing the sigma G promoter sequence. For example, the promoter can have one of the sequences shown in Table 5 below. The consensus sequence for binding transcription factor sigma G is CATNNTA, where N is any nucleotide. The sigma G promoter sequences in the promoters shown in Table 5 are indicated in bold and underlined text.

Table 5. Promoter sequences having sigma G sequences

promoter Nucleic acid sequence GPR protease, B. subtilis 168 (SEQ ID NO: 235) GTAACTAAAGCTTCTACAGTTTTAACAGCTGAACGCATGTCAGACTTGATAGAAGCGTTATGTGCACGACGCTCTTCGCTAAGTTTAGCGCGTTTGATAGCAGATTTAATGTTTGC CATACTT TTCACCTCCCTGGTGCGATCGAGTGACTCGATACTTA CATAGAA CAAGTGATATTCTATCAAACGGAGAAGAGAATTGCAATAGCGAGATCAATGAAATTT CATGTAA AGGAAAGAATGACCTTATATATTTTTGGGGAATCTAACTATATTTACTATGAATTGCGGAGGAGATACG Minimal GPR Protease Promoter, B. subtilis 168 (SEQ ID NO: 236) GCAATAGCGAGATCAATGAAATTT CATGTAA AGGAAAGAATGACCTTATATATTTTTGGGGAATCTAACTATATTTACTATGAATTGCGGAGGAGATACG GPR protease, B. subtilis 168 (SEQ ID NO: 237) TTTCACCTCCTAAGATACAACCTGTAGCACAGTGTCTTAAGGTTAAATCTTCTTCACAATAGAACAAATTGTATTCTATCAAAACACCTTTAGATTGCAATATAAATGTAAAGTATTTTT CATTGAA GGTTCTCTTTTTAG CATGATT TATTCAGCAAATGGCAACAATATAGGTACTTAATGTGAAGGAGGCCCCTGT Minimal GPR Protease Promoter, B. subtilis 168 (SEQ ID NO: 238) GAAGGTTCTCTTTTTAG CATGATT TATTCAGCAAATGGCAACAATATAGGTACTTAATGTGAAGGAGGCCCCTGT SASPα, B. subtilis 168 (SEQ ID NO: 239) GCTTTGTTGATTTCGAGCCGTATATTCAAGAAGCGGTAGATAACATTGAGACAATGACCCTTTATAGCGAACAAGAAGCTAACGATAAATTCGCTGAACTCTTTTAAATCAATTTTCAGCTCCTGTATACAATTACCAAAGTTTTTCTGAATGAAGCCATGTGTTTTGACA CATTCTA TACTCACAAGGAGGTGAGACAC Minimal promoter SASPα, B. subtilis 168 (SEQ ID NO: 240) GAATGAAGCCATGTGTTTTGACA CATTCTA TACTCACAAGGAGGTGAGACAC SASPβ, B. subtilis 168 (SEQ ID NO: 241) AAACGGCTAAGCTTTTTTTATTTCTCAAGATTTACCACAATTCTCCGCATGATTTTCCGGCCATTTTAACATAATACGTAGTAACAAGCCGGCAAAGCATTGGGTTACGCCGAGGCGGCAGTGACACCCGAGAAGGGTTCACAGATTGGTGCAACTCCAGTTAACCCAAC CATACTA AATAAAAAGGAGATTTTACAC Minimal promoter SASPβ, B. subtilis 168 (SEQ ID NO: 242) GATTGGTGCAACTCCAGTTAACCCAAC CATACTA AATAAAAAGGAGATTTTACAC SASPγ, B. subtilis 168 (SEQ ID NO: 243) TTCGCTTCTCCCACTTAATCTGATTTACATTCCAAGGAATCCAATGATTTATATGGAGATCTGAAACATAATCAATTTTCATTTTGTCTCCACCTTTCTTAATGAAAAATTTATTTCTTTGGCGTGTATAAATTAAAATAATCTCTC CATAATA TGATTCAAACAAGCTTGTTTTCATTACACTTTAGGAGATGAATAAG Minimal promoter SASPγ, B. subtilis 168
(SEQ ID NO: 244) GTATAAATTAAAATAATCTCTC CATAATA TGATTCAAACAAGCTTGTTTTCATTACACTTTAGGAGATGAATAAG SASPδ, B. subtilis 168 (SEQ ID NO: 245) TACAGTCCTTCTCCATTTTGACATTCCATATTCAGGCAACCGCACATAAAATGACAGCAGA CATTCTA TAGTCTGCGCCACCCCGGCTCAGAGGCCGGGGTTTTATTTTTCTCCACAACAATTGCCAGCATAAATAAACCCCGTATATTTCAAACTAAATACGCGTTAAGAATTTCTTTATCGAAAAAGGAGATGAAAAAG Minimal promoter SASPδ, B. subtilis 168 (SEQ ID NO: 246) GCAACCGCACATAAAATGACAGCAGA CATTCTA TAGTCTGCGCCACCCCGGCTCAGAGGCCGGGGTTTTATTTTTCTCCACAACAATTGCCAGCATAAATAAACCCCGTATATTTCAAACTAAATACGCGTTAAGAATTTCTTTATCGAAAAAGGAGATGAAAAAG

[00271] Expression of the nuclease or protease under the control of the sigma-G promoter allows site-specific expression of the nuclease or protease in the prospore, where the enzyme activity is directed to the prospore and the region where the bacterial target DNA is located. Excessive cleavage of the prospore DNA is lethal to the bacterial spore once it begins to germinate.

[00272] For example, as further illustrated in the examples described herein below, overexpression of spore germinating protease (GPR) in its active form in the prospore of a member of the Bacillus cereus family during sporulation results in proteolytic cleavage of proteins in the prospore and inactivation of the spore and/or makes the spore more susceptible to inactivation by ultraviolet or gamma irradiation. Similarly, overexpression of a non-specific endonuclease in the prospore during sporulation disrupts the DNA in the spore, resulting in a high number of inactivated spore particles. These methods for inactivating spores of members of the Bacillus cereus family can be used alone or in conjunction with each other and/or with other methods for inactivating spores.

[00273] Gene expression in Bacillus spores is tightly regulated by the expression of sporulation-specific sigma factors that direct RNA polymerase to the genes to be expressed during each stage of sporulation. Late gene expression in the prospore, where bacterial DNA and essential proteins are packaged, is regulated by factor sigma G. During late sporulation, bacterial bacterial DNA is packaged with protective proteins called small acid-soluble proteins (SASPs). These SASP proteins include, among others, SASPα, SASPβ and SASPγ. SASP proteins protect bacterial DNA from UV radiation and other threats. During germination of proproteins, the spore germination protease is activated and cleaves these SASP proteins.

[00274] By expressing GPR under the control of the sigma G promoter, GPR is expressed in the prospore and SASP protective proteins are degraded after sporulation has begun, leaving bacterial DNA more susceptible to degradation. Similarly, expression of a non-specific nuclease under the control of the sigma G promoter leads to cleavage of host DNA. Because the spore is unable to repair large-scale damage to its DNA, this eventually leads to the death of the spore. Overexpression of GPR and a non-specific endonuclease can be used together to degrade both SASP protective proteins and host DNA.

[00275] The protease or nuclease can be expressed under the control of any promoter containing the sigma G promoter sequence.

[00276] Thus, a 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 expressed under the control of a promoter having a high degree of sequence identity with any of the promoter sequences shown in Table 5.

[00277] For example, the promoter may contain a nucleic acid sequence having at least 95% identity with any of the nucleic acid sequences of SEQ ID NO: 235-246.

[00278] The promoter may include a nucleic acid sequence having at least 98% identity with any of the nucleic acid sequences of SEQ ID NO: 235-246.

[00279] The promoter may contain a nucleic acid sequence having at least 99% identity with any of the nucleic acid sequences of SEQ ID NO: 235-246.

[00280] The promoter may contain a nucleic acid sequence having 100% identity with any of the nucleic acid sequences of SEQ ID NO: 235-246.

[00281] In any of the recombinant bacteria of the genus Bacillus that express a protease or nuclease, the spores of the recombinant bacterium of the genus Bacillus may be more susceptible to inactivation, for example, by ultraviolet irradiation, gamma irradiation, or by treatment with bleach, hydrogen peroxide, chloroform, phenol or acetic acid, compared to the same spores that do not express the protease or nuclease at an increased level compared to the expression of the protease or nuclease in wild-type bacterium of the genus Bacillus treated under the same conditions.

b. Genetic inactivation by mutation of a gene encoding a sprouting receptor, spore core lytic enzyme, small acid soluble spore protein (SASP), or spore coat protein

[00282] Spores of any recombinant member of the Bacillus cereus family that express a fusion protein containing a targeting sequence, an exosporium protein, or an exosporium protein fragment that targets the fusion protein to the exospore of the recombinant member of the Bacillus cereus family can also be genetically inactivated or can be sensitized to physical or chemical inactivation by modifying a member of the Bacillus cereus family so that it contains the mutation.

[00283] Such mutations include knockout or other inactivating mutations in one or more of the genes encoding the sprouting receptor. Sprouting receptor genes include, for example, GerA, GerB, GerK, GerH, GerI, GerG, GerL, GerQ, GerR, GerS, GerN, GerU, or GerX.

[00284] Such mutations also include knockout or other inactivating mutations in the lytic enzymes of the spore bark. For example, spore bark lytic enzymes SleB and CwJ can be mutated to inactivate spores. Such mutations prevent spore growth during germination and effectively inactivate spores.

[00285] In addition, such mutations include knockout or other inactivating mutations of SASP genes (eg, SASPα, SASPβ, or SASPγ). Such mutations remove the protection of spores from UV radiation and make them more susceptible to inactivation by UV radiation and other methods.

[00286] Such methods also include knockout or other inactivating mutations in genes encoding spore coat or cortex proteins (eg, CotA, CotB, or CotC). Such mutations make the spores more susceptible to inactivation by physical or chemical means, such as exposure to ultraviolet radiation, gamma radiation, or treatment with solvents such as bleach, hydrogen peroxide, chloroform, phenol, or acetic acid.

[00287] Thus, the present invention relates to a recombinant member of the Bacillus cereus family that expresses a fusion protein containing at least one protein or peptide of interest and a targeting sequence, an exosporium protein or an exospore protein fragment, which targets the fusion protein into the exospore of the recombinant member of the Bacillus cereus family. A recombinant member of the Bacillus cereus family contains a mutation that partially or completely inactivates spores of a recombinant member of the Bacillus cereus family or increases the sensitivity of spores of a recombinant member of the Bacillus cereus family to physical or chemical inactivation compared to the same spores that do not contain the mutation. The mutation includes a mutation in a gene encoding a sprouting receptor, a mutation in a gene encoding a spore bark 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 bark protein.

[00288] In addition, the present invention provides a recombinant member of the Bacillus cereus family that expresses a fusion protein as described in section I above. A recombinant member of the Bacillus cereus family contains a mutation that partially or completely inactivates spores of a recombinant member of the Bacillus cereus family or increases the sensitivity of spores of a recombinant member of the Bacillus cereus family to physical or chemical inactivation compared to the same spores that do not contain the mutation.

[00289] Any of the recombinant members of the Bacillus cereus family described in section VA above that express a protease or nuclease may also contain a mutation that partially or completely inactivates the spores of the recombinant member of the Bacillus cereus family or renders the spores of the recombinant member of the Bacillus cereus family more sensitive to physical or chemical inactivation compared to the same spores that do not contain the mutation. For example, a mutation may include a mutation in a gene encoding a sprouting receptor, a mutation in a gene encoding a spore bark lytic enzyme, a mutation in a gene encoding a small acid soluble spore protein (SASP), or a mutation in a gene encoding a coat or spore bark protein.

[00290] For example, the mutation may include a mutation in a gene encoding a sprouting receptor, such as a knockout of a gene encoding a sprouting receptor. The sprouting receptor may include GerA, GerB, GerK, GerH, GerI, GerG, GerL, GerQ, GerR, GerS, GerN, GerU, or GerX.

[00291] For example, a mutation may include a mutation in a gene encoding a spore cortex lytic enzyme, such as a knockout of a gene encoding a spore cortex lytic enzyme. The spore bark lytic enzyme may include SleB or CwlJ.

[00292] For example, a mutation may include a mutation in a gene encoding SASP, such as a mutation in the SspA gene, a mutation in the SspB gene, a mutation in the SspC gene, a mutation in the SspD gene, a mutation in the SspE gene, a mutation in the SspF gene, a mutation in SspG gene, SspH mutation, SspI mutation, SspJ mutation, SspK mutation, SspL mutation, SspM mutation, SspN mutation, SspO mutation, SspP mutation, or a combination thereof . SASP may include SASPα, SASPβ or SASPγ. Spores of a recombinant member of the Bacillus cereus family may be more susceptible to inactivation by ultraviolet or gamma irradiation compared to the same spores that do not contain a mutation in the gene encoding SASP.

[00293] For example, a mutation may include a mutation in a gene encoding a spore coat or bark protein, such as a knockout of a gene encoding a spore coat or bark protein. The spore coat or bark protein may include CotA, CotB or CotC. Spores of a recombinant member of the Bacillus cereus family may be more susceptible to inactivation by exposure to ultraviolet radiation, gamma radiation, or by treatment with bleach, hydrogen peroxide, chloroform, phenol, or acetic acid, compared to the same spores that do not contain a mutation in the coat protein or spore bark treated under the same conditions.

VI. Recombinant members of the family Bacillus cereus , which overexpress exosporium enzymes that have beneficial effects on plants or slow the germination of spores of a member of the family Bacillus cereus

[00294] Also contemplated are recombinant members of the Bacillus cereus family that overexpress various exospore proteins to provide beneficial effects on the plant or slow spore germination.

[00295] Provided is a recombinant member of the Bacillus cereus family that expresses the exosporium protein, wherein the expression of the exospore protein is increased compared to the expression of the exospore protein in a wild-type member of the Bacillus cereus family under the same conditions. The exosporium protein may contain an exosporium enzyme, where the exosporium enzyme includes an enzyme involved in nutrient solubilization, inosinuridine hydrolase, a protease, an enzyme that catalyzes free radical degradation, arginase, or alanine racemase. Alternatively, the exosporium protein may include BclA protein, BclB protein, CotE protein, CotO protein, ExsY protein, ExsFA/BxpB protein, CotY protein, ExsFB protein, ExsJ protein, ExsH protein, YjcA protein, YjcB protein, BclC protein, BxpA protein, BclE, BetA/BAS3290 protein, ExsA protein, ExsK protein, ExsB protein, YabG protein, or Tgl protein.

[00296] The exospore protein is preferably not part of a fusion protein.

[00297] Exemplary amino acid sequences for AcpC, InhA1, InhA2, InhA3, SODA1, and SODA2 are provided in Tables 1 and 2 above. Exemplary sequences for alanine racemase 1, alanine racemase 2, arginase, IunH1, and IunH2 correspond to the SEQ ID NOs shown in the table below.

Table 6 Exemplary amino acid sequences of exospore enzymes

Protein and Strain SEQID NO. Alanine racemase 1, B. anthracis ΔSterne 247 Alanine racemase 2, Bacillus cereus F837/78 248 Arginase, Bacillus thuringiensis pondicheriensis 4BA1 249 IunH1, B. cereus Str. CI 250 IunH2, Bacillus thuringiensis 251

[00298] Overexpression of inosinuridine hydrolases and alanine racemases interferes with the ability of spores to germinate and thereby maintains spores at the dormant stage and increases spore stability.

[00299] The enzymes SODA and arginase degrade free radicals. Spores that overexpress these enzymes have increased resistance to stress caused by free radicals.

[00300] When the exospore protein includes an exospore enzyme and the exospore enzyme includes an enzyme involved in nutrient solubilization, the enzyme involved in nutrient solubilization may include an enzyme involved in phosphate solubilization, such as acid phosphatase (e.g., AcpC). The acid phosphatase may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 137.

[00301] The acid phosphatase may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 137.

[00302] The acid phosphatase may comprise an amino acid sequence having at least 98% identity with SEQ ID NO: 137.

[00303] The acid phosphatase may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 137.

[00304] The acid phosphatase may contain an amino acid sequence that has 100% identity with SEQ ID NO: 137.

[00305] When the exosporium protein includes an exospore enzyme and the exosporium enzyme includes inosinuridine hydrolase, the inosinuridine hydrolase may include IunH1 or IunH2. The inosinuridin hydrolase may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 250 or 251.

[00306] The inosinuridin hydrolase may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 250 or 251.

[00307] The inosinuridin hydrolase may comprise an amino acid sequence that shares at least 95% identity with SEQ ID NO: 250 or 251.

[00308] The inosinuridin hydrolase may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 250 or 251.

[00309] The inosinuridin hydrolase may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 250 or 251.

[00310] The inosinuridin hydrolase may contain an amino acid sequence that has 100% identity with SEQ ID NO: 250 or 251.

[00311] When the exospore protein includes an exospore enzyme and the exospore enzyme includes a protease, the protease may be a metalloproteinase (eg, InhA1, InhA2, or InhA3). The metalloproteinase may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

[00312] The metalloproteinase may comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

[00313] The metalloproteinase may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

[00314] The metalloproteinase may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

[00315] The metalloproteinase may comprise an amino acid sequence having at least 98% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

[00316] The metalloproteinase may comprise an amino acid sequence having at least 99% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

[00317] The metalloproteinase may contain an amino acid sequence having 100% identity with SEQ ID NO: 114, 121, 122, 129, 130, or 138.

[00318] When the exosporium protein includes an exospore enzyme and the exosporium enzyme includes an enzyme that catalyzes free radical degradation, the enzyme that catalyzes free radical degradation may include superoxide dismutase (e.g., superoxide dismutase 1 (SODA1) or superoxide dismutase 2 (SODA2)). Superoxide dismutase may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 155 or 156.

[00319] Superoxide dismutase may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 155 or 156.

[00320] Superoxide dismutase may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 155 or 156.

[00321] Superoxide dismutase may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 155 or 156.

[00322] Superoxide dismutase may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 155 or 156.

[00323] Superoxide dismutase may contain an amino acid sequence having 100% identity with SEQ ID NO: 155 or 156.

[00324] When the exospore protein includes an exospore enzyme and the exospore enzyme includes arginase, the arginase may include Bacillus thuringiensis arginase. The arginase may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 249.

[00325] The arginase may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 249.

[00326] The arginase may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 249.

[00327] The arginase may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 249.

[00328] The arginase may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 249.

[00329] Arginase may contain an amino acid sequence with 100% identity with SEQ ID NO: 249.

[00330] When the exosporium protein includes an exospore enzyme and the exosporium enzyme includes an alanine racemase, the alanine racemase may include alanine racemase 1 (ALR1) or alanine racemase 2 (ALR2). The alanine racemase may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 247 or 248.

[00331] The alanine racemase may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 247 or 248.

[00332] The alanine racemase may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 247 or 248.

[00333] The alanine racemase may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 247 or 248.

[00334] The alanine racemase may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 247 or 248.

[00335] Alanine racemase may contain an amino acid sequence having 100% identity with SEQ ID NO: 247 or 248.

[00336] The exosporium protein may include BclA protein, BclB protein, CotE protein, CotO protein, ExsY protein, ExsFA/BxpB protein, CotY protein, ExsFB protein, ExsJ protein, ExsH protein, YjcA protein, YjcB protein, BclC protein, BxpA protein , BclE protein, BetA/BAS3290 protein, ExsA protein, ExsK protein, ExsB protein, YabG protein, or Tgl protein. The exospore protein preferably comprises a BclA protein, a BclB protein, a CotE protein, or a CotO protein. Exemplary amino acid sequences for these exospore proteins can be found in Table 2 above.

[00337] The exospore protein may include a BclA protein. The BclA protein may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 141 or 142.

[00338] The BclA protein may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 141 or 142.

[00339] The BclA protein may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 141 or 142.

[00340] The BclA protein may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 141 or 142.

[00341] The BclA protein may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 141 or 142.

[00342] The BclA protein may contain an amino acid sequence having 100% identity with SEQ ID NO: 141 or 142.

[00343] The exosporium protein may include the BclB protein. The BclB protein may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 143 or 144.

[00344] The BclB protein may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 143 or 144.

[00345] The BclB protein may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 143 or 144.

[00346] The BclB protein may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 143 or 144.

[00347] The BclB protein may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 143 or 144.

[00348] The BclB protein may contain an amino acid sequence that has 100% identity with SEQ ID NO: 143 or 144.

[00349] The exospore protein may include a CotE protein. The CotE protein may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 149.

[00350] The CotE protein may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 149.

[00351] The CotE protein may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 149.

[00352] The CotE protein may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 149.

[00353] The CotE protein may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 149.

[00354] The CotE protein may contain an amino acid sequence that has 100% identity with SEQ ID NO: 149.

[00355] The exospore protein may include the CotO protein. The CotO protein may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 126.

[00356] The CotO protein may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 126.

[00357] The CotO protein may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 126.

[00358] The CotO protein may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 126.

[00359] The CotO protein may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 126.

[00360] The CotO protein may contain an amino acid sequence having at least 100% identity with SEQ ID NO: 126.

[00361] The exosporium protein may include an ExsY protein. The ExsY protein may 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.

[00362] The exosporium protein may include an ExsFA/BxpB protein. The ExsFA/BxpB protein may 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: 124.

[00363] The exosporium protein may include CotY protein. The CotY protein may contain 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.

[00364] The exosporium protein may include an ExsFB protein. The ExsFB protein may 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: 127 or 128.

[00365] The exosporium protein may include an ExsJ protein. The ExJ protein may contain 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.

[00366] The exosporium protein may include an ExsH protein. The ExsH protein may 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.

[00367] The exosporium protein may include the YjcA protein. The YjcA protein may 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.

[00368] The exosporium protein may include the YjcB protein. The YjcB protein may contain 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.

[00369] The exosporium protein may include a BclC protein. The BclC protein may contain 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: 136.

[00370] The exosporium protein may include a BxpA protein. The BxpA protein may contain 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: 145.

[00371] The exosporium protein may include a BclE protein. The BclE protein may contain 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.

[00372] The exosporium protein may include the BetA/BAS3290 protein. The BetA/BAS3290 protein may contain 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: 148.

[00373] The exosporium protein may include an ExsA protein. The ExsA protein may contain 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.

[00374] The exosporium protein may include an ExsK protein. The ExsK protein may 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.

[00375] The exosporium protein may include an ExsB protein. The ExsB protein may contain 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.

[00376] The exosporium protein may include the YabG protein. The YabG protein may contain 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.

[00377] The exosporium protein may include a Tgl protein. The Tjl protein may 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.

[00378] The recombinant member of the Bacillus cereus family can also express 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 into the exospore of the recombinant member of the Bacillus cereus family.

VII. Expression of fusion proteins in endophytic members of the family Bacillus cereus , in representatives of the family bacillus cereus, capable of degrading herbicides or pesticides, or in probiotic members of the family Bacillus cereus

[00379] Any of the fusion proteins comprising a protein or peptide of interest and a targeting sequence, exosporium protein or exospore protein fragment that targets the fusion protein to the exospore of a recombinant member of the Bacillus cereus family can be expressed in an endophytic member of the Bacillus cereus family, a bacterial strain that capable of degrading a herbicide or pesticide, or in a probiotic strain of bacteria.

[00380] Expression of fusion proteins in an endophytic bacterial strain provides the ability to deliver the protein or peptide of interest directly to the plant. Endophytic strains can be delivered to plants using a variety of methods, for example, endophytic strains can be delivered by seed treatment, treatment of the plant growth medium (eg soil), irrigation, application to the plant itself (eg foliar application to aerial parts of the plant). Once inside the plant, the bacteria increase in number and colonize the internal tissues of the plant.

[00381] As further explained herein below, probiotic bacterial strains that express fusion proteins, and in particular strains that are both probiotic and endophytic that express fusion proteins, are useful in methods for delivering proteins or peptides of interest. (e.g. enzymes) to animals.

[00382] Although any of the fusion proteins comprising a protein or peptide of interest and a targeting sequence, an exosporium protein or exospore protein fragment that targets the fusion protein to the exospore of a recombinant Bacillus cereus can be expressed in a strain of a Bacillus cereus that is capable of degrading herbicide or pesticide, as further explained herein below, these strains are particularly useful in methods for decontaminating environments contaminated with herbicide and/or pesticide.

[00383] Thus, the present invention relates to a recombinant member of the Bacillus cereus family that expresses a fusion protein containing at least one protein or peptide of interest and a targeting sequence, an exosporium protein or exosporium protein fragment, which targets the fusion protein into the exospores of the recombinant family member Bacillus cereus , wherein the recombinant member of the Bacillus cereus family comprises an endophytic bacterial strain, a bacterial strain that is capable of degrading the herbicide or pesticide, or a probiotic bacterial strain.

[00384] The endophytic bacterial strain may include Bacillus cereus EE349, Bacillus cereus EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, or Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus cereus EE-B00377, Bacillus pseudomyidesco B00366 or Bacillus mycoides EE-B00363.

[00385] For example, an endophytic bacterial strain may include a member of the Bacillus cereus family EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444 or Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, a member of the Bacillus cereus family EE-B00377, Bacillus pseudomycoides EE- B00366 EE-B00363.

[00386] The bacterial strain that is capable of degrading the herbicide or pesticide may include Bacillus cereus EE349, Bacillus cereus EE-B00377, Bacillus pseudomycoides EE-B00366, or Bacillus mycoides EE-B00363.

[00387] The probiotic bacterial strain may include a member of the Bacillus cereus EE349 family, a member of the Bacillus cereus EE439 family, Bacillus thuringiensis EE417, or Bacillus cereus EE444.

[00388] In addition, the present invention relates to a recombinant member of the Bacillus cereus family that expresses a fusion protein containing at least one protein or peptide of interest and a targeting sequence, an exosporium protein or an exospore protein fragment, which targets the fusion protein into the exospore of the recombinant member of the Bacillus cereus family, wherein the recombinant member of the Bacillus cereus family comprises an endophytic strain of bacteria, and the fusion protein comprises any of the fusion proteins described in section I above.

VIII. Targeting sequences, exospore proteins and fragments of exospore proteins for use in: (a) recombinant members of the family Bacillus cereus that express the fusion protein and co-express the modulator protein; (b) recombinant members of the family Bacillus cereus which involve a mutation or other genetic change that allows the collection of free exospores; (c) recombinant members of the family Bacillus cereus that overexpress a protease or nuclease; (d) recombinant members of the family Bacillus cereus that express the fusion protein and overexpress the exosporium protein, which has beneficial effects on plants; or (e) or endophytic recombinant members of the family Bacillus cereus , which express fusion proteins

[00389] Any of the targeting sequences, exospore proteins, or exospore proteins described in this section can be found in any of the fusion proteins in:

(a) any of the recombinant members of the Bacillus cereus family that express the fusion protein and overexpress the modulator protein described in section II above;

(b) any of the recombinant members of the Bacillus cereus family that express the fusion protein and contain a mutation or other genetic change that allows the collection of free exospore described in section IV above;

(c) any of the recombinant members of the Bacillus cereus family that express the fusion protein and overexpress the protease or nuclease described in section VA;

(d) any of the recombinant members of the Bacillus cereus family which express the fusion protein and overexpress the exosporium protein which has the beneficial effects on plants described in section VI above; and

(e) any of the endophytic recombinant members of the Bacillus cereus family that express the fusion protein described in section VII above.

[00391] For example, the targeting sequence may comprise an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 63%.

[00392] For example, the targeting sequence may comprise an amino acid sequence having at least about 56% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 63%.

[00393] For example, the targeting sequence may comprise an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 72%.

[00394] For example, the targeting sequence may comprise an amino acid sequence having at least about 62% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 72%.

[00395] For example, the targeting sequence may comprise an amino acid sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 72%.

[00396] For example, the targeting sequence may comprise an amino acid sequence having at least about 68% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%.

[00397] For example, the targeting sequence may comprise an amino acid sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%.

[00398] For example, the targeting sequence may comprise an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%.

[00399] For example, the targeting sequence may comprise an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 90%.

[00400] For example, a targeting sequence may consist of: (a) a 16 amino acid sequence with at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1, where 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.

[00401] The targeting sequence may consist of an amino acid sequence as described in these examples.

[00402] The fusion protein may comprise an exosporium protein or an exosporium protein fragment containing an amino acid sequence 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 and 122.

[00403] The fusion protein may comprise an exosporium protein or an exosporium protein fragment containing an amino acid sequence 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 and 122.

[00404] The fusion protein may comprise an exosporium protein or an exosporium protein fragment containing 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 and 122.

[00405] The fusion protein may comprise an exosporium protein or an exosporium protein fragment containing an amino acid sequence having at least 99% identity with 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.

[00406] The fusion protein may comprise an exosporium protein or an exosporium protein fragment containing 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 and 122.

[00407] The fusion protein may comprise an exosporium protein containing an amino acid sequence having at least 90% identity with SEQ ID NOs: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 122.

[00408] The fusion protein may comprise an exosporium protein comprising an amino acid sequence having at least 95% identity with SEQ ID NOs: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 122.

[00409] The fusion protein may comprise an exosporium protein comprising an amino acid sequence having at least 98% identity with SEQ ID NOs: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 122.

[00410] The fusion protein may comprise an exosporium protein comprising an amino acid sequence having at least 99% identity with SEQ ID NOs: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 122.

[00411] The fusion protein may comprise an exosporium protein containing an amino acid sequence having 100% identity with SEQ ID NOs: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90 , 92, 94 or 122.

[00412] The targeting sequence, exosporium protein, or exosporium protein fragment of the fusion protein may contain the amino acid sequence GXT at the C-terminus, where X is any amino acid.

[00413] The targeting sequence, exosporium protein, or exosporium protein fragment may contain an alanine residue at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1.

[00414] The targeting sequence, exosporium protein, or exosporium protein fragment may 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 corresponds to the amino acid 20 SEQ ID NO: 1.

IX. Fusion proteins containing a protein or peptide of interest and a spore coat protein, recombinant spore coat bacteria, and seeds coated with recombinant spore coat bacteria

A. Spore coat proteins that can be used to target a fusion protein containing spore coat protein to the spore surface of a recombinant spore forming bacterium

[00415] A number of spore coat proteins can be used to display proteins or peptides of interest on the spore surface of a recombinant spore-forming bacterium. Such bacteria include any spore-forming bacteria, and in particular include bacteria of the genera Bacillus , Lysinibacillus , Virginibacillus , Clostridia , and Paenibacillus . Spore-forming bacteria of the genus Bacillus include members of the Bacillus cereus family as well as other Bacillus species that are not members of the Bacillus cereus family (for example, bacteria of the Bacillus species that lack exospores). These spore coat proteins include CotB, CotC, CgeA, CotB/H, CotG, spore coat protein X, and CotY. For ease of reference, a description of the amino acid sequences for exemplary spore coat proteins that can be used to target proteins or peptides of interest to the spore surface of a recombinant spore-forming bacterium is shown in Table 7 below along with their SEQ ID NO.

Table 7. Spore envelope protein sequences used to target proteins and peptides of interest to the spore surface of a recombinant spore-forming bacterium

Spore coat protein SEQID NO. CotB ( Bacillus subtilis ) 252 CotC ( Bacillus subtilis ) 253 CgeA ( Bacillus amyloliquefaciens ) 254 CotB/H ( Bacillus amyloliquefaciens ) 255 CotG ( Bacillus subtilis ) 256 X spore coat protein ( Bacillus megaterium ) 257 CotY ( Bacillus amyloliquefaciens ) 258 CotY ( Bacillus licheniformis ) 259

b. Fusion proteins containing a protein or peptide of interest and a spore coat protein

[00416] The present invention also provides fusion proteins comprising at least one protein or peptide of interest and a spore coat protein, wherein the spore coat protein comprises a CotB/H protein, a spore X protein, or a CotY protein, wherein the CotY protein comprises the amino acid sequence, having at least 80% identity with SEQ ID NO: 258 or 259.

[00417] For example, the spore coat protein may include a CotB/H protein.

[00418] For example, the spore coat protein may include X spore protein.

[00419] For example, the spore coat protein may include a CotY protein, wherein the CotY protein contains an amino acid sequence that shares at least 80% identity with SEQ ID NO: 258 or 259.

[00420] The spore coat protein may comprise an amino acid sequence having at least 85% identity with SEQ ID NO: 255, 257, 258, or 259.

[00421] The spore coat protein may comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 255, 257, 258, or 259.

[00422] The spore coat protein may comprise an amino acid sequence having at least 95% identity with SEQ ID NO: 255, 257, 258, or 259.

[00423] The spore coat protein may comprise an amino acid sequence having at least 98% identity with SEQ ID NO: 255, 257, 258, or 259.

[00424] The spore coat protein may comprise an amino acid sequence having at least 99% identity with SEQ ID NO: 255, 257, 258, or 259.

[00425] The spore coat protein may comprise an amino acid sequence having at least 100% identity with SEQ ID NO: 255, 257, 258, or 259.

C. Recombinant spore coat bacteria that express fusion proteins and plant seeds coated with recombinant spore coat bacteria

[00426] Provided are recombinant spore-forming bacteria that express any of the fusion proteins described in section IX.B. The recombinant spore-forming bacteria may include an endophytic bacterial strain, a plant growth promoting bacterial strain, or a bacterial strain that is both endophytic and plant growth promoting.

[00427] In addition, the present invention provides 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 spore surface of the bacterium, wherein the spore coat protein comprises a CotB protein , CotC protein, CgeA protein, CotB/H protein, CotG protein, X coat protein, or CotY protein; and wherein the recombinant spore-forming bacterium comprises an endophytic bacterial strain, a plant growth promoting bacterial strain, or a bacterial strain that is both endophytic and plant growth promoting.

[00428] Expression of the fusion protein in an endophytic bacterial strain allows delivery of the protein or peptide of interest to the interior of the plant. Endophytic strains can be delivered to plants using a variety of methods, for example, endophytic strains can be delivered by seed treatment, treatment of the seed growth medium (eg soil), watering, application directly to the plant (eg foliar application to aerial parts of a plant). After entering the plant, the bacteria increase in number and colonize the internal tissues of the plant.

[00429] The present invention also relates to plant seeds coated with a recombinant spore-forming bacterium, 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 bacterial spore surface, wherein the protein the spore envelope includes cotB protein, CotC protein, CgeA protein, CotB/H protein, Cot G protein, X spore protein, or cotY protein.

[00430] The recombinant spore coat forming bacterium may include a bacterium of the genus Bacillus or Lysinibacillus.

[00431] In addition, the present invention provides a recombinant bacterium of the genus Bacillus , wherein the recombinant bacterium comprises a recombinant spore-forming bacterium, and 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 bacterial spore, wherein the spore coat protein comprises a CotB protein, a CotC protein, a CgeA protein, a CotB/H protein, a Cot G protein, an X coat protein, or a CotY protein. The recombinant spore-forming bacterium expresses a protease or nuclease, wherein the expression of the protease or nuclease is increased compared to the expression of the protease or nuclease in wild-type Bacillus bacteria under the same conditions, and where the increased expression of the protease or nuclease partially or completely inactivates spores of the recombinant Bacillus bacterium or increases sensitivity of spores of recombinant bacteria of the genus Bacillus to physical or chemical inactivation. The protease or nuclease may be any of the proteases or nucleases described in section VA above and may be expressed under the control of any of the promoters described in section VA above. In addition, the invention relates to plant seeds coated with such spore-forming bacteria. The recombinant bacterium may comprise an endophytic bacterial strain, a plant growth promoting bacterial strain, or a bacterial strain that is both endophytic and plant growth promoting.

[00432] In any of the plant seeds described in this section, the recombinant spore-forming bacterium may include an endophytic bacterial strain, a plant growth promoting bacterial strain, or a bacterial strain that is both endophytic and plant growth promoting.

[00433] In any of the recombinant spore-forming bacteria or seeds, an endophytic bacterial strain, a plant growth promoting bacterial strain, or a bacterial strain that is both endophytic and plant growth promoting may include Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillus fusiformis EE442 or Lysinibacillus sphaericus EE443, Bacillus pumilus EE-B00143, Bacillus subtilis EE148, Bacillus subtilis EE218 or Bacillus megaterium EE281. For example, an endophytic bacterial strain may include Bacillus subtilis EE405 or Bacillus megaterium EE385.

[00434] Alternatively, an endophytic strain, a plant growth promoting bacterial strain, or a bacterial strain that is both endophytic and plant growth promoting may include a member of the Bacillus cereus EE349 family, a member of the Bacillus cereus EE439 family, a Bacillus thuringiensis EE417, a Bacillus cereus EE444, or Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus cereus EE-B00377, Bacillus pseudomycoides EE-B00366, Bacillus mycoides EE-B00363, Bacillus mycoides BT155, Bacillus mycoides EE118, Bacillus mycoides EE141, BTacillus mycoides , family member Bacillus cereus EE128, Bacillus thuringiensis BT013A or a member of the Bacillus cereus EE349 family.

[00435] In any of the recombinant spore-forming bacteria or seeds, the spore coat protein may comprise an amino acid sequence having at least 85% identity with any of SEQ ID NOs: 252-259.

[00436] The spore coat protein may comprise an amino acid sequence having at least 90% identity with any of SEQ ID NOs: 252-259.

[00437] The spore coat protein may comprise an amino acid sequence having at least 95% identity with any of SEQ ID NOs: 252-259.

[00438] The spore coat protein may comprise an amino acid sequence having at least 98% identity with any of SEQ ID NOs: 252-259.

[00439] The spore coat protein may comprise an amino acid sequence having at least 99% identity with any of SEQ ID NOs: 252-259.

[00440] The spore coat protein may contain an amino acid sequence that has 100% identity with any of SEQ ID NOs: 252-259.

[00441] Also provided 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 spore surface of the bacterium. The recombinant spore-forming bacterium is not a recombinant member of the Bacillus cereus family. The protein that targets the fusion protein to the bacterial spore surface 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.

[00442] The protein that targets the fusion protein to the bacterial spore surface may contain an amino acid sequence that shares 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.

[00443] The protein that targets the fusion protein to the bacterial spore surface may contain an amino acid sequence that shares 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.

[00444] The protein that targets the fusion protein to the bacterial spore surface may contain an amino acid sequence that shares 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.

[00445] The protein that targets the fusion protein to the bacterial spore surface may contain an amino acid sequence that shares 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.

[00446] The protein that targets the fusion protein to the bacterial spore surface may contain an amino acid sequence that shares 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.

[00447] For example, a protein that targets the fusion protein to the surface of a bacterial spore may 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.

[00448] The recombinant spore-forming bacterium includes an endophytic bacterial strain, a plant growth promoting bacterial strain, or a bacterial strain that is both endophytic and plant growth promoting. For example, an endophytic bacterial strain, a plant growth promoting bacterial strain, or a bacterial strain that is both endophytic and plant growth promoting includes Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillus fusiformis EE442, Lysinibacillus sphaericus EE443, Bacillus pumilus EE-B00143, Bacillus subtilis EE148, Bacillus subtilis EE218 or Bacillus megaterium EE281. The endophytic bacterial strain preferably includes Bacillus sp. EE387.

X. Methods for making fusion proteins

[00449] Any of the fusion proteins described herein can be obtained using standard cloning and molecular biology techniques known in the art. For example, a gene encoding a protein or peptide of interest (e.g., a gene encoding a plant growth promoting protein or peptide) can be amplified by polymerase chain reaction (PCR) and ligated to DNA encoding any of the targeting sequences described above, exospore proteins, fragments exosporium proteins or spore coat proteins to produce a DNA molecule that encodes a fusion protein. The DNA molecule encoding the fusion protein can be cloned into any suitable vector, such as a plasmid vector. The vector suitably includes a multiple cloning site into which the DNA molecule encoding the fusion protein can be easily inserted. Suitably, the vector also contains a selectable marker, such as an antibiotic resistance gene, so that bacteria transformed, transfected or crossed with the vector can be readily identified and isolated. When the vector is a plasmid, the plasmid suitably also contains an origin of replication. Alternatively, the DNA encoding the fusion protein can be inserted into the chromosomal DNA of a member of the B. cereus family or a spore-forming host bacterium.

XI. Labels, markers and linkers that can be included in fusion proteins

[00450] Any of the fusion proteins described herein may also include additional polypeptide sequences that are not part of a targeting sequence, an exosporium protein, an exosporium protein fragment, or a plant growth promoting protein or peptide, a protein or peptide that protects a plant from a pathogen , a protein or peptide that enhances plant stress resistance, or a plant-binding protein or peptide. For example, the fusion protein may include labels or markers to facilitate purification or visualization of the fusion protein (eg, a polyhistidine tag or a fluorescent protein such as GFP or YFP) or visualization of recombinant Bacillus cereus spores expressing the fusion protein.

[00451] Expression of fusion proteins on the exospores of a member of the Bacillus cereus family or on the spore surface of a spore-forming bacterium using the targeting sequences, exospore proteins, exospore protein fragments, and spore coat proteins described herein is increased due to the lack of secondary structure at the N-terminus of these sequences , which provides native folding of fused proteins and preservation of activity. Proper folding can furthermore be improved by including a short amino acid linker between the targeting sequence, the exospore protein, the exosporium protein fragment, the spore coat protein, and the protein or peptide of interest.

[00452] Thus, any of the fusion proteins described herein may include an amino acid linker between a targeting sequence, an exospore protein, an exospore protein fragment, or a spore coat protein, and the protein or peptide of interest.

[00453] The linker may include a polyalanine linker or a polyglycine linker. It is also possible to use a linker containing a mixture of both alanine and glycine residues.

[00454] For example, in a fusion protein in which the targeting sequence contains SEQ ID NO: 1, the fusion protein may have one of the following structures:

No linker: SEQ ID NO: 1-POI

Alanine linker: SEQ ID NO: 1-A _n -POI

Glycine linker: SEQ ID NO: 1-G _n -POI

Mixed alanine and glycine linker: SEQ ID NO: 1 - (A/G) _n - POI

where A _n , G _n and (A/G) _n represent any number of alanine residues, any number of glycine residues or any number of a mixture of alanine and glycine residues, respectively. For example, n can be from 1 to 25, and preferably is from 6 to 10. When the linker contains a mixture of alanine and glycine residues, any combination of glycine and alanine residues can be used. In the structures described above, "POI" is a protein or peptide of interest.

[00455] Alternatively or additionally, the linker may include a protease recognition site. Inclusion of a protease recognition site allows targeted removal by a protease that recognizes the protease recognition site of the protein or peptide of interest.

XII. Proteins and peptides of interest

[00456] The protein or peptide of interest may include any protein or peptide.

[00457] A protein or peptide of interest in the fusion proteins described herein may include, for example: (a) a plant growth promoting protein or peptide; (b) a protein or peptide that protects the plant from a pathogen; (c) a protein or peptide that enhances plant stress resistance; (d) a plant-binding protein or peptide; (e) an enzyme that catalyses 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 the plant; (h) an antigen; (i) a reclamation enzyme; (j) an enzyme suitable for breaking the emulsion or gel in the fracturing fluid; or (k) an antibacterial protein or peptide.

A. Plant growth promoting proteins or peptides

[00458] The protein or peptide of interest may include a plant growth promoting protein or peptide.

[00459] The plant growth promoting protein or peptide can include a peptide hormone, a non-hormonal peptide, an enzyme involved in the production or activation of a plant growth promoting compound, or an enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source.

[00460] For example, a plant growth promoting protein or peptide may include a peptide hormone.

[00461] The peptide hormone may include phytosulfokine (eg, phytosulfokin-α), clavata 3 (CLV3), systemin, ZmlGF, or SCR/SP11.

[00462] The plant growth promoting protein or peptide may include a non-hormonal peptide.

[00463] The non-hormonal peptide may include RKN 16D10, Hg-Syv46, eNOD40 peptide, melittin, mastoparan, Mas7, RHPP, POLARIS, or kunitz trypsin inhibitor (KTI).

[00464] The plant growth promoting protein or peptide may include an enzyme involved in the production or activation of the plant growth promoting compound. An enzyme involved in the production or activation of a plant growth stimulating compound can be any enzyme that catalyzes any step in the biological pathway for the synthesis of 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 the compound, which stimulates the growth of a plant or changes the structure of a plant into an active or more active form of the compound.

[00465] A plant growth promoting compound may include a compound produced by bacteria or fungi in the rhizosphere, such as 2,3-butanediol.

[00466] Alternatively, the plant growth stimulating compound may include plant growth hormone.

[00467] Plant growth hormone may include cytokinin or cytokinin derivative, ethylene, auxin or auxin derivative, gibberellic acid or gibberellic acid derivative, abscisic acid or abscisic acid derivative, or jasmonic acid or jasmonic acid derivative.

[00468] When the plant growth promoting compound comprises a cytokinin or a cytokinin derivative, the cytokinin or cytokinin derivative may include kinetin, cis-zeatin, trans-zeatin, 6-benzylaminopurine, dihydroxyzeatin, N6-(D2-isopentenyl)adenine, ribosylzeatin, N6-( D2-isopentenyl)adenosine, 2-methylthio-cis-ribosylzeatin, cis-ribosylzeatin, trans-ribosylzeatin, 2-methylthio-trans-ribosylzeatin, ribosylzeatin-5-monophosphate, N6-methylaminopurine, N6-dimethylaminopurine, 2ʹ-deoxyzeatin riboside, 4- hydroxy-3-methyl-trans-2-butenylaminopurine, ortho-topoline, meta-topoline, benzyladenine, ortho-methyl topoline, meta-methyl topoline, or a combination thereof.

[00469] When the plant growth promoting compound comprises an auxin or an auxin derivative, the auxin or auxin derivative may include 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 may include indole-3-acetic acid, indole-3-pyruvic acid, indole-3-acetaldoxime, indole-3-acetamide, indole-3-acetonitrile, indole-3-ethanol, indole-3- pyruvate, indole-3-acetaldoxime, indole-3-butyric acid, phenylacetic acid, 4-chloroindole-3-acetic acid, glucose-conjugated auxin, or a combination thereof.

[00470] An enzyme involved in the production or activation of a plant growth stimulating compound may include acetoin reductase, indole-3-acetamide hydrolase, tryptophan monooxygenase, acetolactate synthetase, α-acetolactate decarboxylase, pyruvate decarboxylase, diacetyl reductase, butanediol dehydrogenase, aminotransferase (e.g. indole-3-pyruvate decarboxylase, indole-3-acetaldehyde dehydrogenase, tryptophan side chain oxidase, nitrile hydrolase, nitrilase, peptidase, protease, adenosine phosphate isopentenyl transferase, phosphatase, adenosine kinase, adenine phosphoribosyl transferase, CYP735A, -O-glucosyltransferase, β-glucosidase, cis-hydroxylase, cis-hydroxylase CK, N-glucosyltransferase CK, 2,5-ribonucleotide phosphohydrolase, adenosine nucleosidase, purine nucleoside phosphorylase, zeatin reductase, hydroxylamine reductase, 2-oxoglutarate dioxygenase, gibberello 2B/3B hydrolase, gibberallin-3-oxidase, gibberallin-20-oxidase, chitosanase, chitinase, β-1,3-glucanase, β-1,4-glucanase, β-1,6-glucanase, aminocyclopropane-1 deaminase -carboxylic acid or an enzyme involved in the production of the nod factor (eg nodA, nodB or nodI).

[00471] When the enzyme contains a protease or peptidase, the protease or peptidase may be a protease or peptidase that cleaves proteins, peptides, proproteins, or preproproteins to form a biologically active peptide. A biologically active peptide can be any peptide that exhibits biological activity.

[00472] Examples of biologically active peptides include RKN 16D10 and RHPP.

[00473] A protease or peptidase that cleaves proteins, peptides, proproteins, or preproproteins to form a biologically active peptide may include subtilisin, acid protease, alkaline protease, proteinase, endopeptidase, exopeptidase, thermolysin, papain, pepsin, trypsin, pronase, carboxylase, serine protease, glutamine protease, aspartate protease, cysteine protease, threonine protease or metalloproteinase.

[00474] A protease or peptidase can cleave proteins in a protein-rich material (eg, soy flour or yeast extract).

[00475] When the enzyme includes chitosanase, the chitosanase may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 313.

[00476] Chitosanase may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 313.

[00477] Chitosanase may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 313.

[00478] Chitosanase may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 313.

[00479] Chitosanase may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 313.

[00480] Chitosanase may contain an amino acid sequence having at least 100% identity with SEQ ID NO: 313.

[00481] For example, a fusion protein may contain amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1) as a targeting sequence and an amino acid sequence containing SEQ ID NO: 313 as an enzyme that is specific for a cellular component bacteria or fungus. In addition, the fusion protein may contain a linker (eg, a polyalanine linker) between the targeting sequence and the enzyme.

[00482] The plant growth-promoting protein or peptide may include an enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source.

[00483] An enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source may include cellulase, lipase, lignin oxidase, protease, glycoside hydrolase, phosphatase, nitrogenase, nuclease, amidase, nitrate reductase, nitrite reductase, amylase, ammonia oxidase, ligninase, glucosidase , phospholipase, phytase, pectinase, glucanase, sulfatase, urease, xylanase, or siderophore.

[00484] When added to a plant growth medium or applied to a plant, seed, or area surrounding a plant or plant seed, fusion proteins containing enzymes that degrade or modify a bacterial, fungal, or plant nutrient source can facilitate nutrient processing near plants and result in increased uptake of nutrients by the plant or beneficial bacteria or fungi near the plant.

[00485] An enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source may include cellulase.

[00486] Cellulase may include endocellulase (for example, an endogluconase such as Bacillus subtilis endoglucanase, Bacillus thuringiensis endoglucanase, Bacillus cereus endogluconase, or Bacillus clausii endogluconase), exocellulase (for example, Trichoderma reesei exocellulase ), or β-glucosidase (for example, Bacillus β-glucosidase subtilis , Bacillus thuringiensis β-glucosidase, Bacillus cereus β-glucosidase, or Bacillus clausii β-glucosidase). The cellulase preferably comprises Bacillus subtilis endoglucanase.

[00487] The endoglucanase may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 311.

[00488] The endoglucanase may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 311.

[00489] Endoglucanase may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 311.

[00490] The endoglucanase may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 311.

[00491] The endoglucanase may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 311.

[00492] Endoglucanase may contain an amino acid sequence that has 100% identity with SEQ ID NO: 311.

[00493] For example, a fusion protein may contain amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1) as a targeting sequence and an amino acid sequence containing SEQ ID NO: 311 as an enzyme that degrades or modifies a nutrient source. substances of bacteria, fungi or plants. In addition, the fusion protein may contain a linker (eg, a polyalanine linker) between the targeting sequence and the enzyme.

[00494] An enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source may include a lipase (eg, Bacillus subtilis lipase, Bacillus thuringiensis lipase, Bacillus cereus lipase, or Bacillus clausii lipase).

[00495] An enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source may include lignin oxidase. For example, lignin oxidase may include lignin peroxidase, laccase, glyoxal oxidase, ligninase, or manganese peroxidase.

[00496] An enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source may include a protease. For example, a protease may include subtilisin, acid protease, alkaline protease, proteinase, peptidase, endopeptidase, exopeptidase, thermolysin, papain, pepsin, trypsin, pronase, carboxylase, serine protease, glutamine protease, aspartate protease, cysteine protease, threonine protease, or metalloproteinase.

[00497] An enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source may include a phosphatase. For example, the phosphatase may include phosphoric acid monoester hydrolase, phosphomonoesterase (e.g., PhoA4), phosphoric acid diester hydrolase, phosphodiesterase, triphosphoric acid monoester hydrolase, phosphoryl anhydride hydrolase, pyrophosphatase, phytase (e.g., Bacillus subtilis EE148 phytase or Bacillus thuringiensis BT013A phytase) , trimetaphosphatase or triphosphatase.

[00498] An enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source may include nitrogenase. For example, the nitrogenase may include a nitrogenase of the Nif family (eg, NifBDEHKNXV Paenibacillus massiliensis ).

[00499] An enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source may include a phospholipase. For example, the phospholipase may include phospholipase A1, phospholipase A2, phospholipase C, phospholipase D, or lysophospholipase. The phospholipase preferably includes phospholipase C.

[00500] Phospholipase C may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 312.

[00501] Phospholipase C may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 312.

[00502] Phospholipase C may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 312.

[00503] Phospholipase C may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 312.

[00504] Phospholipase C may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 312.

[00505] Phospholipase C may contain an amino acid sequence that has 100% identity with SEQ ID NO: 312.

[00506] For example, a fusion protein may contain amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1) as a targeting sequence and an amino acid sequence containing SEQ ID NO: 312 as an enzyme that degrades or modifies a nutrient source. substances of bacteria, fungi or plants. In addition, the fusion protein may contain a linker (eg, a polyalanine linker) between the targeting sequence and the enzyme.

b. Proteins or peptides that protect plants from pathogens

[00507] The protein or peptide of interest may include a protein or peptide that protects the plant from a pathogen.

[00508] A protein or peptide that protects a plant from a pathogen may include a protein or peptide that enhances the plant's immune system.

[00509] For example, a plant immune enhancing protein or peptide can include harpin, a harpin-like protein, α-elastin, β-elastin, systemin, phenylalanine ammonia lyase, elicitin, defensin, cryptogain, flagellin protein, or a flagellin peptide (e.g., flg22 ).

[00510] A 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 antifungal activity. Examples of such proteins and peptides include bacteriocins, lysozymes, lysozyme peptides (eg, LysM), siderophores, avidins, streptavidins, nonribosomal active peptides, conalbumins, albumins, lactoferrins, lactoferrin peptides (eg, LfcinB), and TasA.

[00511] A protein or peptide that protects a plant from a pathogen can be a protein or peptide that has insecticidal activity, helminthicidal activity, insect or worm predation control, or a combination thereof. For example, a protein or peptide that protects a plant from a pathogen may include an insecticidal bacterial toxin (e.g., VIP insecticidal protein), an endotoxin, a Cry toxin (e.g., Cry toxin from Bacillus thuringiensis ), a protease-inhibiting protein or peptide (e.g., a trypsin inhibitor or arrowhead protease inhibitor), cysteine protease or chitinase. When the Cry toxin contains the Cry toxin from Bacillus thuringiensis , the Cry toxin may be a Cry5B protein or a Cry21A protein. Cry5B and Cry21A have insecticidal and nematocidal activity.

[00512] A protein that protects a plant from a pathogen may include an enzyme. For example, the enzyme may include a protease or lactonase. Proteases and lactonases can be specific for a bacterial signaling molecule (eg, the bacterial signaling molecule homoserine lactone).

[00513] When the enzyme includes lactonase, the lactonase may include 1,4-lactonase, 2-pyrone-4,6-dicarboxylate lactonase, 3-oxoadipate enol lactonase, actinomycin lactonase, deoxylimonate A-ring lactonase, gluconolactonase, L-rhamnono-1 ,4-lactonase, limonin-D-ring lactonase, steroid lactonase, triacetate lactonase, or xylono-1,4-lactonase.

[00514] An enzyme may include an enzyme that is specific to a cellular component of a bacterium or fungus. For example, the enzyme may include β-1,3-glucanase, β-1,4-glucanase, β-1,6-glucanase, chitosanase, chitinase, chitosanase-like enzyme, lyticase, peptidase, protease, protease (e.g., alkaline protease , acid protease or neutral protease), mutanolysin, stafolysin or lysozyme.

[00515] When the enzyme includes chitosanase, the chitosanase may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 313.

[00516] Chitosanase may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 313.

[00517] Chitosanase may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 313.

[00518] Chitosanase may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 313.

[00519] Chitosanase may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 313.

[00520] Chitosanase may contain an amino acid sequence having at least 100% identity with SEQ ID NO: 313.

[00521] For example, a fusion protein may contain amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1) as a targeting sequence and an amino acid sequence containing SEQ ID NO: 313 as an enzyme that is specific for a cellular component bacteria or fungus. In addition, the fusion protein may contain a linker (eg, a polyalanine linker) between the targeting sequence and the enzyme.

[00522] For any of the proteins or peptides described above that protect a plant from a pathogen, the pathogen may include a protein or peptide of interest that protects the plant from a bacterial, worm, or insect pathogen.

[00523] For example, a bacterial pathogen may include an α-class proteobacterium, a β-class proteobacterium, a γ-class proteobacterium, or a combination thereof; or the bacterial pathogen includes Agrobacterium tumefaciens , Pantoea stewartii , Erwinia carotovora , Ralstonia solanacearum , Pseudomonas syringae , Pseudomonas aeruginosa , Xanthomonas campestris , or a combination thereof.

[00524] A protein or peptide that protects a plant from a pathogen may include a protein or peptide that protects a plant from worm or insect predation by a pathogen.

[00525] The worm or insect pathogen may include caterpillar, epsilon cutworm, corn borer, fall armyworm, armyworm, Japanese beetle, small corn stalk moth, corn weevil, sprout fly larva, web-producing caterpillar, corn stalk moth, southern corn rootworm, southern potato wireworm, stalk borer, sugarcane beetle, beetle grub, cabbage cutworm, cotton weevil, yellow striped caterpillar, red-breasted leech, bed bug, aphid, little armyworm, Mexican bean weevil, soy armyworm, soy stem grinder or their combination.

C. Proteins or peptides that increase plant resistance to stress

[00526] The protein or peptide of interest may include a protein or peptide that enhances plant stress resistance.

[00527] For example, a protein or peptide that enhances plant stress resistance may include an enzyme that degrades a stress-causing compound. Stress-causing compounds include, but are not limited to, aminocyclopropane-1-carboxylic acid (ACC), reactive oxygen species, nitric oxide, oxylipins, and phenols. Specific reactive oxygen species include hydroxyl, hydrogen peroxide, oxygen, and superoxide.

[00528] An enzyme that degrades a stress-causing compound may include superoxide dismutase, oxidase, catalase, aminocyclopropane-1-carboxylic acid deaminase, peroxidase, an antioxidant enzyme, or an antioxidant peptide.

[00529] When the enzyme that degrades the stress-causing compound includes superoxide dismutase, the superoxide dismutase may include superoxide dismutase 1 (SODA1) or superoxide dismutase 2 (SODA2).

[00530] Superoxide dismutase may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 155 or 156.

[00531] Superoxide dismutase may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 155 or 156.

[00532] Superoxide dismutase may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 155 or 156.

[00533] Superoxide dismutase may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 155 or 156.

[00534] Superoxide dismutase may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 155 or 156.

[00535] Superoxide dismutase may contain an amino acid sequence having 100% identity with SEQ ID NO: 155 or 156.

[00536] A protein or peptide that enhances plant stress resistance may include a protein or peptide that protects the plant from environmental stressors. Environmental stressors may include, for example, drought, flood, heat, freezing, salt, heavy metals, low pH, high pH, or a combination thereof. For example, a protein or peptide that protects a plant from environmental stressors may include a protein that induces the formation of microcrystals of ice, prolinase, phenylalanine ammonia lyase, isochorismate synthase, isochorismate pyruvate lyase, or choline dehydrogenase.

D. Plant-binding proteins or peptides

[00537] The protein or peptide of interest may include a plant-binding protein or peptide. A plant-binding protein or peptide can be any protein or peptide that is capable of binding specifically or non-specifically to any part of a plant (eg, a plant root or an aerial part of a plant such as a leaf, stem, flower or fruit) or plant material. Thus, for example, a plant-binding protein or peptide can be a root-binding protein or peptide, or a leaf-binding protein or peptide.

[00538] Suitable plant-binding proteins and peptides include adhesins (e.g., ricadhesin), flagellins, omptins, lectins, expansins, biofilm structural proteins (e.g., TasA or YuaB), pilus proteins, curl proteins, intimins, invasins, agglutinins, and afimbriae proteins.

E. Enzymes that catalyze the production of nitric oxide

[00539] Many plant species are inherently low in germination. For such plants, it is desirable to increase the level of germination. Nitric oxide is a powerful germination promoter that, when near the seed of a plant, enhances germination.

[00540] The present invention provides fusion proteins comprising any of the targeting sequences, exospore proteins, exospore protein fragments, or spore coat proteins described herein, and an enzyme that catalyses the production of nitric oxide synthase. Thus, the protein or peptide of interest may include an enzyme that catalyses the production of nitric oxide. Fusion proteins containing an enzyme that catalyzes the production of nitric oxide can be expressed in recombinant members of the Bacillus cereus family or recombinant spore-forming bacteria to deliver the enzyme that catalyzes the production of nitric oxide to a plant seed, plant, plant growth medium, or area surrounding the plant, or plant seed, and thereby to stimulate germination.

[00541] For example, an enzyme that catalyses the production of nitric oxide can include nitric oxide synthase (e.g., Bacillus thuringiensis nitric oxide synthase or Bacillus subtilis nitric oxide synthase, e.g., nitric oxide synthase from Bacillus thuringiensis BT013A or Bacillus subtilis 168) or arginase.

[00542] For example, nitric oxide synthase may include one of the amino acid sequences described in Table 8 below.

Table 8 Exemplary nitric oxide synthase sequences

Nitric oxide synthase SEQID NO. Nitric oxide synthase Bacillus subtilis 260 Nitric oxide synthase Bacillus thuringiensis 261

[00543] The nitric oxide synthase may also contain a sequence having a high degree of sequence identity with the nitric oxide synthase sequences shown in Table 8 above. For example, nitric oxide synthase may contain an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 260 or 261.

[00544] The nitric oxide synthase may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 260 or 261.

[00545] The nitric oxide synthase may comprise an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 260 or 261.

[00546] The nitric oxide synthase may comprise an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 260 or 261.

[00547] The nitric oxide synthase may comprise an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 260 or 261.

[00548] The nitric oxide synthase may comprise an amino acid sequence having at least 100% sequence identity with SEQ ID NO: 260 or 261.

[00549] When the protein or peptide of interest includes nitric oxide synthase, the fusion protein may contain one of the amino acid sequences shown in Table 9 below. In the sequences shown in Table 9 below, the targeting sequence is shown in bold, the six amino acid linker is shown underlined, and the nitric oxide synthase sequence is shown in plain text. Thus, the fusion protein may comprise SEQ ID NO: 262 or 263.

Table 9. Exemplary nitric oxide synthase-containing fusion proteins

Fusion protein (SEQ ID NO) Amino acid sequence Met+amino acids 20-35 BclA, alanine linker and Bacillus subtilis nitric oxide synthase (SEQ ID NO: 262) MAFDPNLVGPTLPPIPP AAAAAA MEEKEILWNEAKAFIAACYQELGKEEEVKDRLADIKSEIDLTGSYVHTKEELEHGAKMAWRNSNRCIGRLFWNSLNVIDRRDVRTKEEVRDALFHHIETATNNGKIRPTITIFPPEEKGEKQVEIWNHQLIRYAGYESDGERIGDPASCSLTAACEELGWRGERTDFDLLPLIFRMKGDEQPVWYELPRSLVIEVPITHPDIEAFSDLELKWYGVPIISDMKLEVGGIHYNAAPFNGWYMGTEIGARNLADEKRYDKLKKVASVIGIAADYNTDLWKDQALVELNKAVLHSYKKQGVSIVDHHTAASQFKRFEEQEEEAGRKLTGDWTWLIPPISPAATHIFHRSYDNSIVKPNYFYQDKPYE Met+amino acids 20-35 BclA, alanine linker and Bacillus thuringiensis nitric oxide synthase (SEQ ID NO: 263) MAFDPNLVGPTLPPIPP AAAAAA MSKTKQLIEEASHFITICYKELSKEHFIEERMKEIQAEIEKTGTYEHTFEELVHGSRMAWRNSNRCIGRLFWSKMHILDAREVNDEEGVYHALIHHIKYATNDGKVKPTITIFKQYQGEENNIRIYNHQLIRYAGYKTEMGVTGDSHSTAFTDFCQELGWQGEGTNFDVLPLVFSIDGKAPIYKEIPKEEVKEVPIEHPEYPISSLGAKWYGVPMISDMRLEIGGISYTAAPFNGWYMGTEIGARNLADHDRYNLLPAVAEMMDLDTSRNGTLWKDKALIELNVAVLHSFKKQGVSIVDHHTAAQQFQQFEKQEAACGRVVTGNWVWLIPPLSPATTHIYHKPYPNEILKPNFFH

[00550] Nitric oxide synthases from a number of species can be used as the protein or peptide of interest in fusion proteins, including Bacillus thuringiensis , Bacillus cereus , Bacillus subtilis , and Bacillus mycoides .

F. Proteins and peptides that bind nucleic acids

[00551] It is desirable to deliver nucleic acids to plants in the field, but is hindered by the instability of nucleic acids that are rapidly degraded in the external environment (eg, plant growth medium such as soil).

[00552] The present invention provides fusion proteins comprising any of the targeting sequences, exospore proteins, exospore protein fragments, or spore envelope proteins described herein and a nucleic acid binding protein or peptide. Such fusion proteins stabilize nucleic acids and can be used to deliver nucleic acids to soil and/or plants.

[00553] Thus, a protein or peptide of interest may include a nucleic acid-binding protein or peptide. For example, a nucleic acid binding protein or peptide may include an RNA binding protein or peptide or a DNA binding protein or peptide.

[00554] An RNA-binding protein or peptide may include a non-specific RNA-binding protein or peptide or a specific RNA-binding protein or peptide.

[00555] For example, the RNA binding peptide may include an Hfq protein (eg, Bacillus thuringiensis Hqf protein).

[00556] The DNA binding protein or peptide may include a small acid soluble spore protein (SASP). For example, SASP may include SASP encoded by SspA gene, SspB gene, SspC gene, SspD gene, SspE gene, SspF gene, SspG gene, SspH gene, SspI gene, SspJ gene, SspK gene, SspL gene, SspM gene, SspN gene, the SspO genome or the SspP genome. For example, SASP may include SASPα, SASPβ or SASPγ. SASP may include Bacillus thuringiensis SASP.

[00557] The nucleic acid binding protein may comprise one of the amino acid sequences described in Table 10 below.

Table 10 Exemplary SASP and Hfq sequences

Fusion protein SEQID NO. SASPα 264 SASPγ 265 hfq 266

[00558] The nucleic acid binding protein may also comprise a sequence having a high degree of sequence identity with any of the sequences shown in Table 10 above. For example, the nucleic acid binding protein may comprise a nucleic acid sequence having at least 85% identity with any of SEQ ID NO: 264-266.

[00559] The nucleic acid binding protein may comprise a nucleic acid sequence having at least 90% identity with any of SEQ ID NOs: 264-266.

[00560] The nucleic acid binding protein may comprise a nucleic acid sequence having at least 95% identity with any of SEQ ID NOs: 264-266.

[00561] The nucleic acid binding protein may comprise a nucleic acid sequence having at least 98% identity with any of SEQ ID NOs: 264-266.

[00562] The nucleic acid binding protein may comprise a nucleic acid sequence having at least 99% identity with any of SEQ ID NOs: 264-266.

[00563] The nucleic acid binding protein may comprise a nucleic acid sequence having at least 100% identity with any of SEQ ID NOs: 264-266.

[00564] For example, when the protein or peptide of interest includes a nucleic acid binding protein or peptide, the fusion protein may comprise one of the amino acid sequences shown in Table 11 below. In the sequences shown in Table 11 below, the targeting sequence is shown in bold, the six alanine amino acid linker is underlined, and the sequence of the nucleic acid binding protein or peptide (SASPα, SASPβ, or Hfq) is shown in plain text. Thus, for example, the fusion protein may comprise SEQ ID NO: 267, 268, or 269.

Table 11. Exemplary fusion proteins containing a nucleic acid binding protein

Fusion protein (SEQ ID NO) Amino acid sequence Met+amino acids 20-35 BclA, alanine linker and SASPα (SEQ ID NO: 267) MAFDPNLVGPTLPPIPP AAAAAAAA MAQQSRSRSNNNNDLLIPQAASAIEQMKLEIASEFGVQLGAETTSRANGSVGGEITKRLVRLAQQNMGGQFH Met+amino acids 20-35 BclA, alanine linker and SASPγ (SEQ ID NO: 268) MAFDPNLVGPTLPPIPP AAAAAAAA MANNNSGNSNNLLVPGAAQAIDQMKLEIASEFGVNLGADTTSRANGSVGGEITKRLVSFAQQNMGGGQF Met+amino acids 20-35 BclA, alanine linker and Hfq (SEQ ID NO: 269) MAFDPNLVGPTLPPIPP AAAAAAAA MKPINIQDQFLNQIRKENTYVTVFLLNGFQLRGQVKGFDNFTVLLESEGKQQLIYKHAISTFAPQKNVQLELE

[00565] Nucleases can also be used to both bind and cleave nucleic acid molecules. Nucleases have high affinity for RNA and DNA molecules and exhibit their enzymatic activity by cleaving RNA and/or DNA molecules into smaller RNA and/or DNA fragments. Nucleases can be specific, recognizing and cleaving specific DNA or RNA sequences, or non-specific, cleaving any DNA and/or RNA with which they come into contact. Nucleases can be divided into exonucleases (nucleases that cleave nucleotides from the ends of RNA and/or DNA molecules) or endonucleases (nucleases that cleave the phosphodiester bond in a polynucleotide chain). Each nuclease enzyme has an active site that contains specific amino acids that act to catalyze the cleavage of the nucleic acid molecule. Mutation of these active sites can inactivate the active site and provide high affinity binding of the nuclease to its nucleic acid substrate without cleavage of the substrate. Thus, such mutants can bind and stabilize the nucleic acid molecule without cleaving the nucleic acid molecule.

[00566] Thus, a nucleic acid-binding protein may include a nuclease (eg, a nuclease having an inactivated active site).

[00567] When the protein or peptide of interest includes a nucleic acid-binding protein or peptide, a nucleic acid molecule may be linked to the nucleic acid-binding protein or peptide. The nucleic acid may include, for example, a modulating RNA molecule; RNA-i molecule; miRNA; an aptamer or a DNA molecule that encodes a modulating RNA molecule, an RNA-i molecule, a microRNA or an aptamer.

XIII. Recombinant members of the family bacillus cereus, hosts

[00568] As described above, a member of the Bacillus cereus family can serve as a host for the expression of fusion proteins containing a targeting sequence, an exosporium protein, or an exospore protein fragment that targets the fusion protein to the exospore of a member of the Bacillus cereus family; serve as a host for the expression of modulator proteins that modulate the expression of the fusion protein; serve as a host for overexpression of the exospore enzyme; may be genetically inactivated; or may contain a mutation or other genetic change that allows the collection of free exospore.

[00569] A recombinant member of the Bacillus cereus family may coexpress two or more of any of the fusion proteins described above. For example, a recombinant member of the Bacillus cereus family can co-express at least one fusion protein that contains a plant-binding protein or peptide, together with a fusion protein that contains a plant growth-promoting protein or peptide, a fusion protein that contains a protein or peptide that protects the plant. from a pathogen, a fusion protein containing a protein or peptide that enhances plant resistance to stress, a fusion protein containing an enzyme that catalyzes the production of nitric oxide, or a fusion protein containing a nucleic acid-binding protein or peptide.

[00570] A recombinant member of the Bacillus cereus family may include any Bacillus species that is capable of producing exospores. For example, a recombinant member of the Bacillus cereus family may include Bacillus anthracis , Bacillus cereus , Bacillus thuringiensis , Bacillus mycoides , Bacillus pseudomycoides , Bacillus samanii, Bacillus gaemokensis, Bacillus weihenstephensis , Bacillus toyoiensis , or a combination thereof. In particular, a recombinant member of the Bacillus cereus family may include Bacillus thuringiensis or Bacillus mycoides .

[00571] To obtain a recombinant member of the Bacillus cereus family expressing a fusion protein, any member of the Bacillus cereus family can be conjugated to, 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, when the vector includes an antibiotic resistance gene, the bacteria can be screened for antibiotic resistance. Alternatively, the DNA encoding the fusion protein can be inserted into the chromosomal DNA of the host member of the B. cereus family. The recombinant member of the Bacillus cereus family can then be exposed to conditions that induce sporulation. Suitable conditions for the induction of sporulation are known in the art. For example, a recombinant member of the Bacillus cereus family can be plated on agar plates and incubated at approximately 30° C. for several days (eg, 3 days).

[00572] Inactivated strains, non-toxic strains or genetically manipulated strains of any of the species described above can also be used where appropriate. For example, Bacillus thuringiensis, which lacks the Cry toxin, can be used. Alternatively or additionally, once the recombinant B. cereus spores expressing the fusion protein are obtained, they can be inactivated to prevent further germination during application. You can use any method of inactivation of bacterial spores, which is known in this field. Suitable methods include, but are not limited to, heat treatment, gamma irradiation, X-ray irradiation, UV-A irradiation, UV-B irradiation, chemical treatment (e.g., treatment with glutaraldehyde, formaldehyde, hydrogen peroxide, acetic acid, bleach, or any their combination), or a combination of them. Alternatively, spores derived from non-toxigenic strains or genetically or physically inactivated strains can be used.

[00573] Many strains of members of the Bacillus cereus family have inherent beneficial qualities. For example, some strains have plant growth promoting effects. Any of the recombinant members of the Bacillus cereus family described herein may include a bacterial strain that promotes plant growth.

[00574] A plant growth promoting bacterial strain may include a bacterial strain that produces an insecticidal toxin (e.g., Cry toxin), produces a fungicidal compound (e.g., β-1,3-glucanase, chitosanase, lyticase, or a combination thereof), produces a nematocidal compound (e.g., Cry toxin), produces a bactericidal compound, is resistant to one or more antibiotics, contains one or more freely replicating plasmids, binds to plant roots, colonizes plant roots, forms biofilms, solubilizes nutrients, secretes organic acids, or has any a combination of these effects.

[00575] For example, when the recombinant member of the Bacillus cereus family comprises a plant growth promoting bacterial strain, the plant growth promoting bacterial strain may include (a) Bacillus mycoides BT155 (NRRL No. B-50921), (b) Bacillus mycoides EE118 (NRRL No. B- 50918), (c) Bacillus mycoides EE141 (NRRL No. B-50916), (d) Bacillus mycoides BT46-3 (NRRL No. B-50922), (e) Bacillus cereus EE128 (NRRL No. B-50917), ( f) Bacillus thuringiensis BT013A (NRRL No. B-50924), (g) Bacillus cereus EE349 (NRRL No. B-50928), (h) Bacillus cereus EE-B00377 (NRRL B-67119), (i) Bacillus pseudomycoides EE-B00366 (NRRL B-67120) or (j) Bacillus mycoides EE-B00363 (NRRL B-67121). Strains (a)-(g) were each deposited with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS), 1815 North University Street, Peoria, Illinois 61604 USA on March 10, 2014, and identified by the NRRL deposit number shown in brackets. Bacillus thuringiensis BT013A is also known as Bacillus thuringiensis 4Q7. Strains (h)-(j) were each deposited with the USDA ARS on August 19, 2015, and identified by the NRRL deposit number shown in parentheses.

[00576] These plant growth promoting strains were isolated from the rhizospheres of various vigorous plants and were identified by their 16S rRNA sequences (shown in Table 12) and by biochemical assays. The strains have been identified at least to their generic designation using commonly accepted biochemical and morphological indicators. Biochemical analyzes for confirmed Gram-positive strains such as Bacillus included growth analysis on PEA and nutrient agar, microscopic examination, growth analysis on media with 5% and 7.5% NaCl, growth analysis at pH 5 and pH 9, growth analysis at 42°C and 50°C, determination of the ability to produce acid during fermentation with cellobiose, lactose, glycerol, glucose, sucrose, d-mannitol and starch; determination of fluorescent pigment production; determination of hydrolysis of gelatin; nitrate recovery analysis; determination of catalase production, starch hydrolysis; determination of oxidase reaction, urease production and mobility. The identification of these strains and the demonstration of their plant growth promoting effects are described in the examples below.

Table 12. Partial 16S rRNA sequences for plant growth accelerating members of the family Bacillus cereus

Strain SEQID NO. for a partial sequence of 16S ribosomal RNA Bacillus mycoides EE118 270 Bacillus mycoides EE141 271 Bacillus mycoides BT46-3 272 Representative of the family Bacillus cereus EE128 273 Bacillus thuringiensis BT013A 274 Representative of the family Bacillus cereus EE349 275 Bacillus mycoides BT155 276

[00577] For example, a recombinant member of the Bacillus cereus family containing a plant growth-promoting bacterial strain may include Bacillus mycoides BT155, Bacillus mycoides EE141, or Bacillus thuringiensis BT013A.

[00578] A recombinant member of the Bacillus cereus family may include an endophytic bacterial strain. For example, the endophytic bacterial strain may include Bacillus cereus EE349, Bacillus cereus EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444 or Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus cereus EE-B00377; Bacillus pseudomycoides EE-B00366 or Bacillus mycoides EE-B00363.

[00579] A member of the Bacillus cereus family EE349 is also a plant growth accelerating bacterial strain and is described above. As further described in the examples below, a member of the Bacillus cereus EE349 family was also found to be endophytic.

[00580] Bacillus cereus EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus cereus EE-B00377; Bacillus pseudomycoides EE-B00366 or Bacillus mycoides EE-B00363 are described further below in section XIV.

[00581] An endophytic bacterial strain may include Bacillus cereus EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus cereus EE-B00377; Bacillus pseudomycoides EE-B00366 or Bacillus mycoides EE-B00363.

[00582] A recombinant member of the Bacillus cereus family may include a bacterial strain that is capable of degrading the herbicide or pesticide. As further discussed in the examples below, Bacillus cereus EE349, Bacillus cereus EE-B00377, Bacillus pseudomycoides EE-B00366, and Bacillus mycoides EE-B00363 have been found to be capable of degrading herbicides and/or pesticides. Thus, when the recombinant member of the Bacillus cereus family includes a bacterial strain that is capable of degrading the herbicide, the bacterial strain that is capable of degrading the herbicide may include a member of the Bacillus cereus family EE349, a member of the Bacillus cereus family EE-B00377, Bacillus pseudomycoides EE-B00366, or Bacillus mycoides EE-B00363.

[00583] A bacterial strain that is capable of degrading a herbicide or pesticide can degrade a sulfonylurea herbicide (e.g., sulfentrazone), an aryltriazine herbicide, dicamba, 2,4-D, a phenoxy herbicide, a pyrethrin, a pyrethroid, or a combination thereof.

[00584] A bacterial strain that is capable of degrading a pesticide may be a bacterial strain that is capable of degrading pyrethrin.

[00585] A recombinant member of the Bacillus cereus family may include a probiotic bacterial strain. For example, the probiotic bacterial strain may include a member of the Bacillus cereus EE349 family, a member of the Bacillus cereus EE439 family, Bacillus thuringiensis EE417, or Bacillus cereus EE444.

[00586] A recombinant member of the Bacillus cereus family may include an inactivating mutation in its BclA gene, its CotE gene, or its CotO gene (eg, knockout of the BclA gene, CotE gene, or CotO gene). For example, a recombinant member of the Bacillus cereus family may include an inactivating mutation in its BclA gene (eg, BclA gene knockout). It has been found that expression of fusion proteins in a recombinant member of the Bacillus cereus family having this mutation results in increased levels of fusion protein expression.

XIV. Endophytic bacterial strains

[00587] In addition, the present invention relates to endophytic bacterial strains. Although many bacteria of the rhizosphere have a symbiotic interaction with the plant, only a small subgroup of these bacteria are capable of internalization into the plant and endophytic growth. As further described in the examples below, several strains of members of the Bacillus cereus family and several bacterial strains that are not members of the Bacillus cereus family have been isolated from corn seedlings and found to have the ability to grow endophytically in plants.

A. Endophytic representatives of the Bacillus cereus family

[00588] The present invention relates to biologically pure bacterial cultures that are capable of endophytic growth. The bacterial strain in each of these bacterial cultures may be: (a) a member of the Bacillus cereus EE439 family (NRRL B-50979); (b) Bacillus 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) a member of the Bacillus cereus family EE-B00377 (NRRL B-67119); (g) Bacillus pseudomycoides EE-B00366 (NRRL B-67120) or (h) Bacillus mycoides EE-B00363 (NRRL B-67121). Strains (a)-(c) were each deposited with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS), 1815 North University Street, Peoria, Illinois 61604 USA on September 10, 2014, and are identified with using the NRRL numbers provided in brackets after 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 brackets after the strain name. Strains (e)-(h) were each deposited with the USDA ARS on August 19, 2015 and identified by NRRL numbers provided in brackets after each strain name.

[00589] The new strains described herein have been identified by 16S ribosomal RNA (rRNA) sequencing. Thus, a member of the Bacillus cereus EE439 family has a 16S ribosomal RNA sequence 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 with at least 98%, at least 99% or 100% sequence identity with sequence of SEQ ID NO: 279. Bacillus thuringiensis EE319 has a 16S ribosomal RNA sequence with at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 280. Bacillus thuringiensis EE-B00184 has a 16S ribosomal RNA sequence RNA having at least 98%, at least 99% or 10 0% sequence identity with the sequence of SEQ ID NO: 301. Member of the Bacillus cereus family EE-B00377 has a 16S ribosomal RNA sequence with at least 98%, at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 304. Bacillus pseudomycoides EE-B00366 has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 303. Bacillus mycoides EE-B00363 (NRRL B-67121) has a 16S sequence ribosomal RNA having at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 302. The 16S rRNA sequences are shown in Table 13 below.

Table 13. Partial 16S rRNA sequences for endophytic strains of family members Bacillus cereus

Strain SEQ ID NO for 16S rRNA partial sequence Representative of the family Bacillus cereus EE439 277 Bacillus thuringiensis EE417 278 Bacillus cereus EE444 279 Bacillus thuringiensis EE319 280 Bacillus thuringiensis EE-B00184 301 Bacillus mycoides EE-B00363 302 Bacillus pseudomycoides EE-B00366 303 Representative of the Bacillus cereus family EE-B00377 304

[00590] In addition, the present invention relates to a biologically pure bacterial culture, where the bacteria in the bacterial culture are mutants of Bacillus cereus EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus cereus EE-B00377, Bacillus pseudomycoides EE-B00366 or Bacillus mycoides EE-B00363 containing one or more mutations where the bacteria are endophytic.

b. Other endophytic bacterial strains

[00591] The present invention also relates to other biologically pure bacterial cultures (not members of the Bacillus cereus family) that have the ability to grow endophytically. These strains were isolated from corn seedlings as detailed in the examples below.

[00592] The bacterial strain of each of these bacterial cultures can be (a) Bacillus megaterium EE385 (NRRL B-50980), (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) Lysinibcaillus sphaericus EE443 (NRRL B-50976) or (g) Bacillus pumilus EE-B00143 (NRRL B-67123). Strains (a)-(f) were each deposited with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS) at 1815 North University Street, Peoria, Illinois 61604 USA on September 10, 2014, and they were identified by NRRL numbers provided in brackets after the names of each strain. The next deposit, Bacillus sp. EE387 has been identified as a strain similar to Bacillus pumilus . Strain (g) was deposited with the USDA ARS on August 19, 2015, and is identified by the NRRL number provided in brackets after the name.

[00593] The new strains described herein were identified by 16S ribosomal RNA (rRNA) sequencing. Thus, Bacillus megaterium EE385 has a 16S ribosomal RNA sequence with at least 98%, at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 281. Bacillus sp. EE387 has a 16S ribosomal RNA sequence with at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 282. Bacillus circulans EE388 has a 16S ribosomal RNA sequence with at least 98%, at least at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 283. Bacillus subtilis EE405 has a 16S ribosomal RNA sequence with at least 98%, at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 284. Lysinibacillus fusiformis EE442 has a 16S ribosomal RNA sequence with 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 with at least 98%, at least 99% or 100% sequence identity with sequence SEQ ID NO: 286. Bacillus pumilus EE-B00143 has a 16S ribosomal RNA sequence having at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 305. The 16s rRNA sequences are shown in the table below fourteen.

Table 14. Partial 16S rRNA sequences for non-family endophytic strains Bacillus cereus

Strain (SEQ ID NO) SEQ ID NO for 16S rRNA partial sequence Bacillus megaterium EE385 281 Bacillus sp. EE387 282 Bacillus circulans EE388 283 Bacillus subtilis EE405 284 Lysinibacillus fusiformis EE442 285 Lysinibcaillus sphaericus EE443 286 Bacillus pumilus EE-B00143 305

[00594] In addition, the present invention relates to a biologically pure bacterial culture, where the bacteria in the bacterial culture are mutants of Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillus fusiformis EE442, or Lysinibcaillus sphaericus EE443 containing one or more mutations where the bacteria are endophytic.

[00595] The present invention also relates to a biologically pure bacterial culture, where the bacteria in the bacterial culture are mutants of Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillus fusiformis EE442 or Lysinibcaillus sphaericus EE443 containing one or more mutations where the bacteria are probiotic.

XV. Inocula

[00596] In addition, the invention relates to inoculum of any of the biologically pure bacterial strains described above in the previous section. The inoculums are intended to be applied to plants, plant seeds, plant growth media, or the area surrounding the plant or plant seed, wherein the inoculum contains an effective amount of any of the biologically pure bacterial cultures and an agriculturally acceptable carrier.

[00597] The inoculum may contain an effective amount of a mixture containing at least two of the biologically pure bacterial cultures described above in the previous section.

[00598] In addition, the inoculum may contain an effective amount of rhizobacteria. Rhizobacteria can be a biologically pure bacterial culture of a strain of rhizobacteria. Rhizobacteria may include bacteria of the genus Bradyrhizobium (eg Bradyrhizobium japonicum ), bacteria of the genus Rhizobium (eg Rhizobium phaseoli , Rhizobium leguminosarum or combinations thereof), or combinations thereof.

XVI. Plant seeds coated with an enzyme that catalyzes the production of nitric oxide or with recombinant bacteria that overexpress an enzyme that catalyzes the production of nitric oxide

[00599] Also provided are plant seeds that are coated with: (i) an enzyme that catalyses the production of nitric oxide; (ii) superoxide dismutase or (iii) a recombinant microorganism that expresses an enzyme that catalyzes the production of nitric oxide, or superoxide dismutase, wherein the expression of the enzyme that catalyzes the production of nitric oxide, or superoxide dismutase, is increased compared to the expression of the enzyme that catalyzes the production of nitric oxide, or superoxide dismutase in a wild-type microorganism under the same conditions.

[00600] An enzyme that catalyses the production of nitric oxide may include nitric oxide synthase or arginase.

[00601] The enzyme that catalyses the production of nitric oxide may include nitric oxide synthase, such as nitric oxide synthase from Bacillus thuringiensis BT013A or Bacillus subtilis 168.

[00602] For example, nitric oxide synthase may contain an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 260 or 261.

[00603] Nitric oxide synthase may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 260 or 261.

[00604] Nitric oxide synthase may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 260 or 261.

[00605] The nitric oxide synthase may comprise an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 260 or 261.

[00606] Nitric oxide synthase may contain an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 260 or 261.

[00607] Nitric oxide synthase may contain an amino acid sequence having 100% sequence identity with SEQ ID NO: 260 or 261.

[00608] Superoxide dismutase may include superoxide dismutase 1 (SODA1) or superoxide dismutase 2 (SODA2).

[00609] Superoxide dismutase contains an amino acid sequence having at least 85% identity with SEQ ID NO: 155 or 156.

[00610] Superoxide dismutase contains an amino acid sequence with at least 90% identity with SEQ ID NO: 155 or 156.

[00611] Superoxide dismutase contains an amino acid sequence having at least 95% identity with SEQ ID NO: 155 or 156.

[00612] Superoxide dismutase contains an amino acid sequence with at least 98% identity with SEQ ID NO: 155 or 156.

[00613] Superoxide dismutase contains an amino acid sequence with at least 99% identity with SEQ ID NO: 155 or 156.

[00614] Superoxide dismutase contains an amino acid sequence having at least 100% identity with SEQ ID NO: 155 or 156.

[00615] When a plant seed is coated with a recombinant microorganism, the recombinant microorganism may include a Bacillus species, Escherechia coli , an Aspergillus species such as Aspergillus niger , or a Sacchromyces species such as Sacchromyces cerevisiae .

[00616] For example, the recombinant microorganism may include a member of the Bacillus cereus , Bacillus subtilis, Bacillus licheniformis, or Bacillus megaterium families.

[00617] The amino acid sequences of exemplary nitric oxide synthase enzymes are shown in Table 8 above. The amino acid sequences of exemplary superoxide dismutases are shown in Table 2 above.

XVII. Lineups

[00618] Compositions are contemplated that contain a recombinant member of the Bacillus cereus family as described herein, exospore fragments derived from spores of a recombinant member of the Bacillus cereus family as described herein, or a recombinant spore-forming bacterium as described herein, and agriculturally acceptable carrier.

[00619] An agriculturally acceptable carrier may include an additive such as an oil, a gum, a resin, a clay, a polyoxyethylene glycol, a terpene, a viscous organic compound, a fatty acid ester, a sulfated alcohol, an alkyl sulfonate, a petroleum sulfonate, an alcohol sulfate, sodium alkyl butanediamate , a polyester of sodium thiobutanedioate, a benzene derivative of acetonitrile, a proteinaceous material, or a combination thereof.

[00620] An agriculturally acceptable carrier may include a thickener such as long chain polyethylene glycol alkyl sulfonate, 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 arylalkyl polyoxyethylene glycol, an alkylamine acetate, an alkylarylsulfonate, a polyhydric alcohol, an alkyl phosphate, or a combination thereof; or an anti-caking agent such as a sodium salt (eg, sodium monomethylnaphthalenesulfonate, sodium dimethylnaphthalenesulfonate, sodium sulfite, sodium sulfate, or a combination thereof), calcium carbonate, diatomaceous earth, or a combination thereof.

[00621] The supplement may include a proteinaceous material such as a dairy product, wheat flour, soy flour, blood, albumin, gelatin, alfalfa flour, yeast extract, or a combination thereof.

[00622] An agriculturally acceptable carrier may include vermiculite, coal, press mud for carbonization in sugar production, rice hulls, carboxymethyl cellulose, peat, perlite, fine sand, calcium carbonate, flour, alum, starch, talc, polyvinylpyrrolidone, or their combination.

[00623] The formulation may include a seed coating formulation, a liquid formulation for application to plants or plant growth media, or a solid formulation for application to plants or plant growth media. The seed coating composition may comprise an aqueous or oily solution for application to the seeds, or a powder or granular composition for application to the seeds. The liquid formulation for application to plants or plant growth media may include a concentrated formulation or a ready-to-use formulation. The solid formulation for application to plants or plant growth media may include a granular formulation or a powder formulation.

[00624] In addition, the composition may contain a fertilizer, a micronutrient fertilizer material, an insecticide, a herbicide, a plant growth altering agent, a fungicide, an insecticide, a molluscicide, an algaecide, a bacterial inoculum, a fungal inoculum, or a combination thereof.

[00625] Бактериальный инокулят может включать бактериальный инокулят рода Rhizobium , бактериальный инокулят рода Bradyrhizobium , бактериальный инокулят рода Mesorhizobium , бактериальный инокулят рода Azorhizobium , бактериальный инокулят рода Allorhizobium , бактериальный инокулят рода Sinorhizobium , бактериальный инокулят рода Kluyvera , бактериальный инокулят рода Azotobacter , бактериальный инокулят рода Pseudomonas , bacterial inoculum of the genus Azospirillium , bacterial inoculum of the genus Bacillus , bacterial inoculum of the genus Streptomyces , bacterial inoculum of the genus Paenibacillus , bacterial inoculum of the genus Paracoccus , bacterial inoculum of the genus Enterobacter , bacterial inoculum of the genus Alcaligenes , bacterial inoculum of the genus Mycobacterium bacterial genus inoculum Gliocladium , a bacterial inoculum of the genus Glomus , a bacterial inoculum of the genus Klebsiella , or a combination thereof.

[00626] The bacterial inoculum may include a plant growth promoting bacterial strain. Plant Growth Accelerator Strain

bacteria may produce an insecticidal toxin, produce a fungicidal compound, produce a nematicidal compound, produce a bactericidal compound, may be resistant to one or more antibiotics, may contain one or more freely replicating plasmids, may bind to plant roots, colonize plant roots, form biofilms, solubilize nutrients, secrete organic acids, or may carry out a combination of them.

[00627] For example, the bacterial inoculum may contain Bacillus aryabhattai CAP53 (NRRL No. B-50819), Bacillus aryabhattai CAP56 (NRRL No. B-50817), Bacillus flexus BT054 (NRRL No. B-50816), Paracoccus kondratievae NC35 (NRRL No. B- 50820), Bacillus mycoides BT155 (NRRL No. B-50921), Enterobacter cloacae CAP12 (NRRL No. B-50822), Bacillus nealsonii BOBA57 (NRRL No. NRRL B-50821), Bacillus mycoides EE118 (NRRL No. B-50918), Bacillus subtilis EE148 (NRRL No. B-50927), Alcaligenes faecalis EE107 (NRRL No. B-50920), Bacillus mycoides EE141 (NRRL No. B-50916), Bacillus mycoides BT46-3 (NRRL No. B-50922), Bacillus cereus EE128 ( NRRL No. B-50917), Bacillus thuringiensis BT013A (NRRL No. B-50924), Paenibacillus massiliensis BT23 (NRRL No. B-50923), Bacillus cereus EE349 (NRRL No. B-50928), Bacillus subtilis EE218 (NRRL No. B- 50926), Bacillus megaterium EE281 (NRRL No. B-50925), Bacillus cereus EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120), Bacillus mycoides EE-B00363 (NRRL B-67121), Bacillus pumilus EE-B00143 (NRRL B-67123) or Bacillus thuringiensis EE-B00184 (NRRL B-67122), or their combination. Each of these strains was deposited with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS), 1815 North University Street, Peoria, Illinois 61604 USA, March 7, 2013 ( Bacillus aryabhattai CAP53, Bacillus aryabhattai CAP56, Bacillus flexus BT054, Paracoccus kondratievae NC35, Enterobacter cloacae CAP12 and Bacillus nealsonii BOBA57), March 10, 2014 ( Bacillus mycoides BT155, Bacillus mycoides EE118, Bacillus subtilis EE148, Alcaligenes faecalis EE107,

Bacillus mycoides EE141, Bacillus mycoides BT46-3, member of the family Bacillus cereus EE128, Bacillus thuringiensis BT013A, Paenibacillus massiliensis BT23, Bacillus cereus family member EE349, Bacillus subtilis EE218 and Bacillus megaterium EE281), or August 19, 2015 (member of the family Bacillus cereus -B00377; Bacillus pseudomycoides EE-B00366, Bacillus mycoides EE-B00363, Bacillus pumilus EE-B00143 or Bacillus thuringiensis EE-B00184), and they are identified by the NRRL numbers provided in parentheses.

[00628] These plant growth promoting strains were isolated from the rhizospheres of various vigorous plants and were identified by their 16S rRNA sequences and by biochemical assays. The strains have been identified at least to their generic designation using commonly accepted biochemical and morphological indicators. Biochemical analyzes for confirmed Gram-negative strains such as Paracoccus kondratievae, Alcaligenes faecalis and Enterobacter cloacae included growth analysis on MacConkey medium and nutrient agar, microscopic examination, growth analysis on medium with 5% and 7.5% NaCl, growth analysis at pH 5 and pH 9, analysis of growth at 42°C and 50°C, determination of the ability to produce acid when fermented with cellobiose, lactose, glycerol, glucose, sucrose, d-mannitol and starch; determination of fluorescent pigment production; determination of hydrolysis of gelatin; nitrate recovery analysis; determination of catalase production, starch hydrolysis; determination of oxidase reaction, urease production and mobility. Similarly, biochemical analyzes for confirmed Gram-positive strains such as Bacillus and Paenibacillus included growth on PEA and nutrient agar, microscopy, growth on 5% and 7.5% NaCl, growth at pH 5 and pH 9 , analysis of growth at 42°C and 50°C, determination of the ability to produce acid when fermented with cellobiose, lactose, glycerol, glucose, sucrose, d-mannitol and starch; determination of fluorescent pigment production; determination of hydrolysis of gelatin; nitrate recovery analysis; determination of catalase production, starch hydrolysis; determination of oxidase response, urease production and motility. The identification of these strains and the demonstration of their plant growth promoting effects are described in the examples below. Partial 16S rRNA sequences for Bacillus mycoides BT155, Bacillus mycoides EE118, Bacillus mycoides EE141, Bacillus mycoides BT46-3, Bacillus cereus EE128, Bacillus thuringiensis BT013A, and Bacillus cereus EE349 are shown in Table 12 above. Частичные последовательности 16S рРНК для штаммов Bacillus aryabhattai CAP53, Bacillus aryabhattai CAP56, Bacillus flexus BT054, Paracoccus kondratievae NC35, Enterobacter cloacae CAP12, Bacillus nealsonii BOBA57, Bacillus subtilis EE148, Alcaligenes faecalis EE107, Paenibacillus massiliensis BT23, Bacillus subtilis EE218 и Bacillus megaterium EE281 приведены in table 15 below.

Table 15. Partial 16S rRNA sequences for additional plant growth-promoting bacterial strains

Strain SEQ ID NO for partial sequence of 16S ribosomal RNA Bacillus aryabhattai CAP53 287 Bacillus aryabhattai CAP56 288 Bacillus flexus BT054 289 Paracoccus kondratievae NC35 290 Enterobacter cloacae CAP12 291 Bacillus nealsonii BOBA57 292 Bacillus subtilis EE148 293 Alcaligenes faecalis EE107 294 Paenibacillus massiliensis BT23 295 Bacillus subtilis EE218 296 Bacillus megaterium EE281 297

[00629] For example, the formulation may comprise a plant growth promoting bacterial strain comprising Paracoccus kondratievae NC35, Bacillus aryabhattai CAP53, or Bacillus megaterium EE281, wherein the formulation further comprises any of the recombinant members of the Bacillus cereus family described herein, including any recombinant plant growth promoting strains of members of the Bacillus cereus family described herein (for example, recombinant Bacillus mycoides BT155, Bacillus mycoides EE141 or Bacillus thuringiensis BT013A).

[00630] The fungal inoculum may include fungal inoculum of the Glomeraceae family, fungal inoculum of the Claroidoglomeraceae family, fungal inoculum of the Gigasporaceae family, fungal inoculum of the Acaulosporaceae family, fungal inoculum of the Sacculosporaceae family, fungal inoculum of the Entrophosporaceae family, fungal inoculum of the Pacidsporaceae family, fungal inoculum of the Diversisporaceae family, fungal inoculum of the family Paraglomeraceae, fungal inoculum of the Archaeosporaceae family, fungal inoculum of the Geosiphonaceae family, fungal inoculum of the Ambisporaceae family, fungal inoculum of the Scutellosporaceae family, fungal inoculum of the Dentiscultataceae family, fungal inoculum of the Racocetraceae family, fungal inoculum of the Basidiomycota division, fungal inoculum of the Ascomycota division, fungal inoculum of the Zygomycota division, or a combination thereof.

[00631] In addition, the spore-forming bacterium, alone or in combination with an insecticide, may contain an effective amount of at least one fungicide.

[00632] Exemplary fungicidal ingredients also include captan (N-trichloromethyl)thio-4-cyclohexane-1,2-dicarboximide), fludioxonium 1 (4-(2,2-difluoro-1,3-benzodioxol-4-yl)- 1-H-pyrrole-3-carbonitrile, iprodione carbendazim (commercially available under the tradename Rovral.RTM.), tebuconazole, thiabendazole, azoxystrobin, prochloraz and oxadixyl (N-(2,6-dimethylphenyl)-2-methoxy-N- (2-oxo-3-oxazolidinyl)acetamide).

[00633] If the formulation, plant seed, or inoculum contains a fungicide, the fungicide may include aldimorph, ampropylphos, ampropylphos potassium, andoprim, anilazine, azoconazole, azoxystrobin, benalaxyl, benodanil, benomyl, benzamacryl, benzamacryl-isobutyl, bialaphos, binapacryl, biphenyl, bitertanol , blasticidin-S, boscalid, bromuconazole, bupirimate, butiobate, calcium polysulfide, capsimycin, captafol, captan, carbendazim, carvone, quinomethionate, chlobentiazone, chlorphenazole, chloroneb, chlorpicrin, chlorothalonil, chlosolinate, closilacone, kufraneb, cymoxanil, cyproconazol, ciprofuram, debacarb, dichlorophen, diclobutrazol, diclofluanid, diclomesin, dicloran, dietofencarb, dimethirimol, dimethomorph, dimoxystrobin, diniconazole, diniconazole-M, dinocap, diphenylamine, dipyrithione, dithalymphos, dithianone, dodemorph, dodin, drazoxolone, edifenphos, epoxiconazole, etirimol, etridiasol, famoxadone, fenapanil, fenarimol, fenbuconazole, fenfuram, fenitropan, fenpiclonil, fenpropidine, fenpropimorph, fenty n acetate, fentin hydroxide, ferbam, ferimzone, fluazinam, flemetover, fluoromide, fluquinconazole, flurprimidol, flusilazole, flusulfamide, flutolanil, flutriafol, folpet, fosetyl-aluminum, fosetyl-sodium, phthalide, fuberidazole, furalaxil, furametpyr, furcarbonyl, furconazole-cis, furmecyclox, guazatin, hexachlorobenzene, hexaconazole, hymexazole, imazalil, imibenconazole, iminoctadine, iminoctadine albesilate, iminoctadine triacetate, iodocarb, iprobenfos (IBP), iprodione, irumamycin, isoprothiolane, isovaledione, kasugamycin, kresoxim-methyl, copper preparations, such as: copper hydroxide, copper naphthenate, copper oxychloride, copper sulfate, copper oxide, copper oxin and Bordeaux liquid, mancuprum, mancozeb, maneb, meferimzone, mepanipyrim, mepronil, metalaxyl, metconazole, metasulfocarb, metfuroxam, metiram, metomeclam, metsulfax , mildiomycin, myclobutanil, myclozolin, nickel dimethyldithiocarbamate, nitrothal-isopropyl, nuarimol, ofuras, oxadixyl, oxamocarb, oxolinic acid, oxycarboxy, ox ifentiin, paclobutrazol, pefurazoate, penconazole, pencycuron, fosdifen, pimaricin, piperalin, polyoxin, polyoxorim, probenazole, prochloraz, procymidone, propamocarb, sodium propanosine, propiconazole, propineb, prothiocinazole, pyrazophos, pyrifenox, pyrimethanil, pyroquilon, piroxifur, quinoconazole, quintozene (PCNB), sulfur and sulfur preparations, tebuconazole, tecloftalam, teknazen, tetcyclasis, tetraconazole, thiabendazole, thithiofen, thifluzamide, thiophanate-methyl, thioximide, tolclofos-methyl, tolylfluanid, triadimefon, triadimenol, triazbutyl, triazoxide, trichlamide, tricyclazole, tridemorph, trifloxystrobin, triflumizole, triforin, uniconazole, validamycin A, vincozolin, viniconazole, zarylamide, zineb, ziram, and Dagger G, OK-8705, OK-8801, a-(1,1-dimethylethyl)-(3-( 2-phenoxyethyl)-1H-1,2,4-triazole-1-ethanol, a-(2,4-dichlorophenyl)-[3-fluoro-3-propyl-1H-1,2,4-triazole-1- ethanol, a-(2,4-dichlorophenyl)-[3-methoxy-a-methyl-1H-1,2,4-triazole-1-ethanol, a-(5-methyl-1,3-dioxane-5- yl)-[3-[[4-(trifluoromethyl)phenyl]methylene]-1H-1,2, 4-triazol-1-ethanol, (5RS,6RS)-6-hydroxy-2,2,7,7-tetramethyl-5-(1 H-1,2,4-triazol-1-yl)-3-octanone , (E)-a-(methoxyimino)-N-methyl-2-phenoxyphenylacetamide, 1-isopropyl{2-methyl-1-[[[1-(4-methylphenyl)ethyl]amino]carbonyl]propyl}carbamate, 1 -(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone-O-(phenylmethyl)oxime, 1-(2-methyl-1-naphthalenyl)-1H-pyrrole -2,5-dione, 1-(3,5-dichlorophenyl)-3-(2-propenyl)-2,5-pyrrolidinedione, 1-[(diiodomethyl)sulfonyl]-4-methylbenzene, 1-[[2- (2,4-dichlorophenyl)-1,3-dioxolan-2-yl]methyl]-1H-imidazole, 1-[[2-(4-chlorophenyl)-3-phenyloxiranyl]methyl]-1H-1,2, 4-triazole, 1-[1-[2-[(2,4-dichlorophenyl)-methoxy]phenyl]ethenyl]-1H-imidazole, 1-methyl-5-nonyl-2-(phenylmethyl)-3-pyrrolidinol, 2',6'-dibromo-2-methyl-4'-trifluoromethoxy-4'-trifluoromethyl-1,3-thiazolecarboxanilide, 2,2-dichloro-N-[1-(4-chlorophenyl)ethyl]-1-ethyl -3-methylcyclopropanecarboxamide, 2,6-dichloro-5-(methylthio)-4-pyrimidinylthiocyanate, 2,6-dichloro-N-(4-trifluoromethylbenzyl)benzamide, 2,6-dichloro-N-[[4-(trifluoromethyl )phenyl]methyl]benzamide, 2-(2,3,3-triiodo-2-prop enyl)-2H-tetrazole, 2-[(1-methylethyl)-sulfonyl]-5-(trichloromethyl)-1,3,4-thiadiazole, 2-[[6-deoxy-4-O-(4-O- methyl-(3-D-glycopyranosyl)-a-D-glucopyranosyl]amino]-4-methoxy-1H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile, 2-aminobutane, 2-bromo-2-(bromomethyl) pentandinitrile, 2-chloro-N-(2,3-dihydro-1,1,3-trimethyl-1H-inden-4-yl)-3-pyridinecarboxamide, 2-chloro-N-(2,6-dimethylphenyl)- N-(isothiocyanatemethyl)acetamide, 2-phenylphenol (OPP), 3,4-dichloro-1-[4-(difluoromethoxy)phenyl]pyrrole-2,5-dione, 3,5-dichloro-N-[cyano[( 1-methyl-2-propynyl)oxy]methyl]benzamide, 3-(1,1-dimethylpropyl-1-oxo-1H-indene-2-carbonitrile, 3-[2-(4-chlorophenyl)-5-ethoxy- 3-isoxazolidinyl]pyridine, 4-chloro-2-cyano-N,N-dimethyl-5-(4-methylphenyl)-1H-imidazole-1-sulfonamide, 4-methyltetrazolo[1,5-a]quinazoline-5( 4H)-one, 8-(1,1-dimethylethyl)-N-ethyl-N-propyl-1,4-dioxaspiro[4,5]decan-2-methanamine, 8-hydroxyquinoline sulfate, 9H-xanthene-2- [(phenylamino)carbonyl]-9-carboxylic hydrazide, bis-(1-methylethyl)-3-methyl-4-[(3-methylbenzoyl)oxy]-2,5-thiophene dicarboxylate, cis-1-(4-chloroph enyl)-2-(1 H-1,2,4-triazol-1-yl)cycloheptanol, cis-4-[3-[4-(1,1-dimethylpropyl)phenyl-2-methylpropyl]-2,6 -dimethylmorpholine hydrochloride, ethyl-[(4-chlorophenyl)-azo]cyanoacetate, potassium bicarbonate, methanetetrathiol-sodium salt, methyl 1-(2,3-dihydro-2,2-dimethylinden-1-yl)-1H-imidazol- 5-carboxylate, methyl N-(2,6-dimethylphenyl)-N-(5-isoxazolylcarbonyl)-DL-alaninate, methyl-N-(chloroacetyl)-N-(2,6-dimethylphenyl)-DL-alaninate, N -(2,3-dichloro-4-hydroxyphenyl)-1-methylcyclohexanecarboxamide, N-(2,6-dimethylphenyl)-2-methoxy-N-(tetrahydro-2-oxo-3-furanyl)acetamide, N-(2 ,6-dimethylphenyl)-2-methoxy-N-(tetrahydro-2-oxo-3-thienyl)acetamide, N-(2-chloro-4-nitrophenyl)-4-methyl-3-nitrobenzenesulfonamide, N-(4- cyclohexylphenyl)-1,4,5,6-tetrahydro-2-pyrimidinamine, N-(4-hexylphenyl)-1,4,5,6-tetrahydro-2-pyrimidinamine, N-(5-chloro-2-methylphenyl) -2-methoxy-N-(2-oxo-3-oxazolidinyl)acetamide, N-(6-methoxy)-3-pyridinyl)cyclopropanecarboxamide, N-[2,2,2-trichloro-1-[(chloroacetyl)amino ]ethyl]benzamide, N-[3-chloro-4,5-bis(2-propynyloxy)phenyl]- N'-methoxymethanenimidamide, N-formyl-N-hydroxy-DL-alanine-sodium salt, O,O-diethyl [2-(dipropylamino)-2-oxoethyl]ethylphosphoramidothioate, O-methyl S-phenylphenylpropylphosphoramidothioate, S-methyl-1 ,2,3-benzothiadiazol-7-carbothioate and spiro[2H]-1-benzopyran-2,1'(3'H)-isobenzofuran]-3'-one, N-trichloromethyl)thio-4-cyclohexane-1, 2-dicarboximide, tetramethylthioperoxydicarboxylic diamide, methyl-N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate, 4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1 -H-pyrrole-3-carbonitrile or a combination thereof.

[00634] In addition, suitable fungicides include the following: (1) a compound capable of inhibiting nucleic acid synthesis such as benalaxyl, benalaxyl-M, bupirimate, chiralaxyl, closilacone, dimethirimol, etirimol, furalaxyl, hymexazole, metalaxyl, metalaxyl-M, ofuras, oxadixyl, oxolinic acid; (2) a compound capable of inhibiting mitosis and cell division such as benomyl, carbendazim, diethofencarb, ethaboxam, fuberidazole, pencycuron, thiabendazole thiophanate-methyl, zoxamide; (3) a compound capable of inhibiting respiration, for example a CI respiration inhibitor such as diflumetorim; a CII respiration inhibitor such as boscalid, fenfuram, flutolanil, furametpyr, furmecyclox, mepronil, oxycarboxin, penthiopyrad, thifluzamide; a CIII respiration inhibitor such as amisulbrom, azoxystrobin, cyazofamide, dimoxystrobin, enestrobin, famoxadone, fenamidone, fluoxastrobin, kresoxim-methyl, methominostrobin, orysastrobin, picoxystrobin, trifloxystrobin; (4) a compound capable of acting as an uncoupler such as dinocap, fluazinam, meptyldinocap; (5) a compound capable of inhibiting ATP production such as fentin acetate, fentin chloride, fentin hydroxide; (6) a compound capable of inhibiting aa biosynthesis. and proteins such as andoprim, blasticidin-S, cyprodinil, kasugamycin, kasugamycin hydrochloride hydrate, mepanipyrim, pyrimethanil; (7) a compound capable of inhibiting signal transmission such as fenpiclonil, quinoxyfen; (8) a compound capable of inhibiting lipid and membrane synthesis such as biphenyl, closolinate, edifenphos, etridiasol, iodocarb, iprobenfos, iprodione, isoprothiolane, procymidone, propamocarb, propamocarb hydrochloride, pyrazophos, tolclofos-methyl, vinclozolin; (9) a compound capable of inhibiting ergosterol biosynthesis such as aldimorph, azaconazole, bitertanol, bromuconazole, cyproconazole, diclobutrazol, diniconazole, diniconazole-M, dodemorph, dodemorph acetate, epoxiconazole, etaconazole, fenarimol, fenbuconazole, fenhexamide, fenpropidine, fenpropimorph, fluquinconazole , flurprimidol, flusilazole, flutriafol, furconazole, furconazole-cis, hexaconazole, imazalil, imazalyl sulfate, imibenconazole, metconazole, myclobutanil, naftifine, nuarimol, oxpoconazole, paclobutrazol, pefurazoate, penconazole, prochloraz, propiconazole, propiconazole, spiroxamine, tebuconazole, terbinafine, tetraconazole, triadimefon, triadimenol, tridemorph, triflumizole, triforin, uniconazole, vinconazole, voriconazole; (10) a compound capable of inhibiting cell wall synthesis such as benthiavalicarb, bialaphos, dimethomorph, flumorph, iprovalicarb, mandipropamide, polyoxins, polyoxorim, validamycin A; (11) a compound capable of inhibiting melanin biosynthesis such as carpropamide, diclocimet, fenoxanil, phthalide, pyroquilon, tricyclazole; (12) a compound capable of inducing host defense such as acibenzolar-S-methyl, probenazole, thiadinil; (13) compound capable of having multiple action such as Bordeaux mixture, captafol, captan, chlorothalonil, copper naphthenate, copper oxide, copper oxychloride, copper preparations such as copper hydroxide, copper sulfate, dichlofluanid, dithianone, dodin, dodin free base , ferbam, ftorofolpet, folpet, guazatin, guazatin acetate, iminoctadine, iminoctadine abesilate, iminoctadine triacetate, mancuprum, mancozeb, maneb, metiram, metiram zinc, oxin-copper, propineb, sulfur and sulfur preparations, including calcium polysulfide, tolylfluanid, zineb, ziram; (14) a compound selected from the following list: (2E)-2-(2-{[6-(3-chloro-2-methylphenoxy)-5-fluoropyrimidin-4-yl]oxy}phenyl)-2-(methoxyimino )-N-methylacetamide, (2E)-2-{2-[({[(1E)-1-(3-{[(E)-1-fluoro-2-phenylvinyl]oxy}phenyl)ethylidene]amino} hydroxy)methyl]phenyl}-2-(methoxyimino)-N-methylacetamide, 1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)cycloheptanol, 1-[(4- methoxyphenoxy)methyl]-2,2-dimethylpropyl-1H-imidazole-1-carboxylate, 2,3,5,6-tetrachloro-4-(methylsulfonyl)pyridine, 2-butoxy-6-iodo-3-propyl-4H- chromen-4-one, 2-chloro-N-(1,1,3-trimethyl-2,3-dihydro-1H-inden-4-yl)nicotinamide, 2-phenylphenol and salts, 3,4,5-trichloropyridine -2,6-dicarbonitrile, 3,4-dichloro-N-(2-cyanophenyl)isothiazole-5-carboxamide, 3-[5-(4-chlorophenyl)-2,3-dimethylisoxazolidin-3-yl]pyridine, 5 -chloro-6-(2,4,6-trifluorophenyl)-N-[(1R)-1,2,2-trimethylpropyl][1,2,4]triazolo[1,5-a]pyrimidine-7-amine , 5-chloro-7-(4-methylpiperidin-1-yl)-6-(2,4,6-trifluorophenyl)[1,2,4]triazolo[1,5-a]pyrimidine, 5-chloro-N -[(1R)-1,2-dimethylpropyl]-6-(2,4,6-trifluorophenyl)[1,2,4]triazolo[1,5-a]pi rimidine-7-amine, 8-hydroxyquinoline sulfate, benthiazole, betoxazine, capsimycin, carvone, quinomethionate, kufraneb, cyflufenamid, cymoxanil, dazomet, debacarb, dichlorophen, diclomesin, dicloran, difenzoquat, diphenzoquat methyl sulfate, diphenylamine, ferimzone, flupimetholover, fluorimide, flusulfamide, fosetyl-aluminum, fosetyl-calcium, fosetyl-sodium, hexachlorobenzene, irumamycin, isothianil, metasulfocarb, methyl-(2E)-2-{2-[({cyclopropyl[(4-methoxyphenyl)imino]methyl}thio )methyl]phenyl}-3-methoxyacrylate, methyl 1-(2,2-dimethyl-2,3-dihydro-1H-inden-1-yl)-1H-imidazole-5-carboxylate, methylisothiocyanate, metrafenone, mildiomycin, N -[2-(1,3-dimethylbutyl)phenyl]-5-fluoro-1,3-dimethyl-1H-pyrazole-4-carboxamide, N-(3',4'-dichloro-5-fluorobiphenyl-2-yl )-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide, N-(3-ethyl-3,5,5-trimethylcyclohexyl)-3-(formylamino)-2-hydroxybenzamide, N-(4 -chloro-2-nitrophenyl)-N-ethyl-4-methylbenzenesulfonamide, N-(4-chlorobenzyl)-3-[3-methoxy-4-(prop-2-in-1-yloxy)phenyl]propanamide, N- [(4-chlorophenyl)(c iano)methyl]-3-[3-methoxy-4-(prop-2-in-1-yloxy)phenyl]propanamide, N-[(5-bromo-3-chloropyridin-2-yl)methyl]-2, 4-dichloronicotinamide, N-[1-(5-bromo-3-chloropyridin-2-yl)ethyl]-2,4-dichloronicotinamide, N-[1-(5-bromo-3-chloropyridin-2-yl)ethyl ]-2-fluoro-4-iodonicotinamide, N-[2-(4-{[3-(4-chlorophenyl)prop-2-yn-1-yl]oxy}-3-methoxyphenyl)ethyl]-N&-lt ;-(methylsulfonyl)valinamide, N-{(Z)-[(cyclopropylmethoxy)imino][6-(difluoromethoxy)-2,3-difluorophenyl]methyl}-2-phenylacetamide, N-{2-[1,1' -bi(cyclopropyl)-2-yl]phenyl}-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide, N-{2-[3-chloro-5-(trifluoromethyl)pyridine-2- yl]ethyl}-2-(trifluoromethyl)benzamide, natamycin, N-ethyl-N-methyl-N'-{2-methyl-5-(trifluoromethyl)-4-[3-(trimethylsilyl)propoxy]phenyl}imidoformamide, N-ethyl-N-methyl-N'-{2-methyl-5-(difluoromethyl)-4-[3-(trimethylsilyl)propoxy]phenyl}imidoformamide, nickel dimethyldithiocarbamate, nitrothal-isopropyl, O-{1-[( 4-methoxyphenoxy)methyl]-2,2-dimethylpropyl}1H-imidazole-1-carbothioate, octilinone, oxamocarb, oxyfentiine, pentachlorophenol and salts, phosphoric acid acid and its salts, piperalin, propamocarb fosetilate, propanosine sodium, proquinazid, pyribencarb, pyrrolnitrin, quintosine, tecloftalam, teknazine, triazoxide, trichlamide, valifenal, zarylamide.

[00635] The fungicide may include substituted benzene, thiocarbamate, ethylene bis-dithiocarbamate, thiophthalidamide, copper compound, organomercury compound, organotin compound, cadmium compound, anilazine, benomyl, cyclohexamide, dodin, etridiazaol, iprodione, metlaxyl, thiamimefon, triforin, or a combination thereof .

[00636] If the formulation, plant seed, or inoculum contains a fungicide, the fungicide may be a foliar fungicide. Foliar fungicide includes copper, mancozeb, penthiopyrad, triazoles, cyproconazole, metconazole, propycnasol, prothioconazole, tebuconazole, azoxystrobin, pyraclastobin, fluoxastrobin, picoxystrobin, trifloxystrobin, sulfur, boscalid, thiophanate methyl, chlorotanonil, penthiopyrad, difenconazole, fluprozinol, cyprodinamol, iprodione, penflufen, cyazofamide, flutolanil, cymoxanil, dimethomorph, pyrimethanil, zoxamide, mandipropamide, metrinam, propamocarb, fenamidone, tetraconazole, chloronab, gimexazole, tolclofos, and fenbuconazole.

[00637] If the composition, plant seed, or inoculum comprises a bacterial inoculum of the genus Bacillus , the bacterial inoculum may include Bacillus argri, Bacillus aizawai, Bacillus albolactis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus coagulans, Bacillus endoparasiticus, Bacillus endorhythmos, Bacillus kurstaki, Bacillus lacticola, Bacillus lactimorbus Bacillus lactis Bacillus laterosporus Bacillus lentimorbus Bacillus licheniformis Bacillus megaterium Bacillus medusa Bacillus metiens Bacillus natto Bacillus nigrificans Bacillus popillae Bacillus pumilus Bacillus siamensis thuringiensis, Bacillus unifagellatu, or a combination thereof, plus the inoculums listed under Bacillus Genus in Bergey's Manual of Systematic Bacteriology, First Ed. (1986), incorporated herein by reference to a greater extent.

[00638] If the formulation, plant seed, or inoculum contains an insecticide, the insecticide may be a nematicide. Suitable nematicides include antibiotic nematicides such as abamectin; carbamate nematicides such as acetoprol, Bacillus chitonosporus , chloropicrin, benclothiaz, benomyl, Burholderia cepacia , carbofuran, carbosulfan, and cleotocard; dazomet, DBCP, DCIP, alanicarb, aldicarb, aldoxycarb, oxamyl, diamidafos, fenamiphos, fostyethane, phosphmidone, cadusafos, chlorpyrifos, diclofenthion, dimethoate, etoprofos, fensuolfothion, fostiazate, harpines, heterophos, imicyafos, isamidophos, isazophos, methomil, Myrothecium verrucaria, Paecilomyces lilacinus , phorate, phosphocarb, terbufos, thionazine, thiazophos, dazomet, 1,2-dicloropropane, 1,3-dichloropropene, furfaral, iodomethane, metam, methyl bromide, methyl isothiocyanate, and xylenols.

[00639] For example, but not limited to, the nematicide and insecticide can be provided in the form of the commercial product Avicta Duo, which is a mixture of abamectin and thiamethoxam commercially available from Syngenta.

[00640] If the formulation, plant seed, or inoculum contains a bactericide, it may include streptomycin, penicillins, tetracyclines, ampicillin, and oxolinic acid.

[00641] The fertilizer may include a liquid fertilizer. The microfertilizer material may contain boric acid, borate, boric frit, copper sulfate, copper frit, copper chelate, sodium tetraborate decahydrate, ferrous sulfate, iron oxide, ammonium ferrous sulfate, iron frit, iron chelate, manganese sulfate, manganese oxide, manganese chelate, manganese chloride, manganese frit, sodium molybdate, molybdic acid, zinc sulfate, zinc oxide, zinc carbonate, zinc frit, zinc phosphate, zinc chelate, or a combination thereof.

[00642] The fertilizer may contain 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, calcium ammonium nitrate, calcium sulfate, calcined magnesite, calcined limestone, calcium oxide, calcium nitrate, dolomitic limestone, slaked lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, magnesium potassium sulfate, potassium sulfate, sodium nitrates, magnesian limestone, magnesia, urea, urea-formaldehydes, ammonium urea nitrate, sulfur-coated urea, polymer-coated urea, isobutylidene diurea, K ₂ SO ₄ -2MgSO ₄ , kainite, sylvinite, kieserite, epsom salt, elemental sulphur, lime clay, powdered shells oysters, fish meal, cake, fish current, blood meal, phosphate ore, superphosphates, slag, bone meal, wood ash, manure, bat guano, peat moss, compost, glauconite sand, cotton seed meal, feather meal, crab meal, fish emulsion, humic acid, or a combination thereof.

[00643] The composition, plant seed or inoculum may also include at least one biological control agent selected from (1) bacteria, in particular spore-forming bacteria, (2) fungi or yeasts, and (3) isoflavones. Preferred are combinations containing as a biological control agent, in particular, a spore-forming root colonizing bacterium or a bacterium suitable as a 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) Bacillus 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 lactis , (1.14) Bacillus laterosporus , (1.15) Bacillus lentimorbus , (1.16) Bacillus licheniformis , (1.17) Bacillus medusa , (1.18) Bacillus megaterium , (1.19) Bacillus metiens , (1.20) Bacillus natto , (1.21) Bacillus nigrificans , (1.22) Bacillus popillae , (1.23) Bacillus pumilus , (1.24) Bacillus siamensis , (1.25) Bacillus sphaericus (products known as VectoLex.sup.S), ( 1.26) Bacillus subtilis or B subtilis variant amyloliquefaciens , (1.27) Bacillus thuringiensis , in particular B. thuringiensis variant israelensis (products known as VectoBac.RTM.) or B. thuringiensis subsp . aizawai strain ABTS-1857 (products known as XenTari) or B. thuringiensis subsp . kurstaki strain HD-1 (products known as Dipel ES), (1.28) Bacillus unflagellatus , (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, 288-296), (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., see Crop Protection 2006, 25, 468-475), (1.35 ) Streptomyces griseoviridis (products known as Mycostop.RTM.).

[00644] In addition, combinations containing, as a biological control agent, a fungus or yeast selected from the group consisting of [group (2)]: (2.1) Ampelomyces quisqualis , in particular strain AQ 10 (a product known as AQ 10.RTM.), (2.2) Aureobasidium pullulans , 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 particular strain ATCC 74040 (products known as Naturalis.RTM.), (2.4) Candida oleophila , in particular strain O (products known as Nexy), (2.5) Cladosporium cladosporioides H39 (see Eur. J. Plant Pathol. 2009, 123 , 401-414), (2.6), (2.7) Dilophosphora alopecuri (products known as Twist Fungus), (2.8) Gliocladium catenulatum , in particular strain J1446 (products known as Prestop), (2.9) Lecanicillium lecanii (formerly known as Verticillium lecanii ), in particular the conidia of strain KV01 (products known to aka Mycotal.RTM., Vertalec.RTM.), (2.10) Metarhizium anisopliea (products known as BIO 1020), (2.11) Metschnikovia fructicola , in particular strain NRRL Y-30752 (products known as Shemer.TM.) , (2.12) Microsphaeropsis ochracea (products known as Microx), (2.13), (2.14) Nomuraea rileyi , (2.15), (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, for example, as Trichodex) .

[00645] Also preferred are combinations comprising, as a 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) coumarin, (3.10) ambiol-(2-methyl-4-dimethylaminomethyl-5-hydroxybenzimidazole dihydrochloride), (3.11) ascorbate and (3.12) pratensein and their salts and esters.

[00646] If the formulation, plant seed, or inoculum contains an insecticide, the insecticide may include pyrethroids, organophosphates, caramoyl oximes, pyrazoles, amidines, halogenated hydrocarbons, neonicotinoids, and carbamates and their derivatives. In particular, suitable classes of insecticides include organophosphates, phenylpyrazoles, and pyrethroids. Preferred insecticides are those known as terbufos, chlorpyrifos, chloroxyphos, tefluthrin, carbofuran and tebupyrimphos. Commercially available insecticides include thiomethoxam (commercially available from Syngenta under the brand name Cruiser).

[00647] The insecticide may include an organophosphorus compound, a carbamate, a pyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate, fluoride, sulfur, a haloaromatic substituted urea, a hydrocarbon ester, a biobased insecticide, or a combination thereof.

[00648] Suitable insecticides for use in the present invention include the following: (1) acetylcholine receptor agonists/antagonists such as chloronicotinyls/neonicotinoids, nicotine, bensultap or cartap. Suitable examples of chloronicotinyls/neonicotinoids include acetamiprid, dinotefuran, nitenpyram, nithiazine, thiacloprid, thiamethoxam, imidaclotiz and (2E)-1-[(2-chloro-1,3-thiazol-5-yl)methyl]-3,5-dimethyl -N-nitro-1,3,5-triazinan-2-imine; (2) acetylcholinesterase (ACNE) inhibitors such as carbamates and organophosphates. Suitable examples of carbamates include alanicarb, aldicarb, aldoxycarb, allixicarb, aminocarb, bendiocarb, benfuracarb, bufencarb, butacarb, butocarboxym, butoxycarboxym, carbaryl, carbofuran, carbosulfan, chloetocarb, dimethylfuran, ethiofencarb, fenobucarb, fenotiocarb, isomthiocarb, formetanate sodium, methomyl, metolcarb, oxamyl, phosphocarb, pirimicarb, promecarb, propoxur, thiophanox, triazamate, trimetacarb, XMC, and xylicarb. Suitable examples of organophosphorus compounds include acephate, azamethifos, azinphos (-methyl, -ethyl), bromophos-ethyl, bromfenvinphos (-methyl), butathiophos, cadusafos, carbophenotion, chlorethoxyphos, chlorphenvinphos, chlormephos, chlorpyrifos (-methyl/-ethyl), coumaphos , cyanofenphos, cyanophos, demeton-S-methyl, demeton-S-methylsulfone, dialyphos, diazinon, dichlorfenthion, dichlorphos/DDVP, dicrotophos, dimethoate, dimethylvinphos, dioxabenzophos, disulfotone, EPN, ethion, etoprofos, etrymphos, famfur, fenamiphos, fenitrothion , fensulfothion, fenthion, flupyrazophos, fonofos, formotion, fosmethylan, fostiazate, heptenophos, fodofenfos, iprobenfos, issophos, isofenphos, isopropyl-O-salicylate, isoxathion, malathion, mecarbam, metakryphos, methamidophos, metidathione, mevinphos, monocrotophos, naled, omethoate , oxydemeton-methyl, parathion (-methyl/-ethyl), phentoate, phorate, fosalone, phosmet, phosphamidon, phosphocarb, phoxim, pyrimphos (-methyl/-ethyl), profenophos, propafos, propetamphos, prothiophos, protoate, pyraclophos, pyridafenthion , pyridation, quinalphos, sebufos, sulfotep, sulprofos, tebupyrimphos, temephos, terbufos, tetrachlorvinphos, thiometon, triazophos, trichlorfon, and vamidothion; (3) sodium channel modulators/voltage-gated sodium channel blockers such as pyrethroids and oxadiazines. Suitable examples of pyrethroids include acrinathrin, allethrin (d-cis-trans, d-trans), beta-cyfluthrin, bifenthrin, bioallethrin, bioallethrin-S-cyclopentyl isomer, bioethanomethrin, biopermethrin, bioresmethrin, chlovaportrin, cis-resmethrin, cis-permethrin , clocitrin, cycloprothrin, cyfluthrin, cyhalothrin, cyfenotrin, DDT, deltamethrin, empentrin (1R-isomer), esfenvalerate, etofenprox, fenfluthrin, fenpropatrin, fenpyrythrin, fenvalerate, flubrocitrinate, flucitrinate, flufenprox, flumethrin, fluvalinate, fubfenprox, gamma-cyhalothrin, imiprothrin, cadetrin, lambda-cyhalothrin, metofluthrin, permethrin (cis-, trans-), phenothrin (1R-trans-isomer), pralletrin, profluthrin, protrifenbut, pyresmethrin, resmetrin, RU 15525, silafluofen, tau-fluvalinate, tefluthrin, teralletrin , tetramethrin (1R-isomer), tralocitrin, tralometrin, transfluthrin, ZXI 8901 and pyrethrins (feverfew). Suitable examples of oxadiazines include indoxacarb; (4) acetylcholine receptor modulators such as spinosyns. Suitable examples of spinosyns include spinosad; (5) GABA-dependent chloride channel antagonists such as cyclodiene organochlorines and fiprols. Suitable examples of cyclodiene organochlorines include camphechlor, chlordane, endosulfan, gamma-HCH, HCH, heptachlor, lindane, and methoxychlor. Suitable examples of fiprols include acetoprol and vaniliprol; (6) chloride channel activators such as mectins. Suitable examples of mectins include abamectin, avermectin, emamectin, emamectin benzoate, ivermectin, lepimectin, milbemectin and milbemycin; (7) juvenile hormone mimetics such as diofenolan, epofennonan, fenoxycarb, hydroprene, kinoprene, methoprene, pyriproxyfen, triprene; (8) ecdysone agonists/decomposers such as diacylhydrazines. Suitable examples of diacylhydrazines include chromafenoside, halofenoside, methoxyfenoside, and tebufenoside; (9) chitin biosynthesis inhibitors such as benzoylureas, buprofezin and cyromazine. Suitable examples of benzoylureas include bistrifluoron, chlofluazuron, diflubenzuron, fluazuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, noviflumuron, penfluron, teflubenzuron and triflumuron; (10) oxidative phosphorylation inhibitors, ATP degraders such as organotin compounds and diafenthiuron. Suitable examples of organotin compounds include azocyclotin, cyhexatin and fenbutatin oxide; (11) agents that uncouple oxidative phosphorylation by disrupting the H proton gradient, such as pyrroles and dinitrophenols. A suitable example of pyrroles includes chlorfenapyr. Suitable examples of dinitrophenols include binapacryl, dinobuton, dinocap and DNOC; (12) Site I electron transport inhibitors such as METI, hydramethylnon and dicofol. Suitable examples of METI include phenazquin, fenpyroximate, pyrimidifen, pyridaben, tebufenpyrad, tolfenpyrad; (13) site II electrode transport inhibitors such as rotenone; (14) site III electron transport inhibitors such as acequinocil and fluacripyrim; (15) microbial degraders of the intestinal lining of insects, such as strains of Bacillus thuringiensis ; (16) lipid synthesis inhibitors such as tetronic acids and tetramic acids. Suitable examples of tetronic acids include spirodiclofen, spiromesifen and spirotetramat. A suitable example of tetramic acids includes cis-3-(2,5-dimethylphenyl)-8-methoxy-2-oxo-1-azaspiro[4.5]dec-3-en-4-ylethyl carbonate (aka: 3-(2.5 -dimethylphenyl)-8-methoxy-2-oxo-1-azaspiro[4.5]dec-3-en-4-yl-ethyl carbonic acid ester (CAS Registry No. 382608-10-8) (17) carboxamides such as flonicamid; (18) octopaminergic agonists such as amitraz (19) magnesium-stimulated ATPase inhibitors such as propargitis (20) ryanodine receptor agonists such as phthalamides and rinaxapir A suitable example of phthalamides includes N ² -[1,1- dimethyl-2-(methylsulfonyl)ethyl]-3-iodo-N ¹ -[2-methyl-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]phenyl]-1,2-benzenedicarboxamide (i.e. flubendiamide, CAS Accession No. 272451-65-7), (21) non-reistoxin analogs such as thiocyclam hydrogen oxalate and sodium thiosultap, (22) biological agents, hormones or pheromones such as azadiractin, Bacillus spec. , Beauveria spec., Caudlemont, Metarrhizium spec., Paecilomyces spec., thuringiensis and Vertic illium spec.; (23) active compounds having unknown or unspecified mechanisms of action, such as fumigants, selective feeding inhibitors, mite growth inhibitors, amidoflumet; benclothiaz, benzoximate, biphenazate, bromopropylate, buprofezin, quinomethioate, chlordimeform, chlorbenzilate, chlorpicrin, clothiazoben, cycloprene, cyflumethophen, dicyclanil, phenoxacrim, fentrifanil, flubenzimine, flufenerim, flutensin, gossyplur, hydramethylonone, japonylurine, potassium oil derivatives , pyrafluprol, pyridalil, pyriprole, sulfuramide, tetradifon, tetrasul, triaraten, verbutin, moreover, 3-methylphenylpropylcarbamate compound (Tsumacide Z), 3-(5-chloro-3-pyridinyl)-8-(2,2,2 -trifluoroethyl)-8-azabicyclo[3.2.1]octane-3-carbonitrile (CAS Registry No. 185982-80-3) and the corresponding 3-endo isomer (CAS Registry No. 185984-60-5) (see WO 96/ 37494, WO 98/25923), as well as preparations containing effective plant extracts, nematodes, fungi or viruses. Suitable examples of fumigants include aluminum phosphide, methyl bromide and sulfuryl fluoride. Suitable examples of selective feeding inhibitors include cryolite, flonicamid and pymetrozine. Suitable examples of mite growth inhibitors include clofentezin, ethoxazole, and hexythiazox.

[00649] Commercially available nematicidal ingredients include abamectin (commercially available from Syngenta under the tradename Avicta).

[00650] If the formulation, plant seed, or inoculum contains a herbicide, the herbicide may include 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametrin, atrazine, aminopyralid, benefin, bensulfuron, bensulide, bentazone, bromacyl, bromxinil, butylate, carfentrazone, chlorimuron, chlorsulfuron, clethodim, clomazone, clopiralid, cloransulam, cycloate, DCPA, desmedifam, dicamba, dichlobenil, diclofop, diclosulam, diflufenzopyr, dimethenamid, diquat, diuron, DSMA, endothal, EPTC, ethalfluralin, etofumesate, fenoxaprop, fluazifop-P, flucarbazone, flufanecet, flumetsulam, flumiclorac, flumioxazine, fluometuron, fluroxypyr, fomesafen, foramsulfuron, glufosinate, glyphosate, halosulfuron, hexazinon, imazamethabenz, imazamox, imazapic, imazaquin, lactofen, isoxaflu, MCPA, MCPB, mesotrione, metolachlor-s, metribuzin, metsulfuron, molinate, MSMA, napropamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxydiazone, oxyfluorfen, paraquat, pelargonic acid, pendimethalin, fe nmedipham, picloram, primisulfuron, prodiamine, promethrin, pronamide, propanil, prosulfuron, pyrazon, pyrithiobac, quinclorac, quizalofop, rimsulfuron, sethoxydim, siduron, simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, tebacil, thiazopyr, thifenesulfuron, thiobenesulfuron, thiobensulfuron triallat, triasulfuron, tribenuron, triclopyr, trifluralin, triflusulfuron, or a combination thereof.

[00651] The herbicide may include chlorophenoxy compound, nitrophenol compound, nitrocresol compound, dipyridyl compound, acetamide, aliphatic acid, anilide, benzamide, benzoic acid, benzoic acid derivative, anisic acid, anisic acid derivative, benzonitrile, benzothiadiazinon dioxide, thiocarbamate, carbamate, carbanylate , chlorpyridinyl, cyclohexenone derivative, dinitroaminobenzene derivative, fluoronitrotoluidine compound, isoxazolidinone, nicotinic acid, isopropylamine, isopropylamine derivative, oxadiazolinone, phosphate, phthalate, picolinic acid compound, triazine, triazole, uracil, urea derivative, endotall, sodium chlorate, sulfonylurea, or aryltriazine their combination.

[00652] The composition may contain a herbicide and a bacterial strain that is capable of degrading the herbicide.

[00653] The bacterial strain that is capable of degrading the herbicide may include Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus EE-B00377 (NRRL B-67119), Bacillus pseudomycoides EE-B00366 (NRRL B-67120) ) or Bacillus mycoides EE-B00363 (NRRL B-67121) or a combination thereof.

[00654] The herbicide to be degraded may include a sulfonylurea such as sulfentrazone, aryl triazine, dicamba, phenoxy herbicide, 2,4-D, pyrethrin, pyrethroid, or a combination thereof.

[00655] The formulations may include binders such as carboxymethyl cellulose and natural and synthetic polymers in the form of powders, granules or latexes such as gum arabic, chitin, polyvinyl alcohol and polyvinyl acetate, as well as natural phospholipids such as cephalins and lecithins, and synthetic phospholipids. Binders include binders consisting preferably of an adhesive polymer, which may be natural or synthetic, without phytotoxic effect on the seed to be coated. Additional binders that may be included, either alone or in combination, include, for example, polyethers, polyester esters, polyanhydrides, polyetherurethanes, polyesteramides; polyvinyl acetates; polyvinyl acetate copolymers; polyvinyl alcohols and tylose; copolymers of polyvinyl alcohols; polyvinylpyrrolidones; polysaccharides, including starches, modified starches and starch derivatives, dextrins, maltodextrins, alginates, chitosans and celluloses, cellulose esters, cellulose ethers, and cellulose ether esters, including ethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, and carboxymethylcellulose; fats; oils; proteins, including casein, gelatin, and zeins; gum arabic; shellacs; vinylidene chloride and vinylidene chloride copolymers; lignosulfonates, in particular calcium lignosulfonates; polyacrylates, polymethacrylates and acrylic copolymers; polyvinyl acrylates; polyethylene oxide; polybutenes, polyisobutenes, polystyrene, polybutadiene, polyethyleneamines, polyethyleneamides; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers and polychloroprene.

[00656] Various dyes can be used, including organic chromophores subdivided into nitroso, nitro, azo, including monoazo, biazo and polyazo, diphenylmethane, triarylmethane, xanthene, methane, acridine, thiazole, thiazine, indamine, indophenol, azine, oxazine , anthraquinone and phthalocyanine.

[00657] Other additives that can be added include micronutrients such as iron, manganese, boron, copper, cobalt, molybdenum, and zinc salts.

[00658] One or more preservatives (eg, antimicrobial agents or other biocidal agents) may also be included to preserve and stabilize the composition. Examples of suitable germicides include dichlorophene and benzyl alcohol hemiformal germicides (Proxel® from ICI or Acticide® RS from Thor Chemie and Kathon® MK from Dow Chemical) and isothiazolinone derivatives such as alkylisothiazolinones and benzisothiazolinones (Acticide® MBS from Thor Chemie ). As further examples, suitable preservatives include MIT (2-methyl-4-isothiazolin-3-one), BIT (l,2-benzisothiazolin-3-one, which can be purchased from Avecia, Inc. as Proxel GXL as solution in sodium hydroxide and dipropylene glycol), 5-chloro-2-(4-chlorobenzyl)-3(2H)-isothiazolone, 5-chloro-2-methyl-2H-isothiazol-3-one, 5-chloro-2-methyl -2H-isothiazol-3-one, 5-chloro-2-methyl-2H-isothiazol-3-one hydrochloride, 4,5-dichloro-2-cyclohexyl-4-isothiazolin-3-one, 4,5-dichloro- 2-octyl-2H-isothiazol-3-one, 2-methyl-2H-isothiazol-3-one, 2-methyl-2H-isothiazol-3-one-calcium chloride complex, 2-octyl-2H-isothiazol-3- he and benzyl alcohol hemiformal.

[00659] Examples of suitable formulation thickeners include polysaccharides, organic clays, or a water-soluble polymer that exhibits pseudoplastic properties in an aqueous environment, such as, for example, gum arabic, karaya gum, gum tragacanth, guar gum, locust bean gum, xanthan gum, carrageenan, alginate salt, casein, dextran, pectin, agar, 2-hydroxyethyl starch, 2-aminoethyl starch, 2-hydroxyethylcellulose, methylcellulose, carboxymethylcellulose salt, cellulose sulfate salt, polyacrylamide, alkali metal salts of maleic anhydride copolmers, alkali metal salts poly(meth) acrylate.

[00660] Suitable antifreeze ingredients for the formulation include, for example, but not limited to, ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1 ,4-pentanediol, 3-methyl-l,5-pentanediol, 2,3-dimethyl-2,3-butanediol, trimethylolpropane, mannitol, sorbitol, glycerol, pentaerythritol, 1,4-cyclohexanedimethanol, xylenol, bisphenols such as bisphenol A or similar. In addition, ether alcohols such as diethylene glycol, triethylene glycol, tetraethylene glycol, polyoxyethylene or polyoxypropylene glycols with a molecular weight up to 4000, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, triethylene glycol monomethyl ether, butoxyethanol, butylene glycol monobutyl ether, dipentaerythritol, tripentaerythritol, tetrapentaerythritol, diglycerin , triglycerol, tetraglycerol, pentaglycerol, hexaglycerol, heptaglycerol, octaglycerol, and combinations thereof.

XVIII. Plants' seeds

[00661] In addition, the present invention relates to plant seeds coated with any of the recombinant members of the Bacillus cereus family described in the present description, any of the recombinant spore-forming bacteria described in the present description, any of the biologically pure bacterial cultures described in the present description, any of the inoculums described herein, any enzyme that catalyzes nitric oxide production, any recombinant microorganism that expresses an enzyme that catalyzes nitric oxide production, or any of the formulations other than vaccines as described herein.

XIX. Methods relating to plants and plant seeds, methods of slowing down the germination of spores of a recombinant member of the family Bacillus cereus and methods for obtaining and using exosporium fragments

[00662] In addition, the present invention relates to methods of promoting plant growth, methods of protecting a plant from a pathogen or increasing plant resistance to stress, methods of immobilizing spores of a recombinant member of the Bacillus cereus family or recombinant spore-forming bacteria on a plant, methods of stimulating the germination of a plant seed, methods delivery of nucleic acids to plants, methods for slowing the germination of spores of a recombinant member of the Bacillus cereus family, methods for obtaining and using exospore fragments, and methods for delivering beneficial bacteria to animals.

A. Methods for promoting plant growth

[00663] The present invention relates to methods for promoting plant growth.

[00664] One method of promoting plant growth of the present invention comprises adding to the plant growth medium any of the recombinant Bacillus cereus described above, or any of the formulations containing the recombinant Bacillus cereus described above. Alternatively, any of the recombinant members of the Bacillus cereus family described above, or any of the formulations containing the recombinant member of the Bacillus cereus family described above, may be applied to the plant, plant seed, or area surrounding the plant or plant seed. In such methods, a recombinant member of the Bacillus cereus family expresses a fusion protein containing a plant growth promoting protein or peptide. The plant growth promoting protein or peptide may be physically associated with the exospore of a recombinant member of the Bacillus cereus family.

[00665] Another method of promoting plant growth includes adding to the plant growth medium any of the recombinant spore-forming bacteria described above, or any of the formulations containing the recombinant spore-forming bacteria described above. Alternatively, any of the recombinant spore-forming bacteria described above, or any of the formulations containing the recombinant spore-forming bacterium described above, can be applied to the plant, plant seed, or the area surrounding the plant or plant seed. In such methods, the recombinant spore-forming bacterium expresses a fusion protein containing a plant growth promoting protein or peptide. The plant growth-promoting protein or peptide may be physically associated with the spore coat of the recombinant spore-forming bacterium.

[00666] Another method of promoting plant growth includes adding a recombinant member of the Bacillus cereus family or a formulation containing a recombinant member of the Bacillus cereus family to the plant growth medium. Alternatively, the recombinant member of the Bacillus cereus family or composition can be applied to the plant, plant seed, or the area surrounding the plant or plant seed. Recombinant member of the Bacillus cereus family expresses an enzyme involved in nutrient solubilization, a protease, BclA protein, BclB protein, CotE protein, CotO protein, ExsY protein, ExsFA/BxpB protein, CotY protein, ExsFB protein, ExsJ protein, ExsH protein, YjcA protein , YjcB protein, BclC protein, BxpA protein, BclE protein, BetA/BAS3290 protein, ExsA protein, ExsK protein, ExsB protein, YabG protein, or Tgl protein, where expression of an enzyme involved in nutrient solubilization, protease, BclA protein, BclB protein , CotE protein, CotO protein, ExsY protein, ExsFA/BxpB protein, CotY protein, ExsFB protein, ExsJ protein, ExsH protein, YjcA protein, YjcB protein, BclC protein, BxpA protein, BclE protein, BetA/BAS3290 protein, ExsA protein, ExsK protein, ExsB protein, YabG protein, or Tgl protein is increased compared to the expression of an enzyme involved in nutrient solubilization, protease, BclA protein, BclB protein, CotE protein, CotO protein, ExsY protein, ExsFA/BxpB protein, CotY protein, protein ExsFB, ExsJ protein, ExsH protein, Y protein jcA protein, YjcB protein, BclC protein, BxpA protein, BclE protein, BetA/BAS3290 protein, ExsA protein, ExsK protein, ExsB protein, YabG protein or Tgl protein in a wild-type Bacillus cereus under the same conditions.

[00667] Additional plant growth promotion methods involving the use of exospore fragments derived from a recombinant member of the Bacillus cereus family are described below.

B. Ways to protect a plant from a pathogen or increase plant resistance to stress

[00668] The present invention also relates to methods for protecting a plant from a pathogen or increasing plant resistance to stress.

[00669] One method of protecting a plant from pathogens or increasing a plant's stress tolerance includes adding to the plant growth medium any of the recombinant Bacillus cereus described above, or any of the formulations containing the recombinant Bacillus cereus described above. Alternatively, any of the recombinant members of the Bacillus cereus family described above and any of the formulations containing the recombinant member of the Bacillus cereus family described above may be applied to the plant, plant seed, or the area surrounding the plant or plant seed. In such methods, a recombinant member of the Bacillus cereus family expresses a fusion protein containing a protein or peptide that protects the plant from a pathogen, or a protein or peptide that enhances the plant's resistance to stress. A protein or peptide that protects the plant from a pathogen, or a protein or peptide that enhances the plant's resistance to stress, can be physically associated with the exospore of a recombinant member of the Bacillus cereus family.

[00670] Another method of protecting a plant from a pathogen or increasing a plant's resistance to stress includes adding any of the recombinant spore-forming bacteria described above or any of the formulations containing the recombinant spore-forming bacteria described above to the plant growth medium. Alternatively, any of the recombinant spore-forming bacteria described above, or any of the formulations containing the recombinant spore-forming bacteria described above, can be applied to the plant, plant seed, or the area surrounding the plant or plant seed. In such methods, the recombinant spore-forming bacterium expresses a fusion protein containing a protein or peptide that protects the plant from a pathogen, or a protein or peptide that enhances the plant's resistance to stress. A protein or peptide that protects the plant from a pathogen, or a protein or peptide that enhances the plant's resistance to stress, can be physically associated with the spore coat of the recombinant spore-forming bacterium.

[00671] In any of the methods for protecting a plant from a pathogen, plants grown in a plant growth medium containing a recombinant member of the Bacillus cereus family or recombinant spore-forming bacteria are preferably less susceptible to pathogen infection compared to plants grown under the same conditions in an identical plant growth medium that does not contain a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium.

[00672] In any of the methods for improving plant stress tolerance, plants grown in a plant growth medium containing a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium are less sensitive to stress compared to plants grown under the same conditions in an identical plant growth medium that does not contain a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium.

[00673] Another method of increasing plant stress tolerance includes adding a recombinant member of the Bacillus cereus family or a formulation containing a recombinant member of the Bacillus cereus family to the plant growth medium. Alternatively, the recombinant Bacillus cereus or formulation can be applied to the plant, plant seed, or the area surrounding the plant or plant seed. The recombinant member of the Bacillus cereus family expresses superoxide dismutase or arginase, wherein the expression of superoxide dismutase or arginase is increased compared to the expression of superoxide dismutase or arginase in a wild type member of the Bacillus cereus family under the same conditions.

[00674] Another method of protecting a plant from a pathogen involves adding a recombinant member of the Bacillus cereus family or a formulation containing a recombinant member of the Bacillus cereus family to the plant growth medium. Alternatively, the recombinant Bacillus cereus or formulation can be applied to the plant, plant seed, or the area surrounding the plant or plant seed. The recombinant member of the Bacillus cereus family expresses a protease, wherein the expression of the protease is increased compared to the expression of the protease in a wild-type member of the Bacillus cereus family under the same conditions.

[00675] Additional methods for protecting a plant from a pathogen or increasing plant stress tolerance, involving the use of exosporium fragments derived from a recombinant member of the Bacillus cereus family, are described below.

C. Methods for immobilizing spores of a recombinant member of the Bacillus cereus family or recombinant spore-forming bacteria on a plant

[00676] In addition, the present invention relates to methods for immobilizing spores of a recombinant member of the Bacillus cereus family or recombinant spore-forming bacteria on a plant.

[00677] One method of immobilizing recombinant Bacillus cereus spores on a plant includes adding to the plant growth medium any of the recombinant Bacillus cereus described above, or any of the formulations containing the recombinant Bacillus cereus described above. Alternatively, any of the recombinant members of the Bacillus cereus family described above, or any of the formulations containing the recombinant member of the Bacillus cereus family described above, can be applied to the plant, plant seed, or the area surrounding the plant or plant seed. In such methods, a recombinant member of the Bacillus cereus family expresses a fusion protein containing a plant-binding protein or peptide. The plant-binding protein or peptide may be physically associated with the exospore of a recombinant member of the Bacillus cereus family.

[00678] Another method of immobilizing spores of a recombinant spore-forming bacterium on a plant comprises adding to the plant growth medium any of the recombinant spore-forming bacteria described above or any of the formulations containing the recombinant spore-forming bacterium described above. Alternatively, any of the recombinant spore-forming bacteria described above, or any of the formulations containing the recombinant spore-forming bacterium described above, can be applied to the plant, plant seed, or the area surrounding the plant or plant seed. In such methods, the recombinant spore-forming bacterium expresses a fusion protein containing a plant-binding peptide, and the plant-binding protein or peptide can be physically linked to the spore envelope of the recombinant spore-forming bacterium.

[00679] The plant-binding protein or peptide preferably selectively targets and retains the recombinant member of the Bacillus cereus family or the recombinant spore-forming bacterium on the plant. For example, a plant-binding protein or peptide can selectively target and retain a recombinant member of the Bacillus cereus family to plant roots, root substructures, an aerial part of a plant, or a substructure of an aerial part of a plant.

D. Methods for Promoting Plant Seed Germination

1. Methods for promoting germination involving the use of a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium

[00680] The present invention also relates to methods for promoting the germination of a plant seed.

[00681] One method of promoting germination of a plant seed includes adding to the plant growth medium any of the recombinant members of the Bacillus cereus family described above, or any of the formulations containing the recombinant member of the Bacillus cereus family described above. Alternatively, any of the recombinant members of the Bacillus cereus family described above, or any of the formulations containing the recombinant member of the Bacillus cereus family described above, may be applied to the plant, plant seed, or area surrounding the plant or plant seed. In such methods, a recombinant member of the Bacillus cereus family expresses a fusion protein containing an enzyme that catalyses the production of nitric oxide. An enzyme that catalyses the production of nitric oxide can be physically associated with the exospore of a recombinant member of the Bacillus cereus family.

[00682] Another method of promoting germination of a plant seed includes adding to the plant growth medium any of the recombinant spore-forming bacteria described above, or any of the formulations containing the recombinant spore-forming bacteria described above. Alternatively, any of the recombinant spore-forming bacteria described above, or any of the formulations containing the recombinant spore-forming bacterium described above, can be applied to the plant, plant seed, or the area surrounding the plant or plant seed. In such methods, the recombinant spore-forming bacterium expresses a fusion protein containing an enzyme that catalyzes the production of nitric oxide, and the enzyme that catalyzes the production of nitric oxide may be physically associated with the spore coat of the recombinant spore-forming bacterium.

[00683] The methods described above for promoting germination of a plant seed preferably include applying a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, and a formulation to the plant seed.

[00684] In addition, any of the methods for promoting germination of a plant seed may include incorporating a substrate for an enzyme that catalyzes the production of nitric oxide into a plant growth medium, onto a plant seed, plant, or area surrounding the plant or plant seed. For example, as appropriate, the method further comprises adding L-arginine to the plant growth medium, to the plant seed, plant, or area surrounding the plant or plant seed. For example, L-arginine can be applied to the aerial part of the plant. Preferably L-arginine is applied to the seed of the plant.

[00685] The presence of L-arginine enhances the response and results in more NO production by nitric oxide synthase. Moreover, L-arginine on plant seeds, in the plant growth medium or in the area surrounding the plant, can serve as a substrate for the production of nitric oxide by native bacterial enzymes.

[00686] In any of the above methods for promoting plant seed germination, seeds in a plant growth medium containing a recombinant Bacillus cereus or a recombinant spore-forming bacterium, or seeds coated with a recombinant Bacillus cereus or a recombinant spore-forming bacterium, preferably have an increased level of germination compared to seeds germinated under the same conditions in an identical plant growth medium that does not contain a recombinant Bacillus cereus or a recombinant spore-forming bacterium, or seeds coated with a recombinant Bacillus cereus or a recombinant spore-forming bacterium germinated in the same conditions.

[00687] In any of the above methods for promoting plant seed germination, seeds in a plant growth medium containing a recombinant Bacillus cereus or a recombinant spore-forming bacterium, or seeds coated with a recombinant Bacillus cereus or a recombinant spore-forming bacterium, preferably have longer main root after germination compared to seeds germinated under the same conditions in an identical plant growth medium that does not contain a recombinant Bacillus cereus or a recombinant spore-forming bacterium, or seeds not coated with a recombinant Bacillus cereus or recombinant spore-forming bacterium under the same conditions.

[00688] Additional methods for promoting plant seed germination involving the use of exosporium fragments derived from a recombinant member of the Bacillus cereus family are described below.

2. Methods for promoting germination by delivering to plants enzymes that catalyze nitric oxide production or recombinant microorganisms that overexpress such enzymes

[00689] Another method of promoting the germination of plant seeds includes applying to the plant growth medium or applying to the plant, plant seed or area surrounding the plant or plant seed: (i) an enzyme that catalyzes the production of nitric oxide; (ii) superoxide dismutase; or (iii) a recombinant microorganism that expresses an enzyme that catalyzes the production of nitric oxide or superoxide dismutase, wherein the expression of the enzyme that catalyzes the production of nitric oxide or superoxide dismutase is increased compared to the expression of the enzyme that catalyzes the production of nitric oxide or superoxide dismutase in the wild-type microorganism under the same conditions.

[00690] Preferably, the method includes applying the enzyme or microorganism to the plant seed.

[00691] In addition, the method may include applying a substrate of an enzyme that catalyzes the production of nitric oxide to a plant growth medium, to a plant seed, plant, or to an area surrounding the plant or plant seed. For example, as appropriate, the method further comprises adding L-arginine to the plant growth medium, to the plant seed, plant, or area surrounding the plant or plant seed. For example, L-arginine can be applied to the aerial part of the plant. Preferably L-arginine is applied to the seed of the plant.

[00692] Seeds in a plant growth medium containing an enzyme or microorganism, or seeds coated with an enzyme or microorganism, preferably have an increased level of germination compared to seeds germinated under the same conditions in an identical plant growth medium that does not contain enzyme or microorganism, or seeds not coated with the enzyme or microorganism, germinated under the same conditions.

[00693] Seeds in a plant growth medium containing an enzyme or microorganism, or seeds coated with an enzyme or microorganism, preferably have a longer main root after germination compared to seeds germinated under the same conditions in an identical plant growth medium, which does not contain an enzyme or microorganism, or by seeds not coated with an enzyme or microorganism, under the same conditions.

[00694] An enzyme that catalyses the production of nitric oxide synthase may include nitric oxide synthase or arginase. When the enzyme that catalyses the production of nitric oxide includes nitric oxide synthase, the nitric oxide synthase may include, for example, nitric oxide synthase from Bacillus thuringiensis BT013A or Bacillus subtilis 168. For example, nitric oxide synthase may have at least 85% sequence identity with SEQ ID NO: 260 or 261.

[00695] Nitric oxide synthase may have at least 90% sequence identity with SEQ ID NO: 260 or 261.

[00696] Nitric oxide synthase may have at least 95% sequence identity with SEQ ID NO: 260 or 261.

[00697] Nitric oxide synthase may have at least 98% sequence identity with SEQ ID NO: 260 or 261.

[00698] Nitric oxide synthase may have at least 99% sequence identity with SEQ ID NO: 260 or 261.

[00699] Nitric oxide synthase may have 100% sequence identity with SEQ ID NO: 260 or 261.

[00700] Superoxide dismutase may include superoxide dismutase 1 (SODA1) or superoxide dismutase 2 (SODA2). Superoxide dismutase may contain an amino acid sequence having at least 85% identity with SEQ ID NO: 155 or 156.

[00701] Superoxide dismutase may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 155 or 156.

[00702] Superoxide dismutase may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 155 or 156.

[00703] Superoxide dismutase may contain an amino acid sequence having at least 98% identity with SEQ ID NO: 155 or 156.

[00704] Superoxide dismutase may contain an amino acid sequence having at least 99% identity with SEQ ID NO: 155 or 156.

[00705] Superoxide dismutase may contain an amino acid sequence having at least 100% identity with SEQ ID NO: 155 or 156.

[00706] The recombinant microorganism that expresses an enzyme that catalyses the production of nitric oxide may include a Bacillus species (e.g., a member of the Bacillus cereus family, Bacillus subtilis, Bacillus licheniformis, or Bacillus megaterium ), Escherechia coli , an Aspergillus species (e.g., Aspergillus niger ), or species of Sacchromyces (e.g. Sacchromyces cerevisiae ).

[00707] In any of the above methods, the enzyme or recombinant microorganism can be introduced into the plant growth medium, or can be applied to the plant, plant seed, or area surrounding the plant, in an agriculturally acceptable formulation containing the enzyme or recombinant microorganism. carrier. The composition may contain any of the agriculturally acceptable carriers and other components described in the present description.

[00708] The enzyme that catalyzes the production of nitric oxide can be delivered purified or unpurified and can be delivered alone or in combination with other beneficial proteins, inoculums, or chemicals to a plant seed, plant growth medium, or the area surrounding the plant or plant seed.

E. Methods for delivering nucleic acids to plants

[00709] The present invention also provides methods for delivering nucleic acids to plants.

[00710] One method of delivering nucleic acids to a plant includes adding to the plant growth medium any of the recombinant Bacillus cereus described above, or any of the formulations containing the recombinant Bacillus cereus described above. Alternatively, any of the recombinant members of the Bacillus cereus family described above, or any of the formulations containing the recombinant member of the Bacillus cereus family described above, may be applied to the plant, plant seed, or area surrounding the plant or plant seed. In such methods, a recombinant member of the Bacillus cereus family expresses a fusion protein containing a nucleic acid-binding protein. A nucleic acid-binding protein or peptide is linked to a nucleic acid molecule. The nucleic acid-binding protein or peptide may be physically associated with the exospore of a recombinant member of the Bacillus cereus family.

[00711] In such methods, the recombinant member of the Bacillus cereus family may include an endophytic bacterial strain. The endophytic bacterial strain may include Bacillus cereus EE349, Bacillus cereus EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus cereus EE-B00377, Bacillus or B003 pseudomycoides mycoides EE-B00363. For example, an endophytic bacterial strain may include Bacillus cereus EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus cereus EE-B00377, Bacillus pseudomycoides EE-B00366, or Bacillus mycoides EE-B00366 or Bacillus mycoides6. .

[00712] Another method for delivering nucleic acids to a plant involves adding to the plant growth medium any of the recombinant spore-forming bacteria described above, or any of the formulations containing the recombinant spore-forming bacteria described above. Alternatively, any of the recombinant spore-forming bacteria described above, or any of the formulations containing the recombinant spore-forming bacteria described above, can be applied to the plant, plant seed, or the area surrounding the plant or plant seed. In such methods, the recombinant spore-forming bacterium expresses a fusion protein containing a nucleic acid-binding protein. A nucleic acid-binding protein or peptide is linked to a nucleic acid molecule. The nucleic acid-binding protein or peptide may be physically linked to the spore coat of the recombinant spore-forming bacterium.

[00713] The recombinant spore-forming bacterium may include an endophytic bacterial strain. For example, an endophytic bacterial strain may include Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillus fusiformis EE442, Lysinibacillus sphericus EE443 or Bacillus pumilus EE-B00143.

[00714] In any of the methods described above for delivering nucleic acids to a plant, the nucleic acid molecule may include a modulating RNA molecule; RNA-i molecule; miRNA; aptamer; or a DNA molecule that encodes a modulating RNA molecule, an RNA-i molecule, a microRNA or an aptamer.

[00715] Nucleic acid molecules to be delivered to a plant can be obtained by any means known in the art (eg, chemical synthesis, recombinant production by a microorganism, etc.). A nucleic acid-binding protein or peptide that is part of the fusion proteins described herein can be linked to nucleic acid molecules in a formulation to deliver such nucleic acids to a plant or plants. Nucleic acid-binding proteins and peptides immobilize and stabilize nucleic acids and allow their delivery to the plant intact. The nucleic acid molecules to be delivered to the plant may be in an active form or in an inactive form that can be processed into an active form by the plant.

[00716] To effect the binding of nucleic acid molecules to a nucleic acid-binding protein or peptide, nucleic acid molecules can be incubated with any of the recombinant Bacillus cereus or recombinant spore-forming bacteria described herein that express a fusion protein containing a nucleic acid-binding protein or peptide .

[00717] Additional methods for delivering nucleic acids to a plant involving the use of exospore fragments derived from a recombinant member of the Bacillus cereus family are described below.

F. Methods for inhibiting spore germination of a recombinant member of the Bacillus cereus family

[00718] In addition, the present invention relates to a method for slowing down the germination of a spore of a member of the Bacillus cereus family. The method includes modifying a member of the Bacillus cereus family to express inosinuridine hydrolase or alanine racemase, where the expression of inosinuridine hydrolase or alanine racemase is increased compared to the expression of inosinuridine hydrolase or alanine racemase in a wild type member of the Bacillus cereus family under the same conditions.

G. Inactivation of a member of the Bacillus cereus family or a recombinant spore-forming bacterium prior to use

[00719] In any of the methods described above that use a recombinant Bacillus cereus or a recombinant spore-forming bacterium, the method may further comprise inactivating the recombinant Bacillus cereus or the recombinant spore-forming bacterium prior to being introduced into the plant growth medium or applied to the a plant, a plant seed, or the area surrounding a plant or plant seed.

[00720] For example, inactivation may include exposing a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium to a heat treatment; exposure to gamma radiation; exposure to x-rays; exposure to UV-A radiation; exposure to UV-B radiation; treatments with glutaraldehyde, formaldehyde, hydrogen peroxide, acetic acid, bleach, chloroform or phenol, or combinations thereof.

[00721] Alternatively or additionally, inactivation may include modifying a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium to express a spore germinating protease or non-specific endonuclease, wherein the expression of the spore germinating protease or non-specific endonuclease is increased relative to the expression of the spore germinating protease or non-specific endonuclease in the member. of the wild-type Bacillus cereus family under the same conditions, and wherein the recombinant spore-forming bacterium includes a recombinant bacterium of the genus Bacillus .

H. Methods for Obtaining and Using Exosporium Fragments

[00722] In addition, the present invention relates to methods for obtaining and using exosporium fragments. These methods relate to recombinant members of the Bacillus cereus family described in section IV of the present description above, i. recombinant members of the Bacillus cereus family that contain a mutation or other genetic change that allows the collection of free exospores.

[00723] Thus, the present invention relates to a method for removing exospores from spores of a recombinant member of the Bacillus cereus family. The method includes subjecting a suspension containing any of the recombinant members of the Bacillus cereus family described in section IV of the present description above to centrifugation or filtration to obtain exospore fragments that are separated from the spores. Fragments of exosporium contain a fusion protein.

[00724] A method for removing exospore from spores of a recombinant member of the Bacillus cereus family may include subjecting the suspension containing spores to centrifugation and collecting the supernatant, wherein the supernatant contains exospore fragments and is substantially free of spores.

[00725] Alternatively, a method for removing exospore from spores of a recombinant member of the Bacillus cereus family may include filtering the suspension containing the spores and collecting the filtrate, wherein the filtrate contains exospore fragments and is substantially free of spores.

[00726] Before centrifugation or filtration, the spore suspension can be shaken or mechanically broken.

[00727] Exospore fragments can also be separated from spores by gradient centrifugation, affinity purification, or by allowing the spores to precipitate out of suspension.

[00728] In addition, the present invention relates to methods of using exospore fragments.

[00729] Provides a method of stimulating plant growth. The method includes introducing exosporium fragments or a composition containing exosporium fragments and an agriculturally acceptable carrier into a plant growth medium. Alternatively, the exospore fragments or composition can be applied to the plant, plant seed, or the area surrounding the plant or plant seed. The exosporium fragments are derived from spores of the recombinant member of the Bacillus cereus family described in Section IV of this specification above and contain a fusion protein. The fusion protein contains a plant growth promoting protein or peptide.

[00730] Also provided is a method of protecting a plant from pathogens or increasing a plant's resistance to stress. The method includes introducing exosporium fragments or a composition containing exosporium fragments and an agriculturally acceptable carrier into a plant growth medium. Alternatively, the exospore fragments or composition can be applied to the plant, plant seed, or the area surrounding the plant or plant seed. The exospore fragments are derived from spores of the recombinant member of the Bacillus cereus family described in section IV above and contain a fusion protein. The fusion protein contains a protein or peptide that protects the plant from a pathogen, or a protein or peptide that increases the plant's resistance to stress.

[00731] When the method is a method of protecting a plant from a pathogen, the fusion protein contains a protein or peptide that protects the plant from the pathogen.

[00732] In methods of protecting a plant from a pathogen, plants grown in a plant growth medium containing exosporium fragments are preferably less susceptible to pathogen infection compared to plants grown under the same conditions in an identical plant growth medium that does not contain the fragments. exospore.

[00733] When the method is a method of increasing plant stress tolerance, the fusion protein comprises a protein or peptide that enhances plant stress tolerance.

[00734] In methods for increasing plant stress tolerance, plants grown in a plant growth medium containing exosporium fragments are preferably less stress sensitive than plants grown under the same conditions in an identical plant growth medium that does not contain exospore fragments.

[00735] A method for immobilizing exospore fragments on a plant is also provided. The method includes introducing exosporium fragments or a composition containing exosporium fragments and an agriculturally acceptable carrier into a plant growth medium. Alternatively, the exospore fragments or composition can be applied to the plant, plant seed, or the area surrounding the plant or plant seed. The exosporium fragments are derived from spores of the recombinant member of the Bacillus cereus family described in Section IV of this specification above and contain a fusion protein. The fusion protein contains a plant-binding protein or peptide.

[00736] The plant-binding protein or peptide preferably selectively targets and retains exospore fragments on the plant. For example, a plant-binding protein or peptide can selectively target and retain exospore fragments on plant roots, root substructures, an aerial part of a plant, or an aerial substructure of a plant.

[00737] A method for promoting germination of a plant seed is also provided. The method includes introducing exosporium fragments or a composition containing exosporium fragments and an agriculturally acceptable carrier into a plant growth medium. Alternatively, the exospore fragments or composition can be applied to the plant, plant seed, or the area surrounding the plant or plant seed. The exospore fragments are derived from spores of the recombinant member of the Bacillus cereus family described in section IV of the present description above and contain a fusion protein. The fusion protein contains superoxide dismutase, or an enzyme that catalyses the production of nitric oxide.

[00738] In methods for promoting germination, the method preferably comprises applying exospore fragments to the plant seed.

[00739] In addition, methods for promoting germination may include applying a substrate for an enzyme that catalyzes nitric oxide production to a plant growth medium, to a plant seed, plant, or to an area surrounding the plant or plant seed. For example, as appropriate, the method further comprises adding L-arginine to the plant growth medium, to the plant seed, plant, or area surrounding the plant or plant seed. For example, L-arginine can be applied to the aerial part of the plant. Preferably L-arginine is applied to the seed of the plant.

[00740] The presence of L-arginine enhances the response and results in more NO production by nitric oxide synthase. Moreover, L-arginine on a plant seed, plant growth medium, or area surrounding the plant can serve as a substrate for nitric oxide production by native bacterial enzymes.

[00741] In methods for promoting germination of a plant seed, seeds in a plant growth medium containing exosporium fragments, or seeds to which exosporium fragments are applied, preferably have an increased level of germination compared to the same seeds germinated under the same conditions in an identical medium. for plant growth that does not contain exosporium fragments, or with the same seeds germinated under the same conditions on which exospore fragments were not applied.

[00742] In methods for promoting germination of a plant seed, seeds in a plant growth medium containing exosporium fragments or seeds to which exosporium fragments are applied preferably have a longer main root after germination compared to the same seeds germinated under the same conditions in identical plant growth media that does not contain exospore fragments, or with the same seeds germinated under the same conditions to which no exospore fragments were applied.

[00743] A method for delivering nucleic acids to a plant is also provided. The method includes adding exosporium fragments or a composition containing exosporium fragments to a plant growth medium. Alternatively, the exospore fragments or composition can be applied to the plant, plant seed, or the area surrounding the plant or plant seed. The exosporium fragments are derived from spores of the recombinant member of the Bacillus cereus family described in Section IV of this specification and contain a fusion protein. The fusion protein contains a nucleic acid-binding protein or peptide. A nucleic acid-binding protein or peptide is linked to a nucleic acid molecule.

[00744] In a method for delivering nucleic acids to a plant, the nucleic acid molecule may comprise a modulating RNA molecule; RNA-i molecule; miRNA; an aptamer or a DNA molecule that encodes a modulating RNA molecule, an RNA-i molecule, a microRNA or an aptamer.

[00745] Nucleic acid molecules to be delivered to a plant can be obtained by any means known in the art (eg, chemical synthesis, recombinant production by a microorganism, etc.). The nucleic acid molecules can then be coupled to a nucleic acid-binding protein or peptide that is part of the fusion proteins described herein in a formulation to deliver such nucleic acids to a plant or plants. Nucleic acid-binding proteins and peptides immobilize and stabilize nucleic acids and allow their delivery to the plant intact. The nucleic acid molecule to be delivered to the plant may be in an active form or in an inactive form which can be processed by the plant into an active form.

[00746] To effect binding of nucleic acid molecules to a nucleic acid-binding protein or peptide, nucleic acid molecules can be incubated with exosporium fragments containing a fusion protein containing a nucleic acid-binding protein or peptide.

I. Plant Growth Environment

[00747] In any of the methods described herein involving the use of a plant growth medium, the plant growth medium may comprise soil, water, aqueous solution, sand, gravel, polysaccharide, mulch, compost, peat moss, straw, logs, clay, soy flour, yeast extract, or a combination thereof.

[00748] Moreover, the plant growth medium can be supplemented with an enzyme substrate or cofactor. For example, the substrate or cofactor may include tryptophan, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate (e.g., adenosine-3-triphosphate), indole, trimetaphosphate, ferrodoxin, acetoin, diacetyl, pyruvate, acetolactate, pectin, cellulose, methylcellulose, starch, chitin, pectin, flour, cellulose derivative, phosphate, acetoin, chitosan, inactive indole-3-acetic acid derivative, inactive gibberellic acid derivative, xylan, arabinoxylan, fat, wax, oil, phytic acid, lignin, humic acid, choline, choline derivative, proline, polyproline, proline-rich protein, proline-rich flour, phenylalanine, chorismate, L-arginine, NADH, NADPH, ATP, GTP, cytochrome C, cytochrome p450, or a combination thereof.

J. Methods of application

[00749] The methods described herein may include coating seeds with a recombinant Bacillus cereus , recombinant spore-forming bacterium, or exosporium fragments, or a formulation containing a recombinant Bacillus cereus , recombinant spore-forming bacterium, or exosporium fragments prior to sowing.

[00750] The methods described herein may include applying a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or exosporium fragments, or a composition containing a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or exosporium fragments, to the aerial part of the plant.

[00751] In the methods described herein, adding a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or exosporium fragments to a plant growth medium may include adding a liquid or solid formulation containing a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or exosporium fragments, on Wednesday. The plant growth medium may include soil (eg, potting mix), compost, peat moss, sand, seed mix, or a combination thereof. The method may include applying the composition to the plant growth medium before, at the same time as, or after sowing the seeds, seedlings, cuttings, bulbs, or plants into the plant growth medium.

K. Agrochemicals

[00752] In the methods described herein, the method may further include applying at least one agrochemical to a plant growth medium or applying at least one agrochemical to plants or plant seeds.

[00753] The agrochemical may include fertilizer (e.g., liquid fertilizer), micronutrient material (e.g., boric acid, borate, boric frit, copper sulfate, copper frit, copper chelate, sodium tetraborate decahydrate, ferrous sulfate, iron oxide, ammonium ferrous sulfate, iron frit, iron chelate, manganese sulfate, manganese oxide, manganese chelate, manganese chloride, manganese frit, sodium molybdate, molybdic acid, zinc sulfate, zinc oxide, zinc carbonate, zinc frit, zinc phosphate, zinc chelate, or a combination thereof), insecticide (e.g. organophosphorus compound, carbamate, pyrethroid, acaricide, alkyl phthalate, boric acid, borate, fluoride, sulfur, haloaromatic substituted urea, hydrocarbon ester, biobased insecticide, or combination thereof), herbicide (e.g. chlorophenoxy compound, nitrophenolic compound, nitrocresol compound, dipyridyl compound, acetamide, aliphatic acid, anilide, benzamide, benzoic acid, derivative oe benzoic acid, anisic acid, anisic acid derivative, benzonitrile, benzothiadiazinone dioxide, thiocarbamate, carbamate, carbanylate, chlorpyridinyl, cyclohexenone derivative, dinitroaminobenzene derivative, fluoronitrotoluidine compound, isoxazolidinone, nicotinic acid, isopropylamine, isopropylamine derivative, oxadiazolidinone, phosphate, compound, picolinic acid, triazine, triazole, uracil, urea, endotall, sodium chlorate, or a combination thereof), fungicide (eg, substituted benzene, thiocarbamate, ethylene bis-dithiocarbamate, thiophthalidamide, copper compound, organomercury compound, organotin compound, cadmium compound, anilazine , benomyl, cyclohexamide, dodine, etridiasol, iprodione, metlaxyl, thiamimefon, triforin, or a combination thereof), molluscicide, algaecide, plant growth modifier, bacterial inoculum (e.g., bacterial inoculum of the genus Rhizobium , bacterial inoculum of the genus Bradyrhizobium , bacterial inoculum of the genus Mesorh izobium , бактериальный инокулят рода Azorhizobium , бактериальный инокулят рода Allorhizobium , бактериальный инокулят рода Sinorhizobium , бактериальный инокулят рода Kluyvera , бактериальный инокулят рода Azotobacter , бактериальный инокулят рода Pseudomonas , бактериальный инокулят рода Azospirillium , бактериальный инокулят рода Bacillus , бактериальный инокулят рода Streptomyces , бактериальный инокулят рода Paenibacillus , bacterial inoculum of the genus Paracoccus , bacterial inoculum of the genus Enterobacter , bacterial inoculum of the genus Alcaligenes , bacterial inoculum of the genus Mycobacterium , bacterial inoculum of the genus Trichoderma , bacterial inoculum of the genus Gliocladium , bacterial inoculum of the genus Glomus , bacterial inoculum of the genus Klebsiella , or a combination (for example, fungal inoculum), , mushroom inoculum of the Glomeraceae family, mushroom inoculum of the Claroidoglomeraceae family, mushroom inoculum of the Gigasporaceae family, mushroom inoculum of the Acaulosporaceae family, mushroom inoculum yat of the Sacculosporaceae family, fungal inoculum of the Entrophosporaceae family, fungal inoculum of the Pacidsporaceae family, fungal inoculum of the Diversisporaceae family, fungal inoculum of the Paraglomeraceae family, fungal inoculum of the Archaeosporaceae family, fungal inoculum of the Geosiphonaceae family, fungal inoculum of the Ambisporaceae family, fungal inoculum of the Scutellosporaceae family, fungal inoculum of the Dentiscultataceae family, mushroom Racocetraceae family inoculum, Basidiomycota fungal inoculum, Ascomycota fungal inoculum, Zygomycota fungal inoculum, or a combination thereof), or a combination thereof.

[00754] The fertilizer may include 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, calcium ammonium nitrate, calcium sulfate, calcined magnesite, calcined limestone, calcium oxide, calcium nitrate, dolomitic limestone, slaked lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, magnesium potassium sulfate, potassium sulfate, sodium nitrates, magnesian limestone, magnesia, urea, urea-formaldehydes, ammonium urea nitrate, sulfur-coated urea, polymer-coated urea, isobutylidene diurea, K ₂ SO ₄ -2MgSO ₄ , kainite, sylvinite, kieserite, epsom salt, elemental sulphur, lime clay, powdered shells oysters, fish meal, cake, fish current, blood meal, phosphate ore, superphosphates, slag, bone meal, wood ash, manure, bat guano, peat moss, compost, glauconite sand, cotton seed meal, feather meal, crab meal, fish emulsion, humic acid, or a combination thereof.

[00755] The agrochemical may contain any of the fungicides, bacterial inoculums, or herbicides described in section XVII above.

L. Plants and seeds

[00756] In any of the plant methods described above, the plant may be a dicot plant, a monocot plant, or a gymnosperm plant.

[00757] For example, when the plant is a dicotyledonous plant, the dicotyledonous plant may be selected from the group consisting of bean, pea, tomato, pepper, pumpkin, alfalfa, almond, anise, apple, apricot, arakacha, artichoke, avocado, ground beans, beets, bergamot, black pepper, black locust, blackberry, blueberry, bitter orange, bok choy, brazil nuts, breadfruit, broccoli, broad beans, brussels sprouts, buckwheat, cabbage, camelina, bok choy, cocoa, cantaloupe, cumin, Spanish artichoke, carob, carrot, cashew, cassava, castor bean, cauliflower, allspice celery, salad celery, cherry, chestnut, chickpea, chicory, chili pepper, chrysanthemum, cinnamon, citron, clementine, cloves, clover , coffee, cola nut, colza, corn, cottonseed, cottonseed, Chinese cowpea, crambe, cranberry, watercress, cucumber, currant, cream apple tree, cassia puffy, nodule rank, eggplant, endive, fennel, fenugreek, fig, hazel, flax, geranium, gooseberry, gourd, grape, grapefruit, guava, hemp, hemp seed, henna, hops, horse bean, horseradish, indigo, jasmine, Jerusalem artichoke, jute, fodder cabbage, kapok, kenaf, kohlrabi, kumquat, lavender, lemon, lentil, lespedeza, lettuce, lime, licorice, lychee, loquat, lupine, macadamia nut, nutmeg, mandarin, fodder beet, mango, loquat, melon, mint, mulberry, mustard , nectarine, oilseed nougat, torrei, okra, olive, opium, orange, papaya, parsnip, pea, peach, peanut, pear, pecan, persimmon, pigeon pea, pistachio, plantain, plum, pomegranate, pomelo, poppy, potato, sweet potato, plum tree, pumpkin, quebracho, quince, Cinchona trees, quinoa, radish, ramie, rapeseed, raspberry, Chinese nettle, rhubarb, rose, rubber tree, turnip, safflower, sainfoin, goat's beard, sapodilla, unshiu mandarin, goatfish , sesame, butterwood, soy, spinach, pumpkin large-fruited, strawberry, sugar beet, sugar cane, sunflower, rutabaga, sweet pepper, tangerine, tea, Abyssinian panicle, tobacco, tomato, shamrock, tung tree, turnip, urena, vetch, walnut, watermelon, Paraguayan tea tree, colza, shepherd's purse, watercress, bedbug, watercress, yarutka, star anise, laurel, noble laurel, cassia, jamana, dill, tamarind, pepper mat, oregano, rosemary, sage, sausep, shieldwort, calophylla, charantian momordica, lacquer tree, Tahitian chestnut, basil, blueberry, hibiscus, passion fruit, kaimite, sassafras, cactus, St. , Japanese pepper, yellow plum, maduka, Chinese mahogany, New Zealand spinach, clematis, temedy, tansy, gerbil, mombin, Malay apple, Brazilian cress, thistle, Dioscorea batato howl, horse parsley, gulyavnik officinalis, smolevka, agate, Siamese of Alexandria, thistle, crooked bread, Antillean gooseberry, potash, soleros, sorrel, xiphoid pteris, leaf cabbage, primrose, primrose, purslane, burkun, turpentine tree, white pisonia, wild betel , African pepper, glutinous eriodictyon, tarragon, parsley, chervil, spring rape, saxifrage thigh, sweet lippia, butterbur, perilla, pepper grass, perilla, bitter bean, tuberous oxalis, kampong, Chinese celery, lemon basil, Thai basil, water mimosa , kokorysha, cabbage tree, moringa, wide mirabilis, onoclea, fragrant limnophila, yellow limnocharis, lovage, pepper grass, poppies, common gourd, hyacinth bean, water sweet potato, pasnik, hauttuynia, Okinawa spinach, larvae-shaped clay, small-flowered galinsogi, odorous eryngium , arugula, Spanish artichoke, edible cyclantera, mitsuba, chipilina, kritmum a, mampata, ebolo, Indian coccinia, field sow thistle, red algae, aconitolus cnidoscolus, huazontle, Ethiopian mustard, giant mari, whole-leaved mari, Mexican tea, white mari, centella, silver celosia, caper bush, field cabbage, Beijing cabbage, mizuna , Chinese turnip, kai lan, leafy mustard, Malabar spinach, chard, marshmallow, climbing acacia, Chinese jute, paprika, annatto seeds, peppermint, savoy cabbage, marjoram, cumin, chamomile, lemon balm, clove pepper, blueberry, cherimoya , Cloudberries, Blackthorn, Pitahaya, Durian, Elderberry, Feijoa, Breadfruit, Jambul, Jujube, Physalis, Purple Mangosteen, Rambutan, Redcurrant, Blackcurrant, Salal Berries, Mandarin, Agli, Adzuki, Black Bean, Asparagus Bean, Cranberry Bean , common beans, green beans, multicolor beans, lima beans, mung beans, turkish beans, pinto beans, fiery red beans, green peas, mangetot peas, green and cauliflower, asparagus, nettle, bell pepper, red endive, daikon, radish, sugar marshmallow, soy tat, broccolini, black radish, burdock root, garden bean, Italian broccoli, lobia, lupine, sterculia, velvet beans, square Peas, Pachyrhizus, Mulga, Vernonia, Reed Acacia, Murray Acacia, Prickly Acacia, Witchetti Bush, Coriacea Acacia, Chia, Beech Nut, Candlewood, Coloquint, Melikoccus, Maya Walnut, Mongongo, Ogbono Walnut, Paradise Nut and Chempedac.

[00758] When the plant is a monocot plant, the monocot plant can be selected from the group consisting of corn, wheat, rye, rice, barley, millet, banana, onion, garlic, asparagus, chaff, millet, fonio, raishan, palmer grass , turmeric, saffron, galangal, chives, cardamom, date palm, pineapple, shallot, porrey, chives, water chestnut, wild garlic, cuckoo's tears, bamboo, dagussa, rootless wolfia, malanga, Tahitian spinach, abaca, areca, African millet, betel nut, panicled millet, panicled sorghum, citronella, coconut, taro, maize, taro, durra, durum wheat, edo, furcrea, formio, ginger, hedgehog, trailing feather grass, Sudan grass, sorghum guinea, textile banana, henequen, hybrid corn, iovar, lemon sorghum, Mexican agave, cane millet, finger millet, Italian millet, Japanese millet, common millet, New Zealand flax, rye, oil palm, palmyra pa elm, sago palm, white bentgrass, sisal, sorghum, spelled, sweet corn, sweet sorghum, taro, Abyssinian panicle, timothy grass, triticale, vanilla, wheat and yam.

[00759] When the plant is a gymnosperm, the gymnosperm may be from a family selected from the group consisting of Araucariaceae, Boweniaceae, Cephalotaxaceae, Cupressaceae, Cycadaceae, Ephedraceae, Ginkgoaceae, Gnetaceae, Pinaceae, Podocarpaceae, Taxaceae, Taxodiaceae, Welwitschiaceae, and Zamiaceae .

[00760] The plants and plant seeds described herein may include transgenic plants or plant seeds such as transgenic cereals (wheat, rice), maize, soybeans, potatoes, cotton, tobacco, oilseed rape, and fruit plants (apple fruits, pears, citrus fruits and grapes) Preferred transgenic plants include corn, soybeans, potatoes, cotton, tobacco and oilseed rape.

[00761] Suitable transgenic plants and seeds can be characterized by the formation of toxins in the plant, especially from the genetic material of Bacillus thuringiensis (for example, due to the gene CryIA (a), CryIA (b), CryIA (c), CryIIA, CryIIIA, CryIIIB2, Cry9c, Cry2Ab, Cry3Bb, CryIF, or combinations thereof). The formation of toxins in plants increases the resistance of plants to insects, arachnids, nematodes, and larvae and snails (hereinafter referred to as "Bt plants"). Bt plants, e.g. corn varieties, cotton varieties, soybean varieties and potato varieties, are commercially available under the trade name YIELD GARD® (e.g. corn, cotton, soy), Knock0ut® (e.g. maize), StarLink ® (e.g. maize ), Bollgard® (cotton), Nucotn® (cotton) and NewLeaf® (potato). Herbicide-tolerant plants include those trade names Roundup Ready® (glyphosate tolerance, eg corn, cotton, soy), Clearfield® (eg corn), Liberty Link® (glufosinate tolerance, eg oilseed rape), IMI® (with tolerance to imidazolinones) and STS® (tolerance to sulfonylurea, eg maize).

[00762] Plant seeds as described herein may be genetically modified (e.g., any seed that results in a genetically modified plant or plant part that exhibits herbicide tolerance, tolerance to environmental factors such as lack of water, drought , viruses and nitrogen production, or bacterial, fungal or insect resistance to toxins). Suitable genetically modified seeds include seeds of cabbage crops, vegetables, fruits, trees, fibrous crops, oilseeds, tuber crops, coffee, flowers, legumes, cereals, and other monocots and dicots. Preferably, the genetically modified seeds include peanuts, tobacco, grasses, wheat, barley, rye, sorghum, rice, rapeseed, sugar beet, sunflower, tomato, pepper, beans, lettuce, potatoes and carrots. Most preferably, the genetically modified seeds include cotton, soybean, and corn (sweet, field, seed, or popcorn) seeds.

[00763] Particularly suitable transgenic plants that can be treated according to the invention are plants that include transformation events or a combination of transformation events, which are listed, for example, in the databases of various national or regional regulatory authorities (see, for example, 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

[00764] In addition, the present invention relates to methods for delivering beneficial bacteria and/or proteins or peptides to animals.

[00765] Introducing bacterial strains that are both probiotic and endophytic into a plant allows the bacteria to enter the plant where they divide and increase in number. Endophytic and probiotic strains can be delivered to plants using various methods, for example, endophytic and probiotic strains can be delivered through seed treatment, treatment of the plant growth medium (e.g. soil), irrigation, application directly to the plant (e.g., foliar application to aerial parts plants). After entering the plant, the bacteria increase in number and colonize the internal tissues of the plant. The plant can then be fed to an animal, allowing the delivery of probiotic bacteria to the animal. Cost is reduced compared to traditional methods of delivering probiotic bacteria to animals because the endophytic nature of the bacteria allows them to divide and increase in number in the plant. By initially delivering a small amount of the probiotic and endophytic strain of bacteria to the plant and allowing the bacteria to increase in numbers in the plant, the dose is increased. In addition, the probiotic and endophytic strain can be propagated in the target culture before harvesting and consumption.

[00766] Also for these purposes, you can use bacterial strains that are able to colonize the phylloplana of the plant and are also probiotic. Strains that are able to clone a plant's phylloplana can initially be delivered to plants in small doses, and then they divide and colonize the outer surfaces of plants.

[00767] Suitable strains of bacteria that are both endophytic or phylloplan colonizing and probiotic include strains that can both replicate in the field in or on a plant and provide benefits to animals when consumed. Benefits of probiotic bacteria in animals include, but are not limited to, regulation of the microbiome of the animal's digestive tract, secretion of enzymes that aid in the digestion of plant material, and stimulation of the animal's immune system. Examples of digestive enzymes that may provide benefits include, but are not limited to, cellulases, endoglucanases, exoglucanases, β-glucosidases, amylases, proteases, pectinases, xylanases, xylosidases, lipases, phospholipases, and lignases.

[00768] The Bacillus and Lysinibacillus genera are unique in that they contain a large number of species that are both endophytic and thus colonize plants and act as probiotics in vertebrates. Thus, Bacillus and Lysinibacillus species are highly suitable for the delivery of probiotics to animals by passage and cultivation in plants. Common Bacillus species that can be both endophytic and probiotic include Bacillus subtilis , Bacillus firmus , Bacillus amyloliquefaciens , Bacillus cereus , Bacillus toyocerin, Bacillus megaterium, Bacillus pumilus , and Bacillus licheniformis . Lysinibacillus species that are both endophytic and probiotic can also be used.

[00769] A method for delivering beneficial bacteria to an animal is also provided. The method includes feeding an animal with a plant modified to contain a level of endophytic and probiotic bacterial strain that exceeds the level of endophytic and probiotic bacterial strain in the same unmodified plant grown under the same conditions.

[00770] The plant fed to the animal may include a plant grown in a plant growth medium containing an endophytic and probiotic bacterial strain or a composition containing an endophytic and a probiotic bacterial strain, a plant that has been coated with an endophytic and a probiotic bacterial strain, a plant grown from a seed of a plant to which an endophytic and a probiotic bacterial strain has been applied, a plant grown in an area to which an endophytic and a probiotic bacterial strain has been applied, or a seed grown in an area to which an endophytic and a probiotic bacterial strain has been applied.

[00771] The endophytic and probiotic bacterial strain may include Bacillus or Lysinibacillus species . For example, a Bacillus species may include Bacillus subtilis , Bacillus firmus , Bacillus amyloliquefaciens , Bacillus cereus , Bacillus toyocerin, Bacillus megaterium, Bacillus pumilus, Bacillus licheniformis , or a combination thereof.

[00772] The endophytic and probiotic strain of bacteria may include a member of the Bacillus cereus EE349 family, a member of the Bacillus cereus EE439 family, Bacillus thuringiensis EE417, Bacillus cereus EE444, Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillus fusiformis EE442, Lysinibacillus sphericus EE443, Bacillus pumilus EE-B00143, or a combination thereof.

[00773] In addition, proteins or peptides (e.g., enzymes) can be delivered to animals by feeding animals recombinant members of the Bacillus cereus family expressing a fusion protein containing a protein or peptide, exospore fragments containing such fusion proteins, or recombinant spore-forming bacteria expressing such fused proteins. The recombinant member of the Bacillus cereus family or the recombinant spore-forming bacteria may be an endophytic bacterial strain or a bacterial strain that is capable of colonizing the phylloplana of a plant that allows delivery of a protein or peptide to an animal by ingestion of a plant that is colonized by the bacteria. It is also possible to use probiotic recombinant Bacillus cereus strains or recombinant spore-forming bacteria strains such that an animal that ingests the recombinant Bacillus cereus or recombinant spore-forming bacteria benefits from both the probiotic bacteria and the protein or peptide. Also, recombinant strains of members of the Bacillus cereus family and recombinant strains of spore-forming bacteria that are both endophytic and phylloplan colonizing can be used to deliver proteins or peptides to animals.

[00774] Thus, a method for delivering proteins or peptides to an animal is also provided. The method includes feeding an animal with a recombinant member of the Bacillus cereus family expressing a fusion protein containing a protein or peptide of interest and a targeting sequence, an exosporium protein or an exosporium protein fragment that targets the fusion protein into the exospore of the recombinant member of the Bacillus cereus family. Alternatively, the method comprises feeding the animal with exosporium fragments derived from a recombinant member of the Bacillus cereus family expressing a fusion protein containing a protein or peptide of interest and a targeting sequence, an exosporium protein, or an exospore protein fragment that targets the fusion protein into the exospore of the recombinant member of the Bacillus cereus family.

[00775] A recombinant member of the Bacillus cereus family may include any of the recombinant members of the Bacillus cereus family described herein that express a fusion protein.

[00776] Exospore fragments may include exospore fragments derived from any of the members of the Bacillus cereus family described in section IV above.

[00777] A recombinant member of the Bacillus cereus family may include an endophytic bacterial strain. The endophytic bacterial strain may include Bacillus cereus EE349, Bacillus cereus EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus cereus EE-B00377, Bacillus pseudomycoides 6, or Bacillus mycoides EE-B00363. For example, the endophytic bacterial strain includes Bacillus cereus EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus cereus EE-B00377, Bacillus pseudomycoides EE-B00366, or Bacillus mycoides EE-B00366, or Bacillus mycoides EE-B00366.

[00778] A recombinant member of the familyBacillus cereus may include a probiotic strain of bacteria. The probiotic bacterial strain may include a member of the familyBacillus cereus EE349 (NRRL #B-50928), member of the familyBacillus cereus EE439 (NRRL B-50979),Bacillus thuringiensis EE417 (NRRL B-50979),Bacillus cereus EE444 (NRRL B-50977),Bacillus thuringiensis BT013A (NRRL #B-50924), or a combination of them.

[00779] The recombinant member of the Bacillus cereus family may be present in the plant fed to the animal.

[00780] Alternatively, a recombinant member of the Bacillus cereus family may include a bacterial strain that is capable of colonizing the plant's phylloplana. For example, a bacterial strain that is capable of colonizing a plant phylloplana may include Bacillus mycoides BT155, Bacillus mycoides EE118, Bacillus mycoides EE141, Bacillus mycoides BT46-3, Bacillus cereus EE218, Bacillus thuringiensis BT013A, Bacillus cereus EE-B00377, Bacillus pseudomycoides EE-B00366 or Bacillus mycoides EE-B00363.

[00781] A recombinant member of the Bacillus cereus family may be present on the phylloplane of the plant fed to the animal.

[00783] For example, the targeting sequence may comprise or consist of an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 63%.

[00784] The targeting sequence may comprise or consist of an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 72%.

[00785] The targeting sequence may comprise or consist of an amino acid sequence having at least about 56% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 63%.

[00786] The targeting sequence may comprise or consist of an amino acid sequence having at least about 62% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 72%.

[00787] The targeting sequence may comprise or consist of an amino acid sequence having at least about 68% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%.

[00788] The targeting sequence may comprise or consist of an amino acid sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 72%.

[00789] The targeting sequence may comprise or consist of an amino acid sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%.

[00790] The targeting sequence may comprise or consist of an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%.

[00791] The targeting sequence may comprise or consist of an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 90%.

[00792] The targeting sequence may 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, where the identity with amino acids 25-35 is at least approximately 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.

[00793] The exosporium protein or exosporium protein fragment may contain an amino acid sequence 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, 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.

[00794] The exosporium protein or exosporium protein fragment may contain an amino acid sequence 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, 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.

[00795] The exosporium protein or exosporium protein fragment may comprise an amino acid sequence having at least 98% identity with 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, 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.

[00796] The exosporium protein or exosporium protein fragment may comprise an amino acid sequence having at least 99% identity with 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, 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.

[00797] The exosporium protein or exosporium protein fragment may comprise an amino acid sequence having at least 100% identity with 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, 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.

[00798] The targeting sequence, exosporium protein, or exosporium protein fragment may contain the amino acid sequence GXT at their C-terminus, where X is any amino acid.

[00799] The targeting sequence, exosporium protein, or exosporium protein fragment may contain an alanine residue at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1.

[00800] The targeting sequence, exosporium protein, or exosporium protein fragment may 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 a position of the targeting sequence that corresponds to amino acid 20 SEQ ID NO: 1.

[00801] In addition, the fusion protein may contain an amino acid linker between the targeting sequence, the exospore protein or exospore protein fragment, and the protein or peptide of interest. The linker may be any of the linkers described in section XI above.

[00802] The fusion protein can be expressed under the control of a sporulation promoter native to the targeting sequence, the exosporium protein or exosporium protein fragment of the fusion protein or portion thereof, and/or under the control of a highly expressed sporulation promoter. The promoter may be any of the promoters described in section III above.

[00803] A method for delivering proteins or peptides to an animal is also provided. The method includes feeding the animal with a recombinant spore-forming bacterium. The recombinant spore-forming bacterium may be any of the recombinant spore-forming bacteria described in section IX above.

[00804] The recombinant spore-forming bacterium may be present in the plant fed to the animal.

[00805] The recombinant spore-forming bacterium may include an endophytic and a probiotic strain of bacteria. For example, an endophytic and probiotic bacterial strain may include Bacillus megaterium EE385 (NRRL B-50980), Bacillus sp. EE387 (NRRL B-50981), Bacillus circulans EE388 (NRRL B-50982), Bacillus subtilis EE405 (NRRL B-50978), Lysinibacillus fusiformis EE442 (NRRL B-50975) or Lysinibacillus sphaericus EE443 (NRRL B-50976), Bacillus pumilus EE-B00143 (NRRL B-67123), or a combination thereof.

[00806] In any of the methods, the plant can be treated prior to feeding the animal.

[00807] In any of the methods described above involving feeding a plant to an animal, the method may further include adding an endophytic bacterial strain or a formulation comprising an endophytic bacterial strain to the plant growth medium. Alternatively, the method may include applying an endophytic bacterial strain or a composition containing an endophytic bacterial strain to a plant, plant seed, or an area surrounding the plant or plant seed. The plant fed to the animal includes a plant grown in a plant growth medium containing an endophytic and a probiotic strain of bacteria or a composition containing an endophytic and a probiotic strain of bacteria, a plant on which an endophytic and a probiotic strain of bacteria has been applied, a plant grown from a plant seed, to which an endophytic and probiotic bacterial strain has been applied, a plant grown in an area to which an endophytic and probiotic bacterial strain has been applied, or a seed grown in an area to which an endophytic and probiotic bacterial strain has been applied.

[00808] In any of the methods described above for delivering proteins or peptides to an animal, the protein or peptide of interest comprises an enzyme. For example, the enzyme may include xylanase, xylosidase, phytase, phosphatase, protease, cellulase, endoglucanase, exogluconase, glucanase, amylase (e.g., α-amylase or β-amylase), lipase, phospholipase, glycosylase, galactanase, α-galactosidase, β- glucosidase, amylase, pectinase, biotinase, polygalacturonase, ligninase, or a combination thereof. The lipase may include phospholipase A1, phospholipase A2, phospholipase C, phospholipase D, lysophospholipase, or a combination thereof. Preferably, the enzyme comprises a xylanase or a phytase.

[00809] In any of the methods involving feeding an animal a plant, the plant may be treated prior to feeding the animal.

[00810] In any of the methods described above, including delivery of bacteria, proteins, or peptides to an animal, the animal may be a mammal (e.g., sheep, goat, cow, pig, deer, alpaca, bison, camel, donkey, horse, mule, llama , rabbit, dog, or cat), poultry (such as chicken, turkey, duck, goose, quail, or pheasant), fish (such as salmon, trout, tilapia, tuna, catfish, or carp), or crustacean (such as small shrimp, large shrimp, lobster, crab or crayfish).

XXI. Methods for delivering useful nucleic acids to animals, insects, worms, fungi and protozoa

[00811] In addition, the invention relates to methods for delivering a nucleic acid molecule to an animal, insect, worm, fungus, or protozoan.

[00812] The method may include feeding the animal, insect, or worm a plant modified there to contain a level of nucleic acid molecule greater than the level of a nucleic acid molecule in the same unmodified plant grown under the same conditions.

[00813] In addition, a method for delivering a nucleic acid molecule to an animal, insect, or worm is provided. The method may include feeding an animal, insect, or worm with a recombinant member of the Bacillus cereus family expressing a fusion protein containing a protein or peptide of interest and a targeting sequence, exosporium protein or exospore protein fragment that targets the fusion protein into the exospore of the recombinant member of the Bacillus cereus family. Alternatively, the method may include feeding the animal, insect, or worm a recombinant spore-forming bacterium that expresses a fusion protein containing at least one protein or peptide of interest and a spore coat protein that targets the fusion protein to the bacterial spore surface. The protein or peptide of interest includes a nucleic acid binding protein or peptide, and a nucleic acid molecule is linked to the DNA or RNA binding protein or peptide. The nucleic acid-binding protein or peptide may be physically associated with the exospore of a recombinant member of the Bacillus cereus family or with the spore envelope of a recombinant spore-forming bacterium.

[00814] Another method is contemplated for delivering a nucleic acid molecule to an animal, insect, or worm. The method includes feeding the animal, insect or worm with exospore fragments derived from a recombinant member of the Bacillus cereus family. The exosporium fragments are derived from spores of the recombinant member of the Bacillus cereus family described in Section IV of this specification above and contain a fusion protein. The fusion protein comprises a nucleic acid-binding protein or peptide, and wherein the nucleic acid-binding protein or peptide is linked to a nucleic acid molecule.

[00815] Preferably the worm is a nematode

[00816] A method is provided for delivering a nucleic acid molecule to a fungus or protozoan. The method includes contacting a fungus or protozoan with a recombinant member of the Bacillus cereus family expressing a fusion protein containing a protein or peptide of interest and a targeting sequence, an exosporium protein or an exosporium protein fragment, which targets the fusion protein into the exospore of the recombinant member of the Bacillus cereus family. Alternatively, the method includes contacting a fungus or 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 bacterial spore surface. The protein or peptide of interest includes a nucleic acid-binding protein or peptide, and the nucleic acid molecule is linked to the nucleic acid-binding protein or peptide.

[00817] In addition, a method for delivering a nucleic acid molecule to a fungus or protozoan is provided. The method includes bringing a fungus or protozoan into contact with exospore fragments. The exospore fragments are derived from spores of the recombinant member of the Bacillus cereus family described in section IV of the present description above and contain a fusion protein. The fusion protein contains a nucleic acid-binding protein or peptide, and the nucleic acid-binding protein or peptide is linked to the nucleic acid molecule.

[00818] A nucleic acid molecule may include a modulating RNA molecule; RNA-i molecule; micro-RNA; aptamer; or a DNA molecule that encodes a modulating RNA molecule, an RNA-i molecule, a microRNA or an aptamer.

[00819] A recombinant member of the Bacillus cereus family may include any of the recombinant members of the Bacillus cereus family that expresses a fusion protein.

[00820] The fusion protein may comprise any of the fusion proteins described herein, which includes a nucleic acid binding protein.

[00821] The spore coat protein includes CotB protein, CotC protein, CgeA protein, CotB/H protein, CotG protein, X spore coat protein, or CotY protein.

[00822] The spore coat protein may comprise an amino acid sequence having at least 85% identity with any of SEQ ID NOs: 252-259.

[00823] The spore coat protein may comprise an amino acid sequence having at least 90% identity with any of SEQ ID NOs: 252-259.

[00824] The spore coat protein may comprise an amino acid sequence having at least 95% identity with any of SEQ ID NOs: 252-259.

[00825] The spore coat protein may comprise an amino acid sequence having at least 98% identity with any of SEQ ID NOs: 252-259.

[00826] The spore coat protein may comprise an amino acid sequence having at least 99% identity with any of SEQ ID NOs: 252-259.

[00827] The spore coat protein may contain an amino acid sequence having 100% identity with any of SEQ ID NOs: 252-259.

[00828] The methods described above can be used for numerous purposes. For example, these methods can be used to deliver RNA or DNA to animals to reduce the animal's susceptibility to a disease or treat a disease in an animal (eg, an organic disease such as stroke, diabetes, heart disease, and degenerative diseases). RNA and DNA have also been shown to be effective in eliminating or treating disease caused by animal pathogens such as bacteria, viruses, worms (eg nematodes) and fungi. The RNA and DNA may act directly on the pathogen, or may act on the animal's immune system to activate or enhance the immune response.

[00829] In addition, the methods described above can be used to eliminate parasites, including insects, worms (eg, nematodes), fungi, and protozoa. Delivery of specific RNA or DNA to a parasite can result in decreased ability of the parasite to infect a host (eg, plant host), reduced feeding on target hosts or plants, outright killing by blocking key genes, or various other effects.

XXII. Vaccines and method for providing an immunogenic response

[00830] A vaccine is provided that contains a pharmaceutically acceptable carrier and spores of a recombinant member of the Bacillus cereus family that express a fusion protein as described in paragraphs [00171], [00173]-[00176] of Section I above, wherein the protein or peptide of interest is antigen or immunogen.

[00831] In addition, a vaccine is provided that contains a pharmaceutically acceptable carrier and exosporium fragments. The exosporium fragments are derived from spores of the recombinant member of the Bacillus cereus family described in Section IV of this specification above and contain a fusion protein. The fusion protein contains the antigen.

[00832] Another vaccine is contemplated which contains a pharmaceutically acceptable carrier and a recombinant member of the Bacillus cereus family. A recombinant member of the Bacillus cereus family is a recombinant member of the Bacillus cereus family as described above in section II.

[00833] In vaccines that contain exosporium fragments or a recombinant member of the Bacillus cereus family as described above in section II, the targeting sequence, exosporium protein, or exosporium protein fragment may contain any of the targeting sequences, exosporium proteins, or exosporium protein fragments described herein. description. In particular, the targeting sequence, exosporium protein, or exosporium protein fragment may comprise any of the targeting sequences, exosporium proteins, or exosporium protein fragments described in paragraphs [00782]-[00801] above.

[00834] The fusion protein can be expressed under the control of a sporulation promoter native to the targeting sequence, the exosporium protein or exosporium protein fragment of the fusion protein or portion thereof, and/or under the control of a highly expressed sporulation promoter. The promoter may be any of the promoters described in section III above.

[00835] When the protein or peptide of interest is an antigen, displaying the antigen on the outside of the spore or on the exospore fragment provides an immune system response to allow vaccination against various pathogens or diseases. Suitable antigens or small molecule compounds are those known or expected to induce a desired immune response that is sufficient to provide a therapeutic or protective effect when expressed outside a Bacillus spore or displayed on an exospore fragment. Suitability is largely determined by the folding of the three-dimensional structure after incorporation of the recombinant antigen into the exospore, i.e. the antigenic portion(s) of the recombinant molecule must be detectable by the immune system.

[00836] Pathogens or diseases from which an antigen may be derived include, but are not limited to, Acinetobacterial infections caused byAcinetobacter baumannii; actinomycosis caused byActinomyces israelii,Actinomyces gerencseriae andPropionibacterium propionicus; African trypanosomiasis caused byTrypanosoma brucei; acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus; amoebiasis caused byEntamoeba histolytica; anaplasmosis caused byAnaplasma genus, anthrax caused byBacillus anthracis; infectionArcanobacterium haemolyticum, calledArcanobacterium haemolyticum; Argentine hemorrhagic fever caused by the Junin virus; ascariasis caused byAscaris lumbricoides, an astrovirus infection caused by the familyAstroviradae; babesiosis, genusBabesia; infectionBacillus cereus, calledBacillus cereus; bacterial pneumonia; bacterial vaginosis; infectionBacteroides, caused by the genusBacteriodes; balantidiasis caused byBalantidium coli; infectionBaylisascaris, caused by the genusBaylisascaris; BK virus infection caused by BK virus; black piedra calledPiedraia hortae; infectionBlastocystis hominis, calledBlastocystis hominis; blastomycosis caused byBlastomyces dermatitidis; Bolivian hemorrhagic fever caused by the Machupo virus; infectionBorrelia, caused by the genusBorrelia; botulism (and infant botulism) caused by ingestion of a toxinClostridium botulinum; brazilian hemorrhagic fever caused bySabia; brucellosis caused by the genusBrucella; infectionBurkholderia, usually calledBurkholderia cepacia and other typesBurkholderia; ulcerBuruli, calledMycobacterium ulcerans; calicivirus infection (norovirus and sapovirus) caused by the familyCaliciviridae; campylobacteriosis caused by the genusCampylobacter; candidiasis (moniliasis; thrush), usually caused bycandida albicans and other typesCandida; cat scratch disease caused byBartonella henselae; cellulitis, usually causedStreptococcus andStaphylococcus groups A; Chagas disease (American trypanosomiasis)Trypanosoma cruzi; chancroid, calledHaemophilus ducreyi; chickenpox caused by the Varicella zoster virus (VZV); chlamydia caused byChlamydia trachomatis; infectionChlamydophila pneumoniae, calledChlamydophila pneumoniae; cholera causedVibrio Cholerae; chromoblastomycosis, usually caused byFonsecaea pedrosoi; clonorchiasis caused byClonorchis sinensis; infectionClostridium difficile, calledClostridium difficile; coccidioidomycosis caused byCoccidioides immitis andCoccidioides posadasii; Colorado tick fever (CTF) caused by the Colorado tick fever virus (CTFV); the common cold (acute viral nasopharyngitis; viral infection of the upper respiratory tract), usually caused by rhinoviruses and coronaviruses; Creutzfeldt-Jakob disease (CJD) caused by the CJD prion; Crimean-Congo hemorrhagic fever (CCHF) caused by the Crimean-Congo hemorrhagic fever virus; cryptococcosis caused byCryptococcus neoformans; cryptosporidiosis caused by the genusCryptosporidium; larva migrans syndrome, cutaneous form (CLM), usually caused byAncylostoma braziliense and many other parasites; cyclosporiasis caused byCyclospora cayetanensis; cysticercosis caused byTaeniasolium; cytomegalovirus infection caused by cytomegalovirus; Dengue fever caused by Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4) - flaviviruses; dienthamoebiasis caused byDientamoeba fragilis; diphtheria causedCorynebacterium diphtheriae; diphyllobothriasis caused byDiphyllobothrium; dracunculiasis caused byDracunculus medinensis; Ebola hemorrhagic fever caused by Ebolavirus (EBOV); echinococcosis caused byEchinococcus; erlichiosis caused by the genusEhrlichia; enterobiasis (oxyurosis) caused byEnterobius vermicularis; infectionEnterococcus, caused by the genusEnterococcus; enterovirus infection caused by the genus Enterovirus; typhus caused byRickettsia prowazekii; erythema infectiosum (fifth disease) caused by parvovirus B19; sudden exanthema caused by human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7); fascilopsidosis caused byFasciolopsis buski; fascioliasis caused byFasciola hepatica andFasciola gigantica; fatal familial insomnia (FFI) caused by the FFI prion; filariasis caused by a superfamilyFilarioidea; food poisoning causedClostridium perfringens; infection with free-living amoebae; infectionFusobacterium, caused by the genusFusobacterium; gas gangrene (Clostridial myonecrosis), usually calledClostridium perfringens or other typesClostridium; geotrichosis caused byGeotrichum candida; the Gerstmann-Streussler-Scheinker syndrome (GSS) caused by the GSS prion; giardiasis caused byGiardia intestinalis; sap calledBurkholderia mallei; gnathostomiasis caused byGnathostoma spinigerum andGnathostoma hispidum; gonorrhea caused byNeisseria gonorrhoeae; inguinal granuloma (donovanosis) caused byKlebsiella granulomatis; group A streptococcal infection caused byStreptococcus pyogenes; group B streptococcal infection caused byStreptococcus agalactiae; Haemophilus influenzae infection caused byhaemophilus influenzae; viral pemphigus of the mouth and extremities (HFMD) caused by enteroviruses, mainly Coxsackie A virus and enterovirus 71 (EV71); hantavirus pulmonary syndrome (HPS) caused by the Sin Nombre virus; infectionHelicobacter pylori, calledHelicobacter pylori; hemolytic-uremic syndrome (HUS) caused byEscherichia coli O157:H7; hemorrhagic fever with renal syndrome (HFRS), caused by the familyBunyaviridae; hepatitis A, caused by the hepatitis A virus; hepatitis B, caused by the hepatitis B virus; hepatitis C caused by the hepatitis C virus; hepatitis D, caused by the hepatitis D virus; hepatitis E, caused by the hepatitis E virus; herpes simplex caused by herpes simplex virus 1 and 2 (HSV-1 and HSV-2); histoplasmosis caused byHistoplasma capsulatum; hookworm caused byAncylostoma duodenale andNecator americanus; human bocavirus infection caused by human bocavirus (HBoV); human erlihoz, calledEhrlichia ewingii; human granulocytic anaplasmosis (HGA) caused byAnaplasma phagocytophilum; human metapneumovirus infection caused by human metapneumovirus (hMPV); human monocytic ehrlichosis, caused byEhrlichia chaffeensis; human papillomavirus (HPV) infection caused by human papillomavirus (HPV); human parainfluenza virus infection caused by human parainfluenza viruses (HPIV); hymenolepiasis caused byHymenolepis nana andHymenolepis diminuta; Epstein-Barr infectious mononucleosis (Mono) caused by the Epstein-Barr virus (EBV); influenza (flu), caused by the familyOrthomyxoviridae; isosporiasis caused byIsospora Belli; Kawasaki disease (cause unknown, but evidence suggests it is contagious) keratitis; infectionKingella kingae, calledKingella kingae; kuru caused by the prion Kuru; Lassa fever caused by the Lassa virus; legionellosis (legionnaires' disease) caused byLegionella pneumophila; legionellosis (Pontiac fever) caused byLegionella pneumophila; leishmaniasis caused by the genusLeishmania; leprosy caused byMycobacterium leprae andMycobacterium lepromatosis; leptospirosis caused byLeptospira; listeriosis caused byListeria monocytogenes; Lyme disease (Lyme borreliosis), usually caused byBorrelia burgdorferi and other typesBorrelia; lymphatic filariasis (elephantiasis), caused byWuchereria bancrofti andBrugia malayi; lymphocytic choriomeningitis caused by lymphocytic choriomeningitis virus (LCMV); malaria caused byPlasmodium; Marburg hemorrhagic fever (MHF) caused by the Marburg disease virus; measles caused by the measles virus; melioidosis (Whitmore's disease)Burkholderia pseudomallei; meningitis; meningococcal disease caused byNeisseria meningitidis; metagonimiasis, usually caused byMetagonimus yokagawai; microsporidiosis caused by a typeMicrosporidia; molluscum contagiosum (MC) caused by a virusMolluscum contagiosum (MCV); mumps caused by the mumps virus; rat typhus (endemic typhus) caused byRickettsia typhi; infectionMycoplasma pneumonia, calledMycoplasma pneumoniae; mycetoma caused by numerous types of bacteria (Actinomycetoma) and mushrooms (Eumycetoma); myiasis caused by parasitic larvae of Diptera flies; neonatal conjunctivitis (ophthalmia neonatorum), most commonly caused byChlamydia trachomatisandNeisseria gonorrhoeae; (new) variant of Creutzfeldt-Jakob disease (vCJD, nvCJD) caused by the vCJD prion; nocardiosis, usually causedNocardia asteroides and other typesNocardia; onchocerciasis (river blindness) caused byOnchocerca volvulus; paracoccidioidomycosis (South American blastomycosis) caused byParacoccidioides brasiliensis; paragonimiasis, usually caused byParagonimus westermani and other typesParagonimus; pasteurellosis caused by the genusPasteurella; head lice (head lice) caused byPediculus humanus capitis; head lice (body lice) caused byPediculus humanus corporis; pubic pediculosis (pubic lice, flatheads) caused byPhthirus pubis; pelvic inflammatory disease (PID); whooping cough (whooping cough)Bordetella pertussis; the plague causedYersinia pestis; pneumococcal infection caused byStreptococcus pneumoniae; pneumocystis pneumonia (PCP) caused byPneumocystis jirovecii; pneumonia; poliomyelitis caused by poliovirus; infectionPrevotella, caused by the genusPrevotella; primary amoebic meningoencephalitis (PAM), usually caused byNaegleria fowleri; progressive multifocal leukoencephalopathy caused by the JC virus; psittacosis caused byChlamydophila psittaci; Q fever caused byCoxiella burnetii; rabies caused by the rabies virus; rat bite fever caused byStreptobacillus moniliformis andSpirillum minus; respiratory syncytial virus infection caused by respiratory syncytial virus (RSV); rhinosporidiosis caused byRhinosporidium seeberi; rhinovirus infection caused by rhinovirus; rickettsial infection caused byRickettsia; smallpox rickettsiosis caused byRickettsia akari; Rift Valley fever (RVF) caused by Rift Valley fever virus; Rocky Mountain spotted fever (RMSF) is caused byRickettsia rickettsii; rotavirus infection caused by rotavirus; rubella caused by the rubella virus; salmonellosis caused by the genusSalmonella; SARS (severe acute respiratory syndrome) caused by the SARS coronavirus; scabies causedSarcoptes scabiei; schistosomiasis due toSchistosoma; sepsis; shigellosis (bacillary dysentery) caused byShigella genus; shingles (herpes zoster) caused by the Varicella zoster virus (VZV); smallpox (variola) caused byVariola major orVariola minor; sporotrichosis caused bySporothrix schenckii; staphylococcal food poisoning caused byStaphylococcus; staphylococcal infection caused by the genusStaphylococcus; strongyloidiasis caused byStrongyloides stercoralis; syphilis caused byTreponema pallidum; taeniasis due to genusTaenia; tetanus (lockjaw) caused byClostridium tetani; dermatophytosis of the beard and mustache (parasitic sycosis), usually caused by the genusTrichophyton; athlete's foot (ringworm of the scalp), usually caused byTrichophyton tonsurans; mycosis of the smooth skin of the trunk (ringworm of the trunk), usually caused byTrichophyton; ringworm inguinal (inguinal epidermophytosis), usually caused byEpidermophyton floccosum,Trichophyton rubrum andTrichophyton mentagrophytes; ringworm of the hands (ringworm of the hands) caused byTrichophyton rubrum; black lichen, usually causedHortaea werneckii; tinea pedis (athlete's foot), usually caused byTrichophyton; mycosis of the nail (onychomycosis), usually caused byTrichophyton; versicolor versicolor (Pitiriasis versicolor) caused by the genusMalassezia; toxocariasis (larva migrans syndrome, ocular form (OLM)), caused byToxocara canis orToxocara cati; toxocariasis (larva migrans syndrome, visceral form (VLM)), caused byToxocara canis orToxocara cati; toxoplasmosis caused byToxoplasma gondii; trichinosis caused byTrichinella spiralis; trichomoniasis caused byTrichomonas vaginalis; trichiuriasis (whipworm infection) caused byTrichuris trichiura; tuberculosis, usually causedMycobacterium tuberculosis; tularemia caused byFrancisella tularensis; infectionUreaplasma urealyticum, calledUreaplasma urealyticum; Venezuelan equine encephalitis caused by the Venezuelan equine encephalitis virus; Venezuelan hemorrhagic fever caused by the Guanarito virus; viral pneumonia; West Nile fever caused by West Nile virus; white piedra (white lichen), calledTrichosporon beigelii; infectionYersinia pseudotuberculosis, calledYersinia pseudotuberculosis; yersiniosis caused byYersinia enterocolitica; yellow fever caused by the yellow fever virus; zygomycosis caused by detachmentMucorales (mucormycosis) and detachmentEntomophthorales (entomophoramycosis).

[00837] When the protein or peptide of interest is an antigen, any member of the Bacillus cereus family can be used to express the fusion protein. Bacillus thuringiensis or Bacillus mycoides are preferred.

[00838] To obtain a vaccine, the antigen of interest is incorporated into a fusion protein by known methods such as PCR splicing by overlapping extension, restriction endonuclease digestion, and de novo gene ligation or synthesis. The fusion protein is then introduced into a recombinant member of the Bacillus cereus family by transfection, transformation, conjugation, electroporation, or other known methods. The recombinant member of the Bacillus cereus family is then grown in a culture medium (eg, minimal liquid medium) and allowed to sporulate. Preferably, sporulation continues to completion and then the spores are collected and stored. Spores can be collected either by centrifugation or by removing the spores from the growth plates and inoculating into a liquid medium (eg PBS or water) and then centrifuging and washing the resulting spore pellet in the liquid medium. Prior to use, the spore pellet may be resuspended in liquid medium to the desired concentration for use or injection. When the vaccine contains exosporium fragments, exosporium fragments can be obtained using any of the methods described in section XIX.H above.

[00839] The desired concentration of recombinant Bacillus cereus spores or exosporium fragments in a vaccine is based on the size of the individual, the amount of active antigen on the surface of the spores, and the presence and concentration of adjuvants in the vaccine formulation. The vaccine of the invention may contain conventional adjuvants, including pharmaceutically acceptable carriers.

[00840] Provides a method of inducing an immunogenic response in an individual. The method includes administering a vaccine containing spores of a recombinant member of the Bacillus cereus family expressing fusion proteins or exospore fragments containing fusion proteins, as described herein, to an individual.

[00841] The vaccine as described herein is suitable for intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, topical, oral, intranasal, intradermal, transepithelial, or inhalation administration.

[00842] The vaccine may be administered to an individual, which may be a human, mouse, bird, pig, ruminant, sheep, cat, dog, horse, goat, reptile, or non-human primate. Preferably the individual is a mammal and most preferably a human.

XXIII. Reclamation

[00843] When the protein or peptide of interest is a remediation protein or peptide, the toxic substance is catalytically converted by the remediation protein or peptide to a non-toxic or less toxic substance.

[00844] When the remediation protein or peptide includes an enzyme, the enzyme is exposed and stabilized on the outer region of the spore and can be delivered to contaminated soil or contaminated water in a form that is active against the target pollutant or chemical.

[00845] Suitable enzymes depend on the pollutant or chemical targeted by remediation.

[00846] To obtain a recultivation composition, the enzyme of interest is incorporated into the fusion protein by known methods such as PCR splicing by overlap extension, restriction endonuclease digestion and ligation, or de novo gene synthesis. The fusion protein gene is then introduced into a recombinant member of the Bacillus cereus family by transfection, transformation, conjugation, electroporation, or other known methods. The recombinant member of the Bacillus cereus family is then grown in a culture medium (eg, minimal liquid medium) and allowed to sporulate. Preferably, sporulation continues to completion and then the spores are collected and stored. Spores can be collected either by centrifugation or by removing the spores from the growth plates and inoculating into a liquid medium (eg PBS or water) and then centrifuging and washing the resulting spore pellet in the liquid medium. Before use, the spore pellet can be resuspended in a liquid medium to the desired concentration for use. Alternatively, the sludge of the spore formulation can be formulated into granules at the desired concentration for application and incorporation into a contaminated environment. When exospore fragments are used for reclamation, exospore fragments can be obtained using any of the methods described in section XIX.H above.

[00847] Provides a method of reducing the amount of pollutants in the environment. The method includes exposing the polluted environment to spores of a recombinant member of the Bacillus cereus family that express a fusion protein as described above in paragraphs [00171], [00173]-[00175] and [00177] of section I, where the protein or peptide of interest includes a reclamation enzyme .

[00848] There is another way to reduce the amount of pollutants in the environment. The method includes exospore fragments exposing the polluted environment. The exosporium fragments are derived from the recombinant member of the Bacillus cereus family described in Section IV of this specification above and contain a fusion protein. The fusion protein contains a reclamation enzyme.

[00849] There is another way to reduce the amount of impurities in the environment. The method includes exposing the polluted environment to spores of a recombinant member of the Bacillus cereus family. A recombinant member of the Bacillus cereus family is a recombinant member of the Bacillus cereus family as described above in section II.

[00850] In methods for reducing contaminants that include exposing the contaminated environment to fragments of an exosporium or a recombinant member of the Bacillus cereus family, as described above in section II, the targeting sequence, exosporium protein, or exosporium protein fragment may be any of the targeting sequences, proteins exospore or fragments of exospore proteins described in the present description. In particular, the targeting sequence, exosporium protein, or exosporium protein fragment may comprise any of the targeting sequences, exosporium proteins, or exosporium protein fragments described in paragraphs [00782]-[00801] above.

[00851] The fusion protein can be expressed under the control of a sporulation promoter native to the targeting sequence, the exosporium protein, or exosporium protein fragment of the fusion protein or portion thereof, and/or under the control of a highly expressed sporulation promoter. The promoter may be any of the promoters described in section III above.

[00852] When the protein or peptide of interest is a reclamation enzyme, any member of the Bacillus cereus family can be used to express the fusion protein. Preferred are Bacillus thuringiensis, Bacillus cereus or Bacillus mycoides .

[00853] Spores of a recombinant member of the Bacillus cereus family may include an endophytic bacterial strain for phytoremediation, such as a member of the Bacillus cereus EE349 family, a Bacillus cereus EE439 family member, a Bacillus thuringiensis EE417, a Bacillus cereus EE444, or a Bacillus thuringiensis EE319.

[00854] The contaminated environment to be treated may be a gas, liquid, semi-liquid, gel, film, semi-solid, or solid. Solid environment can be soil such as surface soil and subsoil, compost, crop residues, leaves, mulch, felled trees, biofilm, mud layer, humus, sludge, sand, slag, sediment, sewage, waste rock , nuclear waste, ammunition and shells, hospital waste, press car parts, metal trim, insulation waste, food waste, asbestos, batteries, metal scrap, landfill waste, woodworking waste, textile production waste, cullet, leather waste, rubber waste, plastic waste, electronic component waste, agricultural waste, photographic waste, ceramic waste, pharmaceutical waste, wax, spent catalysts, or combinations thereof. The liquid environment can be drinking water, groundwater, surface water, saline solutions, reservoirs, lagoons, aquatic ecosystems, industrial wastewater, acid mine water, spent automotive fluid, spent electrolytic baths, degreasing solutions, dry cleaning solutions, engine coolants, drilling fluid waste, cutting fluid waste, hydraulic fracturing fluid waste, lubricant waste, paint, domestic waste water, oily effluents, pulp mill effluent, water treatment systems, wastewater treatment system, sewerage, sedimentation sump, sewage reservoir, lake, river, or a combination thereof. The gaseous environment may be air, exhaust gas such as emissions from power plants, incinerators, crematoria or refineries, process effluent, landfill gas, natural gas, propane, or a combination thereof.

[00855] A polluted environment can be contaminated with various contaminants, including, but not limited to, a chemical warfare agent, including sarin (GB; o-isopropylmethylphosphonofluoridate); soman (GD; o-pinacolylmethylphosphonofluoridate); cyclosarin (GF; o-cyclohexylmethylphosphonofluoridate); VX (O-ethyl-S-[2-(diisopropylamino)ethyl]methylphosphonothioate); tabun (GA; N,N-dimethylethylphosphoroamidocyanidate), DFP (diisopropylphosphorofluoridate) or mustard gas; an inorganic compound containing arsenic, antimony, barium, beryllium, cadmium, chromium, copper, iron, lead, manganese, mercury, nickel, selenium, silver, tin, thallium, uranium, zinc, or a combination thereof; an organic compound containing a polycyclic aromatic hydrocarbon (PAH), a chlorinated aromatic compound, a chlorinated aliphatic compound, a nitroaromatic compound (NAC), a phenolic compound, a cyano compound, a dioxin, or a combination thereof; crude oil, refined oil, fuel oil, diesel fuel, gasoline, hydraulic oil and kerosene or a volatile component thereof such as benzene, toluene, ethylbenzene, xylene or naphthalene; explosive, fertilizer, pesticide, insecticide or herbicide.

[00856] The concentration of recombinant spores or exosporium fragments required to treat a contaminated environment is based on factors including the volume or area to be treated, the prevalence of a given chemical, pollutant, or organic material present, the amount of time available for treatment, and the amount of active enzyme on the surface of the spores.

[00857] Recombinant Bacillus cereus spores or exosporium fragments can be brought into contact with a contaminated environment by incorporating the spores or exospore fragments into a contaminant-containing stream, contacting the contaminant-containing stream with an immobilizing material containing spores or exosporium fragments ( e.g. filter, membrane, sponge or cassette), incorporating spores or exospore fragments into pellets to be mixed with a contaminated environment, spraying spores or exospore fragments onto or into a contaminated environment, injecting spores or exospore fragments into a contaminated environment, or irrigating a contaminated environment. environment with spores or exosporium fragments.

[00858] Spores can be combined with bacterial inoculums, chemicals, solvents, and other products that can accelerate the decomposition process.

[00859] The reclaiming enzyme includes, but is not limited to, phosphate binding protein, protease, carbohydrate hydrolyase, lipase, phospholipase, nuclease, nutrient binding protein, cellulase, oxidoreductase, monooxygenase, dioxygenase, laccase, lignin peroxidase, manganese peroxidase, peroxidase, dehalogenase, catalase, amylase, reductase, oxidase, amidase, ligninase, xylanase, pectinase, xylosidase, endoglucanase, exoglucanase, glucosidase, biofilm-inhibiting peptide, herbicide-degrading enzyme, pesticide-degrading enzyme (eg, pyrethrinase), or a combination thereof.

[00860] When the enzyme includes a herbicide-degrading enzyme or a pesticide-degrading enzyme, the recombinant member of the Bacillus cereus family suitably includes a bacterial strain that is capable of degrading the herbicide or pesticide. For example, a bacterial strain that is capable of degrading a herbicide or pesticide may include Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120) or Bacillus mycoides EE-B00363 (NRRL B-67121).

[00861] A method for phytoremediation of the soil is also provided. The method includes introducing spores of a recombinant member of the Bacillus cereus family into contaminated soil; or applying spores of a recombinant member of the Bacillus cereus family to a plant planted in contaminated soil, or a plant seed to be planted in contaminated soil, or to an area of contaminated soil surrounding the plant or plant seed; wherein the spores of the recombinant member of the Bacillus cereus family express a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, an exosporium protein or exosporium protein fragment, which targets the fusion protein into the exospore of a spore of the recombinant member of the Bacillus cereus family, wherein the fusion protein is a fusion protein as described above, wherein the protein or peptide of interest comprises a reclamation enzyme, and wherein the recombinant member of the Bacillus cereus family comprises an endophytic bacterial strain or a root colonizing bacterial strain. For example, a recombinant spore-forming bacterium may include an endophytic bacterial strain.

[00862] Another method of phytoremediation of contaminated soil is also provided. The method includes expression of a reclamation enzyme in a spore of a member of the Bacillus cereus family, where the expression of the reclamation enzyme in a spore of a recombinant member of the Bacillus cereus family is increased compared to the expression of the reclamation enzyme in a spore of a wild-type member of the Bacillus cereus family.

[00863] Another method of phytoremediation of contaminated soil is also provided. The method includes introducing a recombinant spore-forming bacterium into contaminated soil, or applying a recombinant spore-forming bacterium to a plant planted in contaminated soil, or a plant seed to be planted in contaminated soil, or to an area of contaminated soil surrounding a plant or plant seed. 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 spore surface of the bacterium. The spore coat protein includes CotB protein, CotC protein, CgeA protein, CotB/H protein, CotG protein, X spore coat protein, or CotY protein. The recombinant spore forming bacterium includes an endophytic bacterial strain or a root colonizing bacterial strain. The protein or peptide of interest includes a reclamation enzyme.

[00864] Another method of phytoremediation of contaminated soil is also provided. The method includes introducing exospore fragments into contaminated soil or applying exospore fragments to a plant planted in contaminated soil, or a plant seed for planting in contaminated soil, or to an area of contaminated soil surrounding a plant or plant seed. Fragments of exosporium originate from spores of a recombinant member of the familyBacillus cereusdescribed in section IV of the present description above and contain a fusion protein. The fusion protein contains a reclamation enzyme.

[00865] Another method of phytoremediation of contaminated soil is contemplated. The method includes introducing spores of a recombinant representative of the Bacillus cereus family into contaminated soil. Alternatively, the method comprises applying spores of a recombinant member of the Bacillus cereus family to a plant planted in contaminated soil, or a plant seed to be planted in contaminated soil, or to an area of contaminated soil surrounding the plant or plant seed. The recombinant member of the Bacillus cereus family is a recombinant member of the Bacillus cereus family as described in section II above, and the fusion protein includes a reclamation enzyme.

[00866] In methods for phytoremediation of contaminated soil that involve the use of exosporium fragments or a recombinant member of the Bacillus cereus family, as described above in section II, the targeting sequence, exosporium protein, or exosporium protein fragment may comprise any of the targeting sequences, exospore proteins, or exospore protein fragments described in the present description. In particular, the targeting sequence, exosporium protein, or exosporium protein fragment may include any of the targeting sequences, exosporium proteins, or exosporium protein fragments described in paragraphs [00782]-[00801] above.

[00867] The fusion protein can be expressed under the control of a sporulation promoter native to the targeting sequence, the exosporium protein or exosporium protein fragment of the fusion protein or portion thereof, and/or under the control of a highly expressed sporulation promoter. The promoter may be any of the promoters described in section III above.

[00868] The remediation enzyme is exposed on the outer region of the spores and in the plant so that both the plant and the spores can convert the target contaminant. The plant can capture the target pollutant while the spores convert the pollutant to a non-toxic or less toxic form in the plant or root system.

[00869] Spores of a recombinant member of the Bacillus cereus family may include an endophytic bacterial strain such as Bacillus cereus EE349, Bacillus cereus EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, or Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus cereus EE-B00377, Bacillus pseudomycoides EE-B00366 or Bacillus mycoides EE-B00363.

[00870] Spores or exospore fragments can be applied to a plant or plant seed, and the plant or plant grown from the plant seed is tolerant of the target contaminant to be eliminated from the contaminated soil.

[00871] In the phytoremediation method, recombinant members of the Bacillus cereus family undergo sporulation in the plant.

[00872] The spores of the recombinant member of the Bacillus cereus family can be introduced into the plant growth medium in various ways, such as by irrigating the soil at the time of planting. The spores can also be applied to the seed of a plant by treating the seed.

[00873] Preferably, the plant to be treated with the reclamation enzyme is tolerant of the target pollutant such that the plant is not damaged by the target pollutant.

[00874] The concentration of recombinant spores required for a phytoremediation method is based on factors including the volume or area to be treated, the ability of endophytic strains to colonize plant roots, the extent to which the target contaminant is present, and the amount of active enzyme on the surface of the spores.

[00875] There is another way to reduce the amount of impurities in the environment. The method includes exposing the polluted environment to spores of a strain of a member of the Bacillus cereus family, which is capable of degrading the herbicide or pesticide. Environmental contaminants include a herbicide, a pesticide, or a combination thereof. The Bacillus cereus strain that is capable of degrading the herbicide or pesticide includes Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120); Bacillus mycoides EE-B00363 (NRRL B-67121) or a combination thereof.

[00876] A strain of a member of the Bacillus cereus family that is capable of degrading a herbicide or pesticide may include a recombinant member of the Bacillus cereus family that expresses a fusion protein containing at least one protein or peptide of interest and a targeting sequence, an exospore protein or an exospore protein fragment, which target the fusion protein to the exospores of a recombinant member of the Bacillus cereus family. The protein or peptide of interest preferably comprises a herbicide-degrading enzyme, a pesticide-degrading enzyme, or a combination thereof.

[00877] In this way, dual pesticide or herbicide degrading activity can be achieved since both strains of members of the Bacillus cereus family and herbicide degrading or pesticide degrading enzymes in the fusion protein exhibit pesticide and/or herbicide degradation activity. Herbicides and/or pesticides that are degraded by a strain of the Bacillus cereus family that is capable of degrading the herbicide or pesticide may be the same or different from herbicides and/or pesticides that are degraded by the herbicide degrading enzyme or the pesticide degrading enzyme. Thus, when the environment is polluted with one type of herbicide or pesticide, a dual degrading effect against that one herbicide or pesticide can be achieved. Alternatively, when the environment is contaminated with more than one type of herbicide or pesticide, a dual degrading effect against two or more different herbicides or pesticides can be achieved.

[00878] In methods for reducing contaminants involving the use of one of the strains of members of the Bacillus cereus family described herein that are capable of degrading a herbicide or pesticide, the targeting sequence, exosporium protein, or exosporium protein fragment may comprise any of the targeting sequences, exosporium proteins, or fragments of exosporium proteins described in the present description. In particular, the targeting sequence, exosporium protein, or exosporium protein fragment may include any of the targeting sequences, exosporium proteins, or exosporium protein fragments described in paragraphs [00782]-[00801] above.

XXIV. Breakdown of emulsions or gels in fracturing fluid

[00879] A method of treating a fracturing fluid to break an emulsion or gel in the fluid is provided . The method includes adding spores of a recombinant member of the Bacillus cereus family to a fracturing fluid. A recombinant member of the Bacillus cereus family expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, an exosporium protein or exosporium protein fragment, that targets the fusion protein into the exospore of the spore of the recombinant member of the Bacillus cereus family. Such a fusion protein is described above, wherein the protein or peptide of interest includes an enzyme suitable for breaking an emulsion or gel.

[00880] A recombinant member of the Bacillus cereus family may include any recombinant member of the Bacillus cereus family described herein that expresses a fusion protein.

[00881] Another method of treating a fracturing fluid to break an emulsion or gel in the fluid is contemplated. The method includes adding exospore fragments to a fracturing fluid. The exosporium fragments are derived from the recombinant member of the Bacillus cereus family described in Section IV of this specification above and contain a fusion protein. The fusion protein contains an enzyme suitable for breaking the emulsion or gel.

[00882] The enzyme is selected based on the target emulsion or gel to be treated and the pH of the fracturing fluid. Enzymes include, but are not limited to, hemicellulase, amylase, pectinase, carbohydrate hydrolysis, cellulase, agarase, polygalacturonase, endoglucanase, or a combination thereof.

[00883] The emulsion or gel contains a polymer or other component that the enzyme can break down. The emulsion or gel may contain a polymer, gum arabic, agar, xanthan gum, cellulose, carboxymethylcellulose, carboxymethylhydroxyethylcellulose, hydroxyethylmethylcellulose, guar, a guar derivative, or a combination thereof.

[00884] When the protein or peptide of interest is an emulsion or gel breaking enzyme, any member of the Bacillus cereus family can be used to express the fusion protein. Bacillus thuringiensis or Bacillus mycoides are preferred.

[00885] Spores or exospore fragments can be injected into a well that is in contact with a subterranean hydrocarbon containing formation, such as a sandstone reservoir or a carbonate reservoir.

[00886] The required concentration of spores or exospore fragments 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 exospore fragments, and the presence and concentration of adjuvants delivered by the enzymes.

[00887] Enzymes can break down polymers or other components in the emulsion or gel, or can dissolve such components so that the fracturing fluid can be pumped out of the well.

[00888] Methods for treating a fracturing fluid to break an emulsion or gel in the fluid can use any of the targeting sequences, exospore proteins, or exospore protein fragments described above. In particular, the targeting sequence, exosporium protein, or exosporium protein fragment may include any of the targeting sequences, exosporium proteins, or exosporium protein fragments described in paragraphs [00759]-[00778] above.

[00889] The fusion protein can be expressed under the control of a sporulation promoter native to the targeting sequence, the exosporium protein or exosporium protein fragment in the fusion protein or portion thereof, and/or under the control of a highly expressed sporulation promoter. The promoter may be any of the promoters described in section III above.

XXV. Processing of raw materials

[00890] Raw materials are obtained from plants that are harvested for their biomass and processed into feed (draining, ensiling, extrusion, pelletizing, 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 facilitate the degradation of this fibrous material, resulting in a more degradable and easier to process material. Traditionally, enzymes are added after the raw material has been processed and delivered to the organism that consumes the raw material. Enzymes delivered in raw materials can improve the health and weight gain of target animals, as well as reduce the environmental impact of the waste products of animals fed such enzyme-supplemented feeds.

[00891] These same systems can be used to pre-treat feedstock destined for biofuel production, including processing into bioethanol, biodiesel, or other biofuels.

[00892] Many spore species have the ability to persist on aboveground plant surfaces such as leaves, stems and fruits for an extended period of time. Using spore display technologies as described herein to display enzymes on these spores, an active enzyme is delivered to the feedstock that will be present when the feedstock is harvested. These target enzymes can also be delivered to the raw plant at seeding, either by delivering the recombinant spores to the plant seeds or by delivering the recombinant spores to the plant growth medium or to the area around the plant.

[00893] A method for delivering enzymes to a plant is provided. The method includes introducing into a plant growth medium a recombinant member of the Bacillus cereus family that expresses a fusion protein containing at least one protein or peptide of interest and a targeting sequence, an exosporium protein or an exosporium protein fragment that targets the fusion protein into the exospore of the recombinant member of the Bacillus family cereus , or a formulation containing a recombinant member of the Bacillus cereus family as described herein; or applying to the plant, plant seed, or area surrounding the plant or plant seed, a recombinant member of the Bacillus cereus family or a formulation containing a recombinant member of the Bacillus cereus family. The protein or peptide of interest includes an enzyme. The enzyme may be physically associated with the exospore of a recombinant member of the Bacillus cereus family.

[00894] Another method for delivering enzymes to a plant is contemplated. The method includes adding to the plant growth medium a recombinant spore-forming bacterium or a composition containing a recombinant spore-forming bacterium; or applying to the plant, plant seed, area surrounding the plant or plant seed, a recombinant spore-forming bacterium or a composition comprising a recombinant spore-forming bacterium. 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 bacterial spore surface. The spore coat protein includes CotB protein, CotC protein, CgeA protein, CotB/H protein, CotG protein, X spore coat protein, or CotY protein. The recombinant spore-forming bacterium includes an endophytic strain of bacteria. The protein or peptide of interest includes an enzyme, and the enzyme is physically associated with the spore coat of the recombinant spore-forming bacterium.

[00895] Another method for delivering enzymes to a plant is contemplated. The method includes adding exosporium fragments or a composition containing exosporium fragments to a plant growth medium; or applying exosporium fragments or a composition containing exosporium fragments to the plant, plant seed, or an area surrounding the plant or plant seed. The exosporium fragments are derived from spores of the recombinant member of the Bacillus cereus family described in Section IV of this specification above and contain a fusion protein. The protein or peptide of interest includes an enzyme.

[00896] When a method for delivering enzymes to a plant involves the use of exosporium fragments, the method may further include treating the plant with a penetration enhancer, surfactant, detergent, oil, or a combination thereof.

[00897] Optimal bacterial strains for these methods include, but are not limited to, members of the Bacillus cereus family, including Bacillus cereus, Bacillus mycoides, Bacillus thuringiensis , and Bacillus pseudomycoides , as well as other spore-forming Bacillus , including Bacillus megaterium, Bacillus firmus, Bacillus flexus, members of the clade Bacillus subtilis , Bacillus pumilus, Bacillus licheniformis , and Bacillus subtilis .

[00898] The application can be carried out directly on the plant material, optionally in conjunction with adjuvants such as non-ionic or other surfactants. The recombinant member of the Bacillus cereus family can be applied to the aerial part of the plant prior to harvest, for example by spraying to the aerial part of the plant.

[00899] Application to a plant seed is typically in the form of a seed dipping liquid, slurry, or polymer-based seed coating. Optionally, the application can be carried out in conjunction with seed-applied inoculums, fungicides, insecticides, or nematicides.

[00900] Application to the plant growth medium or application to the area around the plant can be carried out before sowing, at sowing or after sowing the seeds, optionally in conjunction with fertilizers, fungicides, herbicides or insecticides.

[00901] The enzyme is useful for degradation of biomass, breakdown of cellulosic material, facilitating digestion in the digestive system of a target animal that can be plant fed, or for biofuel production (eg, bioethanol or biodiesel production).

[00902] The enzyme includes, but is not limited to, a non-specific protease, metalloproteinase, cellulase, xylanase, phosphatase, endoglucanase, exoglucanase, β-glucosidase, amylase, pectinase, xylosidase, lipase, phospholipase, or a combination thereof.

[00903] The choice of enzyme may depend on the feedstock and the intended use of the feedstock. Preferably the enzymes are degrading enzymes.

[00904] Protease family enzymes of interest include non-specific proteases such as serine proteases, histidine proteases, aspartate proteases, and metalloproteases.

[00905] Cellulase family enzymes of interest include exoglucanases, endoglucanases, β-1,3-glucosidases, cellulases, hemicellulases, a-glucosidases.

[00906] Enzymes of the xylanase family of interest include xylosidases, endoxylanases, exoxylanases, pectinases, methyl pectinases, polygalacturonase.

[00907] Phosphatase enzymes of interest include acid phosphatases, alkaline phosphatases, polyphosphatases, phytases, monophosphatases, and diphosphatases.

[00908] Many of these enzymes are also useful for plant growth.

[00909] These enzymes can not only "pre-digest" a portion of the feedstock to increase absorption of key nutrients by the target animal fed with the feedstock, but also promote digestion in the target animal's digestive system.

[00910] The "pre-cleavage" of cellulosic material upon collection can release free cellulose during processing for bioethanol and biofuel production, as well as pretreatment of oils intended for biofuel production.

[00911] The bacterium may be an endophytic bacterium. The choice of endophytic recombinant bacteria allows the bacteria to enter the plant, as well as to colonize and grow within the plant tissues. This will result in the establishment of an increasing number of recombinant spore-forming organisms within the plant as it grows after applying a relatively small amount of recombinant spores to seeds or seed application at planting. When harvesting plant biomass material, the bacteria will undergo sporulation, creating new enzymes in plants that will be active on the raw material when it is harvested, transported and used, for example, either as animal feed or biofuel production. This can significantly reduce the production cost of degrading enzymes compared to existing technologies. This is a unique way to deliver degrading enzymes to biomass prior to industrial processing.

[00912] Although the optimal bacterial strains are as described above, the selection of endophytic strains will increase efficiency. Preferably, the endophytic bacteria include Bacillus cereus EE349, Bacillus cereus 439, Bacillus thuringiensis EE417, Bacillus cereus EE444, Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillus fusiformis EE442, Lysinibacillus sphaericus EE443, or a combination thereof.

[00913] The plant may be a crop selected from corn, alfalfa, wheat, pasture crop, forage crop, soybean, millet, jicama, sugar sorghum, sugarcane, or a combination thereof, and other biofuel and bioethanol feedstocks.

[00914] For methods of delivering enzymes to a plant, any of the targeting sequences, exospore proteins, or exospore protein fragments described herein can be used.

XXVI. Application of spores to change the properties of target plants

[00915] Recombinant members of the Bacillus cereus family and recombinant spore-forming bacteria, as described herein, allow the interaction of surface-exposed signaling molecules that affect biochemical cascades and a number of other proteins that are beneficial to plant health. The presence of spore-exposed proteins or peptides can lead to a change in the metabolism of the target plant, resulting in a change in the composition of the plant, its fruits, or other properties or characteristics.

[00916] The expression of fusion proteins can be directly used to change the composition of the target plant. The choice of different enzymes results in different effects on the target plant.

[00917] A method for changing a property of a plant is provided. The method includes introducing into a plant growth medium a recombinant member of the Bacillus cereus family that expresses a fusion protein containing at least one protein or peptide of interest and a targeting sequence, an exosporium protein or an exosporium protein fragment that targets the fusion protein into the exospore of the recombinant member of the Bacillus family cereus, or a formulation containing a recombinant member of the Bacillus cereus family as described herein; or applying to the plant, plant seed, or area surrounding the plant or plant seed, a recombinant member of the Bacillus cereus family or a formulation containing a recombinant member of the Bacillus cereus family. The protein or peptide of interest includes a plant signaling molecule or an enzyme that affects plant composition, and the protein or peptide of interest may be physically associated with the exospore of a recombinant member of the Bacillus cereus family.

[00918] Another way of changing the properties of a plant is contemplated. The method includes adding to the plant growth medium a recombinant spore-forming bacterium or a composition containing a recombinant spore-forming bacterium; or applying to the plant, plant seed, or area surrounding the plant or plant seed, a recombinant spore-forming bacterium or a composition comprising a recombinant spore-forming bacterium. 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 spore surface of the bacterium. The spore coat protein includes CotB protein, CotC protein, CgeA protein, CotB/H protein, CotG protein, X spore coat protein, or CotY protein. The recombinant spore-forming bacterium includes an endophytic strain of bacteria. The protein or peptide of interest includes a plant signaling molecule or an enzyme that influences plant composition, and the protein or peptide of interest may be physically associated with the spore envelope of the recombinant spore-forming bacterium.

[00919] Another way of changing the properties of a plant is contemplated. The method includes adding exosporium fragments or a composition containing exosporium fragments to a plant growth medium; or applying exosporium fragments or a composition containing exosporium fragments to the plant, plant seed, or an area surrounding the plant or plant seed. The exosporium fragments are derived from spores of the recombinant member of the Bacillus cereus family described in Section IV of this specification above and contain a fusion protein. The protein or peptide of interest includes a plant signaling molecule or an enzyme that influences plant composition.

[00920] When the method of modifying a property of a plant involves the use of exospore fragments, the method may further include treating the plant with a penetration enhancer, surfactant, detergent, oil, or a combination thereof.

[00921] The target bacterium preferably survives or actively grows in the environment and on the roots of the target plant. Optimal bacterial strains for these methods include, but are not limited to, Bacillus cereus EE349, Bacillus cereus 439, Bacillus thuringiensis EE417 , Bacillus cereus EE444 , Bacillus megaterium EE385 , Bacillus sp. EE387 , Bacillus circulans EE388 , Bacillus subtilis EE405 , Lysinibacillus fusiformis EE442 or Lysinibacillus sphaericus EE443.

[00922] Plant signaling molecules or enzymes can also be delivered to the plant at seeding, either by delivering the recombinant spores to the plant seeds or by delivering the recombinant spores to the plant growth medium or to the area surrounding the plant.

[00923] Application can be carried out directly on the plant material, optionally in conjunction with adjuvants such as non-ionic or other surfactants. The recombinant member of the Bacillus cereus family or the recombinant spore-forming bacterium or exospore fragments can be applied to the aerial part of the plant prior to harvest, for example by spraying to the aerial part of the plant.

[00924] Application to a plant seed is typically in the form of a seed dipping liquid, slurry, or polymer-based seed coating. Optionally, the application can be carried out in conjunction with seed-applied inoculums, fungicides, insecticides, or nematicides.

[00925] Application to the plant growth medium or application to the area around the plant can be carried out before sowing, at sowing or after sowing the seeds, optionally in conjunction with fertilizers, fungicides, herbicides or insecticides.

[00926] The enzyme includes, but is not limited to, endoglucanases, proteases, phospholipases, aminocarboxy-1-propane deaminase, aminocyclopropane-1-carboxylic acid deaminases, lipases, or a combination thereof.

[00927] Plant signaling molecules include, but are not limited to, flg22 and flagellin peptides, cryptogein, harpins, harpin-like proteins, enzymes that degrade or modify a bacterial, fungal, or plant nutrient source, or a combination thereof.

[00928] Enzymes or plant signaling molecules can cause desired metabolic changes in the host plant, including increased uptake of macronutrients and micronutrients or changes in plant tissue composition by increasing root systems, increasing protein content in plants such as cereals, cereals and fruit plants, by modifying metabolism and increasing nitrogen uptake, and modifying the oil content of rapeseed, canola, soybean and sunflower, the sugar (sucrose) content of grapes, sugar cane, millet, sugar sorghum and other biofuel feedstocks, the content of medical compounds and the content of cannabinoids in marijuana. These changes not only increase the value of the plants of interest, but also increase the usefulness of these plants in various industries such as biofuel production, sugar production, and raw material production.

[00929] Any of the targeting sequences, exospore proteins, or exospore protein fragments described above can be used for methods of modifying a plant property.

XXVII. Disinfection

[00930] A method for disinfecting a surface is provided. The method includes exposing the surface of a recombinant member of the Bacillus cereus family that expresses a fusion protein as described in paragraphs [00170]-[00173] and [00177] of Section I above, wherein the protein or peptide of interest comprises an antibacterial protein or peptide.

[00931] Another method of surface disinfection is contemplated. The method includes exposure of the surface of exospore fragments. The exosporium fragments are derived from the recombinant member of the Bacillus cereus family described in Section IV of this specification above and contain a fusion protein. The fusion protein contains an antibacterial protein or peptide.

[00932] Another method of surface disinfection is contemplated. The method includes exposing the surface to a recombinant member of the Bacillus cereus family. A recombinant member of the Bacillus cereus family is a recombinant member of the Bacillus cereus family as described in section II above.

[00933] In surface disinfection methods that include exposing the surface to fragments of an exosporium or a recombinant member of the Bacillus cereus family, 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 fragments exospore proteins described in the present description. In particular, the targeting sequence, exosporium protein, or exosporium protein fragment may include any of the targeting sequences, exosporium proteins, or exosporium protein fragments described in paragraphs [00759]-[00778] above.

[00934] The fusion protein can be expressed under the control of a sporulation promoter native to the targeting sequence, the exosporium protein or exosporium protein fragment of the fusion protein or portion thereof, and/or under the control of a highly expressed sporulation promoter. The promoter may be any of the promoters described in section III above.

[00935] The antibacterial protein or peptide minimizes or prevents viral agents, bacteria, amoebae, parasites, or mold from forming on or binding to a surface.

[00936] An antibacterial protein or peptide includes, but is not limited to, proteases, nucleases, antimicrobial peptides, LysM, LfcinB, lysostaphin, albumin, defensins, bacteriocins, lipopeptides, innate immune system peptides, lysozyme, lyticase, or a combination thereof.

[00937] Recombinant Bacillus cereus spores can be used in conjunction with other antimicrobial agents, including disinfectants, cleansers, antibiotics, antifungals, and antivirals.

[00938] Although any member of the Bacillus cereus family can be used to express the fusion proteins, either Bacillus thuringiensis or Bacillus mycoides is preferred.

[00939] Any of the targeting sequences, exospore proteins, or exospore protein fragments described herein can be used for these methods.

[00940]

XXVIII. Other uses

[00941] Fusion proteins, where the protein or peptide of interest is an enzyme, or a recombinant representative of Bacillus cereus, where the protein or peptide of interest is an enzyme, can be used to process or degumming a lubricant, oil, or fat; processing hides into leather; production of biofuels, biodiesel or bioethanol; processing or conversion of sugars; starch processing; paper or linen processing; processing animal or fungal by-products or isolating amino acids; directed splitting of technical waste; food and feed additives; dietary supplements; animal feed; industrial cleaning; grain processing; cosmetic production; deodorization; food or beverage processing; optimizing brewing or brewing additives; detergent additives or fabric or yarn processing.

[00942] By displaying the enzyme on the outside of the spore or on exospore fragments, the enzyme can be stabilized, immobilized, or reapplied.

[00943] Industrial processes typically involve harsh conditions, including high temperatures, the presence of solvents, and large amounts of organic material. These conditions inhibit traditional enzymes. Expression of the target enzyme on the surface of spores or exosporium fragments provides resistance to high temperatures and harsh conditions and allows reisolation and reuse of enzymes.

[00944] Key enzymes of interest for such applications include: β-lactamases, proteases, lipases, phospholipases, cellulases, endoglucanases, exoglucanases, pectinases, ligninases, amylases (e.g., α-amylases, β-amylases, or glucoamylases), polygalacturonases, glucosidases , galactosidases, carbohydrate hydrolases, cell wall hydrolases, nucleases, hemicellulases, xylanases, mannases, laccases, lactases, esterases (e.g., pectin methylesterases), phytases, phosphatases, invertases, glucose oxidases, catalases, lyticases, acetolactate decarboxylases, and ureases.

[00945] Preferred enzymes for processing or degumming grease, oil or fat, or for cosmetic production include lipases, phospholipases, esterases, and proteases.

[00946] Preferred hide-to-leather enzymes include lipases, proteases, peptidases, collagenases, and phospholipases.

[00947] Preferred biofuel, biodiesel, or bioethanol production enzymes may include, but are not limited to, lipases and esterases.

[00948] Preferred sugar processing or conversion enzymes, for grain processing, and for textile and yarn processing include carbohydrate hydrolases, amylases, mannases, glucoamylases, invertases, cellulases, hemicellulases, pectinases, pectin methylesterases, xylanases, endoglucanases, exoglucanases, glucosidases, galactosidases, laccases, lactases, catalases and glucose oxidases.

[00949] Preferred starch processing enzymes include amylases and glucoamylases.

[00950] Preferred paper and linen processing enzymes include cellulases, hemicellulases, xylanases, endoglucanases, laccases, ligninases, exoglucanases, phytases, catalases, and glucosidases.

[00951] Preferred enzymes for processing animal or fungal by-products or isolating amino acids include proteases, peptidases, lipases, lyticases, cell wall hydrolases, phospholipases, endoglucanases, cellulases, glucanases, and carbohydrate hydrolases.

[00952] Preferred enzymes for targeting industrial waste, industrial cleaning, detergent additives, and deodorization include lipases, phospholipases, proteases, peptidases, amylases, lyticases, cell wall hydrolases, glucoamylases, cellulase, hemicellulases, xylanases, esterases, glucosidases, galactosidases, laccases , lactases, ureases, phosphatases and carbohydrate hydrolases.

[00953] Preferred food and feed additives, dietary supplements, animal feed, brewing additives, beverage additives or wine processing enzymes include mannases, laccases, lyticases, proteases, peptidases, carbohydrate hydrolases, pectinases, pectin methylesterases, esterases, lipases, cellulases, hemicellulases, xylanases, phytase, phosphatase, invertase, glucosidase, galactosidase, lactase, catalase, glucanase, endoglucanase, acetolactate decarboxylase and glucose oxidase.

[00954] While any member of the Bacillus cereus family can be used to express fusion proteins for these applications, either Bacillus thuringiensis or Bacillus mycoides is preferred.

[00955] The use of fusion proteins containing the enzyme as the protein or peptide of interest, or a recombinant member of the Bacillus cereus family expressing the fusion protein containing the enzyme as the protein or peptide of interest is contemplated. The fusion protein may be any of the fusion proteins described in paragraphs [00169]-[00173], [00175] and [00177] of Section I above. The application may be an application for treating or degumming a lubricant, oil or fat; processing hides into leather; production of biofuels, biodiesel or bioethanol; processing or conversion of sugars; starch processing; paper or linen processing; processing animal or fungal by-products or isolating amino acids; directed splitting of technical waste; food and feed additives; dietary supplements; animal feed; industrial cleaning; grain processing; cosmetic production; deodorization; food or beverage processing; optimizing brewing or brewing additives; detergent additives or fabric or yarn processing.

[00956] The use of exospore fragments is also contemplated. The application may be an application for treating or degumming a lubricant, oil or fat; processing hides into leather; production of biofuels, biodiesel or bioethanol; processing or conversion of sugars; starch processing; paper or linen processing; processing animal or fungal by-products or isolating amino acids; directed splitting of technical waste; food and feed additives; dietary supplements; animal feed; industrial cleaning; grain processing; cosmetic production; deodorization; food or beverage processing; optimizing brewing or brewing additives; detergent additives or fabric or yarn processing. The exosporium fragments are derived from the recombinant member of the Bacillus cereus family described in Section IV of this specification above and contain a fusion protein. The fusion protein contains an enzyme.

[00957] In addition, the use of a recombinant member of the Bacillus cereus family is contemplated. A recombinant member of the Bacillus cereus family is a recombinant member of the Bacillus cereus family as described above in section II. The application may be an application for treating or degumming a lubricant, oil or fat; processing hides into leather; production of biofuels, biodiesel or bioethanol; processing or conversion of sugars; starch processing; paper or linen processing; processing animal or fungal by-products or isolating amino acids; directed splitting of technical waste; food and feed additives; dietary supplements; animal feed; industrial cleaning; grain processing; cosmetic production; deodorization; food or beverage processing; optimizing brewing or brewing additives; detergent additives or fabric or yarn processing.

[00958] In the uses of exosporium fragments or recombinant members of the Bacillus cereus family 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 fragments described herein. In particular, the targeting sequence, exosporium protein, or exosporium protein fragment may include any of the targeting sequences, exosporium proteins, or exosporium protein fragments described in paragraphs [00759]-[00778] above.

[00959] The fusion protein can be expressed under the control of a sporulation promoter native to the targeting sequence, the exosporium protein or exosporium protein fragment of the fusion protein or portion thereof, and/or under the control of a highly expressed sporulation promoter. The promoter may be any of the promoters described in section III above.

[00960] After a detailed description of the invention, it will be obvious that modifications and variations are possible without deviating from the scope of the invention defined in the attached claims.

EXAMPLES

[00961] The following non-limiting examples are provided to further illustrate the present invention.

Example 1 Use of a Recombinant Family Member bacillus cereus, exhibiting lipase or endoglucanase, to stimulate the growth of soybean plants

[00962] Bacillus subtilis lipase and endoglucanase genes were amplified by polymerase chain reaction (PCR) using the following primers shown in Table 16:

TABLE 16

Lipase endoglucanase Straight ggatccatggctgaacacaatcc (SEQ ID NO: 37) ggatccatgaaacggtcaatc (SEQ ID NO: 39) Back ggatccttaattcgtattctggcc (SEQ ID NO: 38) ggatccttactaatttggttctgt (SEQ ID NO: 40)

[00963] To create fusion constructs, the genes were fused to the native bclA promoter from Bacillus thuringiensis DNA encoding the first 35 amino acids of BclA (amino acids 1-35 of SEQ ID NO: 1) using the overlapping extension (SOE) splicing technique. The correct amplicons were cloned into the E. coli/Bacillus pHP13 shuttle vector and the correct clones were screened by DNA sequencing. The correct clones were electroporated into Bacillus thuringiensis (Cry-, plasmid-) and screened for chloramphenicol resistance. The correct transformants were grown in brain-heart broth overnight at 30°C, plated on nutrient agar plates, and incubated at 30°C for 3 days. Spores expressing the fusion construct (BEMD spores) were collected from the plates by washing in phosphate buffered saline (PBS) and purified by centrifugation and additional washes in PBS. Untransformed control spores of Bacillus thuringiensis ( Bt .) were obtained in an identical manner.

[00964] Soybeans (Jake line 011-28-04) were sown to a depth of 2.54 cm in 10 cm deep pots filled with standard topsoil clay soil. The spores were diluted to a concentration of 1×10 ⁴ /ml in 50 ml of water and applied to each seed at sowing. Plants were grown under perfect light using T5, 54 watt lamps and exposed to light for 11 hours per day under controlled temperature conditions of 15.5-25.5°C. Plants were watered to saturation every three days throughout the two week trial. At the end of two weeks, the height of each plant was measured and the measurement results were normalized to those of control Bacillus thuringiensis spores. Conducted two independent tests.

[00965] The results are presented in Table 17 along with the standard error of the mean. In both trials, soybean plants grown in the presence of BEMD spores exhibiting either lipase or endoglucanase grew significantly taller than soybean plants treated with control Bt spores. (statistical analysis was performed using a t-test).

TABLE 17

soy plants Treatment Average height, cm Comparison with control SEM Trial #1 Control Bt 14.034 100.0% 0.521 Lipase, BEMD 17.93 127.8% 0.395 Endocellulase, BEMD 16.31 116.2% 0.411 Trial #2 Control Bt 15.39 100.0% 0.749 Lipase, BEMD 19.15 124.4% 0.428 Endocellulase, BEMD 17.65 114.7% 0.313

Example 2 Use of a Recombinant Family Member bacillus cereus, exhibiting endoglucanase, to stimulate the growth of corn plants.

[00966] Endoglucanase-expressing BEMD spores were prepared identically to that described in Example 1. Field corn was sown to a depth of 3.8 cm in 10 cm deep pots filled with standard top layer clay soil. Control spores and BEMD spores expressing endoglucanase were diluted to a concentration of 1×10 ⁴ /ml in 50 ml of water and applied to each plant at seeding. A control containing only water was also included. Plants were grown under perfect light using T5, 54 watt lamps and exposed to light for 11 hours per day under controlled temperature conditions of 15.5-25.5°C. The plants were watered to saturation every three days during the one week trial. At the end of one week, the growth of each plant was measured and the measurement results were normalized to those of control Bacillus thuringiensis spores.

[00967] The results are presented in Table 18 along with the standard error of the mean. Maize plants grown in the presence of BEMD spores exhibiting endoglucanase grew significantly taller than both soybean plants treated with Bt .

TABLE 18

Height, cm Comparison SEM _H2O 15.44 100% 0.318 bt 18.92 122.50% 0.645 BEMD, endoglucanase 22.71 143.40% 0.616

Example 3 Use of a Recombinant Family Member bacillus cereus, exhibiting endoglucanase or protease, to stimulate the growth of wheat plants.

[00968] BEMD spores expressing endoglucanase were prepared identically to that described in Example 1. BEMD spores expressing E. coli PtrB protease were prepared using methods similar to those described in Example 1 above and the following primers: ggatccatgctaccaaaagcc ( direct, SEQ ID NO: 41) and ggatccttagtccgcaggcgtagc (reverse, SEQ ID NO: 42).

[00969] Winter durum wheat was sown to a depth of 2.54 cm in 10 cm deep pots filled with standard top layer clay soil. Control spores and BEMD spores expressing endoglucanase or protease were diluted to a concentration of 1×10 ⁴ /ml in 50 ml of water and applied to each plant at seeding. A control containing only water was also included. Plants were grown under perfect light using T5, 54 watt lamps and exposed to light for 11 hours per day under controlled temperature conditions of 15.5-25.5°C. The plants were watered to saturation every three days during the one week trial. At the end of one week, the growth of each plant was measured and the measurement results were normalized to those of control plants treated with water only.

[00970] The results are presented in Table 19 along with the standard error of the mean. Wheat plants grown in the presence of BEMD spores exhibiting endoglucanase or protease grew significantly taller than soybean plants treated with control Bt spores. or control plants treated with water only (statistical analysis was performed using a t-test).

TABLE 19

Height, cm Comparison SEM _H2O 18.11 100% 0.721 Control Bt 19.96 110.33% 0.752 BEMD, endoglucanase 24.76 136.80% 0.21 BEMD, protease 22.35 123.40% 0.354

Example 4 Use of Recombinant Family Members Bacillus cereus , exhibiting endoglucanase, to stimulate the growth of chaff plants

[00971] Endoglucanase-expressing BEMD spores were prepared identically to that described in Example 1. Perennial chaff were sown at a depth of 6.4 mm in 10 cm deep pots filled with standard top layer clay soil. Control spores and BEMD spores expressing endoglucanase were diluted to a concentration of 1×10 ⁴ /ml in 50 ml of water and applied to each plant at seeding. A control containing only water was also included. Plants were grown under perfect light using T5, 54 watt lamps and exposed to light for 11 hours per day under controlled temperature conditions of 15.5-25.5°C. Plants were watered to saturation every three days throughout the two week trial. At the end of two weeks, the growth of each plant was measured and the measurement results were normalized to those of control plants treated with water only.

[00972] The results are presented in Table 20 along with the standard error of the mean. Chaff plants grown in the presence of endocellulase-exposing BEMD spores grew significantly taller than chaff plants treated with control Bt spores. or control plants treated with water only (statistical analysis was performed using a t-test).

TABLE 20

Height, cm Comparison SEM _H2O 11.43 100.0% 0.137 Control Bt 12.29 107.7% 0.128 BEMD, endoglucanase 12.78 111.9% 0.137

Example 5 Use of Recombinant Family Members Bacillus cereus , displaying enzymes involved in the synthesis or activation of plant hormones to stimulate plant growth

[00973] The BEMD system can also be used to display enzymes involved in plant hormone synthesis. For example, the plant hormone indole-3-acetic acid is a powerful plant growth stimulant. Indole-3-acetic acid is synthesized in vivo from tryptophan by the enzymes tryptophan monooxygenase and indole-3-acetamide hydrolase. Indole-3-acetic acid and other auxin hormones can also be synthesized in vivo from tryptophan and/or indole by the enzymes nitrilase, tryptophan aminotransferase, indole-3-acetaldehyde dehydrogenase, indole-3-pyruvate decarboxylase, amine oxidase, tryptophan decarboxylase, and tryptophan side chain oxidases.

[00974] The BEMD system can also be used to expose enzymes involved in the modification of plant growth hormones into biologically active or inactive forms. For example, nitrilase can be expressed on a BEMD system to catalyze the conversion of indole-3-acetonitrile to biologically active indole-3-acetic acid. In addition, inactive forms of plant hormones, such as indole-3-acetonitrile, can be added to the plant growth medium with BEMD-expressed nitrilase to provide a gradual release of the active hormone into the plant growth medium. Many other inactive or less active forms of plant hormones can be modified using appropriate enzymes.

[00975] Suitable plant growth hormones (auxins) include indole-3-pyruvic acid, indole-3-acetaldoxime, indole-3-acetamide, indole-3-acetonitrile, indole-3-ethanol, indole-3-pyruvate, indole- 3-butyric acid, phenylacetic acids, 4-chloroindole-3-acetic acid and indole-3-acetaldoxime. These hormones are synthesized from tryptophan and/or indole in vivo by the enzymes tryptophan monooxygenase, indole-3-acetamide hydrolase, nitrilase, nitrile hydrolase, acetolactate synthetase, alpha-acetolactate decarboxylase, tryptophan aminotransferase, indole-3-acetaldehyde dehydrogenase, indole-3-pyruvate decarboxylase, amine oxidase, and tryptophan decarboxylase. and tryptophan side chain oxidases.

[00976] It is also possible to synthesize growth hormones of the cytokine family using enzymes expressed in the BEMD system. Examples of cytokines include kinetin, zeatin (cis and trans), 6-benzylaminopurine, dihydroxyzeatin, N6-(D2-isopentenyl)adenine, ribosylzeatin, N6-(D2-isopentenyl)adenosine, 2-methylthio-cis-ribosylzeatin, cis-ribosylzeatin, ribosylzeatin-5-monophosphate, N6-methylaminopurine, N6-dimethylaminopurine, 2'-deoxyseatin riboside, 4-hydroxy-3-methyl-trans-2-butenylaminopurine, ortho-topolin, meta-topolin, benzyladenine, oprto-methyltopolin and meta-methyltopolin. These plant growth promoting compounds are synthesized in vivo from mevalonate or adenosine mono/di/triphosphate by enzymes including adenosine phosphate isopentenyltransferases, phosphatases, adenosine kinases, adenine phosphoribosyltransferase, CYP735A, 5'-ribonucleotide phosphohydrolase, adenosine nucleosidases, zeatin-cis-trans isomerase, zeafertinase-O-glucosyltransferases , β-glucosidases, cis-hydroxylases, CK cis-hydroxylases, CK N-glucosyltransferases, 2,5-ribonucleotide phosphohydrolases, adenosine nucleosidases, purine nucleoside phosphorylases, and zeatin reductases.

[00977] Using methods similar to those described in Example 1 above, any of these enzymes can be incorporated into a BEMD system for exhibiting BEMD spores by creating a fusion construct containing the enzyme and a targeting sequence that targets the expressed enzyme into the exospore when fused the construct is expressed in a member of the Bacillus cereus family. A recombinant member of the Bacillus cereus family expressing such a construct can then be added to soil or other plant growth medium or applied directly to the foliar part of the plant using methods similar to those described in Example 1 above to promote plant growth.

[00978] The plant growth medium can be supplemented with enzyme precursors or substrates. For example, the plant growth medium can be supplemented with tryptophan, adenosine monophosphates, adenosine diphosphates, adenosine triphosphates, or indole. Suitable concentrations of these substrates are from 100 nM to 100 μM.

Example 6 Use of Recombinant Family Members Bacillus cereus exhibiting proteases or peptidases that cleave proteins, peptides, proproteins, or preproproteins into biologically active peptides to promote plant growth

[00979] Proteases and peptidases can be expressed in a BEMD system that can enzymatically cleave available proteins in the plant growth medium into biologically active peptides that can act directly or indirectly on the plant. Examples include the enzymatic degradation of soy flour, yeast extract, or other protein-rich material added to a plant growth medium into active peptides that can directly stimulate plant growth. Bioactive peptides obtained by enzymatic digestion of proteinaceous material include RHPP and RKN 16D10, potent stimulators of plant root development. In addition, proproteins or preproproteins can be cleaved into active forms by BEMD-expressed proteases and peptidases into their biologically active forms. Inactive proproteins or preproproteins can be added to the plant growth medium to facilitate their gradual degradation by BEMD proteases and slow release of biologically active proteins.

[00980] Using methods similar to those described in Example 1 above, any of these proteases or peptidases can be included in the BEMD system for display on BEMD spores by creating a fusion construct containing the protease or peptidase and a targeting sequence that targets the expressed enzyme into exospores when the fusion construct is expressed in a member of the Bacillus cereus family. A recombinant member of the Bacillus cereus family expressing such a construct can then be added to soil or other plant growth medium supplemented with soy flour, yeast extract, or other protein-rich material to promote plant growth. Suitably, soy flour, yeast extract, or other protein-rich material is added to the plant growth medium in the form of a liquid composition containing from about 10 μg/L to about 100 mg/L of protein flour, yeast extract, or other protein-rich material.

Example 7 Use of BEMD Spores Expressing PtrB Protease to Promote Plant Growth.

[00981] BEMD spores expressing E. coli PtrB protease were prepared as described in Example 3 above. Soybean seeds were sown at a depth of 2.54 cm in 10 cm deep pots filled with standard top layer clay soil. Control spores and BEMD spores expressing the protease were diluted to a concentration of 1×10 ⁴ /ml in 50 ml of water and applied to each plant at seeding. A control containing only water was also included. Plants were grown under perfect light using T5, 54 watt lamps and exposed to light for 13 hours a day under controlled temperature conditions of 15.5-25.5°C. The plants were watered to saturation every three days during the one week trial. At the end of one week, the growth of each plant was measured and the measurement results were normalized to those of control plants treated with water only.

[00982] The results are presented in Table 21 along with the standard error of the mean as a percentage of the water-only control. Soybean plants grown in the presence of protease-exposing BEMD spores grew significantly taller than soybean plants treated with Bt . spore control or water-only control plants (statistical analysis was performed using t-test). The addition of soy flour to control plants treated with water or B. thuringiensis had little effect. In contrast, in the presence of soybean meal and the BEMD protease system, soybean plants responded substantially to all other treatments.

TABLE 21

Treatment soy flour Height (cm) After normalization to treatment with water only SEM, as a percentage of water-only treatment Only water Not 12.10 100% 3.1% Only water 25mg/pot 12.43 102.7% 7.4% B. thuringiensis Not 12.52 103.5% 5.2% B. thuringiensis 25mg/pot 11.99 99.1% 5.0% BEMD with protease Not 12.97 107.2% 6.1% BEMD with protease 25mg/pot 14.44 119.3% 4.8%

Example 8 Use of Recombinant Family Members Bacillus cereus expressing proteins or peptides involved in plant growth stimulation

[00983] The BEMD system can also be used to display proteins or peptides that are directly involved in promoting plant growth. For example, plant peptide hormones and non-hormonal peptides that stimulate plant growth can be expressed in a BEMD system. For example, non-hormonal peptides that directly bind and activate plant receptors can be expressed in the BEMD system to directly affect receptors in the target plant and roots. Such peptide hormones and non-hormonal peptides include phytosulfokine, calcalva 3 (CLV3), systemin, RKN 16D10, Hg-Syv46, eNOD40, NOD family proteins, ZmlGF, SCR/SP11 family proteins and peptides, RHPP, POLARIS and KTI. These peptides and related peptides can be expressed in a BEMD system and delivered to a plant growth medium or directly applied to the foliar portion to promote plant growth.

[00984] Using methods similar to those described in Example 1 above, any of these proteins or peptides can be incorporated into a BEMD system for display on BEMD spores by generating a fusion construct containing an enzyme and a targeting sequence that targets the expressed enzyme into exospores. when the fusion construct is expressed in a member of the Bacillus cereus family. A recombinant member of the Bacillus cereus family expressing such a construct can then be added to soil or other plant growth medium, or applied directly to the foliar part of the plant using methods similar to those described in Example 1 above to promote plant growth.

Example 9 Use of BEMD Spores Expressing POLARIS or KTI to Promote Plant Growth

[00985] BEMD spores expressing POLARIS plant peptide and KTI soybean peptide were generated by synthesizing genes encoding POLARIS or KIT peptides linked to the targeting sequence of SEQ ID NO: 96. The genes were then introduced into Bacillus thuringiensis and spores were obtained as described in Example 1. Soybean seeds were sown to a depth of 2.54 cm in 10 cm deep pots filled with standard top layer clay soil. Spores, BEMD expressing POLARIS or KTI, were diluted to a concentration of 1×10 ⁴ /ml in 50 ml of water and applied to each plant at planting. A control treated with water only was also included. Pure peptides POLARIS and KTI were also tested for their effects on soy at 0.05 mg/pot. Plants were grown under perfect light using T5, 54 watt lamps and exposed to light for 13 hours a day under controlled temperature conditions of 15.5-25.5°C. Plants were watered to saturation every three days throughout the two week trial. At the end of two weeks, the height of each plant was measured, the roots were measured, and the measurement results were normalized to those of control plants treated with water only.

[00986] The results are presented in Table 22 along with the standard error of the mean as a percentage of the result for the water-only control. Soybean plants grown in the presence of BEMD spores exhibiting POLARIS grew taller and had a slight increase in root development relative to water-treated control soybean plants. The presence of free KTI peptide led to a significant slowdown in plant growth with a loss of 6-8% of their height, but an increase of 15% in root length. Expression of KTI on the BEMD system resulted in a plant growth benefit, but no plant growth retardation effect. Importantly, the presence of Bacillus thuringiensis control spores with free KTI peptide did not interfere with the growth retardation effect of KTI, while BEMD with KTI did not exhibit such growth retardation.

TABLE 22

Treatment Peptide Roots after normalization to the results of water treatment SEM Height after normalization to the results of water treatment SEM Water Not 100% 6.8% 100% 4.3% Water KTI, 0.05mg/pot 115% 8.4% 91.8% 3.1% BEMD, POLARIS Not 106.3% 7.9% 107.3% 1.7% BEMD, KTI Not 113.3% 5.8% 99.4% 3.4% B. thuringiensis KTI, 0.05mg/pot 115% 7.7% 93.4% 4.2%

Example 10 Use of Recombinant Family Members Bacillus cereus that exhibit enzymes that degrade or modify a bacterial, fungal, or plant nutrient source to promote plant growth and/or nutrient utilization

[00987] The BEMD system can also be used to expose enzymes that degrade or positively modify a bacterial, fungal, or plant nutrient source found in soil or other plant growth media. Such enzymes degrade products present in the soil or other plant growth medium into forms that can be easily taken up by plants and/or beneficial bacteria and/or fungi in the rhizosphere. Such enzymes include, for example, glucoside hydrolases for the degradation of complex carbohydrates, cellulases for the degradation of cellulose; lipases for the degradation of lipids, including oils, fats and waxes; lignin oxidase for degradation of lignin and humic acids; proteases for degradation of polypeptides; phospholipases for membrane degradation; amidases and nitrogenases for nitrogen replenishment; amylases for starch processing; nucleases to replenish nucleotides, pectinases to destroy pectin, sulfatase to replenish sulfur, and xylanases to destroy xylans and arabinoxylans. The resulting products, including simple sugars, amino acids, fatty acids, and other nutrients, will be more readily available for direct uptake by plants and/or for stimulating beneficial bacteria and/or fungi to grow and develop in plant rhizospheres.

[00988] In addition, enzymes and other biological molecules can be used to release or store phosphate, nitrogen, and other key elemental nutrients for plant uptake from their various organic and inorganic forms in the soil. For example, phosphatases can be used to degrade phosphates in the environment into usable inorganic phosphates for plant utilization. The phosphates may be naturally occurring phosphates present in the plant growth medium. Alternatively or additionally, the plant growth medium can be supplemented with phosphates such as trimetaphosphate, a common agricultural additive. Examples of suitable phosphatases include phosphoric acid monoester hydrolases, phosphomonoesterases, phosphoric acid diesters hydrolases, phosphodiesterases, triphosphoric acid monoester hydrolases, phosphorylanhydride hydrolase, pyrophosphatase, phytase, trimetaphosphatase and triphosphatase. For example, the enzymes trimetaphosphatase, triphosphatase, and pyrophosphatase sequentially break down trimetaphosphate to usable inorganic phosphate.

[00989] The nitrogenase family of enzymes converts atomic nitrogen (N ₂ ) to ammonia, thereby converting nitrogen that would otherwise be inaccessible to plants into a usable form. Suitable enzymes belong to the Nif family of nitrogenases.

[00990] Chemical energy, such as adenosine-3-triphosphate, ferrodoxin, or additional enzymes that create such energy in the BEMD system, can also be added to the plant growth medium. They are cofactors for nitrogenases and are limited in soil. Thus, such cofactors can be added to the soil to enhance the reactions described above.

[00991] Other additives that can be added to the plant growth medium include starches, cellulose and cellulose derivatives, pectins, xylans and arabinoxylans, fats, waxes, oils, phytic acids, lignins, humic acids and other nutrient sources, in relation to which the enzymes described above are active.

[00992] Using methods similar to those described in Example 1 above, any of these enzymes can be incorporated into a BEMD system for display on BEMD spores by creating a fusion construct containing the enzyme and a targeting sequence to target the fusion construct to the exospore of a member of the Bacillus family cereus . The fusion construct can then be expressed in a member of the Bacillus cereus family, and the recombinant member of the Bacillus cereus family can be added to soil or other plant growth media using methods similar to those described in Example 1 above to promote plant growth.

Example 11 Use of Phosphatase-Expressing BEMD Spores to Promote Plant Growth

[00993] BEMD spores expressing Bacillus subtilis phosphatase A4 (PhoA4) were obtained by synthesizing the gene encoding PhoA4 linked to the targeting sequence of SEQ ID NO: 96. This gene was then introduced into Bacillus thuringiensis and spores were obtained as described in Example 1. Corn was sown to a depth of 2.54 cm in 10 cm deep pots filled with standard top layer clay soil. BEMD spores expressing PhoA4 were diluted to a concentration of 1×10 ⁴ /ml in 50 ml of water and applied to each plant at seeding. A control treated with water only was also included. Polyphosphate was added to the pots in liquid form at 0.5 mg/pot. Plants were grown under perfect light using T5, 54 watt lamps and exposed to light for 13 hours a day under controlled temperature conditions of 15.5-25.5°C. Plants were watered to saturation every three days throughout the two week trial. At the end of two weeks, the height of each plant was measured and the measurement results were normalized to those of control plants treated with water only.

[00994] The results are shown in Table 23. Corn grown in the presence of BEMD spores exhibiting PhoA4 shows enhanced growth, especially in the presence of added polyphosphate. This effect was higher than the effect of polyphosphate alone.

TABLE 23

Treatment Additive Growth, comparison with water Water Not 100% Water Polyphosphate 110.8% BEMD, PhoA4 Not 108.3% BEMD, PhoA4 Polyphosphate 114.8%

Example 12 Use of Recombinant Family Members Bacillus cereus exhibiting enzymes involved in the synthesis of 2,3-butanediol or the synthesis or activation of gibberellic acid to stimulate plant growth

[00995] The BEMD system can also be used to display enzymes involved in the synthesis of the plant growth promoting compound 2,3-butanediol. In vivo , 2,3-butanediol is synthesized by beneficial bacteria and fungi in the rhizosphere from acetoin, diacetyl, acetolactate, or pyruvate by the enzymes acetolactate synthetase, α-acetolactase decarboxylase, pyruvate decarboxylase, diacetyl reductase, butanediol dehydrogenases, and acetoin reductase.

[00996] The BEMD system can also be used to display enzymes involved in the synthesis or activation of the gibberellic acid plant growth promoting compound. Gibberellic acid can be produced from inactive or less active forms by the action of enzymes including, but not limited to, hydroxylamine reductase, 2-oxoglutarate dioxygenase, gibberelin 2B/3B hydrolase, gibberelin 3 oxidase, and gibberelin 20 oxidase.

[00997] Any of these enzymes can be incorporated into a BEMD system for display on BEMD spores using methods similar to those described in Example 1 above. A fusion construct can be made that contains the enzyme and a targeting sequence that targets the enzyme to the exospore when the fusion construct is expressed in a member of the Bacillus cereus family. The fusion construct is then expressed in a member of the Bacillus cereus family, and the member of the Bacillus cereus family is added to soil or other plant growth medium to promote plant growth.

[00998] To enhance the effect of enzymes exposed to BEMD, the soil can be supplemented with enzyme substrates. For example, 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 substrate for acetolactate decarboxylase. Alternatively or additionally, the soil or other plant growth medium may be supplemented with less potent or inactive forms of gibberellic acid, which is converted to more active forms by the enzymes described above in the soil or other plant growth medium.

Example 13 Use of Recombinant Family Members Bacillus cereus exhibiting proteases to protect plants from pathogens

[00999] The BEMD system can also be used to expose proteases that protect plants from one or more pathogens. For example, individual members of certain bacterial pathogens may interact through the secretion of bacterial homoserine lactones or related signaling molecules. Thus, proteases specific to the bacterial signaling molecules homoserine lactones can protect plants from such bacterial pathogens by disrupting interactions between bacteria, a step required for bacteria to secrete toxins and activate virulence factors. Suitable proteases specific for bacterial homoserine lactone signaling molecules include endopeptidases and exopeptidases.

[001000] Proteases specific to bacterial homoserine lactone signaling molecules can be incorporated into the BEMD system using methods similar to those described in Example 1 above. A fusion construct can be made that contains the protease and a targeting sequence that targets the protease to the exospore when the fusion construct is expressed in a member of the Bacillus cereus family. The fusion construct is then expressed in a member of the Bacillus cereus family and the member of the Bacillus cereus family is added to soil or other plant growth medium. The protease can then degrade the bacterial signaling molecules homoserine lactones, blocking a key step in the virulence of these organisms and thereby helping to protect the plant from these pathogens. The BEMD system is effectively affected by other proteases and peptidases with this ability, as demonstrated in Examples 6 and 7 above.

Example 14 Use of Recombinant Family Members Bacillus cereus exhibiting antimicrobial proteins and peptides to protect plants from pathogens

[001001] The BEMD system can also be used to display enzymes that exhibit antibacterial and/or antifungal activity, which can help protect plants from one or more pathogens. For example, antifungal proteins and peptides such as bacteriocins, lysozymes (eg, LysM), siderophores, avidins, streptavidins, conalbumin, albumin, lactoferrins (eg, LfcinB), or TasA can be expressed in the BEMD system to achieve their effect on bacterial and fungal plant pathogens. Bacteriocins, albumin, conalbumin, lysozymes, and lactoferrin show direct antimicrobial effects on their targets, while siderophores, avidins, and streptavidins bind essential nutrients that pathogens require for virulence. For example, the LfcinB peptide of lactoferrin, when expressed on the surface of a BEMD system, will lyse bacterial cells that are sensitive to lactoferrin peptides in plant growth media. These proteins and peptides have a specific effect on individual microorganisms and can selectively target a group of pathogens without killing all microorganisms in the plant growth medium.

[001002] Any of these proteins or peptides can be incorporated into a BEMD system for display on BEMD spores using methods similar to those described in Example 1 above. A fusion construct can be made that contains the enzyme and a targeting sequence that targets the enzyme to the exospore when the fusion construct is expressed in a member of the Bacillus cereus family. The fusion construct is then expressed in a member of the Bacillus cereus family and the member of the Bacillus cereus family is added to soil or other plant growth medium to protect the plants from one or more pathogens.

Example 15 Use of BEMD Spores Expressing Antimicrobial Peptides to Protect Plants from Bacteria

[001003] Genes were synthesized that encode 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 control of the BclA promoter (SEQ ID NO: 215). The genes were introduced intoBacillus thuringiensisBT013A and spores were obtained by growing a night culture of transformedbacillus into broth with brain-heart extract, plated on nutrient agar at 30°C and allowed to grow for 3 days. Spores were washed off the plates and rinsed with 3X PBS. culturesStaphylococcus epidermidis grown overnight in TSB broth at 37°C. The overnight culture was then pelleted, washed with PBS and resuspended in PBS at Abs595=0.2. 1×10⁴ BEMDs expressing LysM or LfcinB peptides were incubated in PBS withS. epidermidis for 3 hours at 37°C, with shaking. Control sampleS. epidermidis left untreated (no BEMD spores). After incubation for 3 hours, dilution plates were obtainedS. epidermidis and incubated at 37°C overnight. culturesS. epidermidis were counted the following day and percentage kill was quantified. Table 24 below shows kill activity data. BEMD Expressed Peptides Destroy Significant Numbers of CellsS. epidermidis. In the same way, bacteria will be destroyed in the rhizosphere, on seeds or other plant material. The choice of peptides that are specific to certain classes of bacteria can also provide a favorable shift of the microbial population near the plant or selective targeting of key pathogens.

Table 24

Treatment Survival % killed Not 100% 0% BEMD, LysM 71% 29% BEMD, LfcinB 23% 77%

Example 16 Use of Recombinant Family Members Bacillus cereus exhibiting enzymes to protect plants from pathogens

[001004] The BEMD system can also be used to display enzymes that protect plants from one or more pathogens. For example, yeast and mold cell walls are degraded by enzymes such as β-1,3-glucanases, β-1,4-glucanases, β-1,6-glucanases, chitosanases, chitinases, chitosanase-like proteins, and lyticases. Bacterial cell walls are degraded by enzymes selected from proteinases, proteases, mutanolysin, stafolysin, and lysozymes. Each of these cell wall degrading enzymes can be expressed in the BEMD system and added to plant growth media to selectively inhibit pathogens in the rhizosphere.

[001005] The BEMD system can also be used to display enzymes or proteins that protect plants from pathogenic insects or worms, for example by suppressing insect and/or worm predation on desired plants. Examples of such proteins and enzymes of interest include endotoxins, Cry toxins, other insecticidal protein toxins, protease inhibitors, cysteine proteases, Cry5B protein, Cry 21A protein, chitinase, protease inhibitor proteins, protease inhibitor peptides, trypsin inhibitors, arrowhead protease inhibitors.

[001006] Any of these proteins or peptides can be incorporated into a BEMD system for display on BEMD spores using methods similar to those described in Example 1 above. A fusion construct can be made that contains the enzyme and a targeting sequence that targets the enzyme to the exospore when the fusion construct is expressed in a member of the Bacillus cereus family. The fusion construct is then expressed in a member of the Bacillus cereus family and the member of the Bacillus cereus family is added to soil or other plant growth medium to protect the plants from pathogens.

Example 17 Use of BEMD Spores Expressing Antifungal Enzyme for Plant Protection and Demonstration of Efficacy Against Sacchromyces .

[001007] A gene was synthesized that encodes the antifungal enzyme β-1,3-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 introduced into Bacillus thuringiensis BT013A and pores were provided by growing an overnight culture of the transformed Bacillus in brain-heart broth, plating on nutrient agar at 30° C. and allowing to grow for 3 days. Spores were washed off the plates and rinsed with 3X PBS. Cultures of Saccharomyces cerevisiae were grown overnight in YZ broth at 37°C. The overnight culture was then pelleted, washed with PBS and resuspended in PBS at Abs595=0.2. 1×10 ⁴ BEMD expressing β-1,3-glucanase were incubated in PBS with Saccharomyces for 1 hour at 37°C, with shaking. The Saccharomyces control sample was left untreated (no BEMD spores). After incubation for 3 hours, Saccharomyces dilution plates were prepared and incubated at 37° C. overnight. Saccharomyces cultures were counted the following day and percentage kill was quantified. Table 25 below shows the killing activity of BEMD spores expressing β-1,3-glucanase. The enzyme expressed by BEMD killed a significant number of Saccharomyces cells. In the same way, the destruction of fungal microorganisms in the rhizosphere, on seeds or other plant material will occur. The choice of peptides specific to certain classes of fungi can also provide for shifting the microbial population near the plant in a favorable manner or selectively targeting key fungal pathogens.

Table 25

Treatment Survival % killed Not 100% 0% BEMD, β-1,3-glucanase 83% 17%

Example 18 Use of Recombinant Family Members Bacillus cereus exhibiting peptides or proteins that stimulate the plant immune system to protect plants from pathogens

[001008] The BEMD system can also be used to display peptides and proteins that enhance the immune system of plants. These proteins can be expressed on the outer surface of the BEMD spore and delivered to the plant growth medium to stimulate the plant's immune system to allow the plant to defend itself against plant pathogens. Exemplary proteins and peptides include harpin, α-elastins, β-elastins, systemins, phenylalanine ammonia lyase, elicitins, defensins, cryptogaine, and flagellin proteins and peptides. Exposure of plants to these proteins or peptides will stimulate plant resistance to many plant pathogens.

[001009] Any of these proteins or peptides can be incorporated into a BEMD system for display on BEMD spores using methods similar to those described in Example 1 above. A fusion construct can be made that contains the enzyme and a targeting sequence that targets the enzyme to the exospore when the fusion construct is expressed in a member of the Bacillus cereus family. The fusion construct is then expressed in a member of the Bacillus cereus family and the member of the Bacillus cereus family is added to soil or other plant growth medium to protect the plants from pathogens.

Example 19 Use of Recombinant Family Members Bacillus cereus exhibiting a protein or peptide that binds to roots or leaves to immobilize a recombinant member of the family Bacillus cereus on the root system of a plant or on the leaves of plants.

[001010] Root and leaf binding proteins and peptides can also be included in the BEMD system to allow BEMD spores to be immobilized on the root system or on the leaves of the plant. Exposure of such root or leaf-binding ligands to BEMD spores allows targeting of spores to the plant root system, or root system substructures, or leaves, or leaf substructures to maintain the BEMD litter in an optimal position so that other exposed biological molecules and enzymes are efficient.

[001011] For example, ricadhesin is a root-binding ligand that binds to root hairs. Thus, exposure of ricadhesin to BEMD spores targets the spores to root hairs. Additional proteins that can be used to selectively bind to plant roots or leaves include adhesins, flagellin, omptins, lectins, pilus proteins, curl proteins, intimins, invasins, agglutinin, afimbriae proteins, TasA or YuaB.

[001012] Such root or leaf binding proteins or peptides can be incorporated into the BEMD system using methods similar to those described in Example 1 above. A fusion construct can be prepared that contains a root or leaf binding protein or peptide and a targeting sequence, which targets a protein or peptide to exospores when the construct is expressed in a member of the Bacillus cereus family. The fusion construct containing the root- or leaf-binding ligand is then expressed in a member of the Bacillus cereus family. Such fusion constructs can be co-expressed with one or more additional fusion constructs containing any of the beneficial enzymes described herein (e.g., an enzyme involved in plant hormone synthesis, an enzyme that degrades a nutrient source, or proteases that protect a plant from pathogen). The recombinant member of the Bacillus cereus family is added to soil or other plant growth medium or applied to the leaves of a plant. The root- or leaf-binding ligand targets and immobilizes a member of the Bacillus cereus family to the plant root system or plant leaves, thereby allowing the co-expressed fusion construct to exhibit its effects near the root or leaf system.

Example 20 Use of Recombinant Family Members Bacillus cereus exhibiting proteins or enzymes to increase plant resistance to stress

[001013] Proteins, peptides, and enzymes that enhance plant stress tolerance can be incorporated into a BEMD system and delivered to target plants by application to roots, leaves, or addition to plant growth media. During the period of stress factors, plants release stress-related compounds, including aminocyclopropane-1-carboxylic acid (ACC), reactive oxygen species and others, which leads to a negative effect on plant growth. The BEMD system can be used to display enzymes that degrade stress-related compounds such as aminocyclopropane-1-carboxylic acid deaminase, superoxide dismutases, oxidases, catalase, and other enzymes that attack reactive oxygen species. Such enzymes reduce the amount of these compounds associated with exposure to stress factors and allow plants to continue to grow and even thrive under stressful conditions.

[001014] Any of these proteins or peptides can be incorporated into a BEMD system for display on BEMD spores using methods similar to those described in Example 1 above. A fusion construct can be made that contains the enzyme and a targeting sequence that targets the enzyme to the exospore when the fusion construct is expressed in a member of the Bacillus cereus family. The fusion construct is then expressed in a member of the Bacillus cereus family and the member of the Bacillus cereus family is added to the soil or other plant growth medium or applied to the leaves of the plant to increase the stress resistance of the target plant.

Example 21 Preparation of BEMD Spores Expressing the Protective Enzyme Catalase

[001015] Synthesized a gene that encodes a protective enzyme catalase fromBacillus cereusassociated with the BetA targeting sequence (SEQ ID NO: 97) under the control of the BetA promoter (SEQ ID NO: 197). This gene was introduced intoBacillus thuringiensisBT013A. Spores were obtained by growing a night culture of transformedbacillus and wild-type strain in brain-heart broth, plated on nutrient agar at 30° C. and allowed to grow for 3 days. Spores were washed off the plates and rinsed with 3X PBS. 3 drops of hydrogen peroxide were added to each sediment with spores. The enzyme catalase converts hydrogen peroxide into water and gaseous O₂. Control spores had no vesicles, while catalase BEMD spores had vesicles, demonstrating enzyme activity on the surface of the spores. Other protective enzymes can be similarly exposed and delivered to the plant to act on free radicals produced by plants when exposed to stressors.

Example 22 Use of Recombinant Family Members Bacillus cereus , displaying proteins or enzymes that protect seeds or plants from environmental stressors

[001016] Proteins, peptides, and enzymes that protect the plant from environmental stressors can be incorporated into the BEMD system and delivered to target plants by application to roots, leaves, fruits, or addition to the plant growth medium. During periods of frost, plants can be damaged by ice. The BEMD system can be used to expose peptides, proteins, or enzymes that protect plants from such effects. For example, the BEMD system can be used to expose choline dehydrogenases, which act to produce defense products that protect plants or seeds from frost. Substrates of these enzymes (eg choline and/or choline derivatives) can also be added to the plant growth medium. The addition of such substrates can increase the amount of the protective agent (betaine and related chemicals) produced in the plant environment by BEMD-expressed enzymes. Betaine derivatives are known to protect seeds from cold stress.

[001017] Any of these proteins or peptides can be incorporated into a BEMD system for display on BEMD spores using methods similar to those described in Example 1 above. A fusion construct can be made that contains the enzyme and a targeting sequence that targets the enzyme to the exospore when the fusion construct is expressed in a member of the Bacillus cereus family. The fusion construct is then expressed in a member of the Bacillus cereus family, and the member of the Bacillus cereus family is added to soil or other plant growth media or applied to the leaves of the plant to protect the plant from stress and environmental factors.

Example 23 Increased expression of fusion constructs on a BEMD system using boosted or alternative promoter elements

[001018] The BEMD system can display a wide range of proteins, peptides, and enzymes using one or more of the targeting sequences described herein. Some of these targeting sequences have high affinity for exospores, which may be useful for fusion protein expression, however, their low level of fusion protein expression limits their use in the BEMD system. For such fusion proteins and sequences, alternative highly expressed sporulation promoters can be used instead of native promoters.

[001019] For example, SEQ ID NO: 13 (amino acids 1-39 of the 3572 gene productB. weihenstephensis KBAB4) provides a highly efficient N-terminal sequence for protein delivery to exospores of members of the familyBacillus cereusas shown in Table 26 below. All genes were synthesized in their complete form (including promoter regions and regions encoding fusion proteins) as described herein. When elements of the native promoter for gene 3572B. weihenstephensis KBAB4 (SEQ ID NO: 217) was used to express a fusion protein containing the targeting sequence of SEQ ID NO: 13 fused to the enzyme β-galactosidase (fromE. coli), a low level of fusion protein was expressed, resulting in reduced enzyme activity on the spore surface. Enzyme activity was measured by converting 0.5 M o-nitrophenylgalactoside in solution for 10 minutes. Enzyme conversion was measured with a spectrophotometer in ABS₅₄₀. Replacement of native promoter elements of the 3572 geneB. weihenstephensis KBAB4 promoters highly expressed SEQ ID NO: 197 (BetA/BAS3290B. anthracis) or SEQ ID NO: 218 (β-propeller protein YVTNB. weihenstephensis KBAB4) led to a significant increase in the enzymatic activity of spores. On the other hand, the replacement of native promoter elements of the 3572 geneB. weihenstephensis KBAB4 promoter native toB. anthracis Sterne BAS1882 (SEQ ID NO: 216) resulted in a decrease in spore enzymatic activity. The expression level of the targeting sequence SEQ ID NO: 13 fused to β-galactosidase was significantly lower (0.38X) under the control of the BAS1882 promoter (SEQ ID NO: 216), and significantly increased under the control of the BetA promoter (SEQ ID NO: 197 ) or a YVTN protein promoter (SEQ ID NO: 218).

TABLE 26

promoter Fusion protein β-galactosidase activity on the BEMD system, normalized Multiplicity of change SEQ ID NO: 217 SEQ ID NO: 13 - β-galactosidase 100% SEQ ID NO: 197 SEQ ID NO: 13 - β-galactosidase 213.4% 2.13X SEQ ID NO: 218 SEQ ID NO: 13 - β-galactosidase 220.7% 2.21X SEQ NO:ID 216 SEQ ID NO: 13 - β-galactosidase 38.1% 0.38X

Example 24 Isolation and Identification of Plant Growth Promotion Bacterial Strains

[001020] Soil samples from the rhizospheres of the healthiest and most resistant potato ( Solanum tuberosum ), squash ( Cucurbita pepo ), tomato ( Solanum lycopersicum ), and vine bean ( Phaseolus coccineus ) plants were collected, diluted with sterile water, and plated onto nutrient agar plates. Bacterial isolates that exhibited the highest growth rates and were capable of passage and expansion were selected for further study. The selected strains were grown in a minimal medium (KH ₂ PO ₄ 3 g, Na ₂ HPO ₄ 6 g, NH ₄ Cl 1 g, NaCl 0.50 g, MgSO ₄ 7H ₂ O 0.15 g, CaCl ₂ 2H ₂ O 0.013 g and glucose 1 g per liter of dry weight). Overnight cultures (30°C) of the selected strains were pelleted by centrifugation, the medium was decanted, and they were resuspended in an equal amount of distilled water. Ten seeds of lettuce were seeded per treatment to a depth of 1 cm in topsoil clay soil (Columbia, MO) that had been sifted to remove large residues. Seeds were inoculated at inoculation in 4 cm pots with 0.5 µl of resuspended bacteria in water mixed with 10 ml of H ₂ O. Ten ml of H ₂ O was sufficient to deliver the bacteria to 3 in ³ (7.62 cm ³ ) of soil, and also to saturate the soil for proper seed germination. Plants were grown at 65-75°F (18-24°C) with 11 hours of light/day, and watered with 5 ml of water every 3 days. After one week, data were collected on plant growth and leaf diameter, as well as general plant health. Initial screening of rhizosphere isolates resulted in over 200 different species of bacteria and fungi from the rhizosphere of four plants. Some of the bacterial species are described in Table 27. The strains identified are indicated by their proper bacterial designations. Other strains are indicated by their unknown identification number. Inoculums that gave results close to the control (+/- 2%) are not included in the table.

TABLE 27

head lettuce Bacterial inoculum Average height (cm) Comparison SEM Without inoculation 1.8 The control 0.07 Paracoccus kondratiavae NC35 2 111.1% 0.05 B. aryabhattai CAP53 3.65 202.8% 0.45 B. flexus BT054 2.45 136.1% 0.11 Bacillus mycoides strain BT155 2.17 120.4% 0.21 B. aryabhattai CAP56 2.1 116.7% 0.20 B.nealsonii BOBA57 2.8 155.6% 0.03 E. cloacae CAP12 2.4 133.3% 0.41 Unknown 8 1.77 77.8% 0.65 Unknown 122 1.9 105.6% 0.11 Unknown 15 1.4 77.8% 0.41 Unknown 39 1.8 100.0% 0.20 Unknown 401 2 111.1% 0.21 Unknown 402 1.53 85.2% 0.27 Unknown 41 1.45 80.6% 0.31 Unknown 42 1.4 77.8% 0.15 Unknown 44 2.2 133.3% 0.08 Unknown 51 1.83 102.9% 0.21

[001021] The bacterial strains that caused the greatest effect on overall plant health and plant height in the initial lettuce trial were further identified. The bacterial strains were grown overnight in Luria-Bertani broth at 37° C. and the overnight cultures were centrifuged. The medium was decanted and the remaining bacterial pellet was subjected to chromosomal DNA isolation using a Qiagen bacterial chromosomal DNA isolation kit. Chromosomal DNA was subjected to PCR amplification of 16S rRNA-coding regions using primers E338F 5'-ACT CCT ACG GGA GGC AGC AGT-3' (SEQ ID NO: 298), E1099R A 5'-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 the Promega PCR Purification Kit and the resulting amplicons were diluted and sent to the University of Missouri DNA Core for DNA sequencing. The DNA sequences were compared to the NCBI BLAST database of bacterial isolates, and the genus and species were identified by direct comparison with known strains. The best species identified are shown in Table 27. In many cases, the 16S rRNA sequences were only able to indicate the genus of the selected bacterial strain. In cases where direct identification was not appropriate, additional biochemical analyzes were performed using methods standard in the art to distinguish between strains at the species and strain levels, and these are shown in Table 28.

TABLE 28

Trial E. cloacae CAP12 P. kondratiavae NC35 B. aryabhattai CAP53 B. flexus BT054 B. mycoides BT155 B. aryabhattai CAP56 B.nealsoni BOBA57 Urease - - - - - - + Catalase + + + + + + + oxidase - + + + - - - Nitrate + + - + + - + Height, 5% NaCl + - + + - + + Height, 7.5% NaCl - - + + - + - Altitude, 42°C + + + + + + + Altitude, 50°C - - + + - + - Height, pH 5 + - + + - + - Height, pH 9 + + + + + + + acid, cellobiose + - + + + + - acid, lactose + - + + + - + acid, starch - - - + - + -

Example 25 Isolation and Identification of Additional Plant Growth Promoting Bacterial Strains

[001022] Soil samples from agricultural fields near Gas, Kansas were collected, diluted with sterile water, and plated on nutrient agar plates. Bacterial isolates that exhibited the highest growth rates and were capable of passage and expansion were selected for further study. The selected strains were grown in minimal media (KH ₂ PO ₄ 3 g, Na ₂ HPO ₄ 6 g, NH ₄ Cl 1 g, NaCl 0.50 g, MgSO ₄ 7H ₂ O 0.15 g, CaCl ₂ 2H ₂ O 0.013 g and glucose 1 g per liter of dry weight). Overnight cultures (30°C) of the selected strains were pelleted by centrifugation, the medium was decanted, and they were resuspended in an equal amount of distilled water. Corn seeds were coated with commercial seed polymer mixed with water only (total 1.6 μl per seed) or commercial polymer containing selected bacterial strains (total 1.6 μl per seed). Coated seeds were sown in pots with a diameter (3 inches) of 7.62 cm to a depth of 1 inch (2.54 cm) in topsoil clay soil (Columbia, MO) that had been sifted to remove large residues. Plants were grown at 18-24°C (65-75°F) with 11 hours of light/day, and watered with 50 ml of water at seeding and every 3 days. After two weeks, data were collected on plant growth and leaf diameter, as well as general plant health. For germination and root length assays after 3 days, the seeds were coated as described above and evenly distributed 10 seeds per paper towel. Paper towels were moistened with 10 ml of water, wrapped, placed in a small plastic bag and incubated at 30°C or placed on a heated germination mat at 27-30°C (80-85°F). Root sizes were recorded after 3 days. Initial screening of rhizosphere isolates resulted in over 100 different species of bacteria and fungi from the rhizosphere. Some of the bacterial species are described in Table 29. The strains identified are indicated by their proper bacterial designations.

TABLE 29

Corn Seed Processing Bacterial inoculum Mean height (2 weeks) normalized to control polymer (%) Average root length (3 days) normalized to control polymer Control polymer 100 100 B. mycoides EE118 111.1 189.1 B. subtilis EE148 99.4 172.8 Alcaligenes faecalis EE107 111.5 129.2 B. mycoides EE141 109.2 143.5 B. mycoides BT46-3 105.6 141.3 Representative of the family B. cereus EE128 105.6 - B. thuringiensis BT013A 101.8 103.8 Paenibacillus massiliensis BT23 104.2 139.4 Representative of the family B. cereus EE349 105.2 - B. subtilis EE218 106.6 - B. megaterium EE281 107.8 -

[001023] The bacterial strains that caused the greatest effect on the overall health of the plant are described in Table 29. The bacterial strains were grown overnight in Luria-Bertani broth at 37°C and the overnight cultures were centrifuged. The medium was decanted and the remaining bacterial pellet was subjected to chromosomal DNA isolation using a Qiagen bacterial chromosomal DNA isolation kit. Chromosomal DNA was subjected to PCR amplification of 16S rRNA-coding regions using primers E338F 5'-ACT CCT ACG GGA GGC AGC AGT-3' (SEQ ID NO: 298), E1099R A 5'-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 the Promega PCR Purification Kit and the resulting amplicons were diluted and sent to the University of Missouri DNA Core for DNA sequencing. The DNA sequences were compared to the NCBI BLAST database of bacterial isolates, and the genus and species were identified by direct comparison with known strains. The best species identified are shown in Table 16. In many cases, the 16S rRNA sequences were only able to indicate the genus of the selected bacterial strain. In cases where direct identification was not appropriate, additional biochemical analyzes were performed using methods standard in the art to distinguish between strains at the species and strain level, and the identified strains are shown in Table 30.

TABLE 30

Trial B. thuringiensis BT013A Representative of the family B. cereus EE349 B. subtilis EE148 B. subtilis EE218 B. megaterium EE281 Paenibacillus massiliensis BT23 B. mycoides BT46-3 Alcaligenes faecalis EE107 B. mycoides EE118 Representative of the family B. cereus EE128 B. mycoides EE141 Mobility + + + + + + - + - - - Rhizoid colony - - - - - + + - + - + catalase + + + + + + + + + + + oxidase + - - - - - - + - - - Nitrate + + wk - - - + + + + + Height, 5% NaCl + wk - + + - + + - + - Height, 7.5% NaCl wk - - + + - - - - - - Altitude, 42°C - + + + + + + + - + - Height, 50°C - - - - - - - - - - - Height, pH 5 wk - + + + - wk + - + - Height, pH 9 + + - + + - wk + + + - acid, cellobiose - - wk + - + + wk + - wk acid, lactose - + + + + - + + - + wk acid, starch - + - + + - + wk + + -

wk=growth within a week or low rise

Example 26 Study of Plant Growth Stimulating Bacterial Strains on Alfalfa

[001024] Selected strains were grown in minimal medium (KH ₂ PO ₄ 3 g, Na ₂ HPO ₄ 6 g, NH ₄ Cl 1 g, NaCl 0.50 g, MgSO ₄ 7H ₂ O 0.15 g, CaCl ₂ 2H ₂ O 0.013 g and glucose 1 g per liter of dry weight). Overnight cultures (30°C) of the selected strains were pelleted by centrifugation, the medium was decanted, and the bacteria were resuspended in an equal amount of distilled water. Ten Zeba-coated alfalfa seeds were sown per treatment to a depth of 0.6 cm in top layer clay soil (Columbia, MO) that had been sieved to remove large residues. Seeds were inoculated at plating with 0.5 µl of resuspended bacteria in water mixed with 10 ml of H ₂ O. Ten ml of H ₂ O was sufficient to deliver the bacteria to 3 in ³ (7.62 cm ³ ) of soil and also to saturate the soil for proper seed germination. Plants were grown at 65-75°F (18-24°C) with 11 hours of light/day and watered with 5 ml of water every 3 days. Alfalfa was allowed to grow for 1 week to analyze the emergence and initial germination of plants under the described conditions. Identified strains, indicated by their proper bacterial designations, and final height data are shown in Table 31.

TABLE 31

Alfalfa Bacterial inoculum Average height (cm) Comparison SEM Without inoculation 4.82 - 0.008 B. aryabhattai CAP56 4.85 101.20% 0.016 B.nealsonii BOBA57 4.86 101.70% 0.021 E. cloacae CAP12 5.6 116.23% 0.020

Example 27 Study of Plant Growth Stimulating Strains on Cucumbers

[001025] Selected strains were grown in minimal medium (KH ₂ PO ₄ 3 g, Na ₂ HPO ₄ 6 g, NH ₄ Cl 1 g, NaCl 0.50 g, MgSO ₄ 7H ₂ O 0.15 g, CaCl ₂ 2H ₂ O 0.013 g and glucose 1 g per liter of dry weight). Overnight cultures (30°C) of the selected strains were pelleted by centrifugation, the medium was decanted and resuspended in an equal amount of distilled water. Ten cucumber seeds were sown per treatment to a depth of 1 cm in topsoil clay soil (Columbia, MO) that had been sifted to remove large residues. Seeds were inoculated at plating with 0.5 µl of resuspended bacteria in water mixed with 10 ml of H ₂ O. Ten ml of H ₂ O was sufficient to deliver the bacteria to 3 in ³ (7.62 cm ³ ) of soil and also to saturate the soil for proper seed germination. Plants were grown at 65-75°F (18-24°C) with 11 hours of light/day, and watered with 5 ml of water every 3 days. Cucumbers were allowed to grow for 2 weeks to analyze the emergence and initial germination of plants under the described conditions. Identified strains, indicated by their proper bacterial designations, and final height data are shown in Table 32.

TABLE 32

cucumbers Bacterial inoculum Average height (cm) Comparison SEM Without inoculation 11.23 - 0.067 B. aryabhattai CAP53 11.5 102.00% 0.023 B. aryabhattai CAP56 11.35 101.20% 0.035 B.nealsonii BOBA57 11.33 101.10% 0.014

Example 28. Study of bacterial strains that accelerate plant growth on squash

[001026] Selected strains were grown in minimal medium (KH ₂ PO ₄ 3 g, Na ₂ HPO ₄ 6 g, NH ₄ Cl 1 g, NaCl 0.50 g, MgSO ₄ 7H ₂ O 0.15 g, CaCl ₂ 2H ₂ O 0.013 g, and glucose 1 g per liter of dry weight). Overnight cultures (30°C) of the selected strains were pelleted by centrifugation, the medium was decanted, and they were resuspended in an equal amount of distilled water. Squash seeds were sown per treatment to a depth of 1 cm in a topsoil clay soil (Columbia, MO) that had been screened to remove large residues. Seeds were inoculated at inoculation with 0.5 µl of resuspended bacteria in water mixed with 10 ml of H ₂ O. Ten ml of H ₂ O was sufficient to deliver the bacteria to 3 in ³ (7.62 cm ³ ) of soil and also to saturate the soil for proper seed germination. Plants were grown at 65-75°F (18-24°C) with 11 hours of light/day, and watered with 5 ml of water every 3 days. The squash was allowed to grow for 2 weeks to analyze the emergence and initial germination of plants under the described conditions. The identified strains, indicated by their proper bacterial designations, end height data, and end leaf diameter data (based on the width of two leaves) are shown in Table 33.

TABLE 33

Squash Bacterial inoculum Average height (cm) Comparison SEM Leaf diameter (cm) Comparison Without inoculum 10.16 - 0.028 5.08 - B. aryabhattai CAP53 11.75 115.60% 0.055 7.25 142.60% B. flexus BT054 11.88 116.90% 0.017 6.36 125.20% Bacillus mycoides BT155 11.92 117.20% 0.051 6.33 124.60% B. aryabhattai CAP56 11.95 117.60% 0.027 6.33 124.60% B.nealsonii BOBA57 11.89 117.00% 0.118 6.42 126.40% E. cloacae CAP12 11.42 112.30% 0.039 6.83 134.40%

Example 29. Study of bacterial strains that stimulate plant growth on a chaff.

[001027] Selected strains were grown in minimal medium (KH ₂ PO ₄ 3 g, Na ₂ HPO ₄ 6 g, NH ₄ Cl 1 g, NaCl 0.50 g, MgSO ₄ 7H ₂ O 0.15 g, CaCl ₂ 2H ₂ O 0.013 g, and glucose 1 g per liter of dry weight). Overnight cultures (30°C) of the selected strains were pelleted by centrifugation, the medium was decanted, and they were resuspended in an equal amount of distilled water. Thirty chaff seeds were sown per treatment to a depth of 0.3 cm in top layer clay soil (Columbia, MO) that had been sifted to remove large residues. Seeds were inoculated at inoculation with 0.5 µl of resuspended bacteria in water mixed with 10 ml of H ₂ O. Ten ml of H ₂ O was sufficient to deliver the bacteria to 3 in ³ (7.62 cm ³ ) of soil and also to saturate the soil for proper seed germination. Plants were grown at 65-75°F (18-24°C) with 11 hours of light/day, and watered with 5 ml of water every 3 days. The tares were allowed to grow for 1.5 weeks to analyze the emergence and initial germination of plants under the described conditions. Identified strains, indicated by their proper bacterial designations, and height data are shown in Table 34.

TABLE 34

Chaff Bacterial inoculum Average height (cm) Comparison SEM Without inoculum 1.61 - 0.023 B. aryabhattai CAP53 2.01 124.70% 0.012 B. flexus BT054 2.21 137.30% 0.034 Bacillus mycoides BT155 2.29 142.20% 0.049 B. aryabhattai CAP56 2.19 136.00% 0.009 B.nealsonii BOBA57 2.29 142.40% 0.045 E. cloacae CAP12 1.98 122.50% 0.015

Example 30 Study of Plant Growth-Promoting Bacterial Strains in Maize

[001028] Selected strains were grown in minimal medium (KH ₂ PO ₄ 3 g, Na ₂ HPO ₄ 6 g, NH ₄ Cl 1 g, NaCl 0.50 g, MgSO ₄ 7H ₂ O 0.15 g, CaCl ₂ 2H ₂ O 0.013 g, and glucose 1 g per liter of dry weight). Overnight cultures (30°C) of the selected strains were pelleted by centrifugation, the medium was decanted, and they were resuspended in an equal amount of distilled water. Ten seeds of corn were sown per treatment to a depth of 2.5 cm in topsoil clay soil (Columbia, MO) that was sieved to remove large residues. Seeds were inoculated at plating with 0.5 µl of resuspended bacteria in water mixed with 10 ml of H ₂ O. Ten ml of H ₂ O was sufficient to deliver the bacteria to 3 in ³ (7.62 cm ³ ) of soil and also to saturate the soil for proper seed germination. Plants were grown at 65-75°F (18-24°C) with 11 hours of light/day, and watered with 5 ml of water every 3 days. The corn was allowed to grow for 2 weeks to analyze the emergence and initial germination of plants under the described conditions. Identified strains, indicated by their proper bacterial designations, and final height data are shown in Table 35.

TABLE 35

Corn Bacterial inoculum Average height (cm) Comparison SEM Without inoculation 8.9 - 0.039 B. aryabhattai CAP53 11.01 123.60% 0.081 B. flexus BT054 9.96 112.00% 0.095 Bacillus mycoides strain BT155 9.6 107.90% 0.041 B. aryabhattai CAP56 9.54 107.10% 0.088 B.nealsonii BOBA57 9.23 103.70% 0.077

Example 31 Study of Plant Growth Promotion Bacterial Strains on Soy

[001029] Selected strains were grown in minimal medium (KH ₂ PO ₄ 3 g, Na ₂ HPO ₄ 6 g, NH ₄ Cl 1 g, NaCl 0.50 g, MgSO ₄ 7H ₂ O 0.15 g, CaCl ₂ 2H ₂ O 0.013 g, and glucose 1 g per liter of dry weight, or for Bradyrhizobium or Rhizobium on yeast medium with mannitol). Overnight cultures (30°C) of the selected strains were pelleted by centrifugation, the medium was decanted, and they were resuspended in an equal amount of distilled water. Ten soybean seeds were sown per treatment to a depth of 2.5 cm in topsoil clay soil (Columbia, MO) that had been screened to remove large residues. Seeds were inoculated at inoculation with 0.5 µl of resuspended bacteria in water mixed with 10 ml of H ₂ O. In the study of two bacterial strains, 0.5 µl of each resuspended bacteria was mixed with 10 ml of H ₂ O. Ten ml of H ₂ O was sufficient for delivering bacteria to 3 inches ³ (7.62 cm ³ ) of soil, as well as to saturate the soil for proper seed germination. Plants were grown at 65-75°F (18-24°C) with 11 hours of light/day, and watered with 5 ml of water every 3 days. Soybeans were allowed to grow for 2 weeks to analyze the emergence and initial germination of plants under the described conditions. The identified strains, indicated by their proper bacterial designations, and the final height data are shown in Table 36. Consistent inoculation of bacterial strains within the scope of the present invention with representatives of Bradyrhizobium sp. or Rhizobium sp. resulted in increased plant growth compared to each inoculum alone.

TABLE 36

Soya Bacterial inoculum Average height (cm) Comparison SEM Without inoculation 13.94 - 0.089 B. aryabhattai CAP53 16.32 117.1% 0.146 B. flexus BT054 17.85 128.0% 0.177 Bacillus mycoides strain BT155 18.93 135.8% 0.117 B. aryabhattai CAP56 17.23 123.6% 0.133 B. aryabhattai CAP53 16.32 117.1% 0.077 B. aryabhattai CAP53 and Bradyrhizobium sp. 16.72 119.9% 0.182 B. aryabhattai CAP53 and Rhizobium sp. 17.32 124.2% 0.086 Bradyrhizobium sp. 14.25 102.2% Rhizobium sp. 14.75 105.8%

Example 32. Representatives of the family Bacillus cereus with signs of plant growth stimulation

[001030] Bacillus mycoides strain BT155, Bacillus mycoides strain EE118, Bacillus mycoides strain EE141, Bacillus mycoides strain BT46-3, Bacillus cereus strain EE349, Bacillus thuringiensis strain BT013A, and Bacillus megaterium strain EE281 were grown in Luria-Bertani broth at 37°C. C and overnight cultures were pelleted by centrifugation, the medium was decanted, and they were resuspended in an equal amount of distilled water. 20 seeds of corn were sown per treatment to a depth of 2.5 cm in topsoil clay soil (Columbia, MO) that had been screened to remove large residues. Seeds were inoculated at plating with 0.5 µl of resuspended bacteria in water mixed with 50 ml of H ₂ O. Fifty ml of H ₂ O was sufficient to deliver the bacteria to 29 in ³ (442.5 cm ³ ) of soil and also to saturate the soil for proper seed germination. Plants were grown at 65-72°F with 13 hours of light/day, and watered with 5 ml of water every 3 days. The seedlings were allowed to grow for 2 weeks to analyze the emergence and initial germination of plants under the described conditions. Identified strains, indicated by their proper bacterial designations, and final height data are shown in Table 37.

TABLE 37

Bacterial inoculum Average height, cm, corn Percent sem, Control as H ₂ O 11.41 100% 0.123 B. mycoides EE118 12.43 108.9% 0.207 B. mycoides EE141 12.84 112.5% 0.231 B. mycoides BT46-3 11.81 103.5% 0.089 Bacillus thuringiensis BT013A 12.05 105.6% 0.148 Representative of the Bacillus cereus EE128 family 13.12 114.9% 0.159 Bacillus mycoides BT155 12.85 112.6% 0.163 Bacillus megaterium EE281 11.99 105.1% 0.098

[001031] All plant growth promoting bacteria tested had a beneficial effect on corn height after two weeks under the described conditions. A member of the Bacillus cereus family, strain EE128, had the best effect in this trial, producing an increase in corn height of more than 14%.

Example 33. Enhanced selection of family members Bacillus cereus as BEMD hosts for expression to screen for plant growth promoting activity and other beneficial activities

[001032] The BEMD system can be used to display 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 achieved by selecting an expression host (a member of the Bacillus cereus family) that has inherent beneficial properties. Many strains of members of the Bacillus cereus family have the benefit of accelerating plant growth. In addition, many strains of members of the Bacillus cereus family have protective effects through direct fungicidal, insecticidal, nematicidal or other protective activity. When such strains are used as expression hosts for the BEMD system, the final spore product will have a combination of agriculturally beneficial traits.

[001033] Table 38 shows the results of an experiment where the fusion protein was expressed in various strains of members of the Bacillus cereus family. All strains expressed a fusion protein containing amino acids 1-35 of SEQ ID NO: 1 and PhoA4 phosphatase from Bacillus subtilis , a useful enzyme for enhancing phosphate uptake in maize. The gene was synthesized, cloned into the pMK4 vector, and introduced into each of the Bacillus spp. listed in Table 38. The strains were sporulated by incubation at 30°C on nutrient agar plates containing 10 μg/ml chloramphenicol for three days. The spores were harvested, washed and applied to corn at seeding at 1×10 ⁵ cfu/ml in 50 ml water per 7.62 cm pot with 5 mg polyphosphate per pot. Corn was grown in silt-clay soil for two weeks. Plants were grown under perfect light using T5, 54 watt lamps and exposed to 13 hours of light per day under controlled temperature conditions of 15.5-25.5°C. Plants were watered to saturation every three days throughout the two week trial. At the end of two weeks, the height of each plant was measured and the measurement results were normalized to those of control Bacillus thuringiensis spores. Expression of the SEQ ID NO: 1-phosphatase fusion protein resulted in an increase in corn height after 2 weeks, regardless of the chosen expression host strain. As shown in Table 38, the use of a growth promoting member of the Bacillus cereus family further increased the height of the corn.

TABLE 38

Species Bacillus Strain Fusion protein Height after 2 weeks, normalized B. thuringiensis Strain BT013A Not 100% B. thuringiensis Strain BT013A SEQ ID NO: 1-phosphatase 117.4% B. mycoides Strain EE141 Not 107.3% B. mycoides Strain EE141 SEQ ID NO: 1-phosphatase 123.3% Member of the B. cereus family Strain EE128 Not 124.1% Member of the B. cereus family Strain EE128 SEQ ID NO: 1-phosphatase 131.7% B. mycoides Strain BT155 Not 104.8% B. mycoides Strain BT155 SEQ ID NO: 1-phosphatase 121.9%

Example 34 Use of different targeting sequences for surface expression of β-galactosidase Bacillus thuringiensis

[001034] A wide variety of targeting sequences can be used to display enzymes, proteins, and peptides on the surface of members of the Bacillus cereus family, which have a high degree of homology to amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1). Several targeting sequences were compared by generating fusion proteins containing targeting sequences associated with Bacillus subtilis lipase. Fusion constructs were synthesized using promoters native to the targeting sequence, cloned into the replicating plasmid pMK4, and introduced into Bacillus thuringiensis BT013A. The strains were sporulated by incubation at 30° C. on nutrient agar plates containing 10 μg/ml chloramphenicol for 3 days. Spores were collected, washed and resuspended in PBS at 1×10 ⁸ /ml. 1x10 ⁵ each spore spores with the fused construct were suspended in 400 μl dH ₂ O. The reaction mixtures were heated with the reaction components to the desired reaction temperature (40°C). 200 μl running buffer (9:1 solution A:solution B) was added. 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 isopropanol. The reaction mixture was incubated at 40° C. for 10 minutes and placed on ice, centrifuged to remove spores, and absorbance recorded at 420 nm. The results are presented in table 39 below. Activity was normalized to that of a control fusion protein containing amino acids 1-35 of SEQ ID NO: 1 fused to Bacillus subtilis lipase.

TABLE 39

Strain Targeting sequence Enzyme Relative Activity B. thuringiensis BT013A Amino acids 1-35 SEQ ID NO: 1 Lipase 100% B. thuringiensis BT013A Amino acids 1-27 SEQ ID NO: 3 Lipase 92.5% B. thuringiensis BT013A Amino acids 1-28 SEQ ID NO: 7 Lipase 13.5% B. thuringiensis BT013A Amino acids 1-24 SEQ ID NO: 9 Lipase 24.8% B. thuringiensis BT013A Amino acids 1-33 SEQ ID NO: 13 Lipase 98.5% B. thuringiensis BT013A Amino acids 1-33 SEQ ID NO: 21 Lipase 107.8% B. thuringiensis BT013A SEQ ID NO: 96 Lipase 137.1% B. thuringiensis BT013A SEQ ID NO: 98 Lipase 146.3% B. thuringiensis BT013A SEQ ID NO: 100 Lipase 115.7% B. thuringiensis BT013A SEQ ID NO: 104 Lipase 81.5%

[001035] Several lipase-associated targeting sequences result in higher levels of enzyme expression and activity on the spore surface. In particular, each of SEQ ID NOs: 96, 98, and 100 containing a shorter targeting sequence resulted in enhanced expression of the fusion construct on the surface of BEMD spores. All fusion proteins containing the tested targeting sequences resulted in surface lipase exposure.

Example 35 Use of different exospore sequences for surface expression of lipase Bacillus thuringiensis and demonstration of the localization of the fusion protein on the surface of the exospore

[001036] A wide variety of exospore proteins can be used to display enzymes, proteins, and peptides on the surface of members of the Bacillus cereus family. Several different exospore proteins were compared by generating fusion proteins containing exospore proteins associated with Bacillus subtilis lipase as described in Example 34. Fusion constructs were synthesized using the exospore protein native promoter listed in Table 40 below, cloned into the replicating plasmid pMK4, and was introduced into Bacillus thuringiensis BT013A. Spores displaying various Bacillus subtilis 168 exosporium-lipase protein fusions were obtained by growing the transformed bacteria in brain-heart extract broth with a selection pressure provided by 10 μg/ml chloramphenicol, plating on a nutrient agar plate, and incubating at 30° C. within 3 days. After 3 days, the spores were washed off the plates, purified by centrifugation and resuspended in PBS at 1×10 ⁸ cfu/ml.

[001037] 1x10 ⁵ spores for each fusion construct were resuspended in 400 μl dH ₂ O. The reaction mixtures were heated with the reaction components to the desired reaction temperature (40°C). 200 μl running buffer (9:1 solution A: solution B) was added. 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 isopropanol. The reaction mixture was incubated at 40° C. for 10 minutes and placed on ice, centrifuged to remove spores, and absorbance recorded at 420 nm. The results are presented in table 40 below. The activity was normalized to the activity of SEQ ID NO: 109 associated with lipase.

TABLE 40

Strain Protein exospore Enzyme Relative Activity B. thuringiensis BT013A SEQ ID NO: 109 Lipase 100% B. thuringiensis BT013A SEQ ID NO: 110 Lipase 134.5% B. thuringiensis BT013A SEQ ID NO: 113 Lipase 17.8% B. thuringiensis BT013A SEQ ID NO: 117 Lipase 19.8% B. thuringiensis BT013A SEQ ID NO: 118 Lipase 8.2%

[001038] The use of exospore proteins of SEQ ID NOs: 109 and 110 resulted in the highest enzyme activity on spores. All fusion proteins containing exospore proteins resulted in active Bacillus subtilis 168 lipase surface exposure, albeit at different levels.

[001039] Using the mCherry fluorescent reporter, it has been demonstrated that additional exospore proteins result in targeting of fusion proteins to the exospore. Fusion constructs were prepared that contained exospore proteins of SEQ ID NOS: 111, 120 and 110 associated with the mCherry reporter. Spores were grown for 1.5 days, collected and resuspended as described above. 7 µl of fluorescent spores were placed under a Nikon E1000 microscope and visualized during late sporulation. Circular localization in the form of a ring indicates localization on the outer layer of the spore, and its features correspond to those of the exospore protein. The results of fluorescence microscopy are presented in Fig.2. Panels A, B, and C of FIG. 2 show fluorescence microscopy images of spores expressing fusion proteins containing exospore proteins of SEQ ID NOs: 111, 120, and 110, respectively, and the mCherry reporter. All three fusion constructs showed high levels of fluorescence and exospore localization, demonstrating their potential utility for expression of foreign proteins on the exospore surface.

Example 36 Use of Various Exospore Targeting Sequences and Proteins for Phosphatase Expression in Spores Bacillus subtilis and effects of phosphatase-expressing spores in soy

[001040] BEMD spores expressing Bacillus subtilis EE148 phosphatase A4 (PhoA4) were generated by synthesizing genes encoding various exospore targeting sequences and proteins under the control of their native PhoA4-related promoters. The synthesized genes were cloned into pMK4 and introduced into Bacillus thuringiensis BT013A. Spores exhibiting various Bacillus subtilis EE148 exosporium-PhoA4 protein fusions were obtained by growing the transformed bacteria in brain-heart extract broth with selective pressure provided by 10 μg/ml chloramphenicol, plating on nutrient agar plates, and incubating at 30°C. within three days. After three days, the spores were washed off the plates, purified by centrifugation, and resuspended in PBS at 1×10 ⁸ cfu/ml.

[001041] Soybean plants were sown to a depth of 2.54 cm in 10 cm deep pots filled with standard top layer clay soil. BEMD spores expressing PhoA4 were diluted to a concentration of 1×10 ⁴ /ml in 50 ml of water and applied to each plant at seeding. A water-only control was also included. The polyphosphate was added to the pots in liquid at 0.5 mg/pot. Plants were grown under perfect light using T5, 54 watt lamps and exposed to 13 hours of light per day under controlled temperature conditions of 15.5-25.5°C. Plants were watered to saturation every three days throughout the two week trial. At the end of two weeks, the height of each plant was measured and the measurement results were normalized to those of control plants treated with water only.

[001042] The results are shown in Table 41. All soybean plants grown in the presence of BEMD spores expressing fusion proteins containing PhoA4 linked to different targeting sequences and exospore proteins with different PhoA4 fusion partners showed increased growth, however, the extent of this The effect varied depending on the targeting sequence or exospore protein used.

TABLE 41

Species Bacillus Targeting sequence or exospore protein associated with PhoA4 Height after 2 weeks, normalized H2O (no bacteria) N/A 100% Bacillus thuringiensis strain BT013A Amino acids 1-35 SEQ ID NO: 1 100% Bacillus thuringiensis strain BT013A Amino acids 1-28 SEQ ID NO: 3 117.4% Bacillus thuringiensis strain BT013A Amino acids 1-33 SEQ ID NO: 21 107.3% Bacillus thuringiensis strain BT013A SEQ ID NO: 96 123.3% Bacillus thuringiensis strain BT013A SEQ ID NO: 98 124.1% Bacillus thuringiensis strain BT013A SEQ ID NO: 109 131.7% Bacillus thuringiensis strain BT013A SEQ ID NO: 110 104.8%

Example 37 Combined Application of BEMD Spores and Seed Treatment, Liquid Fertilizers and Other Additives

[001043] BEMD spores expressing fusion proteins were investigated for compatibility with various seed treatments. BEMD spores expressed fusion proteins containing a targeting sequence of amino acids 1-35 of SEQ ID NO: 1 linked to a phosphatase (PhoA4) from Bacillus subtilis EE148 or a POLARIS peptide. The synthesized genes were cloned into pMK4 and introduced into Bacillus thuringiensis BT013A. Spores displaying various Bacillus subtilis EE148 or POLARIS exosporium-PhoA4 protein fusions were obtained by growing the transformed bacteria in brain-heart extract broth with selective pressure provided by 10 µg/ml chloramphenicol, plating on nutrient agar plates, and incubating at 30°C. C for three days. After three days, the spores were washed off the plates, purified by centrifugation, and resuspended in PBS at 1×10 ⁸ cfu/ml.

[001044] Plants were grown under ideal light using T5, 54 watt lamps and exposed to 13 hours of light per day under controlled temperature conditions of 15.5-25.5°C. Plants were watered to saturation every three days throughout the two week trial. At the end of two weeks, the height of each plant was measured and the measurement results were normalized to those of control plants treated with water only. The results are presented in table 42 below. Watering = applying to the soil in the amount of 50 ml per pot. Polymer = ACCELERON Seed Coating Polymer only. For application during irrigation, BEMD spores were added at 1×10 ⁴ cells/50 ml. For seed coating applications, BEMD spores were added at 1.3×10 ⁴ /cells/seed. 10-34-0 and 6-24-6 are standard commercial starter fertilizer formulations. 10-34-0 is a liquid ammonium phosphate. 6-24-6 is a low salt liquid phosphate fertilizer with an ortho/poly formulation. Dye=red dye for coating Becker Underwood seeds. MACHO, APRON and CRUISER are commercial fungicides used on seeds. MACHO contains the active ingredient imidacloprid, APRON contains 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 treatments and retained the ability to promote plant growth in corn.

TABLE 42

BEMD processing Chemical processing Corn height after 2 weeks, normalized Not No (watering) 100% Not Only polymer 101.3% BEMD, PhoA4 N/A (watering) 111.3% BEMD, POLARIS N/A (watering) 106.7% BEMD, PhoA4 Polymer 109.3% BEMD, POLARIS Polymer 107.3% BEMD, PhoA4 Polymer+dye 102.3% BEMD, PhoA4 Polymer+MACHO 107.9% BEMD, PhoA4 Polymer+APRON 112.3% BEMD, PhoA4 Polymer+CRUISER 116.8% BEMD, PhoA4 Polymer+dye+MACHO+APRON+CRUISER 113.7% Not Starter fertilizer 10-34-0 (watering) 108.5% BEMD, PhoA4 Starter fertilizer 10-34-0 (watering) 114.7% Not Starter fertilizer 6-24-6 (watering) 102.6% BEMD, PhoA4 Starter fertilizer 6-24-6 (watering) 112.9%

[001045] BEMD spores have been found to be compatible with all seed coat variations tested. When BEMD PhA4 spores were combined with dye and polymer alone, there was a slight decrease in activity, however, the spores regained full activity when the dye was combined with other fungicides. BEMD spores also worked well with liquid fertilizers. The starter fertilizer contributed to plant growth, probably through direct nutrient supplementation. BEMD spores worked with both starter fertilizers, indicating that phosphatase activity can still lead to increased plant growth in the presence of excess nutrients. Combinations of BEMD spores with fungicides showed a higher increase in plant growth than BEMD spores alone, probably due to the protection provided to young corn plants during early growth.

Example 38 Use of BEMD Spores as an Aerial Additive to Reduce Stress Inhibition in Maize Growth

[001046] BEMD spores exhibit a system that can be used to deliver enzymes that can mitigate certain stress factors on growing plants in the field or in a greenhouse. For this, enzymes were chosen that selectively act on reactive oxygen species in the soil. Reactive oxygen species are key markers of stress in plants.

[001047] BEMD spores were prepared expressing fusion proteins containing a targeting sequence of amino acids 1-35 of SEQ ID NO: 1 linked to chitosanase, superoxide dismutase, catalase, or β1,3-glucanase from Bacillus thuringiensis BT013A. The synthesized genes were cloned into pMK4 and introduced into Bacillus thuringiensis BT013A. Spores exhibiting various protein fusion constructs were obtained by growing the transformed bacteria in brain-heart extract broth with a selective pressure of 10 μg/ml chloramphenicol, plating on nutrient agar plates, and incubating at 30°C for three days. Three days later, the spores were washed off the plates, purified by centrifugation, and resuspended in PBS at 1×10 ⁸ cfu/ml.

[001048] Three week V5 stage corn plants were grown under ideal light using T5 54 watt lamps and exposed to 13 hours of light per day under controlled temperature conditions of 15.5-25.5°C. Plants were watered to saturation every three days during the test. As plants reached V5, BEMD spores or positive control chemicals were sprayed onto the leaves at either 1×10 ⁵ BEMD spores/ml or the recommended amounts for chemicals. A total of 1 ml of spray was applied to each plant individually. Plant height was measured immediately prior to application of foliar sprays. The corn plants were then stressed by heating to 32.2° C. and reducing the frequency of watering to once a week. Plants were kept under stress conditions for two weeks. At the end of two weeks, the height of the plants was measured again and the appearance was evaluated. Under these stressful conditions, plant growth was minimal in the case of the control treatment. The ability to continue growing under stressful conditions was determined by the increase in plant height within two weeks compared to a control treated with water alone. The results are presented in table 43 below.

TABLE 43

Treatment Quantity Change in plant height after stress exposure within 2 weeks Not Not 0% Bacillus thuringiensis spores BT013A 1 ml/plant -1.6% BEMD, chitosanase 1 ml/plant 0.3% BEMD, chitosanase and chitosan 1 ml/plant and 5 mM 4.7% BEMD, superoxide dismutase 1 ml/plant 8.3% BEMD, B1,3-glucanase 1 ml/plant 4.9% Salicylic acid 1 ml/plant 5.8% Benzothiadiazole (BTH) 1 ml/plant 7.3% BEMD, catalase 1 ml/plant -0.5%

[001049] Several stress-reducing enzymes were applied to corn using the BEMD system as shown in Table 43 above. The control spores had no pronounced effect (plant height reduction -1.6%). The chitosanase enzyme from BEMD had a positive effect when it was combined with the substrate chitosan. The two enzymes with the best performance were β-1,3-glucanase from BEMD and superoxide dismutase from BEMD. β-1,3-glucanase from BEMD had mainly antifungal activity, but could also have a direct effect on plants. Salicylic acid and BTH were positive controls for the foliar assay and a positive response was observed for both of them. This foliar delivery method can be used to deliver stress reducing enzymes to the plant at various times of the season.

Example 39 Fusion Protein Expression Levels Using Different Sigma-K Containing Promoters

[001050] As shown in Example 23 above, substitution of a native targeting sequence promoter, exospore protein, or exospore protein fragment can significantly affect the level of fusion protein expressed on exospore spores of a member of the Bacillus cereus family. For example, replacing the native BclA promoter with the BclB promoter significantly reduces the level of fusion protein on the spore surface of a member of the Bacillus cereus family. Alternatively, replacing the native BclB promoter with the BclA promoter significantly increases fusion protein levels on exospores.

[001051] Relative expression levels of various exosporium proteins under the control of their native sporulation promoters were obtained from microarray data from Bergman et al. active. The sigma-K promoters are the key promoters for the expression of exospore-localized genes and their associated proteins. Relative expression is the increase in the expression level of a gene compared to the average for all other genes on a chromosome at all given time points. Table 44 below shows the relative levels of expression of various sigma K controlled genes in members of the Bacillus cereus family.

TABLE 44

Protein (promoter SEQ ID NO) Relative expression (fold increase in mRNA level) CotO (SEQ ID NO: 226) 79.21 Rhamnose (SEQ ID NO: 225) 75.69 BclC (SEQ ID NO: 179) 14.44 Sigma K (SEQ ID NO: 227) 64 US glycosyltransferase promoter 1 adjacent to BclA (SEQ ID NO: 229) 72.25 DS glycosyltransferase promoter 2 adjacent to BclA (SEQ ID NO: 230) 73.96 BclA (SEQ ID NO: 215) 77.44 Exsy (SEQ ID NO: 220) 32.49 YjcA (SEQ ID NO: 222) 64 YjcB (SEQ ID NO: 223) 70.56 BxpB/ExsFA (SEQ ID NO: 224) 30.25 InhA (SEQ ID NO: 228) 34.25

Example 40 Preparation and study of BEMD spores expressing nitric oxide synthase-containing fusion protein and use of such spores to promote plant seed germination

[001052] BEMD spores were prepared expressing a fusion protein containing amino acids 20-35 of BclA, a 6-alanine linker, and a nitric oxide synthase enzyme from Bacillus subtilis 168. The nitric oxide synthase (NOS) enzyme gene from Bacillus subtilis 168 was synthesized in a fusion construct with BclA promoter, ribosome binding site (RBS), start codon, and BclA amino acids 20-35. A linker region of six alanine residues was included to separate the BclA targeting sequence from the NOS fragments. The amino acid sequences of these fusion proteins, including the methionine encoded by the BclA start codon, BclA amino acids 20-35, the six amino acid linker, and the NOS enzyme, are shown in Table 9 above. . The correct constructs, which were sequenced and validated, were transformed into E. coli cells . The resulting plasmids were transformed into Bacillus thuringiensis BT013A and Bacillus mycoides EE155.

[001053] Recombinant Bacillus thuringiensis BT013A and Bacillus mycoides EE155 transformed with plasmids encoding NOS fusion proteins were then induced to sporulate by removing the bacteria on nutrient agar plates and incubating the plates at 30°C for 72 hours. After 72 hours, bacterial spores were collected from the dish by removing into sterile phosphate-buffered saline (PBS) and purified by density gradient centrifugation three times.

[001054] The spores were then applied to commercial hybrid corn and soybean seeds at 1×10 ⁵ spores/seed. The hybrid soybean variety was BECK 335NR, which contains the cyst nematode protection gene, the ROUNDUP READY glyphosate resistance gene, and the Phytophthora resistance K gene. The hybrid corn variety was BECK 5540RR which contains the ROUNDUP READY glyphosate resistance gene. The seeds were then lightly sprinkled with L-arginine. A control set of seeds were sprinkled with L-arginine, but without spores. The seeds were then placed between two paper towels, which were then moistened with 25 ml H ₂ O. The paper towels were then rolled up, placed in a small sandwich bag, and sealed tightly. These bags were then placed in an incubator at 30° C. and allowed to germinate for 24 or 48 hours. The number of seeds germinating at each time point was measured and the results compared with untreated and control seeds. The results of these experiments are presented in tables 45 and 46 below.

Table 45. Increase in germination rate of hybrid soybean seeds treated with spores of recombinant family members Bacillus cereus , expressing a fusion protein containing nitric oxide synthase

Treatment Germination for 1 day (%) Germination on day 2 (%) Plain Soybean Seeds 15.0% 92.3% Soy Seeds Plus L-Arginine 20.5% 94.9% Soybean plus B. thuringiensis BT013A seeds expressing B. subtilis NOS fusion protein 28.9% 97.5% Soybean seeds with L-arginine and B. mycoides EE155 expressing B. subtilis NOS 30.0% 97.5%

Table 46. Increase in germination rate of hybrid maize seeds treated with spores of recombinant members of the family Bacillus cereus expressing a fusion protein containing nitric oxide synthase

Treatment Germination for 1 day (%) Germination on day 2 (%) Common corn seeds 0.0% 77.5% Corn Seeds Plus L-Arginine 4.1% 80.5% Corn plus B. thuringiensis BT013A seeds expressing B. subtilis NOS fusion protein 6.5% 82.5% Maize seeds with L-arginine and B. mycoides EE155 expressing B. subtilis NOS 4.3% 95.0%

[001055] As can be seen from Tables 45 and 46, seed treatment with L-arginine and a recombinant Bacillus cereus expressing a nitric oxide synthase-containing fusion protein resulted in an increase in both soybean and corn germinated seeds.

Example 41 Preparation and study of BEMD spores expressing a fusion protein containing nucleic acid binding proteins

[001056] BEMD spores were prepared expressing a fusion protein containing amino acids 20-35 of BclA, a linker of eight alanine residues, and non-specific DNA-binding protein SASPα from Bacillus subtilis 168 or non-specific DNA-binding protein SASPγ from Bacillus subtilis 168. DNA encoding the gene SASPα and SASPγ were synthesized in frame with the BclA promoter, RBS, BclA start codon, and BclA amino acids 20-35. A linker region of eight alanine residues was included between the BclA targeting sequence and the RNA/DNA binding proteins. The linker allows for higher mobility and folding of the fusion proteins. The amino acid sequences of these fusion proteins, including the methionine encoded by the BclA start codon, BclA amino acids 20-35, the eight amino acid linker, and the SASPα or SASPγ protein, are shown in Table 11 above. -BclA20-35-SASPα and pHP13-BclA20-25-SASPγ. pHP13 is a well-characterized 5.5 kb shuttle vector plasmid with chloramphenicol and erythromycin resistance cassettes. It was constructed by ligation of plasmids pE194, pC194 and pUC9.

[001057] Correct clones were subjected to DNA sequencing and transformed into E. coli strain SCS110. The plasmid DNA was then purified and transformed into Bacillus thuringiensis BT013A. These bacteria were then induced to sporulate by being plated on nutrient agar plates for 72 hours at 30°C. Spores were collected and purified as described in the immediately preceding example above.

[001058] Then, to assess the ability of recombinant spores to bind nucleic acids, recombinant members of the Bacillus cereus family transformed with plasmids encoding SASPα and SASPγ fusion proteins were incubated in PBS with random DNA primers that contained a fluorescein tag at the 5' end. The experiment also included a control using non-recombinant spores. Spores were incubated for ten minutes with 50 mM labeled 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 PBS. Spores were again besieged and the supernatant was removed after centrifugation, and then they were subjected to analysis. Spores treated with fluorescein-labeled DNA were examined under a Nikon E600 fluorescent microscope and DNA binding was determined by the change in total fluorescence compared to control spores that did not contain DNA-binding fusion proteins. The results of this analysis are presented in table 47 below.

Table 47. DNA binding to spores of a recombinant member of the family bacillus cereus, expressing a fusion protein containing a DNA-binding protein

Treatment DNA binding (normalized) B. thuringiensis BT013A spores (non-recombinant) 100% B. thuringiensis BT013 spores expressing BclA-SASPα fusion protein 341.2% B. thuringiensis BT013A spores expressing BclA-SASPγ fusion protein 250.1%

[001059] In addition, figure 3 shows the binding of DNA to spores as measured by the binding of fluorescein-labeled DNA. 3, "control" refers to B. thuringiensis BT013A non-recombinant spores (non-recombinant), "SASPa" refers to B. thuringiensis BT013A spores expressing the BclA-SASPα fusion protein, and SASPc refers to B. thuringiensis BT013A spores expressing the fusion BclA-SASPγ protein.

[001060] As can be seen from the data shown in Table 47 and Figure 3, spores expressing SASPα or SASPγ fusion proteins bound significantly higher amounts of DNA than non-recombinant spores, demonstrating the high affinity of these spores for DNA.

Example 42 Preparation and Study of BEMD Spores Expressing a Nuclease-Containing Fusion Protein

[001061] In addition to the non-specific DNA and RNA binding proteins described above in the immediately preceding example, nucleases can also be used to bind and cleave nucleic acid molecules. BEMD spores expressing a fusion protein containing amino acids 20-35 of BclA and an endonuclease enzyme were generated and analyzed for their ability to bind and cleave DNA.

[001062] Bacillus subtilis endonuclease 1 was subjected to PCR amplification and in-frame fusion with the BclA promoter, RBS, start codon, and BclA amino acids 20-35. This construct was then cloned into plasmid pHP13 to obtain plasmid pHP13-BclA20-35-endonuclease. This construct was sequenced and transformed and upscaled in E. coli . Plasmid DNA was then isolated from E. coli and introduced into Bacillus thuringiensis BT013A. Spores were obtained and purified as described in Example 40 above.

[001063] Endonuclease activity was assayed by incubating recombinant spores expressing endonuclease fusion protein and non-recombinant control spores in PBS at 1x10 ⁸ spores/mL in PBS with 300 ng salmon sperm DNA and 1 μg/mL DNA dye DAPI (4' ,6-diamidino-2-phenylindole). The reaction was allowed to proceed for 10 minutes at 37°C. After 10 minutes, the supernatant was analyzed for digested DNA using a fluorimeter. As DNA is cleaved, the DAPI dye is released from the individual nucleotides released, and thus cleavage can be determined by the decrease in DAPI staining over time. The results of this analysis are presented in table 48 below.

Table 48. Nuclease activity and DNA binding by BEMD spores expressing endonuclease fusion protein

Treatment Design Decreased DNA signal (supernatant) Spore-associated DNA (fluorescence on spores) Bacillus thuringiensis spores BT013A - 5% 5.3% Bacillus thuringiensis spores BT013A BclA endonuclease 65% 21.9%

[001064] The data provided in Table 48 above demonstrates that the endonuclease fusion protein was expressed on Bacillus thuringiensis BT013A spore exospores and was able to cleave salmon sperm DNA as evidenced by a decrease in DAPI signal in the supernatant. Surprisingly, a portion of the endonuclease bound DNA tightly without cleaving it, retaining the fluorescent DAPI signal on the spores, even after washing the spores to remove excess DNA. This demonstrates that not all DNA was processed and that nucleases expressed outside the spore can bind DNA tightly. To increase this effect, a nuclease having an inactivated active site can be used in the fusion protein, which can result in less DNA cleavage and even more DNA binding on spores.

Example 43 Agricultural Use of Spores Expressing Fusion Proteins Containing Nucleic Acid Binding Proteins or Peptides

[001065] Recombinant members of the Bacillus cereus family or recombinant spore-forming bacteria expressing fusion proteins containing nucleic acid binding proteins or peptides can be used in agriculture to deliver nucleic acids to a plant growth medium (e.g., soil) and/or plants. For example, recombinant members of the Bacillus cereus family or recombinant spore-forming bacteria can be delivered to plants by seed treatment, furrow/soil irrigation or foliar treatment. Moreover, fusion proteins containing nucleic acid-binding proteins or peptides can be expressed in any of the endophytic members of the Bacillus cereus family or any of the other endophytic Bacillus species described herein, allowing delivery of nucleic acids associated with nucleic acid-binding proteins orally. plants where they are more efficient in reaching their target cells. For example, fusion proteins containing nucleic acid binding proteins can be expressed in an endophytic strain of a member of the Bacillus cereus family EE349. Expression of another fusion protein (containing endoglucanase as a protein of interest) in this strain is described in Example 51 below, demonstrating that the fusion proteins expressed in this endophytic strain are delivered into plants. Thus, expression of fusion proteins containing SASPα, SASPγ, Hfq, or a nuclease having an inactivated active site in endophytic strains of members of the Bacillus cereus family, such as member of the Bacillus cereus family EE349, may provide a means for delivering RNA and DNA (e.g., RNA- i or rDNA) inside the plant. Other non-specific nucleic acid binding proteins can also be used for this purpose in fusion proteins.

Example 44 Generation of BEMD spores that express a fusion protein and also overexpress a protein that modulates the expression of fusion proteins

[001066] Overexpression of various exosporium proteins (referred to herein as "modulator proteins") in a recombinant member of the Bacillus cereus family expressing any of the fusion proteins described herein can modulate (increase or decrease) the expression level of the fusion protein. These modulator proteins include ExsY, ExsFA/BxpB, CotY, CotO, ExsFB, InhA1, InhA2, ExsJ, ExsH, YjcA, YjcB, BclC, AcpC, InhA3, alanine racemase 1, alanine racemase 2, BclA, BclB, BxpB, BclE, BetA /BAS3290, CotE, ExsA, ExsK, ExsB, YabG, Tgl, superoxide dismutase 1 (SODA1) and superoxide dismutase 2 (SODA2).

[001067] The ability to control the level of expression of a fusion protein allows control of the amount of protein or peptide of interest in the fusion protein that is exposed on the outside of the spore of the recombinant member of the Bacillus cereus family. For example, when the protein or peptide of interest in the fusion protein includes a plant growth-promoting protein or peptide (e.g., an enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source), a recombinant member of the Bacillus cereus family expressing the fusion protein produces a spore, which, when applied to a seed, a plant, or added to a plant growth medium, has a beneficial effect on the plant due to the action of a plant growth promoting protein or peptide. Modulating the expression level of the fusion protein results in modulating the level of the protein peptide of interest that is exposed outside the spore of the recombinant member of the Bacillus cereus family. In some cases, it is useful to increase the expression level of the fusion protein (for example, when it is desirable to increase the expression of the enzyme and thereby increase the amount of enzyme per spore that can be delivered to the plant). In other cases, it is useful to reduce the expression level of the fusion protein (for example, when it is desirable to reduce protein expression and thereby reduce the amount of protein per spore that is delivered to the plant, for example, when high levels of protein have deleterious effects on the plant).

[001068] To obtain plasmids for expression of fusion proteins in members of the Bacillus cereus family, PCR fragments were obtained that contained the BclA promoter (SEQ ID NO: 85), an initiation codon, and BclA amino acids 20-35 fused in-frame or with the Bacillus subtilis endoglucanase gene 168 or with the β-galactosidase gene from E. coli DH5α. These PCR fragments were digested with XhoI and ligated into the SalI region of the pSUPER plasmid to obtain plasmids pSUPER-BclA 20-35-endoglucanase and pSUPER-BclA 20-35-βgal, respectively. Plasmid pSUPER was generated by fusion of plasmid pUC57 (containing the ampicillin resistance cassette) with plasmid pBC16-1 from Bacillus (containing the tetracycline resistance cassette). This plasmid is 5.5 kb. can replicate in both E. coli and Bacillus spp.

[001069] Plasmids pSUPER-BclA 20-35-endoglucanase and pSUPER-BclA 20-35-βgal were transformed and increased in dam methylase negative E. coli strains. The plasmid sequences pSUPER-BclA 20-35-endoglucanase and pSUPER-BclA 20-35-βgal were confirmed by DNA sequencing.

[001070] Plasmids pSUPER-BclA 20-35-endoglucanase and pSUPER-BclA 20-35-βgal were transformed into host strains of Bacillus thuringiensis BT013A (for pSUPER-BclA 20-35-Endo) or Bacillus mycoides BT155 (pSUPER-BclA 20- 35-βgal). These transformed strains expressed either the β-galactosidase enzyme or the endoglucanase enzyme outside the spore.

[001071] To obtain plasmids for overexpression of modulator proteins, PCR fragments containing native promoter regions and genes encoding ExsFA/BxpB, CotO, ExsFB, YjcB, BclC, AcpC, BclA, BclB, BxpB and CotE were obtained, cleaved with SalI and ligated into plasmid pHP13. The nucleotide sequences for the native promoter regions are shown in Table 3 above. The pHP13 plasmid is a multicopy plasmid and thus results in high levels of expression of the encoded modulator proteins when the plasmids are transformed into a host cell that is a member of the Bacillus cereus family. pHP13 plasmids containing promoter regions and genes encoding ExsFA/BxpB, CotO, ExsFB, YjcB, BclC, AcpC, BclA, BclB, BxpB, BclE, BetA/BAS3290 and CotE are referred to herein as pHP13-ExsFA/BxpB, pHP13 -CotO, pHP13-ExsFB, pHP13-YjcB, pHP13-BclC, pHP13-AcpC, pHP13-BclA, pHP13-BclB, pHP13-BxpB and pHP13-CotE, respectively.

[001072] pHP13 plasmids containing promoter regions and genes encoding modulator proteins were transformed and increased in E. coli strains. The sequences of these plasmids were confirmed by DNA sequencing.

[001073] pHP13 plasmids encoding modulator proteins were transformed into Bacillus thuringiensis BT013A containing pSUPER-BclA 20-35-endoglucanase or Bacillus mycoides BT155 containing pSUPER-BclA 20-35-βgal. The resulting recombinant bacteria were plated on nutrient agar plates containing 10 μg/ml chloramphenicol for the selection of the pHP13 plasmid and 10 μg/ml tetracycline for the selection of the pSUPER plasmids. Bacteria containing both plasmids were then grown in brain-heart extract broth overnight with both tetracycline and chloramphenicol. The overnight cultures were then removed onto nutrient agar and the bacteria were allowed to sporulate at 30° C. for 72 hours. After 72 hours, bacterial spores were collected from the plate by removing into sterile PBS and purified by density gradient centrifugation three times. The clean spores were then diluted to 1×10 ⁸ cfu/ml and analyzed for enzymatic activity using a population of 1×10 ⁸ colony forming units (cf).

Example 45 Increased or Decreased Expression of Fusion Proteins on a BEMD System by Overexpressing a Protein That Modulates the Expression of a Fusion

[001074] Recombinant Bacillus mycoides EE155 spores prepared as described in the previous example above were analyzed for β-galactosidase activity, and recombinant Bacillus thuringiensis BT013A spores prepared as described in the previous example above were analyzed for endoglucanase activity.

[001075] β-galactosidase activity was analyzed by measuring the hydrolysis of the chromogenic substrate ortho-nitrophenyl-β-galactoside (ONPG). A commercial source of β-galactosidase (0.2 μg, 0.4 μg and 0.8 μg from a stock solution of 100 μg/ml) was used to prepare the standards. 250 μl of the spore preparation was pelleted and the spores were resuspended in 50 μl of enzyme dilution buffer (10 mM TRIS, pH 7.6, 0.2 M NaCl ₂ , 5% glycerol). To each sample and to the standard was added 600 µl of a mixture of substrates preheated to 37°C (10 mM KCl, 1 mM MgSO ₄ × 7 H ₂ O, 100 mM NaH ₂ PO ₄ , pH 7.5) containing 1.14 mg /ml ONPG. Each reaction mixture was incubated at room temperature for 2 minutes. To stop the reaction, 250 μl of 1 M sodium carbonate was added. The solution was centrifuged for 5 min at 14,000×g to remove spores for reading absorbance. Absorbance was determined at 420 nm using an IMPLEN model P330 nanospectrophotometer. Samples were analyzed in triplicate with a blank sample for each reaction. The results of this analysis are presented below in table 49.

Table 49. Effects of Overexpression of Exosporium Proteins on BclA 20-35-βgal Expression Levels

Plasmid encoding fusion protein Plasmid encoding modulator protein Enzymatic activity (normalized) - - 0% pSUPER-BclA 20-35-βgal - 100% pSUPER-BclA 20-35-βgal pHP13-CotO 112.8% pSUPER-BclA 20-35-βgal pHP13-CotE 135.4% pSUPER-BclA 20-35-βgal pHP13-YjcB 45.4% pSUPER-BclA 20-35-βgal pHP13-BclA 144.7% pSUPER-BclA 20-35-βgal pHP13-BclB 132.6% pSUPER-BclA 20-35-βgal pHP13-AcpC 76.1% pSUPER-BclA 20-35-βgal pHP13-BxpB 103%

[001076] As can be seen from the results presented in Table 49, overexpression of CotO, CotE, BclA, BclB, and BxpB increased expression of the β-galactosidase-containing fusion protein, resulting in increased activity of the enzyme on spores. Conversely, overexpression of YjcB or AcpC reduced the expression of the β-galactosidase containing fusion protein, resulting in decreased activity of the enzyme on spores.

[001077] Endoglucanase activity assay was performed by cellulase activity using a carboxymethyl cellulose (CMC) substrate and dinitrosalicylic acid (DNS reagent). A commercial source of cellulase enzyme was used to prepare standards in 50 mM citrate buffer, pH 4.8. 1% CMC (sodium salt of carboxymethyl cellulose) was prepared in 50 mM citrate buffer, pH 4.8 to serve as a substrate for the reaction. 250 μl of the spore preparation was pelleted and the spores were resuspended in 150 ml of 50 mM citrate buffer, pH 4.8. The reaction was carried out using a reagent consisting of 1% DNS, 1% NaOH, 0.05% Na ₂ SO ₄ , 0.2% phenol and 18.2% Rochelle's salt. 150 μl of sample was mixed with 250 μl of 1% CMC substrate and incubated in a water bath at 50°C for 15 minutes. Added 300 μl of DNS reagent and the samples were boiled at 100°C for 10 minutes, and then cooled on ice. The solution was centrifuged for 5 minutes at 14,000×g to remove spores for absorbance reading. Absorbance was determined at 540 nm using an IMPLEN model P330 nanospectrophotometer. Samples were analyzed in triplicate with a blank sample for each reaction. The results of this analysis are presented in table 50 below.

Table 50. Effects of overexpression of exosporium proteins on expression levels of BclA 20-35-endoglucanase

Plasmid encoding fusion protein Plasmid encoding modulator protein Enzymatic activity (normalized) - - 0% pSUPER-BclA 20-35-endoglucanase - 100% pSUPER-BclA 20-35-endoglucanase pHP13-CotO 215.7% pSUPER-BclA 20-35-endoglucanase pHP13-CotE 125.5% pSUPER-BclA 20-35-endoglucanase pHP13-YjcB 89.3% pSUPER-BclA 20-35-endoglucanase pHP13-BclB 193.0% pSUPER-BclA 20-35-endoglucanase pHP13-AcpC 33.7% pSUPER-BclA 20-35-endoglucanase pHP13-BxpB 202.3% pSUPER-BclA 20-35-endoglucanase pHP13-BclC 3.2%

[001078] As shown in Table 50, overexpression of CotO, CotE, BclB, and BxpB increased expression of the endoglucanase-containing fusion protein, resulting in increased enzyme activity on spores. Overexpression of YjcB, AcpC, or BclC, on the other hand, reduced fusion protein expression, resulting in reduced enzyme activity on spores.

[001079] Collectively, overexpression of CotO, CotE, BclB, or BxpB increased the expression of both fusion proteins, resulting in increased activity of both β-galactosidase and endoglucanase, on spores expressing the BclA 20-35-βgal or BclA 20- fusion proteins. 35-endoglucanase, respectively. Overexpression of YjcB or AcpC, on the other hand, reduced the expression of both fusion proteins, resulting in decreased β-galactosidase and endoglucanase activity on spores expressing the BclA 20-35- _β gal or BclA 20-35-endoglucanase fusion proteins, respectively. Overexpression of eGFP-tagged BclC and BclA20-35 also reduced expression of the BclA 20-35-endoglucanase fusion protein, while BclA overexpression increased expression of the BclA 20-35-βgal fusion protein.

Example 46 Effects of BEMD Spores Expressing Fusion Protein and Overexpressing Modulator Protein on Maize Growth

[001080] Application of recombinant spores of Bacillus thuringiensis BT103A and Bacillus mycoides BT155 expressing a fusion protein containing Bacillus subtilis 168 endoglucanase to corn results in increased seedling vigor and growth response within two weeks. Changing the expression level of the fusion protein containing endoglucanase, induced by overexpression of the modulator protein in such spores, as described above in the previous example, leads to corresponding changes in the effects of BEMD spores on maize growth.

[001081] To demonstrate this, pSUPER-BclA 20-35-endoglucanase and pHP13-CotO or pHP13-BclB were co-expressed in Bacillus thuringiensis BT013A. Spores were obtained on nutrient agar as described in Example 40 above. Spores were diluted to a concentration of 1×10 ⁴ spores/50 ml of water, and 50 ml of water was added to commercial hybrid corn seed in a potting mix at planting. The hybrid corn variety was BECK 5540RR, which contains the ROUNDUP READY glyphosate resistance gene. Corn seeds were coated with fungicide and biological inoculum.

[001082] Plants were grown under artificial light for 14 hours a day and plant growth was determined over a period of ten days. During the experiment, the plants were watered every three days. Ten days later, plant height was measured and normalized to the height of untreated corn plants. The results of these experiments are presented in Table 51 below.

Table 51. Effects of BEMD spores expressing an endoglucanase-containing fusion protein and overexpressing a modulator protein on hybrid maize growth

Plasmid encoding fusion protein Plasmid encoding modulator protein Expression strain Maize growth (normalized to control as pSUPER-BclA 20-35 endoglucanase alone) pSUPER-BclA 20-35-endoglucanase Not Bacillus thuringiensis BT013A 100% pSUPER-BclA 20-35-endoglucanase pHP13-CotO Bacillus thuringiensis BT013A 103.8% pSUPER-BclA 20-35-endoglucanase pHP13-BclB Bacillus thuringiensis BT013A 107.6%

[001083] As shown in Table 51, overexpression of the exospore proteins CotO and BclB enhanced the effects of the BclA 20-35-endoglucanase fusion protein on corn seedling growth and vigor after 10 days. These effects correlate with levels of fusion protein expression in BEMD spores expressing BclA 20-35-endoglucanase and pHP13-CotO or pHP13-BclB, indicating that alteration of fusion protein expression levels by modulator proteins mediates effects on seedling growth and vigor.

Example 47 Genetic Inactivation of Family Members Bacillus cereus and the use of such inactivated members of the family Bacillus cereus for expression of fusion proteins

[001084] As described above, overexpression of the spore germinating protease (GPR) in its active form in the prospore of a member of the Bacillus cereus family during sporulation results in proteolytic cleavage of proteins in the prospore and inactivation of the spore. Similarly, overexpression of a non-specific endonuclease in the prospore during sporulation degrades the DNA in the spore, resulting in the proportion of inactivated spore particles in the spore population.

[001085] A plasmid encoding a non-specific endonuclease under the control of the sigma G promoter was prepared. The non-specific endonuclease 1 gene from Bacillus subtilis 168 and the sigma G promoter (SEQ ID NO: 235) were synthesized and ligated into plasmid pHP13 using the SalI region to obtain plasmid pHP13-SigG -nuclease. The correct clones were sequenced and transformed and expanded in E. coli cells . Plasmid DNA was isolated from E. coli cells and transformed into Bacillus thuringiensis BT013A. Correct clones were confirmed by PCR. The amino acid sequence for Bacillus subtilis 168 endonuclease 1 is shown in Table 4 above.

[001086] Bacillus thuringiensis BT013A cells expressing endonuclease sigma G were generated and purified on nutrient agar plates as described in Example 40 above. Spores were visually counted using a hemocytometer, diluted and seeded diluted onto nutrient agar plates. The ratio of live spores to killed spores was calculated by determining the change from visual count to plate count. Control spores (untreated) were included in each analysis. In addition, 1×10 ⁸ spores were treated with UV radiation for 10 minutes using a manual UV lamp and the assay was repeated. The visually counted amount and the number on the plate were again compared to assess the death of the spores. The results of these analyzes are presented in table 52 below.

Table 52. Viability of spores of a member of the family bacillus cereus, expressing a non-specific nuclease under the control of the sigma G promoter

Treatment share of the living Proportion alive after UV Bacillus thuringiensis BT013A 100% 61.3% Bacillus thuringiensis BT013A expressing SigG endonuclease 70.4% 24.5%

[001087] As can be seen from Table 52, expression of endonuclease 1 under the control of the sigma G promoter reduced cell viability by approximately 30% in spores that were not exposed to UV irradiation and by approximately 75% in spores that were exposed to UV irradiation. -radiation.

[001088] Co-expression of both a spore germinating protease and a non-specific endonuclease, under the control of sigma G promoters, would be expected to further reduce spore viability.

Example 48 Preparation of exosporium fragments from recombinant members of the family Bacillus cereus having a knockout of the CotE gene

[001089] A plasmid pUCpE was constructed that contained the backbone of the plasmid pUC19 capable of replicating in E. coli as well as the origin of replication of the erythromycin resistance cassette from pE194. This construct is able to replicate in both E. coli and Bacillus spp. A 1 kb DNA region corresponding to the upstream region of the CotE gene and a 1 kb region corresponding to the downstream region of the CotE gene were amplified by PCR from Bacillus anthracis ΔSterne. Then two regions of 1 kb. were spliced using overlapping extension splicing with 15 bp homologous overhangs that corresponded to opposite PCR amplicons. This fragment is 2 kb. cleaved with XhoI (in outer primers) and ligated into the SalI region of pUCpE. The plasmid construct was confirmed by DNA digestion and sequencing. The omega resistance gene of Gram-positive bacteria to kanamycin was digested with BamHI and placed between two 1 kb regions. The final construct was again confirmed by PCR and sequenced, and the final plasmid was introduced into Bacillus anthracis ΔSterne. Correct clones were screened for resistance to both erythromycin and kanamycin.

[001090] Clones were passaged at high temperature (40°C) in brain heart extract broth in the presence of kanamycin (25 μg/ml) and were routinely selected for isolation on LB agar plates containing kanamycin and grown at 30°C . Individual colonies were toothpicked onto LB agar plates containing 5 μg/ml erythromycin and grown at 30°C. Clones that retained kanamycin resistance but lost erythromycin resistance (meaning loss of plasmid but recombination and deletion of the CotE gene) were grown in broth with brain-heart extract plus kanamycin, and chromosomal DNA was isolated using the Qiagen chromosomal DNA isolation kit. . Proper deletion of the CotE gene was determined by PCR amplification of the CotE gene region and by loss of CotE and acquisition of a kanamycin resistance cassette.

[001091] A construct (pHP13-AcpC-eGFP) was generated that encoded the ApcC (acid phosphatase) exospore protein fused in frame to the eGFP (enhanced green fluorescent protein) fluorescent reporter protein. The pHP13-ApcC-eGFP construct included a native ApcC promoter, a ribosome binding site, and a coding sequence for ApcC (from B. anthracis ΔSterne) fused in frame to eGFP (from pGFPuv). This construct was generated by PCR amplification of the individual AcpC and eGFP genes with appropriate primers that contained a 15 bp overlap corresponding to consecutive amplicons. The two PCR amplicons were then purified and combined in a second PCR reaction using external primers that contained XhoI regions. The two amplicons primed each other through their compatible ends and formed confluent PCR amplicons which were purified and digested with XhoI for 1 hour at 37°C. The spliced PCR product was cloned into the SalI region of pHP13 and the correct clones were sequenced and transformed into E. coli SCS110. Plasmid DNA was then isolated from E. coli and introduced into B. anthracis Δ Sterne CotE::Kan, obtained as described above, which were grown in brain-heart extract broth containing 10 μg/ml chloramphenicol overnight at 30°C. C. One milliliter 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 by centrifugation at 10,000×g for 5 minutes, and the supernatant (containing disrupted exospore fragments) was filtered through a 100,000 Da cut-off membrane filter to obtain purified exospore fragments containing fusion proteins.

[001092] A transmission electron microscopy photograph showing CotE knockout spores is shown in Figure 4. Solid arrows indicate exospore fragments that have been separated from spores, and open arrows indicate spores from which the exospore has been removed.

[001093] Purification of exosporium fragments was performed as follows: CotE::kan spores were grown in brain-heart broth overnight at 30°C and plated on nutrient agar plates and grown at 30°C for 3 days. After 3 days, the spores were collected by removing them from the plates with PBS-moistened cotton rolls and resuspended in 1 ml PBS in a microcentrifuge tube. The spores were separated from the culture by centrifugation and the supernatant containing exospore fragments was filtered through a 0.22 μm filter to remove any residual spores. The filtrate was then filtered through a 100 kDa cut-off filter to collect exospore fragments, but allowed free proteins to pass through the filter. The 100 kD cut-off filter was washed and the collected exospore fragments were boiled in SDS buffer for 5 minutes and separated by SDS-PAGE electrophoresis. Figure 5 is a photograph of an SDS-PAGE gel showing purified exosporium fragments (lane 2) and a standard protein marker (lane 1). The exospore fragments shown in lane 2 correspond to the individual proteins that make up the exospore fragments. Only a subset of the bands that are commonly seen in a whole spore SDS-PAGE preparation are visible.

[001094] A ten microliter exospore fragment preparation containing the AcpC-eGFP fusion protein was tested in a phosphatase assay for activity against pNPP (p-nitrophenyl polyphosphate). Acid phosphatase activity was detected by spectrophotometry based on the release of p-nitrophenol from phosphate via phosphatase activity. Briefly, 1 ml of 10 mM pNPP in phosphate buffer at pH 6.0 was incubated with exospore fragments 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 was read on a spectrophotometer at 420 nm for free p-nitrophenol. It was found that the purified exospore fragments were able to efficiently release phosphate groups from pNPP, demonstrating that ApcC was present on the exospore fragments. The results of this analysis are presented in Fig.6. 6, "CotE control spores" refers to CotE knockout spores alone (not expressing AcpC-eGFP fusion protein), "CotE Acp-eGFP" refers to CotE knockout spores expressing AcpC-eGFP fusion protein, and "fragments CotE AcpC-eGFP" refers to exospore fragments obtained as described above from CotE knockout spores expressing the AcpC-eGFP fusion protein.

[001095] These results demonstrate that disruption of the exospore, such as a CotE gene knockate mutation, can be used to generate exospore fragments that are substantially free of spores and demonstrate that these exospore fragments contain fusion proteins that target the exospore.

Example 49 Expression of Fusion Proteins in Recombinant Family Members Bacillus cereus , which are capable of degrading herbicides, and the use of such recombinant members of the family Bacillus cereus to stimulate plant growth

[001096] Recombinant members of the Bacillus cereus family expressing fusion proteins can have pronounced effects on plant health and growth, as illustrated, for example, in examples 1-4, 7, 9, 11, 33, 36, 37 and 38 above. The fusion proteins containing the targeting sequence, exosporium protein or exosporium protein fragment described herein can be used in a number of different species and strains of the Bacillus cereus family, which includes Bacillus anthracis , Bacillus cereus , Bacillus thuringiensis , Bacillus mycoides , Bacillus pseudomycoides , Bacillus samanii , Bacillus gaemokensis, Bacillus weihenstephensis and Bacillus toyoiensis . Many members of the Bacillus cereus family potently degrade organic and inorganic material in the environment, and some members of the Bacillus cereus family have the ability to degrade herbicides. Expression of fusion proteins in such strains is advantageous because it can provide herbicide degradation activity, thereby mitigating plant stress that can be caused by herbicide application, in addition to being able to stimulate plant growth and provide other plant health benefits, depending from the peptide or protein of interest selected for inclusion in the fusion protein.

[001097] A member of the Bacillus cereus EE349 family was isolated, identified and characterized as described in Example 25 above and found to have the ability to stimulate plant growth. In addition, this strain was found to have the ability to degrade a variety of herbicides, including sulfonylurea compounds and aryltriazines.

[001098] To demonstrate the ability of Bacillus cereus EE349 to degrade herbicides, 1×10 ⁵ spores of Bacillus cereus EE349 were applied to lentils sown in soil containing various concentrations of sulfentrazone. Seeds were allowed to grow at 24° C. for 3 weeks on a 13 hour day/night cycle with water every 3 days. After 3 weeks, the plants were measured for root growth. A control set of seeds without Bacillus cereus EE349 was sown under identical conditions.

[001099] The results of this experiment can be seen in Fig.7. 7, "protected" refers to seeds treated with a member of the Bacillus cereus EE349 family, and "unprotected" refers to untreated seeds. The y-axis represents the root length normalized to the root length of the water-only control. Figure 7 shows that as the concentration of the herbicide increased, the inhibition of root growth also increased. However, seed application of Bacillus cereus EE349 mitigated much of this inhibition, even at full soil herbicide concentration. Thus, as can be seen from figure 7, a member of the family Bacillus cereus EE349 can act as an antidote.

[001100] Moreover, the ability of a member of the Bacillus cereus EE349 family to express fusion proteins is demonstrated in Example 51 below. Thus, a member of the Bacillus cereus EE349 family can be used as a dual purpose antidote and host for the expression of fusion proteins containing a targeting sequence, an exospore protein or an exospore protein fragment that targets the fusion protein to the exospore.

Example 50 Preparation of Recombinant Family Members Bacillus cereus , which overexpress exospore enzymes, and the effects of such recombinant members of the family Bacillus cereus on plants

[001101] The exospores of members of the Bacillus cereus family naturally contain various naturally occurring enzymes that may have beneficial effects on plants. For example, exospores from members of the Bacillus cereus family contain enzymes involved in nutrient solubilization (eg, acid phosphatases such as AcpC), inosinuridine hydrolases, proteases (eg, mettaloproteinases such as InhA1, InhA2, and InhA3), enzymes that catalyze free radical degradation. (eg superoxide dismutases such as SODA1 and SODA2), arginases and alanine racemases. Overexpression of such enzymes in members of the Bacillus cereus family can provide a recombinant member of the Bacillus cereus family that will have beneficial effects when applied to seeds, plants, plant growth media, or the area surrounding the plant or plant seed.

[001102] InhA2 and InhA3 metalloproteinases, acid phosphatase (AcpC), and superoxide dismutase 1 and 2 were amplified by PCR with their native promoters using primers that contained XhoI regions (the amino acid sequences for InhA2, InhA3, AcpC, SODA1 and SODA 2 are shown above in tables 1 and 2, and the nucleotide sequences for native promoters for these proteins are presented in table 3). The PCR products were digested with XhoI and cloned into the E. coli/Bacillus pHP13 shuttle vector through their SalI region. The correct clones were confirmed by PCR and DNA sequencing. Plasmids were introduced into Bacillus thuringiensis BT013A and Bacillus mycoides EE155. Correct clones were screened by plating on LB agar plates containing chloramphenicol. Overnight cultures of the correct clones were grown in brain-heart extract 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 confirmed by light microscopy. The spores were then subjected to enzymatic assays.

[001103] Bacillus mycoides EE155 spores overexpressing AcpC (ie, spores containing the plasmid pHP13-AcpC (acid phosphatase)) were analyzed for phosphatase activity. One milliliter of sporulation culture was pelleted and the pellet was resuspended in 1 mL PBS, and tested in a phosphatase assay for activity against pNPP (p-nitrophenyl polyphosphate) as described in Example 48 above. AcpC overexpressing spores had significantly higher phosphatase activity, as illustrated in Fig.8. 8, the y-axis shows the units of phosphatase activity determined from the release of p-nitrophenol.

[001104] Increased acid phosphatase activity seen with Bacillus mycoides EE155 spores modified to overexpress AcpC can solubilize nutrients when such spores are added to a plant growth medium or applied to a plant seed, plant, or area surrounding the plant or seed plants. Since phosphate is a very important nutrient for plant growth and development, it can enhance plant growth and provide beneficial effects on plant health.

[001105] Similarly, superoxide dismutase is a very potent antioxidant protein. Overexpression of superoxide dismutase in a member of the Bacillus cereus family can provide spores having the ability to degrade free radicals that stress plants. The removal of free radicals can mitigate this stress to some extent and result in increased plant vigor under stressful conditions. Bacillus thuringiensis BT013A spores overexpressing SODA1 and SODA2 (ie, spores transformed with plasmids pHP13-SODA1 and pHP13-SODA2, respectively) can be subjected to enzymatic analysis. One milliliter of sporulation culture can be pelleted and the pellet can be resuspended in 1 ml dH ₂ O containing xanthine. Xanthine oxidase as well as cytochrome C can then be added to the reaction mixture. Inhibition of cytochrome C degradation in this assay is indicative of superoxide dismutase activity.

[001106] Bacillus mycoides EE155 spores overexpressing zinc metalloproteinase (i.e., spores transformed with the pHP13-InhA2 plasmid) were subjected to enzymatic analysis. One milliliter of sporulation culture was pelleted and the pellet was resuspended in 1 mL PBS. The spores were then reacted with 0.5% azocasein, a protease substrate, for 5 minutes. These reactions were precipitated with TCA (trichloroacetic acid) to remove undigested casein and the absorbance of the remaining free azo dye was read at ABS595. Spores overexpressing InhA2 had a 211% higher protease activity compared to non-recombinant Bacillus mycoides EE155 spores.

[001107] Examples 3 and 7 above illustrate that protease expression on exospores of a member of the Bacillus cereus family can provide beneficial effects on plants. Bacillus thuringiensis BT013A spores with InhA1, InhA2 or InhA3 can have similar effects when applied to plant growth media or applied to plant seeds, plants or the area surrounding the plant or plant seed.

Example 51 Expression of Fusion Proteins in an Endophytic Strain of the Family Bacillus cereus

[001108] A member of the Bacillus cereus EE349 family has been found to have the ability to grow endophytically and be able to serve as a host strain for the BEMD system. To demonstrate the ability of a member of the Bacillus cereus EE349 family for endophytic growth and the ability to serve as a host strain for the BEMD system, a member of the Bacillus cereus EE349 family was transformed with the pSUPER-BclA 20-35-endoglucanase plasmid (described in Example 44 above). Spores were obtained and purified as described in Example 40 above.

[001109] These spores were diluted to a concentration of 1×10 ⁵ spores/50 ml of water, and then 50 ml of water was added to commercial hybrid corn seeds in potting soil at planting. Corn seeds were coated with fungicide and biological inoculum. The hybrid corn variety was BECK 5475RR, which contains the ROUNDUP READY glyphosate resistance gene and the AQUAMAX drought resistance gene. Plants were grown under artificial light for 14 hours a day and plant growth was determined for ten days. The plants were watered every three days during the experiment. Ten days later, plant height was measured and normalized to the height of untreated corn plants. The results of these experiments are presented in table 53.

Table 53. Effects of an endophytic member of the family bacillus cereus, expressing BclA fusion protein 20-35-endoglucanase, on the growth of corn seedlings

Plasmid Expression strain Corn Growth (Normalized) No (control) Not 100% Not Representative of the family Bacillus cereus EE349 104.1% pSUPER-BclA 20-35-endoglucanase Representative of the family Bacillus cereus EE349 111.5%

[001110] As can be seen from the data shown in Table 53, expression of the pSUPER-BclA 20-35-endoglucanase construct in an endophytic strain of Bacillus cereus EE349 resulted in increased corn growth compared to untreated plants or plants treated with Bacillus only. cereus EE349.

[001111] Then, a member of the Bacillus cereus 349 family expressing BclA 20-35-endoglucanase was isolated from corn plants. Plants growing for ten days were removed from the soil and washed to remove excess debris. The plants were then turned over, exposed to 5% bleach for ten minutes, washed with water, exposed to hydrogen peroxide (10%) for ten minutes, washed again with water, and cut the stems with a sterile razor blade. The separated stem halves were placed face down on nutrient agar plates for two hours. After two hours, the stems were removed and the agar plates were incubated at 30° C. for 48 hours. After 48 hours, the plates were examined for colony morphology and colonies of the Bacillus cereus family found inside the plant were selected with a toothpick for nutrient agar plates and nutrient agar plates plus tetracycline (for selection of bacteria containing the plasmid pSUPER-20-35 BclA-endoglucanase ). The resulting increase in the number of colonies of a member of the Bacillus cereus 349 family is shown in Table 54. These results demonstrate the ability to introduce the BEMD system into a target plant in an endophytic strain of a member of Bacillus cereus .

Table 54: Endophytic analysis of a member of the family Bacillus cereus EE349

Treatment Endophytic bacteria (total) Bacteria of the Bacillus cereus family (all strains) Tetracycline-resistant members of the Bacillus cereus family H ₂ O (control) 156 31 0 Representative of the Bacillus cereus EE349 family transformed with pSUPER-20-35 BclA-endoglucanase 221 64 21

[001112] Tetracycline-resistant Bacillus clones were grown overnight at 30°C in broth with brain-heart extract plus tetracycline and besieged by centrifugation at 10,000×g for 5 minutes. The supernatant was removed and the pellet was frozen overnight at -20°C. Chromosomal DNA was then extracted from each clone and the presence of the pSUPER-20-35 BclA-endoglucanase plasmid was determined by transforming the chromosomal DNA (containing the plasmid) into E. coli DH5α cells and plated with LB plus ampicillin. The correct clones were subjected to DNA sequence analysis, which confirmed that a member of the Bacillus cereus 349 family was inside the plant (was endophytic) and contained the plasmid.

[001113] Many endophytic bacteria have been found in corn seedlings, with a number of different strains and species of the Bacillus cereus family found inside both control and EE349 treated plants. Tetracycline-resistant members of the Bacillus cereus family (indicative of the presence of the pSUPER-20-35 BclA-endoglucanase plasmid) were found only in treated maize seedlings, and all of them had the same colony morphology as the original exercising host, a member of the Bacillus cereus family EE349 . The presence of plasmid pSUPER 20-35 BclA-endoglucanase was confirmed by amplification by PCR using unique primers.

Example 52 Isolation, Identification and Characterization of Endophytic Bacterial Strains of the Family Bacillus cereus

[001114] In addition to the endophytic strain of Bacillus cereus 349 described above in the previous example, several other members of the Bacillus cereus family have also been identified that have the ability to grow endophytically: Bacillus cereus EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444 , Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus mycoides EE-B00363, Bacillus pseudomycoides EE-B00366 and Bacillus cereus EE-B00377.

[001115] To obtain these additional members of the Bacillus cereus family, commercial hybrid corn seeds were sown in potting soil and allowed to grow. Corn seeds were coated with fungicide and biological inoculum. Plants were grown under artificial light for 14 hours a day and plant growth was determined for 14 days. The plants were watered every three days during the experiment. After 14 days, the plants were removed from the soil and washed to remove excess soil residues. The plants were then turned over, exposed to 5% bleach for ten minutes, washed with water, exposed to hydrogen peroxide (10%) for ten minutes, washed again with water, and the stems were separated with a sterile razor blade. The separated stem halves were placed face down on nutrient agar plates for two hours. After two hours, the stems were removed and the agar plates were incubated at 30° C. for 48 hours. After 48 hours, the plates were examined for colony morphology, and colonies of Bacillus cereus found inside the plant were picked with a toothpick on nutrient agar. They were then grown overnight at 30° C. in brain-heart broth and pelleted by centrifugation at 10,000×g for 5 minutes. The supernatant was removed and the pellet was frozen overnight at -20°C. Then, the chromosomal DNA of each clone was extracted and the identity of each colony was confirmed by PCR using 16S rRNA primers, and the amplicons were sent for DNA sequencing and identification. The 16S rRNA sequences for these strains are shown in Table 13 above.

Example 53 Isolation, identification and characterization of additional endophytic bacterial strains (not members of the family Bacillus cereus )

[001116] Endophytic bacterial strains of Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillus fusiformis EE442, Lysinibacillus spp. EE443 and Bacillus pumilus EE-B00143. Corn seedlings grown for two weeks were first sterilized. Plants were extracted from the soil and washed to remove excess soil residue. The plants were then turned over, exposed to 5% bleach for ten minutes, washed with water, exposed to hydrogen peroxide (10%) for ten minutes and washed again with water. The stems were cut with a sterile razor blade. The separated stem halves were placed face down on nutrient agar plates for two hours. After two hours, the plant stems were removed from the cups, and then the cups were incubated at 30°C for 48 hours. Bacilli colonies that were endophytic were selected for further analysis. These strains were grown in brain-heart broth overnight at 30° C. and the cultures were subjected to DNA extraction using the Qiagen Chromosomal DNA Kit. The DNA was amplified by the PCR method to obtain the 16S rRNA gene, which was sent for DNA sequencing. The obtained sequences were searched in BLAST using the NCBI databases to establish the identity of the species Bacilli . The 16S rRNA sequences are shown in Table 14 above.

Example 54 Expression of Spore Coat Protein-Containing Fusion Proteins in Endophytic Bacterial Strains bacillus

[001117] Bacillus thuringiensis EE319, Bacillus firmus A30, and Bacillus lichenformis A4 endophytic bacterial strains were transformed to contain plasmids encoding various spore coat proteins fused to endoglucanase. Received plasmids pHP13-CotC-endoglucanase and pHP13-CgeA-endoglucanase. Each of these plasmids encoded a spore coat protein (CotC or CgeA) fused in frame to a polyalanine linker containing eight alanine residues and endoglucanase. The polyalanine linker and edoglucanase were fused to the C-terminus of the spore coat proteins.

[001118] To obtain plasmids encoding fusion proteins, the endoglucanase gene from Bacillus subtilis 168 was amplified by PCR. The genes encoding the spore coat proteins CotC and CgeA were also amplified by PCR from Bacillus subtilis 168 (CotC) or Bacillus amyloliquefaciens (CgeA) chromosomal DNA. The correct amplicons were then spliced by the overlap PCR method to obtain a DNA fragment of the fusion protein by annealing the 15 bp homologous overhangs. Each of the outer primers was designed to contain XhoI regions. Amplicons were purified with the Promega PCR Purification Kit and the DNA was digested with XhoI and ligated into the SalI region of pHP13. The plasmid DNA was then sequenced, transformed into E. coli cells , and the DNA was introduced into various endophytic Bacillus strains.

[001119] Spores of each of the recombinant Bacillus species expressing fusion proteins were obtained by removing overnight cultures on nutrient agar plates, which were then incubated at 30° C. for 72 hours. After 72 hours, bacterial spores were collected from plants by stripping into sterile PBS. Spores were purified by density gradient centrifugation three times, diluted to 1×10 ⁸ kfu/ml, and analyzed for endoglucanase activity as described in Example 45 above. The results of this analysis are shown in Table 55 below and in FIG. nine.

Table 55. Endoglucanase activity in spores bacillus, expressing CotC-endoglucanase or CgeA-endoglucanase fusion proteins

Plasmid Expression strain Data readout for enzymes / activity Control disputes Bacillus firmus A30 0.201 Control disputes Bacillus thuringiensis BT013A 0.206 pHP13-CgeA-endoglucanase Bacillus firmus A30 0.818 pHP13-CotC-endoglucanase Bacillus thuringiensis EE319 1.738 pHP13-CotC-endoglucanase Bacillus licheniformis A4 0.414

[001120] In Figure 9, CotC1, CotC2 and CotC3 are three separate experimental cultures for sporulation of Bacillus thuringiensis EE319 with pHP13-CotC-Endo.

Example 55 Spore Effects bacillus, expressing CotC-endoglucanase, CotB-endoglucanase, or CgeA-endoglucanase fusion proteins on corn and soybean seed growth

[001121] Recombinant Bacillus spores expressing fusion proteins containing spore coat protein and endoglucanase (e.g., the CotC-endoglucanase, CotB-endoglucanase, or CgeA-endoglucanase fusion proteins described above in the previous example) can be investigated for their effects on plant growth (e.g. corn and soybeans) as follows. Spores can be obtained as described above in the previous example, washed, diluted to 1×10 ⁸ k.o.u./ml in water and applied to plant seeds (for example, corn and soybean seeds) in an amount of 1×10 ^5-7 spore/seed. The spores can then be applied either as a seed treatment or as a soil watering. Plants can be planted to a depth of 1" (2.54 cm) in 4" (10.16 cm) pots, and grown at 18.3°C with a 13 hour light/dark cycle. After two days, you can determine the height of the plants and the length of the roots.

Example 56 Delivery of Probiotic Bacteria to Animals by Feeding Plants Containing Probiotic Bacteria to Animals

[001122] 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 applying to a soil furrow), to a plant (e.g., by foliar application ) or on an area surrounding a plant or plant seed, and then feeding such plants or plants grown from plant seeds to an animal. The bacteria can be applied to the leaves or stems of plants while the plants are growing and will colonize the phylloplana (leaf and stem surface). Plants before feeding the animal can be processed into animal feed.

[001123] The use of endophytic bacterial strains in such methods allows the bacteria to survive and remain in the plant tissue so that they are ingested in significant amounts by the animal when the plant material from the plant is consumed. For example, it is believed that strains of a member of the Bacillus cereus EE349 family, a member of the Bacillus cereus EE439 family, Bacillus thuringiensis EE417, Bacillus cereus EE444, Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillus fusiformis EE442, Lysinibacillus spp. EE443 and Bacillus pumilus EE-B00143 are probiotic and endophytic and can be used in these methods.

[001124] Any of these strains or other probiotic and endophytic strains can be grown and spores produced as described in Example 40 above. The spores can then be introduced into a plant growth medium, applied to a plant seed, plant, or area surrounding the plant or seed plants. Plants grown in a plant growth medium, plants grown from plant seeds, plants coated with bacteria, or plant seeds grown in an area coated with bacteria can be grown and then fed to an animal. Endophytic bacteria can colonize the interior tissue of a plant and can replicate in significant numbers within the plant. The bacteria will sporulate using conventional harvesting methods, allowing long-term storage of plant material (eg as hay or silage) that can later be fed to the target animal.

[001125] Only a small amount of bacteria need be used in these methods because endophytic bacteria naturally colonize plants and proliferate on and in plants.

Example 57 Delivery of Beneficial Enzymes to Animals by Feeding Animals Plants Containing a Recombinant Family Member Bacillus cereus or other recombinant bacteria expressing a fusion protein containing a beneficial enzyme

[001126] Recombinant members of the Bacillus cereus family expressing a fusion protein containing a protein or peptide of interest and a targeting sequence, exosporium protein or exosporium protein fragment that targets the fusion protein to exospores, as described herein, can also be used to deliver beneficial enzymes animals. The recombinant members of the Bacillus cereus family can be fed directly to animals (eg, by mixing the recombinant member of the Bacillus cereus family into an animal feed that is subsequently fed to the animal). Alternatively, the methods for delivering bacteria to animals described in the previous example above can be used in conjunction with a recombinant member of the Bacillus cereus family expressing a fusion protein that contains a protein or peptide that has beneficial effects in the animal (e.g., an enzyme that aids in the digestion of plant material).

[001127] Enzymes present in feed for livestock, fish, and other animals can affect nutrient absorption, production, and health of the animal that consumes the enzymes. Enzymes that are beneficial to animal health include, for example, xylanases, phytases, phosphatases, proteases, cellulases, endoglucanases, glucanases, amylases, lipases, phospholipases, glycosylases, galactanases, α-galactosidases, amylases, pectinases, biotinases, and polygalacturonases, among others. The BEMD system can be used to express such enzymes on the surface of the exospore. Recombinant members of the Bacillus cereus family expressing a fusion protein containing one of these enzymes can be applied to the plant growth medium, to the plant seed, to the plant, or to the area surrounding the plant or plant seed. Similarly, these methods can use recombinant bacteria that express a fusion protein containing one of these enzymes and a spore coat protein that targets the fusion protein to the bacterial spore surface. The recombinant bacteria can be applied to the plant growth medium, to the plant seed or plant, or to the area surrounding the plant or plant seed. Plants grown in a plant growth medium, plants grown from plant seeds, plants coated with bacteria, or plants or plant seeds grown in an area coated with bacteria can be grown and then fed to an animal, and thus way, the animal to deliver a useful enzyme. Bacteria will sporulate using conventional harvesting methods that allow long-term storage of plant material (such as hay or silage) that can later be fed to the target animal.

[001128] Endophytic strains of members of the Bacillus cereus family can be used as hosts for expressing fusion proteins containing a protein or peptide of interest (e.g., an enzyme with beneficial effects in animals) and a targeting sequence, exosporium protein, or exosporium protein fragment, which target fusion protein into exospores. For example, the endophytic strains of Bacillus cereus EE349, Bacillus cereus EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, and Bacillus thuringiensis EE319 described herein can be used as hosts.

[001129] Additional members of the Bacillus cereus family may be selected for application to the aerial part of the plant, as these bacteria do not need to be endophytic for phylloplana to colonize. For example, Bacillus mycoides BT155, Bacillus mycoides EE118, Bacillus mycoides EE141, Bacillus mycoides BT46-3, Bacillus cereus EE218, Bacillus thuringiensis BT013A, Bacillus thuringiensis EE-B00184, Bacillus mycoides EE-B00363, Bacillus pseudomycoides can be used for this purpose. -B00366 or a member of the Bacillus cereus family EE-B00377.

[001130] Similarly, endophytic strains of recombinant bacteria can be used as hosts to express fusion proteins containing a protein or peptide of interest and a spore coat protein that targets the fusion protein to the bacterial spore surface. For example, endophytic strains of Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillus fusiformis EE442, Lysinibacillus spp. EE443 or Bacillus pumilus EE-B00143.

[001131] The use of endophytic bacterial strains in these methods allows the bacteria to survive and remain in the plant tissue such that both the bacteria and the fusion proteins expressed by the bacteria will be ingested in significant amounts by animals upon ingestion of the plant material from the plant. Thus, by simply adding a recombinant member of the Bacillus cereus family or other recombinant bacteria at seeding, beneficial enzymes can be distributed into plant tissues and delivered to animals through their consumption of plant material.

Example 58 : Use of different targeting sequences for the expression of endoglucanase on the surface of spores of a member of the Bacillus cereus family , and the use of such spores to stimulate plant growth

[001132] The pSUPER plasmid was modified by cloning the PCR-derived fragment by homologous recombination, which fused the BclA promoter, start codon, and BclA amino acids 20-35 (amino acids 20-35 of SEQ ID NO: 1) in frame with Bacillus subtilis 168 endoglucanase (pSUPER-BclA 20-35-Endo) as described in Example 44 above. This plasmid was then subjected to reverse PCR to amplify the entire plasmid backbone except for the sequence corresponding to amino acids 20-35 of BclA. This reverse PCR product was combined with a PCR product that amplified an equivalent region from each of SEQ ID NO: 5, 15, 25, 81, 85, 87 or amino acids 20-33 of SEQ ID NO: 1. Thus, constructs were obtained 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: 5; (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 contained a wild-type BclA promoter and a methionine at the initiation codon position, followed by a targeting sequence fused in-frame to the Bacillus subtilis endoglucanase gene. Each of these constructs was transformed into E. coli and plated to form single colonies on Luria plus ampicillin (100 μg/ml) plates. Plasmids from each individual colony were grown in overnight cultures in Luria broth plus ampicillin and purified using the WIZARD SV miniprep kit and sequence verified by Sanger sequencing. DNA was also quantified by spectrophotometry, and DNA was introduced into Bacillus thuringiensis BT013A. In addition, the pSUPER-BclA-20-35 Endo construct was introduced into Bacillus thuringiensis BT013A from which the native BclA protein had been removed by homologous recombination (BclA knockout, "BclA KO"). The correct colonies were screened by plating on nutrient broth plates containing an antibiotic (tetracycline at 10 μg/ml). Each positive colony was grown in brain-heart broth at 30° C. overnight at 300 rpm with antibiotic, and the genomic DNA was purified and re-sequenced to confirm genetic purity. Confirmed colonies were grown overnight in brain heart extract broth with 10 μg/ml tetracycline and induced to sporulate by sporulation in yeast extract medium.

[001133] Each batch produced in yeast extract medium was harvested 48 hours after spore production and subjected to enzyme comparison in the resulting spores using the methodology described in Example 45. Absorbance was determined at 540 nm using an IMPLEN P330 nanophotometer. There were three samples and a blank sample for each reaction. The results of the determination of enzymes are presented in table 56.

[001134] For corn, 1 μl of whole broth in each case for each of the constructs was placed on each seed. For squash, 2 μl of whole broth for each construct was applied to each seed. To do this, 50 seeds were placed in a 50 ml propylene tube with a conical bottom and slightly shaken using a vortex mixer. To the vial of these spinning seeds, 50 µl (for corn) or 100 µl (for squash) of recombinant spore broth was slowly pipetted and the shaking action covered the seeds with an even coating of whole cell broth for each construct. These seeds were then sown to a depth of 1" (2.54 cm) in natural soil using a 39.6 cm ³ (15.6 in ³ ) sowing pot, two seeds per pot. The pots were then watered until saturated and the plants were allowed to Plants were grown in a controlled growth room at 70°F during the day and 60°F in the evening with a photoperiod of 14 hours/day, under artificial light for 14 days Plant growth was measured after 14 days and the results were normalized to the control group in which water-only treatment was performed as a seed treatment.

Table 56. Enzyme levels and plant growth phenotypes

Targeting sequence Endoglucanase enzyme levels (mU/ml) Sequence identity with a.k. 20-35 BcLA Sequence identity with a.k. 25-35 BclA Maize growth phenotype Squash growth phenotype Average change in plant phenotype Control (H ₂ O) 0 IU/ml N/A N/A 100% 100% 100% A.k. 20-35 BclA (SEQ ID NO: 1) 38.2 100% 100% 112% 94.7% 103.4% A.k. 23-38 SEQ ID NO: 5 33.5 50.0% 72.7% 106.7% 102.3% 104.5% A.k. 28-43 SEQ ID NO: 15 16.7 68.8% 81.8% 115.7% 103.4% 109.6% A.k. 9-24 SEQ ID NO: 25 25.7 56.3% 63.6% 118.4% 107.1% 112.8% A.k. 23-38 SEQ ID NO: 81 21.5 50.0% 72.7% 106.7% 98.3% 102.5% A.k. 13-28 SEQ ID NO: 85 38.3 43.8% 54.5% 99.7% 100.5% 100.1% A.k. 13-28 SEQ ID NO: 87 14.4 43.8% 54.5% 102.6% 104.1% 103.4% A.k. 20-33 SEQ ID NO: 1 30.5 N/A 100% 104.6% 100.7% 102.7% AK20-35 SEQ ID NO: 1 in BT013A BclA KO 100.8 100% 100% ND ND ND

a.k.=amino acids

ND=not defined

[001135] 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 control of the expression level of the enzyme outside the spore.

[001136] The use of amino acids 20-35 of SEQ ID NO: 1 or a.k. 13-28 of SEQ ID NO: 85 as the target sequence resulted in the highest levels of enzyme production. This is unexpected given the low degree of identity between these targeting sequences (43.8% identity over the entire length of the targeting sequence). The use of amino acids 28-43 of SEQ ID NO: 15 or amino acids 9-24 of SEQ ID NO: 25 resulted in the highest plant response among the two plant types. Expression of a fusion protein containing amino acids 20-25 of SEQ ID NO: 1 as a targeting sequence in the BT013A BclA KO host resulted in a very large (263.8%) increase in the level of enzymatic activity on the spore surface compared to expression of the same fusion protein in the wild type strain.

Example 59 : Use of different targeting sequences and exosporium proteins to express phospholipase, lipase and endoglucanase on the surface of spores of a member of the Bacillus cereus family and use of such spores to stimulate plant growth

[001137] The pSUPER plasmid was modified by cloning the PCR-derived fragment (XhoI digestion and ligation) in which the BclA promoter, start codon and BclA amino acids 20-35 (amino acids 20-35 of SEQ ID NO: 1) were fused, followed by a linker a sequence of six alanine residues, in frame with the gene for either Bacillus thuringiensis phosphatidylcholine-specific phospholipase C (PC-PLC) (pSUPER-BclA 20-35-PL) or Bacillus subtilis lipase LipA (pSUPER-BclA-20-35-lipase ) or Bacillus subtilis eglS endoglucanase (pSUPER-BclA-20-35-Endo) as described in Example 44 above. These plasmids were then subjected to reverse PCR to amplify the entire plasmid backbone except for the sequence corresponding to amino acids 20-35 of BclA. This reverse PCR product was combined with a PCR product that amplified an equivalent region from each of SEQ ID NO: 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); full-length exospore proteins 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 NOs: 1. Each of these constructs contained the BclA promoter wild-type, methionine at the initiation codon position followed by a targeting sequence or exospore protein fused in-frame to the gene for Bacillus cereus phosphatidylcholine-specific phospholipase C, Bacillus subtilis lipase 168, or Bacillus subtilis endoglucanase eglS 168. Each of these constructs was screened for the correct transformants as described in Example 58 above.

[001138] Each batch produced in yeast extract medium was collected 48 hours after spore production and subjected to a comparison of the resulting spore enzymes. Enzyme data acquisition for endoglucanase was performed as described in Example 58 above. For phospholipase C enzyme assay, 1 ml of recombinant spores was pelleted at 10,000×g for 3 minutes and the supernatant was removed and discarded. The spore pellet was then resuspended in 500 μl reaction buffer (0.25 mM Tris-HCL, 60% glycerol, 20 mM o-nitrophenylphosphorylcholine, pH 7.2). The negative control for enzyme assays contained BT013A spores without enzyme expression. Each sample was incubated at 37° C. for 18 hours, centrifuged again to remove spores, diluted 1:1 in water, and Abs540 was read using a spectrophotometer. This was compared to a standard curve against commercially purchased phospholipase and lipase controls to establish activity in U/ml. The results of these enzyme determinations are shown in Table 57 and Table 58.

Table 57 Endoglucanase Enzyme Levels

Targeting Sequence Experiment #1 Endoglucanase levels (mU/ml) Control (H ₂ O) 0 IU/ml A.k. 20-35 SEQ ID NO: 1 38.2 SEQ ID NO: 120 25.7 SEQ ID NO: 111 29.7 SEQ ID NO: 121 24.4 SEQ ID NO: 108 24.0 SEQ ID NO: 114 11.0 A.k. 20-33 SEQ ID NO: 1 30.5 Targeting sequence experiment #2 Endoglucanase levels (mU/ml) A.k. 20-31 SEQ ID NO: 1 48.22 A.k. 21-33 SEQ ID NO: 1 60.86 A.k. 23-33 SEQ ID NO: 1 19.93 A.k. 23-31 SEQ ID NO: 1 45.31 A.k. 20-35 SEQ ID NO: 1 54.1

A.k. = amino acids

[001139] Many of the exospore targeting sequences and proteins were able to exhibit a large number of active enzymes on the spore surface, including SEQ ID NOS: 108, 111, 114, 120 and 121. Amino acids 20-31, 21-33 and 23-31 SEQ ID NO: 1 provided similar levels of enzyme expression with amino acids 20-35 of SEQ ID NO: 1, indicating that smaller fragments are sufficient to expose the enzymes to the spore surface. Only amino acids 23-33 of SEQ ID NO: 1 showed a reduced level of enzyme exposure to spores.

Table 58 Phospholipase Enzyme Levels

Targeting sequence PC-PLC enzyme levels Lipase enzyme levels Control (H ₂ O) 0.0 0.0 A.k. 20-35 SEQ ID NO: 1 0.787 0.436 A.k. 23-38 SEQ ID NO: 5 0.688 0.602 A.k. 28-43 SEQ ID NO: 15 0.372 0.228 A.k. 9-24 SEQ ID NO: 25 0.247 0.359 SEQ ID NO: 114 0.446 0.798 SEQ ID NO: 120 3.612 0.753 SEQ ID NO: 111 0.738 0.329

A.k. = amino acids

[001140] Similar to the results shown in Table 57 above, the highest levels of phospholipase or lipase on the surface of spores were observed when using 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.

[001141] The effects of these spores expressing several of these constructs on nodulation in soybean are shown in Table 59.

Table 59. Plant responses to phospholipase

Targeting sequence Nodulation per plant (soybean) Control (H ₂ O) 9.8 Control strain ( Bacillus thuringiensis BT013A) 8.2 Bacillus thuringiensis BT013A expressing a fusion protein from a.k. 20-35 SEQ ID NO: 1 and phospholipase 14.0

[001142] Soybean plants were covered as described above, however, the analysis was carried out up to a period of 3 weeks. The plants were carefully removed, dirt was carefully washed off the roots, and nodules were counted for each plant. As shown in Table 59, the application of phospholipase-expressing spores to soybean seeds results in increased nodulation on the plants, which is a positive indication of both early growth and eventual increase in soybean yield.

Example 60 RNA Binding MIR319 and random RNA 1 with spores bacillus cereus, expressing a fusion protein containing a nucleic acid-binding protein and using such spores to deliver RNA to plants

[001143] DNA and RNA can be associated with spores of a member of the Bacillus cereus family that express fusion proteins containing a targeting sequence and a nucleic acid-binding protein or peptide on their exospore, as described in the examples above and in the description. The spores act as a delivery mechanism, delivering the target nucleic acid (eg miRNA) to the target plant. To demonstrate this ability, spores of a recombinant member of the Bacillus cereus spores family with a common miRNA MIR319 were delivered to soybeans using spores expressing a fusion protein containing amino acids 20-35 of SEQ ID NO: 1 fused in-frame to a known DNA-binding protein SspC gene . MIR319 has different effects on plant phenotype in different plants, and even in different parts of the same plant. For example, in some species, treatment of leaves with MIR319 results in leaf curl, while in other species, use of MIR319 results in stress tolerance. MIR319 is ubiquitous among plant genomes, is a common cascade regulator, and its delivery to different plants results in different phenotypes.

Table 60: RNAs used in this study

RNA 3ʹ-sequence 5ʹ-sequence MIR319 UUGGACUGAAGGGUGCUCCC (SEQ ID NO: 306) GAGCUCUCUUCAGUCCACUC (SEQ ID NO: 307) or AGAGCGUCCUUCAGUCCACUC (SEQ ID NO: 308) Random RNA #1 GAGCCCATGGTTGAATGAGT (SEQ ID NO: 309) ACTCATTCAACCATGGGCTC (SEQ ID NO: 310)

[001144] Synthetic miRNAMIR319fromGlycinemax(soy) designed to match the sequenceMIR319,available in miRBase (miRBase.org, central miRNA sequence repository). Two partially complementary single strand sequences were synthesized at Integrated DNA Technologies (IDT, Iowa) to match the 3' and 5' mature gene products known to exist.in vivo (two different versions of the 5'-sequence were used). Similarly, two single-stranded RNAs were synthesized with random sequences that did not match the soybean genome as a control. Double-stranded (ds) gene products were prepared by combining two single-stranded (ss) products at 95° C. for 10 min and then slowly cooling to room temperature to allow annealing.bacillus thuringiensis, expressing a fusion protein containing the BclA promoter, a methionine residue at the initiation codon position, and amino acids 20-35 of SEQ ID NO: 1 fused in-frame to a known DNA-binding protein geneSspC (SASP α/β type, small acid soluble protein C sporeBacillus thuringiensis BT013A) was constructed using standard cloning techniques as described in Example 58 above. This construct (SspC-BclA) were created inE. coli, transformed intoBacillus thuringiensis BT013A and clones were confirmed by DNA sequencing. controversyB. thuringiensis expressingSspC-BclA, was prepared by growing transformed bacteria overnight in Brain Heart Broth (BHI) for 2 days in a medium based on yeast extract until a density of 2×10^eight spores per milliliter (mL) with less than 1% vegetative cells. DNA was extracted from an aliquot of the BHI parent culture and sent for sequencing to confirm plasmid insertion.SspC-BclA. To obtain spores for seed treatment, 1 ml of spore culture in yeast extract medium was pelleted by centrifugation and resuspended in 100 μl of water. This concentrated suspension was counted and the spores used were 6×10^eightdispute / ml. For each spore seed, 1 μl of spores was combined with 10 μl of RNA at a concentration of 10 μM and incubated at 30° C. for 2 hours (large scale for multiple seeds). After this incubation, spores were precipitated (containing bound RNA) and excess unbound RNA in the supernatant was removed and the pellet was resuspended in 10 μl of water. The samples were applied to the seeds as follows: prepared 39.6 cm³ (15.6 inches³) commercial Timberline brand topsoil in each pot and a 1 inch (2.54 CM) hole was made into which 2 ml of water was added and one seed was placed on top. A 10 µl sample of spores+bound RNA was applied with a micropipette directly on top of the seeds. The seeds were left for 30 min, and then the seeds were freely covered with neighboring soil. Seeds were allowed to germinate for 4 days in an artificially lit grow room with a 13/11 hour light/day cycle and a temperature range of 21°C day/15°C night. On the 14th day, the plants were pulled out, photographed and measured. Height was normalized to that of plants treated with water control (see Table 61).

[001145] Example 41 above describes the ability of spores of a recombinant member of the familyBacillus cereus with SspC-BclA bind and hold DNA. To evaluate the RNA binding capacity of spores expressing SspC-BclA, biotin labeled random RNA sequences were synthesized by IDT and incubated with spores exactly as performed for the treatments described above (1 μl of spores in the amount of 6×10^eight spores/ml + 10 µl 10 µM RNA for 2 hours at 30°C, precipitated and resuspended in 10 µl water). Avidin conjugated with fluorescein (FITC) (Life Technologies) at a final concentration of 20 μg/ml was added to a 10 μl spore+RNA sample and incubated for 1 hour at room temperature in the dark. Avidin is known to bind biotin and FITC is a fluorescent label. The spores were re-settled to remove excess unbound avidin-FITC and resuspended in 4% paraformaldehyde in PBS and stored at 4° C. overnight in the dark. The spores were examined for fluorescence and photographed (see Table 62). In addition, as shown in Figure 10, Sspc-BclA labeled spores were able to bind and retain both ssRNA and dsRNA, as shown by FITC-avidin labeling of spores in the presence of biotin-bound ssRNA or dsRNA. To obtain the results shown in Figure 10, spores were incubated with either double-stranded or single-stranded biotin-labeled RNA (random sequence) and detected with fluorescein-conjugated avidin (FITC). No fluorescence was detected on spores incubated with water only. Bright field microscopy images and corresponding fluorescence images were acquired using a 40X objective and a 10X eyepiece.

[001146] As seen in Table 61 below, the main effect of MIR319 as a seed treatment on soybeans is root growth and overall height. Twisting roots were defined as having at least two 180° turns. The height was measured for the main stem. When soybean plants were uprooted and analyzed for the presence of "twisted roots", the specific soybean phenotype observed by the inventors' team did not show signs of curling roots in the water-treated control, control strain BT013A, control treated with double-stranded (dsRNA) RNA alone, or spores alone ( carrier control). The only sign of curling roots was noted when SspC-BclA spores (vehicle) were delivered to the seeds along with dsRNA (60% curling roots) (see also FIG. 10). Figure 11 also shows the phenotypic changes in willow plants exposed to SspC-BclA litter in combination with dsMIR319 RNA. When spores are used to deliver RNA, the effect of the RNA is enhanced resulting in increased growth retardation and the curling root phenotype shown in FIG. To obtain the results shown in Figure 11, soybean seeds were treated with double-stranded (ds) MIR319 with or without prior binding of B. thuringiensis spores expressing SspC-BclA . Application of dsMIR319 resulted in slightly taller plants on average; however, application of spore-associated dsMIR319 resulted in "rolling" roots, defined as having at least two 180° turns, and a lower overall height. A median sample from each experimental condition is shown. Images were taken using a digital camera with plants together in one image.

[001147] As an RNA control, a set of ssRNA (single stranded) and dsRNA was applied to soybeans. In these experiments, random ssRNA had no effect when applied alone, while dsRNA had a growth retardation effect on plant height when delivered to seeds. In both cases, when spores (vehicle) were used in conjunction with a random version of either ssRNA or dsRNA, there was a significant increase in the stunting phenotype (stunting 33% and 27.8%, respectively). This growth retardation is not noticeable in the case of spore samples (vehicle control) alone. Taken together, these data demonstrate the ability of spores to enhance and specifically deliver ssRNA and dsRNA to plants by seed application, and demonstrate the ability of two different RNAs (random #1 and MIR319 ) to influence the phenotype when delivered through Bacillus cereus spores expressing the fusion protein, containing DNA/RNA-binding protein.

Table 61: Effect MIR319 regarding roots and height on soybean development

Seed processing (5 replicas of each) % twisting roots Height (normalized to control) Water (control) 0 100% Water + spores (control) 0 105.21% Random ssRNA #1 No Controversy 0 102.62% Random ssRNA #1+spores 0 69.62% dsMIR319 no dispute 0 125.30% dsMIR319+spores 60% 67.10% Random dsRNA #1 No Controversy 0 82.40% Random dsRNA #1+spores 0 54.61%

Table 62: Fluorescent Detection of SspC-BclA Expressing Spores with Bound Biotin Labeled RNA

Spore handling Fluorescence found on spores Spores + water (control of background fluorescence of spores) Not found Spores + water + avidin-Fitc (spore background fluorescence control + FITC fluorescence) Not found Spores+biotin-labeled ssRNA+avidin-FITC Discovered Spores+biotin-labeled dsRNA+avidin-FITC Discovered

[001148] As can be seen in Table 62, no fluorescence was detected on spores without the presence of RNA. Both single-stranded (ss) and double-stranded (ds) RNA were detected on spores.

Example 61 Delivery of Nucleic Acids to Nematodes Caenorhabditis elegans by feeding them recombinant spores bacillus thuringiensis, expressing a fusion protein

[001149] The delivery of RNA and DNA to nematodes has many applications in plant science, animal health, and basic research. Nematodes cause significant damage and crop losses in commercial and non-commercial key crops, and parasitic nematodes cause high morbidity in humans and other animals in many poor regions of the world. RNA and DNA delivery has the potential to mitigate and treat many nematode problems, and delivery of RNA and DNA constructs has been shown to be useful in targeting nematodes. This example illustrates the applicability of the RNA/DNA delivery mechanism described in Example 60 above to deliver spores to nematodes.

[001150] Wild-type C. elegans nematodes were purchased from Carolina Biological (North Carolina) and maintained at 23° C. on NGM-Lite agar plates coated with E. coli OP50 for nutrition. Two different strains of Bacillus thuringiensis BT013A were constructed using standard cloning techniques to express amino acids 20-35 of SEQ ID NO: 1 fused in frame with green fluorescent protein (GFP) or mCherry to track the presence of spores in the gut. These spores, labeled with green or red fluorescent dye, were obtained by growing overnight in BHI medium (with brain-heart extract) and then for three days in yeast extract medium until a density of approximately 2×10 ⁸ spores per milliliter was obtained. (ml) with less than 1% vegetative cells. To obtain spores for feeding nematodes, 1 ml of spore culture in medium was pelleted by centrifugation and resuspended in 100 μl of water to remove excess medium. This concentrated suspension was counted and diluted to 1×10 ⁸ spores/ml. To feed the worms with spores, 1 µl of a spore suspension containing spores labeled with both red and green fluorescent dye was added to a 60 mm NGM-lite agar plate with 10 µl of PBS (phosphate-buffered saline) to facilitate distribution. Other food sources were unavailable. Twenty wild-type nematodes of various ages were immediately transferred to dishes. Live nematodes were checked after 5 hours for spore consumption using standard fluorescence microscopy.

[001151] As can be seen in Figure 12 and Table 63, the centralized intestine of nematodes was fluorescent when fed with recombinant spores of a member of the Bacillus cereus family expressing a fusion protein containing the targeting sequence and GFP, while the intestine was not fluorescent, when they were fed OP50 E. coli (standard food). Images of live nematodes were obtained with a 4x objective and a 10x eyepiece. This demonstrates the ability of these spores to both be ingested and deliver "cargo" in the form of target proteins, an example of which is the green fluorescent protein. Other exospore proteins and targeting proteins can also be used interchangeably with the targeting sequence to deliver RNA and DNA to the nematode or other target organism. Other recombinant spores of members of the Bacillus cereus family may also be used due to the highly conserved nature of the exospore and its formation on the surface of the spores.

Table 63: Fluorescence C. elegans, found in the intestine

food source Green fluorescence found in the intestine OP50 E. coli (control) Not GFP Expressing B. thuringiensis BT013A Yes (high)

Example 62: Construction, Purification and Use of Exosporium Fragments

[001152] Knockout (KO) mutants: To obtain exsY and cotE (KO) knockout mutant strains of Bacillus thuringiensis BT013A , a pKOKI shuttle plasmid and an insertion vector were constructed that contained a pUC57 backbone that is capable of replicating in E. coli , as well as a resistance cassette to erythromycin with an origin of replication from pE194. This construct is able to replicate in both E. coli and Bacillus spp . A 1 kb region of DNA corresponding to the upstream region of the cotE gene and a 1 kb region corresponding to the downstream region of the cotE gene were amplified by PCR from Bacillus thuringiensis BT013A . A second construct was prepared that contained a 1 kb DNA region that corresponded to the upstream region of the exsY gene and a 1 kb region that corresponded to the downstream region of the exsY gene, both of which were amplified by PCR from Bacillus thuringiensis BT013A. Then, for each construct, two 1 kb regions. were spliced using homologous recombination with overlapping regions with plasmid pKOKI. The plasmid construct was confirmed by DNA digestion and sequencing. Clones were screened for resistance to erythromycin.

[001153] Clones were passaged at high temperature (40°C) in broth with brain-heart extract. Individual colonies were toothpicked onto LB agar plates containing 5 μg/ml erythromycin, grown at 30° C. and screened for the presence of plasmid pKOKI as a free plasmid by colony PCR. Colonies that had an insertion event continued to be passaged to screen for single colonies that lost erythromycin resistance (loss of the plasmid to a large extent, but recombination and deletion of the exsY or cotE genes ). Deletions were confirmed by PCR amplification and sequencing of a given region of the chromosome. The pSUPER-BclA 20-35 Endo plasmid (described in Example 58) was transformed into each of the exsY and cotE KO mutants. As described in Example 48 above, the cotE KO mutant was also transformed with the pSUPER BclA 20-35 eGFP plasmid (produced as described in Example 44 above, but with eGFP replaced with endoglucanase by homologous recombination).

[001154] Dominant negative mutants : To obtain a dominant negative mutant, the present inventors amplified by PCR the N-terminal half and C-terminal half of CotO (SEQ ID NO: 126) containing amino acids 1-81 and 81-199, respectively, and these fragments were cloned into the pHP13 vector using homologous recombination (the pHP13 vector is described above in Example 1). Correct clones were confirmed by Sanger sequencing. Each of the two CotO dominant negative mutants was introduced into Bacillus thuringiensis BT013A, which contained the pSUPER-BclA 20-35 Endo construct, which produces endoglucanase at the spore surface as illustrated in Example 58 above.

[001155] Preparation of exospore fragments : For each of the two KO mutants and for both dominant negative mutants, overnight culture was grown in BHI medium at 30° C., 300 rpm, in baffled flasks with antibiotic selection. One milliliter of this overnight culture was inoculated into yeast extract medium (50 ml) in a baffled flask and grown at 30° C. for 3 days. An aliquot of the spores was removed, 1% Tween was added and the spores were mixed by shaking for one minute. Spores were collected by centrifugation at 10,000×g for 5 minutes and the supernatant containing exospore fragments was filtered through a 0.22 μm filter to remove any residual spores. The supernatant (containing disrupted exospore fragments) was filtered through a 100,000 Da membrane filter to obtain purified exospore fragments containing fusion proteins. Proteins with lower MM were removed by passing through a filter with a cut-off value of 100 kDa. No spores were found in the filtrate or retentate of the supernatant.

[001156] Figure 15 shows transmission electron microscopy photographs showing intact Bacillus thuringiensis BT013A spores (panel A) surrounded by attached exosporium and CotE knockout mutant Bacillus thuringiensis BT013A spores (panel B) from which exospore separated. The arrows in panel A of Fig. 15 indicate exospores of intact spores, while the arrows in panel B of Fig. 15 indicate exospores that are detached from spores. Panel C of FIG. 15 is a transmission electron microscopy photograph of a purified preparation of exospore fragments obtained from CotE knockout spores of Bacillus thuringiensis BT013A (obtained as described above by shaking, centrifuging and filtering the supernatant) visualized by negative staining . Images were acquired on a JEOL JEM 1400 transmission microscope. No visible exospore fragments were observed when control spores ( Bacillus thuringiensis BT013A without CotE knockout expressing BclA 20-35 Endo fusion protein, data not shown) were subjected to the same shaking, centrifugation, filtration procedures as described above.

[001157] The presence of BclA 20-35 endoglucanase in a collection of exosporium fragments from CotE and ExsY knockout mutants and CotO dominant negative mutants : Exospore fragments were generated and purified as described above, which contained the pSUPER BclA 20-35-Endo plasmid, which provides exospore, which contains endoglucanase enzymes on the surface of the spores. Exospore fragments containing this construct were obtained from cotE knockout mutant spores, exsY knockout mutant spores, spores with a dominant CotO N-terminal mutant, or spores with a dominant CotO C-terminal mutant. In each of these experiments, the level of endoglucanase activity on exospore fragments was quantified as a percentage of total enzyme levels. These results were compared against a wild-type construct that did not contain any mutants but contained the pSUPER BclA 20-35-Endo plasmid.

[001158] Effects of exospore fragments on plant growth: These exospore fragments were then applied by seed treatment to soybean seeds (as described in Example 59 above). A wild type control ( B. thuringiensis BT013A expressing the BclA 20-35 Endo construct) was also applied to soybean seeds. For each experiment, 1 μl of exospore fragments from each construct or a 1:2, 1:4, or 1:8 dilution of the fragments was applied to each seed.

Table 64: Enzymatic activity of exospore fragments and plant growth response

Mutation Design Endoglucanase activity, exospore fragments (mU/ml) Growth response of soybean plant, dilution 1:2 Growth response of soybean plant, dilution 1:4 Growth response of soybean plant, dilution 1:8 The presence of spores BT013A wild type BclA 20-35 Endo 10.3 93.1% 92.2% 83.4% Not cotE KO BclA 20-35 Endo 269.0 121.4% 110.7% 90.7% Not exsY KO BclA 20-35 Endo 238.0 107.7% 89.1% 90.7% Not CotO NTD dominant BclA 20-35 Endo 22.4 99.6% N/A N/A Not CotO CTD dominant BclA 20-35 Endo 27.5 95.8% N/A N/A Not

[001159] These results demonstrate that mutations that destroy exospores, such as a knockout mutation in the cotE or exsY gene , or a dominant negative mutation in the CotO protein, can be used to generate exospore fragments that are substantially free of spores and demonstrate, that these exospore fragments contain fusion proteins that target the exospore. These fragments can be used to stimulate plant growth and in other applications. There was a low level of background endoglucanase activity in a preparation of exospore fragments from strain BT013, which does not have mutations and expresses the BclA 20-25 Endo construct (BT013A BclA 20-35 Endo). This was unexpected and may correspond to the low level of unstable exospore that is released from the spores and captured during the collection of exospore fragments. The CotE and ExsY KO strains contain the highest amount of the enzyme in the fraction of exospore fragments. Dominant-negative CotO mutants that express the fusion protein also have elevated levels of the enzyme in the fraction of exospore fragments.

[001160] Exosporium fragments from CotE and ExsY mutants (not expressing BclA 20-35 Endo) applied directly to plants had a negative effect on growth and were excluded from this experiment. When exospore fragments from BT013A BclA 20-35 Endo were applied to soybean, it resulted in a negative growth phenotype. When exospore fragments from CotE or ExsY mutants expressing the BclA 20-35 Endo fusion protein were added to soybeans, there was a significant increase in growth rate (+28.3% and +14.8% for BT013A BclA 20-35 Endo fragments). Exospore fragments with the CotE mutation were still active at a 1:4 dilution, however ExsY exospore fragments no longer provided growth benefits to soybean plants at this dilution. CotO dominant negative mutants expressing the BclA 20-35 Endo fusion protein provided a slight increase in soybean growth compared to fragments from BT013A BclA 20-35 Endo, providing +6.5% and +2.7% growth, respectively.

Example 63: Additional Demonstration of the Suitability of Endophytic Family Members Bacillus cereus and other recombinant species bacillus for endophytic delivery of peptides, proteins and enzymes to the plant

[001161] Bacillus thuringiensis EE417, Bacillus thuringiensis EE-B00184, Bacillus cereus EE439 and Bacillus sp. EE387 are capable of endophytic growth and are capable of 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 serve as a host strain for the BEMD system, each of these strains was transformed with the pMK4-BclA 20-35-eGFP plasmid (described in Example 62 above). Spores were obtained and purified as described in Example 40 above.

[001162] These spores were diluted to a concentration of 1×10 ⁸ /ml, and then 1 μl of whole cell broth was added to commercial hybrid corn seeds in potting soil at planting. Corn seeds were coated with fungicide and biological inoculum. The hybrid corn variety was BECK 6175YE, which contains the ROUNDUP READY glyphosate resistance gene and the AQUAMAX drought resistance gene. Plants were grown under artificial light for 14 hours a day and plant growth was determined for ten days. During the experiment, the plants were watered every three days.

[001163] Then Bacillus thuringiensis EE417, Bacillus thuringiensis EE-B00184, Bacillus cereus EE439 and Bacillus sp. EE387 expressing BclA 20-35-eGFP were isolated from corn plants. Plants grown for ten days were removed from the soil and washed to remove excess soil residue. The plants were inverted, washed with water, exposed to 5% bleach for ten minutes, washed with water, exposed to 70% ethanol for ten minutes, washed again with water, and the stems were separated with a sterile razor blade. The separated stem halves were placed face down on nutrient agar plates for two hours at 30°C. After two hours, the stems were removed and the agar plates were incubated at 30° C. for 48 hours. After 48 hours, the plates were examined for colony morphology, and Bacillus colonies found inside the plant were selected with a toothpick for nutrient agar plates and nutrient agar plus chloramphenicol plates (for selection of bacteria containing the plasmid pMK4-20-35 BclA-eGFP). The results are shown in Table 65. These results demonstrate the possibility of introducing the BEMD system into the target plant by expression in an endophytic strain of a representative of Bacillus cereus . Figure 13 also shows the ability of Bacillus thuringiensis EE-B00184 to express eGFP on spores as evidenced by fluorescence microscopy. In Fig.13, arrows indicate single spores. Figure 14 shows the green fluorescence capability of the isolated bacterial colonies, demonstrating that they actually deliver proteins of interest (in this case, eGFP) into plants. Figure 14 shows the fluorescence of colonies of endophytic bacteria isolated from maize plants on plates irradiated with a GFP filter lamp.

Table 65: Endophytic delivery of "cargo" proteins

Strain Endophytic "Cargo" % Bacillus colonies + plasmid % Bacillus+eGFP colonies Bacillus thuringiensis EE417 Yes BclA 20-35 eGFP 29.8% 29.8% Bacillus thuringiensis EE-B00184 Yes BclA 20-35 eGFP 38.9% 38.9% Bacillus sp. EE387 Yes BclA 20-35 eGFP fifty% fifty% Bacillus cereus EE439 Yes BclA 20-35 eGFP 23.9% 23.9%

[001164] To further demonstrate the ability of these endophytic strains to express proteins on the surface of spores, the following constructs were introduced into the plasmid of Bacillus sp. EE387: pHP13 in which endoglucanase has been fused to any of: BclA 20-35, CotB, CotG, CotC, CgeA, InhA, InhA2, InhA1, CotY or AcpC (amino acids 20-25 SEQ ID NO: 1 or SEQ ID NO: 252, 256, 253, 254, 108, 121, 114, 111 and 120, respectively). The pSUPER BclA-20-35 Endo construct described in Example 58 above was also introduced into Bacillus thuringiensis EE-B00184, another endophytic strain. Transformed cells were screened by PCR and Sanger sequencing. The spores of each of these constructs were obtained by growing an overnight culture in BHI with selection (chloramphenicol) and 500 μl of each culture was scraped onto nutrient agar plates and allowed to incubate at 30° C. for 3 days. After 3 days, the spores were cleared in PBS, diluted to a concentration of 1×10 ⁸ /ml, pelleted by centrifugation to isolate the spores, and the enzyme was quantified on the spores as described in Example 58 above. The enzyme concentration was calculated as mU/ml for each construct. On the ability of Bacillus sp. EE387 express fusion proteins on the surface of their spores indicate enzyme levels. Bacillus sp. EE387 were able to express all of the spore fusion proteins on their surface, however AcpC (SEQ ID NO: 120) was the best fusion protein for this strain. These data were unexpected because Bacillus sp. EE387 is not a strain of a member of the Bacillus cereus family and does not have an exospore, but nevertheless shows surface expression of fusion proteins, including exospore proteins or targeting sequences derived from exospore proteins (e.g., CotY, AcpC and amino acids 20-35 of SEQ ID NO : one).

Table 66: Endophytic strains Bacillus sp. EE387 (EE387) and Bacillus thuringiensis EE-B00184 (EE-B00184) expressing fusion proteins

Fusion partner of exosporium protein or targeting sequence Endophytic host strain Endoglucanase activity (mU/ml) CotB (SEQ ID NO: 252) EE387 4.0 CotG (SEQ ID NO: 256) EE387 4.2 CotC (SEQ ID NO: 253) EE387 4.4 CgeA (SEQ ID NO: 254) EE387 4.1 A.k. 20-35 SEQ ID NO: 1 EE387 16.3 InhA (SEQ ID NO: 108) EE387 7.5 InhA2 (SEQ ID NO: 121) EE387 6.0 CotY (SEQ ID NO: 111) EE387 4.9 AcpC (SEQ ID NO: 120) EE387 36.0 InhA1 (SEQ ID NO: 114) EE387 4.5 A.k. 20-35 SEQ ID NO: 1 EE-B00184 95.8

[001165] These endophytic strains can also be introduced into the plant by addition to the plant growth medium, including soil, irrigation formulations, and granule formulations. Endophytic strains can also enter the target plant through the aerial parts of the plant. This provides a unique and efficient mechanism for delivering proteins and peptides of interest to the plant, or, in the case of DNA and RNA binding proteins, delivering RNA and DNA to the plant.

[001166] These data are for Bacillus sp. EE387 also demonstrate that all of amino acids 20-35 of BclA (SEQ ID NO: 1) and SEQ ID NO: 108, 121 and 120 are distinctly positive in Bacillus strains outside the Bacillus cereus family. Bacillus thuringiensis EE-B00184 is also an exclusive expression host system. These levels are both prominent and positive, indicating that a conserved binding mechanism for these proteins may be present in other Bacillus species.

[001167] Surface expression on spores of Bacillus thuringiensis EE-B00184 . Bacillus thuringiensis EE-B00184 was transformed with pSUPER BclA 20-35 eGFP and allowed to sporulate as described above. The spores were pelleted, washed and subjected to fluorescence microscopy to show the surface of the spores loaded with eGFP proteins in FIG.

Example 64 Expression of Fusion Proteins in Herbicide and Pesticide Degrading Strains of Family Members Bacillus cereus

[001168] Examples 49 and 51 above demonstrate the ability of the herbicide-degrading strain of Bacillus cereus EE349 to both degrade herbicides and serve as a host strain for expression of a fusion protein associated with the exospore of its spores. To further demonstrate the ability of herbicide-degrading strains to produce enzyme-laden exospores on their spores, we introduced pHP13 CotC-Endo (SEQ ID NO: 253), pSUPER AcpC-Endo (SEQ ID NO: 120), pSUPER InhA2-Endo (SEQ ID NO: 120). : 121) and pSUPER 23-38 SEQ ID NO: 5-Endo) in Bacillus cereus EE-B00377. A description of pHP13 CotC-Endo can be found in Example 54, a description of pSUPER AcpC-Endo and pSUPER InhA2-Endo can be found in Example 59, and a description of pSUPER 23-38 SEQ ID NO: 5-Endo can be found in Example 58. Was a member of the Bacillus cereus family EE-B00377 was identified to effectively degrade pyrethrin, dicamba and 2,4-D. The degradation of herbicides and pesticides was confirmed both by growth on the herbicide or pesticide as a source of nutrients, and by the decrease in the level of dicamba and 2,4-D in the presence of the herbicide or pesticide degrading strain. Plasmids were received and the cells were transformed identically to example 48 above. Each construct was confirmed by Sanger sequencing. Spores were obtained using sporulation medium and the conditions are given in Example 48. Enzyme activity was also determined as in Example 58 above.

Table 67: Expression levels of fusion protein enzymes in a pesticide-degrading strain of a member of the family Bacillus cereus EE-B00377

Expression construct Endoglucanase activity (mU/ml) CotC-Endo (SEQ ID NO: 253) 46.9 AcpC Endo (SEQ ID NO: 120) 4.3 pSUPER 23-38 SEQ ID NO: 5 Endo 108

[001169] As can be seen from Table 67, Bacillus cereus EE-B00377 is able to produce endoglucanase and display endoglucanase on its exospore using several different exospore proteins or targeting sequences. Of the constructs tested, amino acids 23-38 of SEQ ID NO: 5 or SEQ ID NO: 253 provided the highest enzyme levels in this strain.

[001170] This example demonstrates the ability of the spore display system to be expressed in herbicide and pesticide degrading strains. This system can be used to express other target proteins on the surface of spores, including proteins that themselves act on herbicides or pesticides, such as herbicide degradation enzymes, pesticide degradation enzymes, metabolic enzymes, reductases, oxidases, and other beneficial enzymes for pesticide degradation. alone or in the presence of plants.

Example 65: Use of Free Nitric Oxide Synthase (NOS) and Spore Bound NOS to Enhance Plant Germination

[001171] Example 40 demonstrates the ability of Bacillus subtilis 168 nitric oxide synthase (NOS) to stimulate germination when associated with exospores of members of the Bacillus cereus family and deliver this NOS-spore protein fusion to or near the seed. In this example, free NOS from Bacillus thuringiensis BT013A (SEQ ID NO: 261) and free eNOS (epithelial NOS from bovine neutrophils, Sigma-Aldrich, catalog number N1533) can also help induce germination and increased growth of NOS-exposed seeds. Plasmids pHP13 BclA-BT NOS, pHP13 BclA-BS NOS and pHP13 BclA-SODA (superoxide dismutase) were prepared identically to pHP13 BclA-BS NOS as described in Example 40 and transformed into Bacillus thuringiensis BT013A. pHP13 BclA-BT NOS contains the BclA promoter, start codon, BclA amino acids 20-35, a 6 alanine linker, and the Bacillus thuringiensis BT013A NOS gene (see Table 9, SEQ ID NO: 263). pHP13 BclA-BS 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 (SODA1) gene (SEQ ID NO: 155).

[001172] Table 68 shows the results of the soil germination assay. In this assay, commercial grade BECK'S 294NR (ROUNDUP READY) was coated with either 1 µl water (control) or 1 µl water supplemented with 34.2 IU bovine neutrophil eNOS. Then 50 seeds from each group were sown and grown as described in example 58, but with 4 seeds per pot. Plant height was measured after 7 days. As shown in Table 68, the presence of eNOS allowed increased seed growth resulting in a 30.7% increase in shoot height of the treated soybeans.

Table 68: Effect of free eNOS on plant growth in soy

Treatment Height normalized to control H ₂ O, 1 µl/seed 100.0% H ₂ O with 34.2 IU eNOS/seed 130.7%

[001173] In addition to the soil germination test described above, standard germination assays were performed as described in Example 40. processed seeds. Treatment was a control with H ₂ O (water), L-arginine, Bacillus thuringiensis BT013A (control strain), Bacillus thuringiensis BT013A with pHP13 BclA-BT NOS, and Bacillus thuringiensis BT013A with pHP13 BclA BS NOS. The results for soy are shown in Table 69 below.

Table 69: Effect of spore-exposed NOS on germination rate in soybean

Treatment Germination rate H ₂ O, 1 µl/seed 38% Control as H ₂ O with L-Arginine 58% Bacillus thuringiensis BT013A (control strain) 52% Bacillus thuringiensis BT013A with BS NOS 82% Bacillus thuringiensis BT013A with BT NOS 54%

[001174] Standard germination assays were also performed as described above for commercial sorghum hybrids. Each of the 50 treated sorghum seeds was coated with 0.5 μl. Treatment was a control with H ₂ O (water), L-arginine, Bacillus thuringiensis BT013A (control strain), Bacillus thuringiensis BT013A with pHP13 BclA-BT NOS, and Bacillus thuringiensis BT013A with pHP13 BclA BS NOS. After 4 days, shoot height and root length were measured to investigate increased seed germination and all data were normalized to water control data. The results are presented in table 70 below. The addition of either BT NOS or BS NOS resulted in significantly increased root length and shoot growth, with the difference most pronounced with BS NOS treatment.

Table 70: Spore-associated NOS and increased germination of sorghum

Treatment Root growth (normalized) Shoot growth (normalized) H ₂ O, 1 µl/seed 100.0% 100.0% Control as H ₂ O with L-Arginine 109% 89% Bacillus thuringiensis BT013A (control strain) 75% 145% Bacillus thuringiensis BT013A with BS NOS 163% 293% Bacillus thuringiensis BT013A with BT NOS 141% 190%

[001175] The experiment described above for sorghum was repeated but with slightly different treatments. Treatments included control H ₂ O (water), Bacillus thuringiensis BT013A (control strain), Bacillus thuringiensis BT013A with pHP13 BclA-SODA, or free bovine eNOS. After 4 days, shoot height and root length were measured to study increased seed germination, and all data were normalized to water control data. The results are presented in table 71 below. The addition of either BT SODA or free NOS (eNOS) results in significantly increased root length and shoot growth.

Table 71: Spore bound SODA and free NOS and increased sorghum growth

Treatment Root growth (normalized) Shoot growth (normalized) H ₂ O, 0.5 µl/seed 100.0% 100.0% Bacillus thuringiensis BT013A (control strain) 79% 117% Bacillus thuringiensis BT013A with pHP13 BclA-SODA 1 125% 228% Free eNOS, 34.2 IU/seed 123% 311%

[001176] Taken together, these results demonstrate that overexpression of nitric oxide synthase from a variety of sources can be imparted to seeds and their germination and outgrowth rates from seeds can be increased both in soil and by conventional germination methods. This effect can also be detected when free NOS is added to seeds. The addition of superoxide dismutase to the spores also results in increased seed germination. L-Arginine contributed to increased germination levels when used alone, or contributed less when mixed with NOS enzymes.

[001177] NOS genes are common in various microorganisms, and these microorganisms can be genetically modified to enhance their ability to express NOS on or near a plant seed in a plant growth medium. Expression of NOS on spores provides a better delivery system because vegetative microorganisms are more unstable and do not live on seeds for long periods of time. Expression on spores using any of the targeting sequences, exospore proteins, fragments of exospore proteins, and spore coat proteins described herein is an effective route to deliver NOS to seeds.

Example 66 Modulation of Enzyme Expression and Plant Growth

[001178] As demonstrated in Examples 44, 45, and 46, overexpression of a modulator protein in a recombinant member of the Bacillus cereus family that coexpresses a fusion protein can result in increased or decreased levels of the fusion protein being incorporated into the exospore. Fusion proteins and constructs were prepared and spores were prepared as described in Examples 44 and 45 above. Growth assay was performed as described in Example 46 above.

[001179] As can be seen in Table 72, expression of the pSUPER BclA 20-35 Endo fusion proteins on the surface of Bacillus thuringiensis BT013A spores using amino acids 20-35 of SEQ ID NO: 1 as the targeting sequence resulted in increased growth in corn, soybean and squash . This effect may be enhanced when the second exosporium protein is overexpressed. Each of the strains overexpressing CotO, BxpB and YjcB had a pronounced effect on the growth of corn, soybean and/or squash, with this increase in growth being most pronounced in corn.

Table 72: Spore Bound SODA and Free NOS and Increase in Sorghum Growth

Treatment corn growth soybean growth Squash growth H ₂ O, 1.0 µl/seed 100.0% 100.0% 100.0% Bacillus thuringiensis BT013A with pSUPER BclA 20-35 Endo (base) 103.8% 108.8% 105.8% Base with pHP13 BclA-CotO 109.6% 106.4% 105.2% Base with pHP13 BclA-BxpB 106.8% 117.2% 113.9% Base with pHP13 BclA-YjcB 110.4% 122.4% 106.7%

[001180] Overexpression of other modulator proteins can also modulate fusion protein expression levels as well as effects on plant growth, including those described in the present example and in examples 44 and 45 above. Each of these can be used to alter or correct enzyme levels to desired effective levels.

Example 67 Overexpression of Exospore Proteins and Effects on Plants

[001181] Overexpression of naturally occurring spore and exospore proteins can influence the effect that plant growth-promoting, endophytic and other members of the Bacillus cereus family have on plants. Expression of various exosporium proteins as part of a fusion protein or as a free enzyme can have beneficial effects on plants, as illustrated above for phosphatases (examples 11 and 36), nitric oxide synthase (example 65) and proteases such as InhA (examples 3, 6, 7, 13). Other exospore and spore proteins, such as alanine racemase and inosine- and uridine-preferring hydrolases, may interfere with or slow down spore germination, and their overexpression will result in spores being less susceptible to rapid germination, which is an undesirable side effect with many types of spores. dispute. Finally, spores that overexpress certain exospore proteins can alter the overall assembly of the exospore, leading to changes in spore binding to plants. An example of this can be seen in Table 73 below.

[001182] Spores were generated as described for Bacillus thuringiensis BT013A in Example 58. Growth assay was performed by placing 1 μl of whole cell broth for each construct per corn seed or 2 μl per squash seed. Seed treatment, sowing and data recording were carried out as described in example 58.

[001183] Bacillus mycoides EE155, a plant growth promoting strain of the Bacillus cereus family, was transformed with expression plasmids as described in Example 44. Overexpression of exosporium proteins in this strain directly resulted in increased spore binding to the plant, and it results in higher plant growth stimulation . In particular, overexpression of BclB, BclA, CotO, CotE led to increased stimulation of plant growth. Other exosporium proteins can be overexpressed that can lead to changes in exospore structure, including ExsY, ExsFA/BxpB, CotY, CotO, ExsFB, InhA1, InhA2, ExsJ, ExsH, YjcA, YjcB, BclC, AcpC, InhA3, alanine racemase 1, alanine racemase 2 , BclA, BclB, BxpA, BclE, BetA/BAS3290, CotE, ExsA, ExsK, ExsB, YabG, Tgl, superoxide dismutase 1 (SODA1) and superoxide dismutase 2 (SODA2). Overexpression or mutation of any of these genes leads to changes in exospore structure and results in increased plant growth benefits associated with members of the Bacillus cereus family.

Table 73: Overexpression of exospore proteins in Bacillus mycoides EE155

bacteria Protein overexpression on plasmid pHP13 Squash growth (normalized to control) Corn growth (normalized to control) Bacillus mycoides B155 N/A (control) 100% 100% Bacillus mycoides B155 BclB 116.3% 101.4% Bacillus mycoides B155 BclA 106.8% 108.5% Bacillus mycoides B155 134.5% 106.3% Bacillus mycoides B155 CotO 118.6% 111.7%

Example 68 Plant Tissue Binding Using Exospore Exposed Binding Proteins

[001184] Spores that are suitable for displaying exogenous and endogenous proteins can be used as fusion partners to enhance spore binding to surfaces, including plant tissues. To demonstrate this trait, Bacillus thuringiensis BT013A spores were transformed with pSUPER BclA 20-35 TasA, pSUPER BclA 20-35 expansin, pSUPER BclA 20-35 Endo and pSUPER BclA 20-35 control plasmids. TasA and expansin are plant-binding proteins. The control plasmid contained the BclA promoter, start codon and amino acids 20-35 of SEQ ID NO: 1, but did not include a fusion partner. These constructs were prepared identically to the other constructs described in Example 58 above.

[001185] For tissue binding assays, corn plants were grown for 2 weeks and soybean plants were grown for 3 weeks as described in Example 58, but without any seed treatment. The primary leaf and first shamrock of the plants were then rubbed with 1 ml of spores containing each of the constructs described above. The leaves were left to dry, cut off from the plants and placed in a 50 ml conical tube with 10 ml of water and shaken vigorously. The spores that were released from the leaf into the water were counted on a hemocytometer and compared with the numbers expected if the spores were not bound to the leaves. This experiment was repeated ten times and a second experiment was performed which involved inoculating this water onto antibiotic plates (tetracycline plus nutrient agar) overnight at 30°C. Final amounts are shown in Table 74.

Table 74: Plant Tissue Binding Increased When Binding Protein Was Expressed on Spores

Processing (construction) culture Total binding, % Binding change relative to control Binding, % (plate analysis) Binding change relative to control Control (control BclA 20-35) Corn 42.9% N/A 0% N/A BclA 20-35 endoglucanase Corn 75.9% +33% 15.6% +15.6% BclA 20-35 expansin Corn 38.4% -4.5% 41.1% +41.1% BclA 20-35 TasA Corn 54.9% +12% 100% +100% Control (control BclA 20-35) Soya 58.3% N/A 65.2% N/A BclA 20-35 endoglucanase Soya 93.7% +35.4% 61% -4.2% BclA 20-35 expansin Soya 87.9% +29.6% 99.1% +33.9 BclA 20-35 TasA Soya 75.7% +20.8% 91.7% +26.5%

[001186] As can be seen from Table 74, the BT013A control spores have a high affinity for soybean with a binding of 58.3% and 65.2% of the control spores. Despite this, expression of endoglucanase, expansin, or TasA on the surface of spores resulted in increased sor binding to soybean leaves, with many spore preparations achieving 100% leaf binding. Significantly less control spore binding occurred in corn, especially in the plate assay. The results of the plate assay are most surprising, showing increased binding for each of the expression constructs, with TasA providing 100% spore binding in this assay.

[001187] These binding proteins can also be used in any of the recombinant spore-forming microorganisms using any of the expression systems or fusion partners described herein. This system is also useful in conjunction with exosporium strips to provide a protein delivery system that is both cell-free and tightly bound to leaves.

Example 69 Use of Recombinant Spore Forming Bacteria Expressing Fusion Proteins Containing Cot/Cge Proteins and Plant Growth Promotion Enzyme

[001188] Spore coat proteins from protein sheets are found on all spores of the Bacillus species described herein, as well as the related genera Virginibacillus, Lysinibacillus, Clostridia, and Paenibacillus . Fusion of proteins or peptides of interest to coat proteins allows expression of foreign proteins on the spore surface and delivery of these proteins or peptides of interest to plants. A series of constructs were created to demonstrate the ability of envelope proteins to deliver enzymes to plants. The pHP13 plasmid from the Bacillus Genetic Stock Culture collection was used to clone each of the constructs described below into a multiple cloning site using homologous recombination with their native promoter elements.

[001189] CotB, CotG and CotC from Bacillus subtilis M01099 or CgeA from Bacillus amyloliquefaciens were fused in frame with the eglS endoglucanase gene from Bacillus subtilis 168, the lipA lipase gene from Bacillus subtilis 168, or the pc-plc gene from Bacillus thuringiensis BT013A. These constructs were cloned into pHP13 by homologous recombination, confirmed by Sanger sequencing, and transformed into Bacillus subtilis EE405, Bacillus subtilis A09, Bacillus cereus EE439 , Bacillus sp. EE398 or Bacillus thuringiensis EE-B00184. Each transformant was also screened for the correct clones by Sanger sequencing. After confirmation of the clones, each clone was grown in brain heart extract (BHI) broth with tetracycline (10 μg/ml) overnight at 30° C. and 100 μl of the overnight culture was scraped onto tetracycline nutrient agar plates. These plates were incubated at 30° C. for 3 days and the spores were collected by brushing them off with a water-moistened cotton roller and resuspended in water.

[001190] The spores for endoglucanase assay were then diluted to 1×10 ⁸ kfu/ml in water and analyzed for enzymatic activity using the chromophore 4-chloro-2 nitrophenyl cellotetrose (4C2NC, 3 mM in water). For this method, 50 μl of spores were placed in a 96-well plate and 50 μl of 300 nM 4C2NC solution was added to each plate. Then the tablet was incubated at 30°C and read the absorbance at 410 nm after 0.5 hours. In all cases, the corresponding absorbance of the control strain was subtracted from the total absorbance of each clone to eliminate any background activity.

[001191] Lipase assay spores were diluted to 1×10 ⁸ kfu/ml in water and analyzed for enzymatic activity in a second method using the 4-nitrophenyl palmitate chromophore (4NP, 3 mM in water). For this method, 50 μl of spores were placed in a 96-well plate and 50 μl of a 300 nM 4NP solution was added to each plate. Then the tablet was incubated at 30°C and read the absorbance at 410 nm after 0.5 hours. In all cases, the corresponding absorbance of the control strain was subtracted from the total absorbance of each clone to eliminate any background activity.

[001192] Phospholipase assay spores were diluted to 1×10 ⁸ kfu/ml in water and analyzed for enzymatic activity as described above for phospholipase in Example 58. In all cases, the corresponding absorbance of the control strain was subtracted from the total absorbance each clone to eliminate any background activity.

[001193] Determination of plant growth responses and processing and collection was carried out as described for the squash in example 58 above. All growth data were normalized to those of the control strain without enzyme exposure on spores.

Table 75: Coat protein fusion constructs and their enzyme expression levels

Processing (construction) Fusion partner for endoglucanase Enzymatic activity of endoglucanase (uptake minus control) Lipase Enzymatic Activity (absorption minus control) Enzymatic activity of phospholipase, (uptake minus control) Growth response of the plant relative to the control strain, squash Control strain Bacillus subtilis A09 N/A 0.0 0.0 0.0 100% A09 CotB 0.01 0.03 0.198 101.5% A09 CotG ND 0.117 0.196 101.0% A09 CotC 0.09 0.069 0.154 99.4% A09 CgeA 0.13 0 0.218 ND Control strain Bacillus subtilis EE405 N/A 0.0 0.0 0.0 100% EE405 CgeA 1.84 ND ND 104.7% Control strain B. thuringiensis EE184 N/A 0.0 0.0 0.0 100% EE184 CotB 2.42 0.262 1.37 95.5% EE184 CotG 2.41 0 0.330 105% EE184 CotC 2.00 0.08 0.373 119.4% EE184 CgeA 2.70 0.520 0 110.1% The control strain of Bacillus sp. EE387 N/A ND 0.0 0.0 100% EE387 CotB ND 0.071 0.163 108.0% EE387 CotG ND 0.104 0.140 105.6% EE387 CotC ND 0.168 0.196 106.3% EE387 CgeA ND 0.124 0.187 108.5%

N/A=not applicable

ND=not defined

[001194] The data in Table 75 demonstrates that coat proteins broadly act on a number of plant beneficial enzymes, both in members of the Bacillus cereus family (EE184, EE439) and non- Bacillus cereus members (EE405, A09 and EE387 of this example). and example 65). The addition of spore-exposed enzymes, in this example endoglucanase, results in a plant growth enhancement phenotype in most cases.

IMPLEMENTATION OPTIONS

[001195] For further illustration, further non-limiting embodiments of the present invention are provided below.

[001196] Embodiment 1 is a fusion protein comprising at least one protein or peptide of interest and: (1) a targeting sequence comprising amino acids 1-30 of SEQ ID NO: 59; (2) a targeting sequence containing SEQ ID NO: 59; (3) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 60; (4) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 59; (5) a targeting sequence containing amino acids 4-30 of SEQ ID NO: 59; (6) a targeting sequence containing amino acids 6-30 of SEQ ID NO: 59; (7) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 61; (8) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 61; (9) a targeting sequence containing SEQ ID NO: 61; (10) an exosporium protein containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 62; (11) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 61; (12) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 61; (13) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 61; (14) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 61; (15) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 63; (16) a targeting sequence containing SEQ ID NO: 63; (17) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 64; (18) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 63; (19) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 63; (20) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 63; (21) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 63; (22) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 63; (23) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 65; (24) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 65; (25) a targeting sequence containing SEQ ID NO: 65; (26) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 66; (27) a targeting sequence containing SEQ ID NO: 107; (28) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 65; (29) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 65; (30) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 67; (31) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 67; (32) a targeting sequence containing SEQ ID NO: 67; (33) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 68; (34) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 67; (35) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 67; (36) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 67; (37) a targeting sequence containing amino acids 1-38 of SEQ ID NO: 69; (38) a targeting sequence containing amino acids 23-38 of SEQ ID NO: 69; (39) a targeting sequence containing SEQ ID NO: 69; (40) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 70; (41) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 69; (42) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 69; (43) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 69; (44) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 69; (45) exosporium protein containing SEQ ID NO: 72; (46) a targeting sequence containing SEQ ID NO: 73; (47) exospore protein containing an amino acid sequence having at least 95% identity with SEQ ID NO: 74; (48) a targeting sequence containing amino acids 1-42 of SEQ ID NO: 75; (49) a targeting sequence containing amino acids 27-42 of SEQ ID NO: 75; (50) a targeting sequence containing SEQ ID NO: 75; (51) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 76; (52) a targeting sequence containing amino acids 2-42 of SEQ ID NO: 75; (53) a targeting sequence containing amino acids 5-42 of SEQ ID NO: 75; (54) a targeting sequence containing amino acids 10-42 of SEQ ID NO: 75; (55) a targeting sequence containing amino acids 15-42 of SEQ ID NO: 75; (56) a targeting sequence containing amino acids 20-42 of SEQ ID NO: 75; (57) a targeting sequence containing amino acids 25-42 of SEQ ID NO: 75; (58) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 77; (59) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 77; (60) a targeting sequence containing SEQ ID NO: 77; (61) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 78; (62) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 77; (63) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 77; (64) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 80; (65) a targeting sequence containing amino acids 1-38 of SEQ ID NO: 81; (66) a targeting sequence containing amino acids 23-38 of SEQ ID NO: 81; (67) a targeting sequence containing SEQ ID NO: 81; (68) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 82; (69) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 81; (70) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 81; (71) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 81; (72) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 81; (73) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 81; (74) a targeting sequence containing amino acids 1-34 of SEQ ID NO: 83; (75) a targeting sequence containing SEQ ID NO: 83; (76) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 84; (77) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 86; (78) a targeting sequence containing amino acids 1-28 of SEQ ID NO: 87; (79) a targeting sequence containing amino acids 13-28 of SEQ ID NO: 87; (80) a targeting sequence containing SEQ ID NO: 87; (81) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 88; (82) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 87; (83) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 87; (84) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 87; (85) a targeting sequence containing amino acids 1-28 of SEQ ID NO: 89; (86) a targeting sequence containing SEQ ID NO: 89; (87) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 90; (88) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 89; (89) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 89; (90) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 89; (91) a targeting sequence containing amino acids 1-93 of SEQ ID NO: 91; (92) a targeting sequence comprising SEQ ID NO: 91; (93) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 92; (94) a targeting sequence containing amino acids 2-93 of SEQ ID NO: 91; (95) a targeting sequence containing amino acids 10-93 of SEQ ID NO: 91; (96) a targeting sequence containing amino acids 20-93 of SEQ ID NO: 91; (97) a targeting sequence containing amino acids 30-93 of SEQ ID NO: 91; (98) a targeting sequence containing amino acids 40-93 of SEQ ID NO: 91; (99) a targeting sequence containing amino acids 50-93 of SEQ ID NO: 91; (100) a targeting sequence containing amino acids 60-93 of SEQ ID NO: 91; (101) a targeting sequence containing amino acids 1-130 of SEQ ID NO: 93; (102) a targeting sequence comprising SEQ ID NO: 93; (103) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 94; (104) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 93; (105) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 93; (106) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 93; (107) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 93; or (108) an exospore protein containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 122.

[001197] Embodiment 2 is a fusion protein according to Embodiment 1, wherein the fusion protein comprises: (1) a targeting sequence comprising amino acids 2-30 of SEQ ID NO: 59; (2) a targeting sequence containing amino acids 4-30 of SEQ ID NO: 59; (3) a targeting sequence containing amino acids 6-30 of SEQ ID NO: 59; (4) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 61; (5) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 61; (6) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 61; (7) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 61; (8) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 63; (9) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 63; (10) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 63; (11) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 63; (12) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 63; (13) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 65; (14) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 65; (15) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 67; (16) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 67; (17) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 67; (18) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 69; (19) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 69; (20) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 69; (21) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 69; (22) a targeting sequence containing amino acids 2-42 of SEQ ID NO: 75; (23) a targeting sequence containing amino acids 5-42 of SEQ ID NO: 75; (24) a targeting sequence containing amino acids 10-42 of SEQ ID NO: 75; (25) a targeting sequence containing amino acids 15-42 of SEQ ID NO: 75; (26) a targeting sequence containing amino acids 20-42 of SEQ ID NO: 75; (27) a targeting sequence containing amino acids 25-42 of SEQ ID NO: 75; (28) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 77; (29) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 77; (30) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 81; (31) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 81; (32) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 81; (33) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 81; (34) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 81; (35) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 87; (36) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 87; (37) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 87; (38) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 89; (39) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 89; (40) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 89; (41) a targeting sequence containing amino acids 2-93 of SEQ ID NO: 91; (42) a targeting sequence containing amino acids 10-93 of SEQ ID NO: 91; (43) a targeting sequence containing amino acids 20-93 of SEQ ID NO: 91; (44) a targeting sequence containing amino acids 30-93 of SEQ ID NO: 91; (45) a targeting sequence containing amino acids 40-93 of SEQ ID NO: 91; (46) a targeting sequence containing amino acids 50-93 of SEQ ID NO: 91; (47) a targeting sequence containing amino acids 60-93 of SEQ ID NO: 91; (48) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 93; (49) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 93; (50) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 93; or (51) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 93.

[001198] Embodiment 3 is a fusion protein comprising at least one protein or peptide of interest and: (1) a targeting sequence consisting of amino acids 20-33 of SEQ ID NO: 1; (2) a targeting sequence 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 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 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 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 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 sequence 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 consisting of amino acids 12-25 of SEQ ID NO: 17; (41) a targeting 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 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 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 consisting 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 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 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: 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 targeting 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 targeting 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 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: 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 sequence 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 sequence consisting of amino acids 18-31 of SEQ ID NO: 53; (88) a targeting sequence 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 containing amino acids 18-31 of SEQ ID NO: 61; (91) a targeting sequence containing amino acids 18-29 of SEQ ID NO: 61; (92) a targeting sequence containing amino acids 19-31 of SEQ ID NO: 61; (93) a targeting sequence containing amino acids 9-22 of SEQ ID NO: 65; (94) a targeting sequence containing amino acids 9-20 of SEQ ID NO: 65; (95) a targeting sequence containing amino acids 10-22 of SEQ ID NO: 65; (96) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 107; (97) a targeting sequence containing amino acids 1-13 of SEQ ID NO: 107; (98) a targeting sequence containing amino acids 12-25 of SEQ ID NO: 67; (99) a targeting sequence containing amino acids 12-23 of SEQ ID NO: 67; (100) a targeting sequence containing amino acids 13-25 of SEQ ID NO: 67; (101) a targeting sequence containing amino acids 15-23 of SEQ ID NO: 67; (102) a targeting sequence containing amino acids 23-36 of SEQ ID NO: 69; (103) a targeting sequence containing amino acids 23-34 of SEQ ID NO: 69; (104) a targeting sequence containing amino acids 24-36 of SEQ ID NO: 69; (105) a targeting sequence containing amino acids 26-34 of SEQ ID NO: 69; (106) a targeting sequence containing amino acids 27-40 of SEQ ID NO: 75; (107) a targeting sequence containing amino acids 27-38 of SEQ ID NO: 75; (108) a targeting sequence containing amino acids 9-22 of SEQ ID NO: 77; (109) a targeting sequence containing amino acids 9-20 of SEQ ID NO: 77; (110) a targeting sequence containing amino acids 10¬¬-22 of SEQ ID NO: 77; (111) a targeting sequence containing amino acids 12-20 of SEQ ID NO: 77; (112) a targeting sequence containing amino acids 23-36 of SEQ ID NO: 81; (113) a targeting sequence containing amino acids 23-34 of SEQ ID NO: 81; (114) a targeting sequence containing amino acids 24-36 of SEQ ID NO: 81; (115) a targeting sequence containing amino acids 26-34 of SEQ ID NO: 81; (116) a targeting sequence containing amino acids 13-26 of SEQ ID NO: 87; (117) a targeting sequence containing amino acids 13-24 of SEQ ID NO: 87; or (118) a targeting sequence containing amino acids 14-26 of SEQ ID NO: 87.

[001199] Embodiment 4 is a fusion protein comprising an enzyme that catalyzes the production of nitric oxide or a nucleic acid binding protein or peptide, and: (1) a targeting sequence comprising an amino acid sequence having at least about 43% amino acid identity 20-35 SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 54%; (2) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 1; (3) a targeting sequence containing amino acids 20-35 of SEQ ID NO: 1; (4) a targeting sequence containing SEQ ID NO: 1; (5) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 2; (6) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 1; (7) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 1; (8) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 1; (9) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 1; (10) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 1; (11) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 3; (12) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 3; (13) a targeting sequence containing SEQ ID NO: 3; (14) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 4; (15) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 3; (16) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 3; (17) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 3; (18) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 3; (19) a targeting sequence containing amino acids 1-38 of SEQ ID NO: 5; (20) a targeting sequence containing amino acids 23-38 of SEQ ID NO: 5; (21) a targeting sequence containing SEQ ID NO: 5; (22) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 6; (23) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 5; (24) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 5; (25) a targeting sequence containing amino acids 8-38 of SEQ ID NO: 5; (26) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 5; (27) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 5; (28) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 5; (29) a targeting sequence containing amino acids 1-28 of SEQ ID NO: 7; (30) a targeting sequence containing amino acids 13-28 of SEQ ID NO: 7; (31) a targeting sequence containing SEQ ID NO: 7; (32) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 8; (33) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 7; (34) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 7; (35) a targeting sequence containing amino acids 8-28 of SEQ ID NO: 7; (36) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 7; (37) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 9; (38) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 9; (39) a targeting sequence containing SEQ ID NO: 9; (40) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 10; (41) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 9; (42) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 9; (43) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 9; (44) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 11; (45) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 11; (46) a targeting sequence containing SEQ ID NO: 11; (47) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 12; (48) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 11; (49) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 11; (50) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 11; (51) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 11; (52) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 11; (53) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 13; (54) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 13; (55) a targeting sequence containing SEQ ID NO: 13; (56) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 14; (57) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 13; (58) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 13; (59) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 13; (60) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 13; (61) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 13; (62) a targeting sequence containing amino acids 1-43 of SEQ ID NO: 15; (63) a targeting sequence containing amino acids 28-43 of SEQ ID NO: 15; (64) a targeting sequence containing SEQ ID NO: 15; (65) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 16; (66) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 15; (67) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 15; (68) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 15; (69) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 15; (70) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 15; (71) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 15; (72) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 15; (73) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 17; (74) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 17; (75) a targeting sequence containing SEQ ID NO: 17; (76) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 18; (77) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 17; (78) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 17; (79) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 17; (80) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 17; (81) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 19; (82) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 19; (83) a targeting sequence containing SEQ ID NO: 19; (84) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 20; (85) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 19; (86) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 19; (87) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 19; (88) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 19; (89) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 19; (90) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 21; (91) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 21; (92) a targeting sequence containing SEQ ID NO: 21; (93) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 22; (94) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 21; (95) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 21; (96) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 21; (97) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 21; (98) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 21; (99) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 23; (100) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 23; (101) a targeting sequence comprising SEQ ID NO: 23; (102) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 24; (103) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 23; (104) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 23; (105) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 23; (106) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 25; (107) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 25; (108) a targeting sequence comprising SEQ ID NO: 25; (109) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 26; (110) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 25; (111) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 25; (112) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 25; (113) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 27; (114) a targeting sequence containing amino acids 15-30 of SEQ ID NO: 27; (115) a targeting sequence comprising SEQ ID NO: 27; (116) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 28; (117) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 27; (118) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 27; (119) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 27; (120) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 27; (121) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 29; (122) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 29; (123) a targeting sequence comprising SEQ ID NO: 29; (124) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 30; (125) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 29; (126) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 29; (127) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 29; (128) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 29; (129) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 29; (130) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 31; (131) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 31; (132) a targeting sequence containing SEQ ID NO: 31; (133) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 32; (134) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 31; (135) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 31; (136) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 31; (137) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 33; (138) a targeting sequence comprising SEQ ID NO: 33; (139) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 34; (140) a targeting sequence containing amino acids 1-16 of SEQ ID NO: 35; (141) a targeting sequence containing SEQ ID NO: 35; (142) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 36; (143) a targeting sequence containing amino acids 1-29 of SEQ ID NO: 43; (144) a targeting sequence containing amino acids 14-29 of SEQ ID NO: 43; (145) a targeting sequence containing SEQ ID NO: 43; (146) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 44; (147) a targeting sequence containing amino acids 2-29 of SEQ ID NO: 43; (148) a targeting sequence containing amino acids 5-29 of SEQ ID NO: 43; (149) a targeting sequence containing amino acids 8-29 of SEQ ID NO: 43; (150) a targeting sequence containing amino acids 10-29 of SEQ ID NO: 43; (151) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 45; (152) a targeting sequence containing amino acids 20-35 of SEQ ID NO: 45; (153) a targeting sequence containing SEQ ID NO: 45; (154) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 46; (155) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 45; (156) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 45; (157) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 45; (158) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 45; (159) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 45; (160) a targeting sequence containing amino acids 1-43 of SEQ ID NO: 47; (161) a targeting sequence containing amino acids 28-43 of SEQ ID NO: 47; (162) a targeting sequence containing SEQ ID NO: 47; (163) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 48; (164) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 47; (165) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 47; (166) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 47; (167) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 47; (168) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 47; (169) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 47; (170) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 47; (171) a targeting sequence containing amino acids 1-32 of SEQ ID NO: 49; (172) a targeting sequence containing amino acids 17-32 of SEQ ID NO: 49; (173) a targeting sequence containing SEQ ID NO: 49; (174) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 50; (175) a targeting sequence containing amino acids 2-32 of SEQ ID NO: 49; (176) a targeting sequence containing amino acids 5-32 of SEQ ID NO: 49; (177) a targeting sequence containing amino acids 8-32 of SEQ ID NO: 49; (178) a targeting sequence containing amino acids 10-32 of SEQ ID NO: 49; (179) a targeting sequence containing amino acids 15-32 of SEQ ID NO: 49; (180) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 51; (181) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 51; (182) a targeting sequence comprising SEQ ID NO: 51; (183) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 52; (184) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 51; (185) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 51; (186) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 51; (187) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 51; (188) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 51; (189) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 53; (190) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 53; (191) a targeting sequence containing SEQ ID NO: 53; (192) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 54; (193) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 53; (194) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 53; (195) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 53; (196) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 53; (197) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 53; (198) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 55; (199) a targeting sequence containing amino acids 15-30 of SEQ ID NO: 55; (200) a targeting sequence comprising SEQ ID NO: 55; (201) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 56; (202) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 55; (203) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 55; (204) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 55; (205) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 55; (206) a targeting sequence containing amino acids 1-130 of SEQ ID NO: 57; (207) a targeting sequence containing amino acids 115-130 of SEQ ID NO: 57; (208) a targeting sequence containing SEQ ID NO: 57; (209) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 58; (210) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 57; (211) a targeting sequence containing amino acids 5-130 of SEQ ID NO: 57; (212) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 57; (213) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 57; (214) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 57; (215) a targeting sequence containing amino acids 40-130 of SEQ ID NO: 57; (216) a targeting sequence containing amino acids 50-130 of SEQ ID NO: 57; (217) a targeting sequence containing amino acids 60-130 of SEQ ID NO: 57; (218) a targeting sequence containing amino acids 70-130 of SEQ ID NO: 57; (219) a targeting sequence containing amino acids 80-130 of SEQ ID NO: 57; (220) a targeting sequence containing amino acids 90-130 of SEQ ID NO: 57; (221) a targeting sequence containing amino acids 100-130 of SEQ ID NO: 57; (222) a targeting sequence containing amino acids 110-130 of SEQ ID NO: 57; (223) an exospore protein fragment containing 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 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 containing SEQ ID NO: 103; (232) a targeting sequence comprising SEQ ID NO: 104; (233) a targeting sequence containing SEQ ID NO: 105; (234) a targeting sequence comprising SEQ ID NO: 106; (235) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (236) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 109; (237) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 110; (238) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 111; (239) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 112; (240) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 113; (241) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 114; (242) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 115; (243) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 116; (244) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 117; (245) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 118; (246) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 119; (247) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 120; (248) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 121; (249) a targeting sequence containing amino acids 22-31 of SEQ ID NO: 1; (250) a targeting sequence containing amino acids 22-33 of SEQ ID NO: 1; (251) a targeting sequence containing amino acids 20-31 of SEQ ID NO: 1; (252) a targeting sequence containing amino acids 14-23 of SEQ ID NO: 3; (253) a targeting sequence containing amino acids 14-25 of SEQ ID NO: 3; or (254) a targeting sequence containing amino acids 12-23 of SEQ ID NO: 3.

[001200] Embodiment 5 is a fusion protein containing a reclamation antigen or enzyme and: (1) a targeting sequence comprising amino acids 2-35 of SEQ ID NO: 1; (2) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 1; (3) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 1; (4) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 1; (5) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 1; (6) a targeting sequence containing amino acids 22-31 of SEQ ID NO: 1; (7) a targeting sequence containing amino acids 22-33 of SEQ ID NO: 1; (8) a targeting sequence containing amino acids 20-31 of SEQ ID NO: 1; (9) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 3; (10) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 3; (11) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 3; (12) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 3; (13) a targeting sequence containing amino acids 14-23 of SEQ ID NO: 3; (14) a targeting sequence containing amino acids 14-25 of SEQ ID NO: 3; (15) a targeting sequence containing amino acids 12-23 of SEQ ID NO: 3; (16) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 5; (17) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 5; (18) a targeting sequence containing amino acids 8-38 of SEQ ID NO: 5; (19) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 5; (20) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 5; (21) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 5; (22) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 7; (23) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 7; (24) a targeting sequence containing amino acids 8-28 of SEQ ID NO: 7; (25) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 7; (26) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 9; (27) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 9; (28) a targeting sequence containing SEQ ID NO: 9; (29) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 10; (30) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 9; (31) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 9; (32) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 9; (33) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 11; (34) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 11; (35) a targeting sequence containing SEQ ID NO: 11; (36) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 12; (37) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 11; (38) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 11; (39) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 11; (40) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 11; (41) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 11; (42) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 13; (43) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 13; (44) a targeting sequence containing SEQ ID NO: 13; (45) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 14; (46) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 13; (47) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 13; (48) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 13; (49) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 13; (50) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 13; (51) a targeting sequence containing amino acids 1-43 of SEQ ID NO: 15; (52) a targeting sequence containing amino acids 28-43 of SEQ ID NO: 15; (53) a targeting sequence containing SEQ ID NO: 15; (54) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 16; (55) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 15; (56) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 15; (57) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 15; (58) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 15; (59) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 15; (60) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 15; (61) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 15; (62) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 17; (63) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 17; (64) a targeting sequence containing SEQ ID NO: 17; (65) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 18; (66) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 17; (67) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 17; (68) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 17; (69) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 17; (70) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 19; (71) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 19; (72) a targeting sequence containing SEQ ID NO: 19; (73) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 20; (74) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 19; (75) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 19; (76) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 19; (77) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 19; (78) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 19; (79) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 21; (80) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 21; (81) a targeting sequence containing SEQ ID NO: 21; (82) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 22; (83) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 21; (84) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 21; (85) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 21; (86) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 21; (87) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 21; (88) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 23; (89) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 23; (90) a targeting sequence containing SEQ ID NO: 23; (91) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 24; (92) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 23; (93) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 23; (94) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 23; (95) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 25; (96) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 25; (97) a targeting sequence containing SEQ ID NO: 25; (98) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 26; (99) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 25; (100) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 25; (101) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 25; (102) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 27; (103) a targeting sequence containing amino acids 15-30 of SEQ ID NO: 27; (104) a targeting sequence containing SEQ ID NO: 27; (105) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 28; (106) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 27; (107) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 27; (108) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 27; (109) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 27; (110) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 29; (111) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 29; (112) a targeting sequence comprising SEQ ID NO: 29; (113) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 30; (114) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 29; (115) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 29; (116) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 29; (117) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 29; (118) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 29; (119) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 31; (120) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 31; (121) a targeting sequence comprising SEQ ID NO: 31; (122) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 32; (123) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 31; (124) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 31; (125) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 31; (126) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 33; (127) a targeting sequence comprising SEQ ID NO: 33; (128) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 34; (129) a targeting sequence containing amino acids 1-16 of SEQ ID NO: 35; (130) a targeting sequence comprising SEQ ID NO: 35; (131) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 36; (132) a targeting sequence containing amino acids 1-29 of SEQ ID NO: 43; (133) a targeting sequence containing amino acids 14-29 of SEQ ID NO: 43; (134) a targeting sequence comprising SEQ ID NO: 43; (135) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 44; (136) a targeting sequence containing amino acids 2-29 of SEQ ID NO: 43; (137) a targeting sequence containing amino acids 5-29 of SEQ ID NO: 43; (138) a targeting sequence containing amino acids 8-29 of SEQ ID NO: 43; (139) a targeting sequence containing amino acids 10-29 of SEQ ID NO: 43; (140) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 45; (141) a targeting sequence containing amino acids 20-35 of SEQ ID NO: 45; (142) a targeting sequence containing SEQ ID NO: 45; (143) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 46; (144) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 45; (145) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 45; (146) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 45; (147) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 45; (148) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 45; (149) a targeting sequence containing amino acids 1-43 of SEQ ID NO: 47; (150) a targeting sequence containing amino acids 28-43 of SEQ ID NO: 47; (151) a targeting sequence comprising SEQ ID NO: 47; (152) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 48; (153) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 47; (154) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 47; (155) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 47; (156) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 47; (157) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 47; (158) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 47; (159) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 47; (160) a targeting sequence containing amino acids 1-32 of SEQ ID NO: 49; (161) a targeting sequence containing amino acids 17-32 of SEQ ID NO: 49; (162) a targeting sequence containing SEQ ID NO: 49; (163) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 50; (164) a targeting sequence containing amino acids 2-32 of SEQ ID NO: 49; (165) a targeting sequence containing amino acids 5-32 of SEQ ID NO: 49; (166) a targeting sequence containing amino acids 8-32 of SEQ ID NO: 49; (167) a targeting sequence containing amino acids 10-32 of SEQ ID NO: 49; (168) a targeting sequence containing amino acids 15-32 of SEQ ID NO: 49; (169) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 51; (170) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 51; (171) a targeting sequence containing SEQ ID NO: 51; (172) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 52; (173) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 51; (174) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 51; (175) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 51; (176) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 51; (177) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 51; (178) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 53; (179) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 53; (180) a targeting sequence comprising SEQ ID NO: 53; (181) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 54; (182) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 53; (183) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 53; (184) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 53; (185) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 53; (186) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 53; (187) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 55; (188) a targeting sequence containing amino acids 15-30 of SEQ ID NO: 55; (189) a targeting sequence comprising SEQ ID NO: 55; (190) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 56; (191) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 55; (192) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 55; (193) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 55; (194) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 55; (195) a targeting sequence containing amino acids 1-130 of SEQ ID NO: 57; (196) a targeting sequence containing amino acids 115-130 of SEQ ID NO: 57; (197) a targeting sequence comprising SEQ ID NO: 57; (198) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 58; (199) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 57; (200) a targeting sequence containing amino acids 5-130 of SEQ ID NO: 57; (201) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 57; (202) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 57; (203) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 57; (204) a targeting sequence containing amino acids 40-130 of SEQ ID NO: 57; (205) a targeting sequence containing amino acids 50-130 of SEQ ID NO: 57; (206) a targeting sequence containing amino acids 60-130 of SEQ ID NO: 57; (207) a targeting sequence containing amino acids 70-130 of SEQ ID NO: 57; (208) a targeting sequence containing amino acids 80-130 of SEQ ID NO: 57; (209) a targeting sequence containing amino acids 90-130 of SEQ ID NO: 57; (210) a targeting sequence containing amino acids 100-130 of SEQ ID NO: 57; (211) a targeting sequence containing amino acids 110-130 of SEQ ID NO: 57; (212) a targeting sequence comprising SEQ ID NO: 97; (213) a targeting sequence containing SEQ ID NO: 98; (214) a targeting sequence comprising SEQ ID NO: 99; (215) a targeting sequence comprising SEQ ID NO: 100; (216) a targeting sequence containing 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 targeting sequence comprising SEQ ID NO: 105; (221) a targeting sequence comprising SEQ ID NO: 106; (222) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (223) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 109; (224) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 110; (225) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 111; (226) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 112; (227) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 113; (228) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 114; (229) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 115; (230) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 116; (231) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 117; (232) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 118; (233) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 119; (234) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 120; or (235) an exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 121.

[001201] Embodiment 6 is a fusion protein according to any one of Embodiments 1-3, wherein the protein or peptide of interest comprises a reclamation antigen or enzyme.

[001202] Embodiment 7 is a fusion protein according to any of embodiments 1-3, wherein the protein or peptide of interest comprises an enzyme suitable for breaking an emulsion or gel in a fracturing fluid, or an antibacterial protein or peptide.

[001203] Embodiment 8 is a fusion protein comprising an enzyme suitable for breaking an emulsion or gel in a fracturing fluid, or an antibacterial 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, where the identity with amino acids 25-35 is at least about 54%; (2) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 1; (3) a targeting sequence containing amino acids 20-35 of SEQ ID NO: 1; (4) a targeting sequence containing SEQ ID NO: 1; (5) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 2; (6) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 1; (7) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 1; (8) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 1; (9) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 1; (10) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 1; (11) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 3; (12) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 3; (13) a targeting sequence containing SEQ ID NO: 3; (14) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 4; (15) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 3; (16) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 3; (17) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 3; (18) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 3; (19) a targeting sequence containing amino acids 1-38 of SEQ ID NO: 5; (20) a targeting sequence containing amino acids 23-38 of SEQ ID NO: 5; (21) a targeting sequence containing SEQ ID NO: 5; (22) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 6; (23) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 5; (24) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 5; (25) a targeting sequence containing amino acids 8-38 of SEQ ID NO: 5; (26) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 5; (27) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 5; (28) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 5; (29) a targeting sequence containing amino acids 1-28 of SEQ ID NO: 7; (30) a targeting sequence containing amino acids 13-28 of SEQ ID NO: 7; (31) a targeting sequence containing SEQ ID NO: 7; (32) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 8; (33) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 7; (34) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 7; (35) a targeting sequence containing amino acids 8-28 of SEQ ID NO: 7; (36) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 7; (37) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 9; (38) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 9; (39) a targeting sequence containing SEQ ID NO: 9; (40) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 10; (41) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 9; (42) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 9; (43) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 9; (44) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 11; (45) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 11; (46) a targeting sequence containing SEQ ID NO: 11; (47) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 12; (48) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 11; (49) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 11; (50) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 11; (51) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 11; (52) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 11; (53) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 13; (54) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 13; (55) a targeting sequence containing SEQ ID NO: 13; (56) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 14; (57) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 13; (58) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 13; (59) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 13; (60) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 13; (61) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 13; (62) a targeting sequence containing amino acids 1-43 of SEQ ID NO: 15; (63) a targeting sequence containing amino acids 28-43 of SEQ ID NO: 15; (64) a targeting sequence containing SEQ ID NO: 15; (65) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 16; (66) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 15; (67) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 15; (68) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 15; (69) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 15; (70) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 15; (71) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 15; (72) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 15; (73) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 17; (74) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 17; (75) a targeting sequence containing SEQ ID NO: 17; (76) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 18; (77) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 17; (78) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 17; (79) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 17; (80) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 17; (81) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 19; (82) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 19; (83) a targeting sequence containing SEQ ID NO: 19; (84) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 20; (85) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 19; (86) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 19; (87) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 19; (88) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 19; (89) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 19; (90) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 21; (91) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 21; (92) a targeting sequence containing SEQ ID NO: 21; (93) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 22; (94) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 21; (95) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 21; (96) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 21; (97) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 21; (98) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 21; (99) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 23; (100) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 23; (101) a targeting sequence comprising SEQ ID NO: 23; (102) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 24; (103) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 23; (104) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 23; (105) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 23; (106) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 25; (107) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 25; (108) a targeting sequence comprising SEQ ID NO: 25; (109) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 26; (110) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 25; (111) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 25; (112) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 25; (113) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 27; (114) a targeting sequence containing amino acids 15-30 of SEQ ID NO: 27; (115) a targeting sequence comprising SEQ ID NO: 27; (116) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 28; (117) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 27; (118) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 27; (119) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 27; (120) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 27; (121) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 29; (122) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 29; (123) a targeting sequence comprising SEQ ID NO: 29; (124) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 30; (125) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 29; (126) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 29; (127) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 29; (128) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 29; (129) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 29; (130) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 31; (131) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 31; (132) a targeting sequence containing SEQ ID NO: 31; (133) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 32; (134) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 31; (135) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 31; (136) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 31; (137) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 33; (138) a targeting sequence comprising SEQ ID NO: 33; (139) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 34; (140) a targeting sequence containing amino acids 1-16 of SEQ ID NO: 35; (141) a targeting sequence containing SEQ ID NO: 35; (142) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 36; (143) a targeting sequence containing amino acids 1-29 of SEQ ID NO: 43; (144) a targeting sequence containing amino acids 14-29 of SEQ ID NO: 43; (145) a targeting sequence containing SEQ ID NO: 43; (146) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 44; (147) a targeting sequence containing amino acids 2-29 of SEQ ID NO: 43; (148) a targeting sequence containing amino acids 5-29 of SEQ ID NO: 43; (149) a targeting sequence containing amino acids 8-29 of SEQ ID NO: 43; (150) a targeting sequence containing amino acids 10-29 of SEQ ID NO: 43; (151) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 45; (152) a targeting sequence containing amino acids 20-35 of SEQ ID NO: 45; (153) a targeting sequence containing SEQ ID NO: 45; (154) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 46; (155) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 45; (156) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 45; (157) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 45; (158) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 45; (159) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 45; (160) a targeting sequence containing amino acids 1-43 of SEQ ID NO: 47; (161) a targeting sequence containing amino acids 28-43 of SEQ ID NO: 47; (162) a targeting sequence containing SEQ ID NO: 47; (163) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 48; (164) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 47; (165) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 47; (166) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 47; (167) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 47; (168) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 47; (169) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 47; (170) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 47; (171) a targeting sequence containing amino acids 1-32 of SEQ ID NO: 49; (172) a targeting sequence containing amino acids 17-32 of SEQ ID NO: 49; (173) a targeting sequence containing SEQ ID NO: 49; (174) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 50; (175) a targeting sequence containing amino acids 2-32 of SEQ ID NO: 49; (176) a targeting sequence containing amino acids 5-32 of SEQ ID NO: 49; (177) a targeting sequence containing amino acids 8-32 of SEQ ID NO: 49; (178) a targeting sequence containing amino acids 10-32 of SEQ ID NO: 49; (179) a targeting sequence containing amino acids 15-32 of SEQ ID NO: 49; (180) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 51; (181) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 51; (182) a targeting sequence comprising SEQ ID NO: 51; (183) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 52; (184) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 51; (185) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 51; (186) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 51; (187) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 51; (188) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 51; (189) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 53; (190) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 53; (191) a targeting sequence containing SEQ ID NO: 53; (192) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 54; (193) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 53; (194) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 53; (195) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 53; (196) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 53; (197) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 53; (198) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 55; (199) a targeting sequence containing amino acids 15-30 of SEQ ID NO: 55; (200) a targeting sequence comprising SEQ ID NO: 55; (201) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 56; (202) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 55; (203) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 55; (204) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 55; (205) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 55; (206) a targeting sequence containing amino acids 1-130 of SEQ ID NO: 57; (207) a targeting sequence containing amino acids 115-130 of SEQ ID NO: 57; (208) a targeting sequence containing SEQ ID NO: 57; (209) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 58; (210) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 57; (211) a targeting sequence containing amino acids 5-130 of SEQ ID NO: 57; (212) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 57; (213) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 57; (214) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 57; (215) a targeting sequence containing amino acids 40-130 of SEQ ID NO: 57; (216) a targeting sequence containing amino acids 50-130 of SEQ ID NO: 57; (217) a targeting sequence containing amino acids 60-130 of SEQ ID NO: 57; (218) a targeting sequence containing amino acids 70-130 of SEQ ID NO: 57; (219) a targeting sequence containing amino acids 80-130 of SEQ ID NO: 57; (220) a targeting sequence containing amino acids 90-130 of SEQ ID NO: 57; (221) a targeting sequence containing amino acids 100-130 of SEQ ID NO: 57; (222) a targeting sequence containing amino acids 110-130 of SEQ ID NO: 57; (223) an exospore protein fragment containing 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 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 containing 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) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (236) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 109; (237) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 110; (238) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 111; (239) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 112; (240) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 113; (241) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 114; (242) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 115; (243) exosporium protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 116; (244) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 117; (245) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 118; (246) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 119; (247) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 120; (248) exospore protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 121; (249) a targeting sequence containing amino acids 22-31 of SEQ ID NO: 1; (250) a targeting sequence containing amino acids 22-33 of SEQ ID NO: 1; (251) a targeting sequence containing amino acids 20-31 of SEQ ID NO: 1; (252) a targeting sequence containing amino acids 14-23 of SEQ ID NO: 3; (253) a targeting sequence containing amino acids 14-25 of SEQ ID NO: 3; or (254) a targeting sequence containing amino acids 12-23 of SEQ ID NO: 3.

[001204] Embodiment 9 is a fusion protein according to any 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 sequence containing amino acids 5-35 of SEQ ID NO: 1; (3) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 1; (4) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 1; (5) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 1; (6) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 3; (7) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 3; (8) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 3; (9) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 3; (10) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 5; (11) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 5; (12) a targeting sequence containing amino acids 8-38 of SEQ ID NO: 5; (13) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 5; (14) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 5; (15) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 5; (16) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 7; (17) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 7; (18) a targeting sequence containing amino acids 8-28 of SEQ ID NO: 7; (19) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 7; (20) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 9; (21) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 9; (22) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 9; (23) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 11; (24) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 11; (25) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 11; (26) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 11; (27) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 11; (28) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 13; (29) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 13; (30) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 13; (31) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 13; (32) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 13; (33) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 15; (34) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 15; (35) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 15; (36) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 15; (37) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 15; (38) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 15; (39) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 15; (40) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 17; (41) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 17; (42) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 17; (43) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 17; (44) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 19; (45) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 19; (46) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 19; (47) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 19; (48) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 19; (49) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 21; (50) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 21; (51) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 21; (52) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 21; (53) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 21; (54) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 23; (55) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 23; (56) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 23; (57) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 25; (58) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 25; (59) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 25; (60) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 27; (61) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 27; (62) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 27; (63) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 27; (64) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 29; (65) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 29; (66) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 29; (67) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 29; (68) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 29; (69) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 31; (70) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 31; (71) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 31; (72) a targeting sequence containing amino acids 2-29 of SEQ ID NO: 43; (73) a targeting sequence containing amino acids 5-29 of SEQ ID NO: 43; (74) a targeting sequence containing amino acids 8-29 of SEQ ID NO: 43; (75) a targeting sequence containing amino acids 10-29 of SEQ ID NO: 43; (76) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 45; (77) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 45; (78) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 45; (79) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 45; (80) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 45; (81) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 47; (82) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 47; (83) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 47; (84) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 47; (85) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 47; (86) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 47; (87) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 47; (88) a targeting sequence containing amino acids 2-32 of SEQ ID NO: 49; (89) a targeting sequence containing amino acids 5-32 of SEQ ID NO: 49; (90) a targeting sequence containing amino acids 8-32 of SEQ ID NO: 49; (91) a targeting sequence containing amino acids 10-32 of SEQ ID NO: 49; (92) a targeting sequence containing amino acids 15-32 of SEQ ID NO: 49; (93) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 51; (94) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 51; (95) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 51; (96) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 51; (97) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 51; (98) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 53; (99) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 53; (100) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 53; (101) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 53; (102) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 53; (103) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 55; (104) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 55; (105) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 55; (106) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 55; (107) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 57; (108) a targeting sequence containing amino acids 5-130 of SEQ ID NO: 57; (109) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 57; (110) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 57; (111) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 57; (112) a targeting sequence containing amino acids 40-130 of SEQ ID NO: 57; (113) a targeting sequence containing amino acids 50-130 of SEQ ID NO: 57; (114) a targeting sequence containing amino acids 60-130 of SEQ ID NO: 57; (115) a targeting sequence containing amino acids 70-130 of SEQ ID NO: 57; (116) a targeting sequence containing amino acids 80-130 of SEQ ID NO: 57; (117) a targeting sequence containing amino acids 90-130 of SEQ ID NO: 57; (118) a targeting sequence containing amino acids 100-130 of SEQ ID NO: 57; or (119) a targeting sequence containing amino acids 110-130 of SEQ ID NO: 57.

[001205] Embodiment 10 is a fusion protein according to Embodiment 9, wherein the targeting sequence comprises: (1) a targeting sequence comprising amino acids 2-24 of SEQ ID NO: 9; (2) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 9; (3) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 9; (4) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 11; (5) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 11; (6) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 11; (7) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 11; (8) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 11; (9) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 13; (10) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 13; (11) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 13; (12) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 13; (13) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 13; (14) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 15; (15) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 15; (16) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 15; (17) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 15; (18) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 15; (19) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 15; (20) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 15; (21) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 17; (22) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 17; (23) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 17; (24) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 17; (25) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 19; (26) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 19; (27) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 19; (28) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 19; (29) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 19; (30) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 21; (31) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 21; (32) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 21; (33) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 21; (34) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 21; (35) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 23; (36) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 23; (37) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 23; (38) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 25; (39) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 25; (40) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 25; (41) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 27; (42) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 27; (43) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 27; (44) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 27; (45) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 29; (46) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 29; (47) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 29; (48) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 29; (49) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 29; (50) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 31; (51) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 31; (52) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 31; (53) a targeting sequence containing amino acids 2-29 of SEQ ID NO: 43; (54) a targeting sequence containing amino acids 5-29 of SEQ ID NO: 43; (55) a targeting sequence containing amino acids 8-29 of SEQ ID NO: 43; (56) a targeting sequence containing amino acids 10-29 of SEQ ID NO: 43; (57) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 45; (58) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 45; (59) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 45; (60) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 45; (61) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 45; (62) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 47; (63) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 47; (64) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 47; (65) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 47; (66) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 47; (67) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 47; (68) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 47; (69) a targeting sequence containing amino acids 2-32 of SEQ ID NO: 49; (70) a targeting sequence containing amino acids 5-32 of SEQ ID NO: 49; (71) a targeting sequence containing amino acids 8-32 of SEQ ID NO: 49; (72) a targeting sequence containing amino acids 10-32 of SEQ ID NO: 49; (73) a targeting sequence containing amino acids 15-32 of SEQ ID NO: 49; (74) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 51; (75) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 51; (76) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 51; (77) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 51; (78) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 51; (79) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 53; (80) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 53; (81) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 53; (82) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 53; (83) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 53; (84) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 55; (85) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 55; (86) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 55; (87) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 55; (88) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 57; (89) a targeting sequence containing amino acids 5-130 of SEQ ID NO: 57; (90) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 57; (91) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 57; (92) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 57; (93) a targeting sequence containing amino acids 40-130 of SEQ ID NO: 57; (94) a targeting sequence containing amino acids 50-130 of SEQ ID NO: 57; (95) a targeting sequence containing amino acids 60-130 of SEQ ID NO: 57; (96) a targeting sequence containing amino acids 70-130 of SEQ ID NO: 57; (97) a targeting sequence containing amino acids 80-130 of SEQ ID NO: 57; (98) a targeting sequence containing amino acids 90-130 of SEQ ID NO: 57; (99) a targeting sequence containing amino acids 100-130 of SEQ ID NO: 57; or (100) a targeting sequence containing amino acids 110-130 of SEQ ID NO: 57.

[001206] Embodiment 11 is a fusion protein according to 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 targeting sequence containing amino acids 8-24 of SEQ ID NO: 9; (3) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 11; (4) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 11; (5) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 11; (6) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 11; (7) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 13; (8) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 13; (9) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 13; (10) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 13; (11) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 15; (12) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 15; (13) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 15; (14) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 15; (15) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 15; (16) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 15; (17) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 17; (18) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 17; (19) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 17; (20) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 19; (21) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 19; (22) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 19; (23) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 19; (24) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 21; (25) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 21; (26) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 21; (27) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 21; (28) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 23; (29) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 23; (30) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 25; (31) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 25; (32) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 27; (33) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 27; (34) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 27; (35) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 29; (36) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 29; (37) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 29; (38) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 29; (39) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 31; (40) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 31; (41) a targeting sequence containing amino acids 5-29 of SEQ ID NO: 43; (42) a targeting sequence containing amino acids 8-29 of SEQ ID NO: 43; (43) a targeting sequence containing amino acids 10-29 of SEQ ID NO: 43; (44) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 45; (45) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 45; (46) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 45; (47) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 45; (48) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 47; (49) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 47; (50) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 47; (51) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 47; (52) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 47; (53) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 47; (54) a targeting sequence containing amino acids 5-32 of SEQ ID NO: 49; (55) a targeting sequence containing amino acids 8-32 of SEQ ID NO: 49; (56) a targeting sequence containing amino acids 10-32 of SEQ ID NO: 49; (57) a targeting sequence containing amino acids 15-32 of SEQ ID NO: 49; (58) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 51; (59) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 51; (60) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 51; (61) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 51; (62) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 53; (63) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 53; (64) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 53; (65) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 53; (66) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 55; (67) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 55; (68) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 55; (69) a targeting sequence containing amino acids 5-130 of SEQ ID NO: 57; (70) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 57; (71) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 57; (72) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 57; (73) a targeting sequence containing amino acids 40-130 of SEQ ID NO: 57; (74) a targeting sequence containing amino acids 50-130 of SEQ ID NO: 57; (75) a targeting sequence containing amino acids 60-130 of SEQ ID NO: 57; (76) a targeting sequence containing amino acids 70-130 of SEQ ID NO: 57; (77) a targeting sequence containing amino acids 80-130 of SEQ ID NO: 57; (78) a targeting sequence containing amino acids 90-130 of SEQ ID NO: 57; (79) a targeting sequence containing amino acids 100-130 of SEQ ID NO: 57; (80) a targeting sequence containing amino acids 110-130 of SEQ ID NO: 57; (81) a targeting sequence containing amino acids 4-30 of SEQ ID NO: 59; (82) a targeting sequence containing amino acids 6-30 of SEQ ID NO: 59; (83) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 61; (84) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 61; (85) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 61; (86) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 63; (87) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 63; (88) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 63; (89) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 63; (90) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 65; (91) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 67; (92) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 67; (93) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 69; (94) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 69; (95) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 69; (96) a targeting sequence containing amino acids 5-42 of SEQ ID NO: 75; (97) a targeting sequence containing amino acids 10-42 of SEQ ID NO: 75; (98) a targeting sequence containing amino acids 15-42 of SEQ ID NO: 75; (99) a targeting sequence containing amino acids 20-42 of SEQ ID NO: 75; (100) a targeting sequence containing amino acids 25-42 of SEQ ID NO: 75; (101) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 77; (102) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 81; (103) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 81; (104) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 81; (105) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 81; (106) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 87; (107) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 87; (108) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 89; (109) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 89; (110) a targeting sequence containing amino acids 10-93 of SEQ ID NO: 91; (111) a targeting sequence containing amino acids 20-93 of SEQ ID NO: 91; (112) a targeting sequence containing amino acids 30-93 of SEQ ID NO: 91; (113) a targeting sequence containing amino acids 40-93 of SEQ ID NO: 91; (114) a targeting sequence containing amino acids 50-93 of SEQ ID NO: 91; (115) a targeting sequence containing amino acids 60-93 of SEQ ID NO: 91; (116) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 93; (117) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 93; or (118) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 93.

[001207] Embodiment 12 is a fusion protein according to any of embodiments 5 and 9-11, wherein the protein or peptide of interest comprises an antigen.

[001208] Embodiment 13 is a fusion protein according to any one of embodiments 5 and 9-11, wherein the protein or peptide of interest comprises a reclamation enzyme.

[001209] Embodiment 14 is a fusion protein according to any one of Embodiments 7-11, wherein the protein or peptide of interest comprises an enzyme suitable for breaking an emulsion or gel in a fracturing fluid.

[001210] Embodiment 15 is a fusion protein according to any one of Embodiments 7-11, wherein the protein or peptide of interest comprises an antibacterial protein or peptide.

[001211] Embodiment 16 is a recombinant member of the Bacillus cereus family that expresses a fusion protein according to any one of Embodiments 1-15.

[001212] Embodiment 17 is a recombinant member of the Bacillus cereus family that expresses:

(i) a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exospore protein or exospore protein fragment, which targets the fusion protein into the exospore of a recombinant member of the Bacillus cereus family; and

(ii) a modulator protein, wherein the expression of the modulator protein is increased compared to the expression of the modulator protein in a member of the Bacillus cereus family under the same conditions; and

where the co-expression of the modulator protein with the fusion protein in a recombinant member of the Bacillus cereus family results in increased or reduced expression of the fusion protein relative to the level of expression of the fusion protein in a recombinant member of the Bacillus cereus family that does not express the modulator protein at an increased level, under the same conditions according to compared with the expression of the modulator protein in a wild-type member of the Bacillus cereus family.

[001213] Embodiment 18 is a recombinant member of the Bacillus cereus family of Embodiment 17, wherein the modulator protein includes ExsY protein, ExsFA/BxpB protein, CotY protein, CotO protein, ExsFB protein, InhA1 protein, InhA2 protein, ExsJ protein, ExsH, YjcA protein, YjcB protein, BclC protein, AcpC protein, InhA3 protein, Alanine racemase 1, Alanine racemase 2, BclA protein, BclB protein, BxpA protein, BclE protein, BetA/BAS3290 protein, CotE protein, ExsA protein, ExsK protein, protein ExsB, YabG protein, Tgl protein, SODA1 protein, SODA2 protein, a variant of any of these, or a combination of any of them.

[001214] Embodiment 19 is a recombinant member of the Bacillus cereus family according to embodiment 17 or 18, wherein the modulator protein, when co-expressed in a recombinant member of the Bacillus cereus family with a fusion protein, results in increased expression of the fusion protein relative to the expression level of the fusion protein in a recombinant member of the Bacillus cereus family that does not express the modulator protein at an increased level, under the same conditions compared to the expression of the modulator protein in a wild-type member of the Bacillus cereus family.

[001215] Embodiment 20 is a recombinant member of the Bacillus cereus family of Embodiment 19, wherein the modulator protein includes a BclB protein, a CotE protein, a BxpB protein, a CotO protein, a BclA protein, a variant of any of these, or a combination of any of them.

[001216] Embodiment 21 is a recombinant member of the Bacillus cereus family according to embodiment 17 or 18, wherein the modulator protein, when co-expressed in a recombinant member of the Bacillus cereus family with a fusion protein, results in reduced expression of the fusion protein relative to the expression level of the fusion protein in a recombinant member of the Bacillus cereus family that does not express the modulator protein at an elevated level, under the same conditions as compared to the expression of the modulator protein in a wild-type member of the Bacillus cereus family.

[001217] Embodiment 22 is a recombinant member of the Bacillus cereus family of Embodiment 21, wherein the modulator protein comprises a BclC protein, an ApcC protein, a YjcB protein, a variant of any of these, or a combination of any of them.

[001218] Embodiment 23 is a recombinant member of the Bacillus cereus family of Embodiment 17 or 18, wherein the modulator protein includes CotO protein, BclB protein, ExsFA/BxpB protein, YjcB protein, a variant of any of these, or a combination of any of them.

[001219] Embodiment 24 is a recombinant member of the Bacillus cereus family according to 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% of at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NOs: 123-156.

[001220] Embodiment 25 is a recombinant member of the Bacillus cereus family according to any one of Embodiments 17-24, wherein the modulator protein comprises SEQ ID NOs: 124, 126, 134, 135, 143, or 144.

[001221] Embodiment 26 is a recombinant member of the Bacillus cereus family according to any one of Embodiments 17-25 containing a vector encoding a modulator protein.

[001222] Embodiment 27 is a recombinant member of the Bacillus cereus family of Embodiment 26, wherein the vector comprises a multicopy plasmid vector.

[001223] Embodiment 28 is a recombinant member of the Bacillus cereus family that expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, an exosporium protein or an exospore protein fragment that targets the fusion protein to the exospore of the recombinant member of the Bacillus family cereus ; where the recombinant member of the Bacillus cereus family contains a mutation that partially or completely inactivates spores of the recombinant member of the Bacillus cereus family or increases the sensitivity of spores of the recombinant member of the Bacillus cereus family to physical or chemical inactivation compared to the same spores that do not contain the mutation, and where the mutation includes a mutation in a gene encoding a sprouting receptor, a mutation in a gene encoding a lytic enzyme of the spore bark, a mutation in a gene encoding a small acid-soluble spore protein (SASP), or a mutation in a gene encoding a coat or spore bark protein.

[001224] Embodiment 29 is a recombinant member of the Bacillus cereus family that expresses a fusion protein according to any one of Embodiments 1-15; where the recombinant member of the Bacillus cereus family contains a mutation that partially or completely inactivates the spores of the recombinant member of the Bacillus cereus family or increases the sensitivity of the spores of the recombinant member of the Bacillus cereus family to physical or chemical inactivation compared to the same spores that do not contain the mutation.

[001225] Embodiment 30 is a recombinant bacterium of the genus Bacillus that expresses a protease or nuclease, where the expression of the protease or nuclease is increased compared to the expression of the protease or nuclease in a wild-type bacterium of the genus Bacillus under the same conditions, and where the increased expression of the protease or nuclease partially or completely inactivates spores of recombinant bacteria of the genus Bacillus or increases the sensitivity of spores of recombinant bacteria of the genus Bacillus to physical or chemical inactivation.

[001226] Embodiment 31 is the recombinant bacterium of Embodiment 30, wherein the recombinant bacterium of the genus Bacillus comprises a recombinant member of the Bacillus cereus family.

[001227] Embodiment 32 is a recombinant bacterium of the genus Bacillus according to embodiment 30 or 31, wherein the recombinant bacterium of the genus Bacillus expresses a protease and a nuclease, wherein the expression of the protease is increased compared to the expression in a wild-type bacterium of the genus Bacillus under the same conditions and expression of the nuclease increased compared to nuclease expression in wild-type bacteria of the genus Bacillus under the same conditions.

[001228] Embodiment 33 is a recombinant bacterium of the genus Bacillus according to any one of Embodiments 30-32, wherein the protease includes a non-specific protease.

[001229] Embodiment 34 is a recombinant bacterium of the genus Bacillus according to any one of embodiments 30-33, wherein the protease includes serine protease, threonine protease, cysteine protease, aspartate protease, glutamic acid protease, alkaline protease, subtilisin, histidine protease, or metalloproteinase.

[001230] Embodiment 35 is a recombinant bacterium of the genus Bacillus according to any one of embodiments 30, 32, and 34, wherein the protease comprises a spore germinating protease.

[001231] Embodiment 36 is a recombinant bacterium of the genus Bacillus according to Embodiment 35, wherein the spore germinating protease comprises an active form of the spore germinating protease.

[001232] Embodiment 37 is a recombinant bacterium of the genus Bacillus according to embodiment 35 or 36, wherein the spore germinating protease comprises Bacillus subtilis spore germinating protease, Bacillus mycoides spore germinating protease, or Bacillus thuringiensis spore germinating protease.

[001233] Embodiment 38 is a recombinant bacterium of the genus Bacillus according to any of embodiments 35-37, wherein the spore germinating protease comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least at least 98%, at least 99% or 100% identity with SEQ ID NO: 233 or 234.

[001234] Embodiment 39 is a recombinant bacterium of the genus Bacillus according to any one of Embodiments 30-38, wherein the nuclease comprises an endonuclease or an exonuclease.

[001235] Embodiment 40 is a recombinant bacterium of the genus Bacillus according to Embodiment 39, wherein the nuclease includes a non-specific endonuclease.

[001236] Embodiment 41 is a recombinant bacterium of the genus Bacillus according to Embodiment 40, wherein the non-specific endonuclease comprises Bacillus subtilis endonuclease 1.

[001237] Embodiment 42 is a recombinant bacterium of the genus Bacillus according to embodiment 40 or 41, wherein the non-specific endonuclease contains 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: 232.

[001238] Embodiment 43 is a recombinant bacterium of the genus Bacillus according to any one of embodiments 30-42, wherein the nuclease or protease is expressed under the control of a promoter containing a sigma G promoter sequence.

[001239] Embodiment 44 is a recombinant bacterium of the genus Bacillus according to Embodiment 43, wherein the promoter contains a nucleic acid sequence having at least 95%, at least 98%, at least 99%, or 100% identity with the nucleic acid sequence according to any one of SEQ ID NO: 235-246.

[001240] Embodiment 45 is a recombinant bacterium of the genus Bacillus according to any one of embodiments 30-44, wherein the spores of the recombinant bacterium of the genus Bacillus are more susceptible to inactivation by ultraviolet irradiation, gamma irradiation, or treatment with bleach, hydrogen peroxide, chloroform, phenol or acetic acid against the same spores that do not express the protease or nuclease at an elevated level compared to the expression of the protease or nuclease in wild-type bacterium of the genus Bacillus treated under the same conditions.

[001241] Embodiment 46 is a recombinant member of the Bacillus cereus family according to any one of embodiments 31-45, wherein the recombinant member of the Bacillus cereus family further comprises a mutation that partially or completely inactivates spores of the recombinant member of the Bacillus cereus family or sensitizes the spores recombinant member of the Bacillus cereus family to physical or chemical inactivation compared to the same spores that do not contain the mutation.

[001242] Embodiment 47 is a recombinant member of the Bacillus cereus family of Embodiment 46, wherein the mutation comprises a mutation in a gene encoding a sprouting receptor, a mutation in a gene encoding a spore bark lytic enzyme, a mutation in a gene encoding a small acid-soluble spore protein (SASP), or a mutation in the gene encoding the spore coat or bark protein.

[001243] Embodiment 48 is a recombinant member of the Bacillus cereus family of Embodiment 28, 29, or 47, wherein the mutation comprises a mutation in a gene encoding a sprouting receptor.

[001244] Embodiment 49 is a recombinant member of the Bacillus cereus family of Embodiment 48, wherein the mutation in the gene encoding the sprouting receptor includes a knockout of the gene encoding the sprouting receptor.

[001245] Embodiment 50 is a recombinant member of the Bacillus cereus family of Embodiment 48 or 49, wherein the sprouting receptor comprises GerA, GerB, GerK, GerH, GerI, GerG, GerL, GerQ, GerR, GerS, GerN, GerU, or GerX.

[001246] Embodiment 51 is a recombinant member of the Bacillus cereus family according to any one of Embodiments 28, 29, and 47-50, wherein the mutation comprises a mutation in a gene encoding a spore bark lytic enzyme.

[001247] Embodiment 52 is a recombinant member of the Bacillus cereus family of Embodiment 51, wherein the mutation in the gene encoding the spore cortex lytic enzyme includes a knockout of the gene encoding the spore cortex lytic enzyme.

[001248] Embodiment 53 is a recombinant member of the Bacillus cereus family according to Embodiment 51 or 52, wherein the spore bark lytic enzyme comprises SleB or CwlJ.

[001249] Embodiment 54 is a recombinant member of the Bacillus cereus family according to any one of Embodiments 28, 29, and 47-53, wherein the mutation comprises a mutation in the gene encoding SASP.

[001250] Embodiment 55 is a recombinant member of the Bacillus cereus family of Embodiment 54, wherein the mutation in the gene encoding SASP includes a knockout of the gene encoding SASP.

[001251] Embodiment 56 is a recombinant member of the Bacillus cereus family of Embodiment 54 or 55, wherein the mutation in the gene encoding SASP includes a mutation in an SspA gene, a mutation in an SspB gene, a mutation in an SspC gene, a mutation in an SspD gene, a mutation in the SspE gene, mutation in the SspF gene, mutation in the SspG gene, mutation in the SspH gene, mutation in the SspI gene, mutation in the SspJ gene, mutation in the SspK gene, mutation in the SspL gene, mutation in the SspM gene, mutation in the SspN gene, mutation in the SspO gene, a mutation in the SspP gene, or a combination thereof.

[001252] Embodiment 57 is a recombinant member of the Bacillus cereus family according to any one of Embodiments 54-56, wherein the SASP includes SASPα, SASPβ, or SASPγ.

[001253] Embodiment 58 is a recombinant member of the Bacillus cereus family according to any one of embodiments 54-57, wherein the spores of the recombinant member of the Bacillus cereus family are more susceptible to inactivation by ultraviolet irradiation or gamma irradiation compared to the same spores, that do not contain a mutation in the gene encoding SASP.

[001254] Embodiment 59 is a recombinant member of the Bacillus cereus family according to any one of Embodiments 28, 29, and 47-58, wherein the mutation comprises a mutation in a gene encoding a spore coat or bark protein.

[001255] Embodiment 60 is a recombinant member of the Bacillus cereus family of Embodiment 59, wherein the mutation in the gene encoding the spore coat or bark protein comprises a knockout of the gene encoding the spore coat or bark protein.

[001256] Embodiment 61 is a recombinant member of the Bacillus cereus family of Embodiment 59 or 60, wherein the spore coat or bark protein comprises CotA, CotB, or CotC.

[001257] Embodiment 62 is a recombinant member of the Bacillus cereus family according to any one of embodiments 59-61, wherein the spores of the recombinant member of the Bacillus cereus family are more susceptible to inactivation by ultraviolet irradiation, gamma irradiation, or by treatment with bleach, hydrogen peroxide , chloroform, phenol, or acetic acid, compared to the same spores that do not contain a mutation in the spore coat or bark protein treated under the same conditions.

[001258] Embodiment 63 is a recombinant member of the Bacillus cereus family according to any one of embodiments 31-62, wherein the recombinant member of the Bacillus cereus family also expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, an exospore protein or an exospore protein fragment that targets the fusion protein to the exospore of a recombinant member of the Bacillus cereus family.

[001259] Embodiment 64 is a recombinant member of the Bacillus cereus family that expresses an exosporium protein, wherein the expression of the exospore protein is increased compared to the expression of the exospore protein in a wild-type member of the Bacillus cereus family under the same conditions, and wherein the exospore protein comprises:

an exosporium enzyme, wherein the exosporium enzyme includes an enzyme involved in nutrient solubilization, inosinuridine hydrolase, a protease, an enzyme that catalyzes free radical degradation, arginase or alanine racemase; or

BclA protein, BclB protein, CotE protein CotO protein, an ExsY protein, ExsFA/BxpB protein, CotY protein, ExsFB protein, ExsJ protein, ExsH protein, YjcA protein, YjcB protein, BclC protein, BxpA protein, BclE protein, BetA/ protein BAS3290, ExsA protein, ExsK protein, ExsB protein, YabG protein, or Tgl protein.

[001260] Embodiment 65 is a recombinant member of the Bacillus cereus family of Embodiment 64 wherein the exospore protein is not part of the fusion protein.

[001261] Embodiment 66 is a recombinant member of the Bacillus cereus family of Embodiment 64 or 65, wherein the exosporium protein comprises an exospore enzyme, wherein the exosporium enzyme comprises an enzyme involved in nutrient solubilization, inosinuridine hydrolase, a protease, an enzyme that catalyzes free radical degradation , arginase or alanine racemase.

[001262] Embodiment 67 is a recombinant member of the Bacillus cereus family of Embodiment 66, wherein the enzyme involved in nutrient solubilization includes an enzyme involved in phosphate solubilization.

[001263] Embodiment 68 is a recombinant member of the Bacillus cereus family according to Embodiment 67, wherein the enzyme involved in phosphate solubilization includes acid phosphatase.

[001264] Embodiment 69 is a recombinant member of the Bacillus cereus family according to Embodiment 68, wherein the acid phosphatase includes AcpC.

[001265] Embodiment 70 is a recombinant member of the Bacillus cereus family of Embodiment 68 or 69, wherein the acid phosphatase 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.

[001266] Embodiment 71 is a recombinant member of the Bacillus cereus family of Embodiment 64 or 65, wherein the exospore enzyme comprises inosinuridine hydrolase.

[001267] Embodiment 72 is a recombinant member of the Bacillus cereus family of Embodiment 71, wherein the inosinuridine hydrolase comprises IunH1 or IunH2.

[001268] Embodiment 73 is a recombinant member of the Bacillus cereus family of Embodiment 71 or 72, wherein the inosinuridine hydrolase contains 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.

[001269] Embodiment 74 is a recombinant member of the Bacillus cereus family of Embodiment 64 or 65, wherein the exospore enzyme comprises a protease.

[001270] Embodiment 75 is a recombinant member of the Bacillus cereus family according to Embodiment 74, wherein the protease comprises a metalloproteinase.

[001271] Embodiment 76 is a recombinant member of the Bacillus cereus family of Embodiment 75, wherein the metalloproteinase comprises InhA1, InhA2, or InhA3.

[001272] Embodiment 77 is a recombinant member of the Bacillus cereus family of Embodiment 75 or 76, wherein the metalloproteinase contains 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.

[001273] Embodiment 78 is a recombinant member of the Bacillus cereus family of Embodiment 64 or 65, wherein the enzyme that catalyzes free radical degradation comprises superoxide dismutase.

[001274] Embodiment 79 is a recombinant member of the Bacillus cereus family of Embodiment 78, wherein the superoxide dismutase comprises superoxide dismutase 1 (SODA1) or superoxide dismutase 2 (SODA2).

[001275] Embodiment 80 is a recombinant member of the Bacillus cereus family of Embodiment 78 or 79, wherein the superoxide dismutase contains 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.

[001276] Embodiment 81 is a recombinant member of the Bacillus cereus family of Embodiment 64 or 65, wherein the exospore enzyme comprises arginase.

[001277] Embodiment 82 is a recombinant member of the Bacillus cereus family of Embodiment 81, wherein the arginase comprises Bacillus thuringiensis arginase.

[001278] Embodiment 83 is a recombinant member of the Bacillus cereus family of Embodiment 81 or 82, wherein the arginase contains 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.

[001279] Embodiment 84 is a recombinant member of the Bacillus cereus family according to Embodiment 64 or 65, wherein the exospore enzyme comprises alanine racemase.

[001280] Embodiment 85 is a recombinant member of the Bacillus cereus family of Embodiment 84, wherein the alanine racemase comprises either alanine racemase 1 (ALR1) or alanine racemase 2 (ALR2).

[001281] Embodiment 86 is a recombinant member of the Bacillus cereus family of Embodiment 84 or 85, wherein the alanine racemase contains 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.

[001282] Embodiment 87 is a recombinant member of the Bacillus cereus family of Embodiment 64 or 65, wherein the exosporium protein includes BclA protein, BclB protein, CotE protein, CotO protein, an ExsY protein, ExsFA/BxpB protein, CotY protein, ExsFB protein, ExsJ protein, ExsH protein, YjcA protein, YjcB protein, BclC protein, BxpA protein, BclE protein, BetA/BAS3290 protein, ExsA protein, ExsK protein, ExsB protein, YabG protein, or Tgl protein.

[001283] Embodiment 88 is a recombinant member of the Bacillus cereus family of Embodiment 87, wherein the exospore protein comprises BclA protein, BclB protein, CotE protein, or CotO protein.

[001284] Embodiment 89 is a recombinant member of the Bacillus cereus family of Embodiment 87 or 88, wherein the exospore protein comprises BclA protein.

[001285] Embodiment 90 is a recombinant member of the Bacillus cereus family of Embodiment 89, wherein the BclA protein contains 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.

[001286] Embodiment 91 is a member of the Bacillus cereus family of Embodiment 87 or 88, wherein the exospore protein comprises BclB protein.

[001287] Embodiment 92 is a member of the Bacillus cereus family 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.

[001288] Embodiment 93 is a recombinant member of the Bacillus cereus family according to Embodiment 87 or 88, wherein the exospore comprises the CotE protein.

[001289] Embodiment 94 is a recombinant member of the Bacillus cereus family of Embodiment 93, wherein the CotE protein contains 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: 149.

[001290] Embodiment 95 is a recombinant member of the Bacillus cereus family according to Embodiment 87 or 88, wherein the exospore protein comprises the CotO protein.

[001291] Embodiment 96 is a recombinant member of the Bacillus cereus family of Embodiment 95, wherein the CotO protein contains 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.

[001292] Embodiment 97 is a recombinant member of the Bacillus cereus family of Embodiment 87, wherein the exosporium protein includes ExsY protein, ExsFA/BxpB protein, CotY protein, ExsFB protein, ExsJ protein, ExsH protein, YjcA protein, YjcB protein, BclC protein , BxpA protein, BclE protein, BetA/BAS3290 protein, ExsA protein, ExsK protein, ExsB protein, YabG protein, or Tgl protein.

[001293] Embodiment 98 is a recombinant member of the Bacillus cereus family of Embodiment 97, wherein the exospore protein comprises:

an ExsY protein, wherein the ExsY protein contains 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 ;

an ExsFA/BxpB protein, wherein the ExsFA/BxpB protein contains 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 contains 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, wherein the ExsFB protein contains 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 ExsJ protein, wherein the ExJ protein contains 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 ExsH protein, wherein the ExsH protein contains 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 ;

a YjcA protein, wherein the YjcA protein contains 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 YjcB protein, wherein the YjcB protein contains 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 BclC protein, wherein the BclC 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 BxpA protein, wherein the BxpA 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: 145;

a BclE protein, wherein the BclE protein contains 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 contains 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: 148;

an ExsA protein, wherein the ExsA protein contains 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 contains 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 ExsB protein, wherein the ExsB protein contains 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, wherein the YabG protein contains 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 Tjl protein, wherein the Tjl protein contains 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 .

[001294] Embodiment 99 is a recombinant member of the Bacillus cereus family according to any one of embodiments 64-98, wherein the recombinant member of the Bacillus cereus family also expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, an exospore protein or an exospore protein fragment that targets the fusion protein to the exospore of a recombinant member of the Bacillus cereus family.

[001295] Embodiment 100 is a recombinant member of the Bacillus cereus family that expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, an exosporium protein or an exospore protein fragment, that targets the fusion protein into the exospore of the recombinant member of the Bacillus family cereus ; where the recombinant member of the Bacillus cereus family contains a mutation or expresses a protein, where the expression of the protein is increased compared to the expression of the protein in a member of the wild-type Bacillus cereus family under the same conditions, and where the mutation or increased expression of the protein provides Bacillus cereus spores having an exospore that is lighter remove wild-type spores from spores compared to exospores.

[001296] Embodiment 101 is a recombinant member of the Bacillus cereus family 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 into the exospore of the recombinant member of the Bacillus family cereus ; where is a recombinant member of the Bacillus cereus family:

(i) contains a mutation in the CotE gene;

(ii) expresses an ExsY protein, wherein the expression of the ExsY protein is increased compared to the expression of the ExsY protein in a wild-type member of the Bacillus cereus family under the same conditions, and where the ExsY protein contains a C-terminal tag containing a globular protein;

(iii) expresses a BclB protein, wherein the expression of the BclB protein is increased compared to the expression of the BclB protein in a wild-type member of the Bacillus cereus family under the same conditions;

(iv) expresses a YjcB protein, wherein the expression of the YjcB protein is increased compared to the expression of the YjcB protein in a wild-type member of the Bacillus cereus family under the same conditions;

(v) contains a mutation in the ExsY gene;

(vi) contains a mutation in the CotY gene;

(vii) contains a mutation in the ExsA gene; or

(viii) contains a mutation in the CotO gene.

[001297] Embodiment 102 is a recombinant member of the Bacillus cereus family according to embodiment 100 or 101, wherein the recombinant member of the Bacillus cereus family contains a mutation in the CotE gene.

[001298] Embodiment 103 is a recombinant member of the Bacillus cereus family of Embodiment 102, wherein a mutation in the CotE gene partially or completely inhibits the ability of CotE to bind exospores to spores.

[001299] Embodiment 104 is a recombinant member of the Bacillus cereus family according to embodiment 102 or 103, wherein the mutation in the CotE gene includes a knockout of the CotE gene or a dominant negative form of the CotE gene.

[001300] Embodiment 105 is a recombinant member of the Bacillus cereus family according to any one of embodiments 100-104, wherein the recombinant member of the Bacillus cereus family expresses an ExsY protein, wherein the expression of the ExsY protein is increased compared to the expression of the ExsY protein in the wild type member of the Bacillus cereus family under the same conditions, and where the ExsY protein contains a C-terminal tag containing a globular protein.

[001301] Embodiment 106 is a recombinant member of the Bacillus cereus family of Embodiment 105, wherein the globular protein has a molecular weight of 25 kDa to 100 kDa.

[001302] Embodiment 107 is a recombinant member of the Bacillus cereus family according to embodiment 105 or 106, wherein the globular protein comprises green fluorescent protein (GFP) or a variant thereof.

[001303] Embodiment 108 is a recombinant member of the Bacillus cereus family according to any of embodiments 105-107, wherein expression of an ExsY protein containing a C-terminal tag containing a globular protein inhibits the binding of the ExsY protein to its targets in the exospore.

[001304] Embodiment 109 is a recombinant member of the Bacillus cereus family according to any one of embodiments 100-108, wherein the recombinant member of the Bacillus cereus family expresses a BclB protein, wherein the expression of the BclB protein is increased compared to the expression of the BclB protein in the wild type member of the Bacillus cereus family under the same conditions.

[001305] Embodiment 110 is a recombinant member of the Bacillus cereus family of Embodiment 109, wherein expression of the BclB protein results in the formation of a fragile exospore.

[001306] Embodiment 111 is a recombinant member of the Bacillus cereus family according to any one of embodiments 100-110, wherein the recombinant member of the Bacillus cereus family expresses the YjcB protein, wherein the expression of the YjcB protein is increased compared to the expression of the YjcB protein in the wild type member of the Bacillus cereus family under the same conditions.

[001307] Embodiment 112 is a recombinant member of the Bacillus cereus family of Embodiment 111, wherein YjcB protein expression results in fragmented exospore formation instead of a complete structure.

[001308] Embodiment 113 is a recombinant member of the Bacillus cereus family according to any one of embodiments 100-112, wherein the recombinant member of the Bacillus cereus family contains a mutation in the ExsY gene.

[001309] Embodiment 114 is a recombinant member of the Bacillus cereus family of Embodiment 113, wherein a mutation in the ExsY gene partially or completely inhibits the ability of ExsY to complete exospore formation or bind exospores to a spore.

[001310] Embodiment 115 is a recombinant member of the Bacillus cereus family according to embodiment 113 or 114, wherein the mutation in the ExsY gene includes a knockout of the ExsY gene.

[001311] Embodiment 116 is a recombinant member of the Bacillus cereus family according to any one of embodiments 100-115, wherein the recombinant member of the Bacillus cereus family contains a mutation in the CotY gene.

[001312] Embodiment 117 is a recombinant member of the Bacillus cereus family of Embodiment 116, wherein the mutation in the CotY gene includes a knockout of the CotY gene.

[001313] Embodiment 118 is a recombinant member of the Bacillus cereus family of Embodiment 116 or 117, wherein a mutation in the CotY gene results in the formation of a fragile exospore.

[001314] Embodiment 119 is a recombinant member of the Bacillus cereus family according to any of embodiments 100-118, wherein the recombinant member of the Bacillus cereus family comprises a mutation in the ExsA gene.

[001315] Embodiment 120 is a recombinant member of the Bacillus cereus family of Embodiment 119, wherein the mutation in the ExsA gene includes a knockout of the ExsA gene.

[001316] Embodiment 121 is a recombinant member of the Bacillus cereus family of Embodiment 119 or 120, wherein a mutation in the ExsA gene results in the formation of a fragile exospore.

[001317] Embodiment 122 is a recombinant member of the Bacillus cereus family according to any of embodiments 100-121, wherein the recombinant member of the Bacillus cereus family contains a mutation in the CotO gene.

[001318] Embodiment 123 is a recombinant member of the Bacillus cereus family of Embodiment 122, wherein the mutation in the CotO gene includes a knockout of the CotO gene or a dominant negative form of the CotO gene.

[001319] Embodiment 124 is a recombinant member of the Bacillus cereus family of Embodiment 122 or 123, wherein a mutation in the CotO gene results in exosporium streaks.

[001320] Embodiment 125 is a recombinant member of the Bacillus cereus family that expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, an exosporium protein or an exospore protein fragment that targets the fusion protein to the exospore of the recombinant member of the Bacillus family cereus , wherein the recombinant member of the Bacillus cereus family comprises an endophytic bacterial strain, a bacterial strain that is capable of degrading the herbicide or pesticide, or a probiotic bacterial strain.

[001321] Embodiment 126 is a recombinant Bacillus cereus of Embodiment 125, wherein the endophytic strain comprises Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus 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 EE-B00377 (NRRL B- 67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120) or Bacillus mycoides EE-B00363 (NRRL B-67121).

[001322] Embodiment 127 is a recombinant member of the Bacillus cereus family of embodiment 125, wherein the endophytic bacterial strain comprises a member of the Bacillus cereus family 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 EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120); or Bacillus mycoides EE-B00363 (NRRL B-67121).

[001323] Embodiment 128 is a recombinant Bacillus cereus of Embodiment 125, wherein the bacterial strain that is capable of degrading the herbicide or pesticide comprises Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120) or Bacillus mycoides EE-B00363 (NRRL B-67121).

[001324] Embodiment 129 is the recombinant Bacillus cereus of Embodiment 125, wherein the probiotic bacterial strain comprises Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus EE439 (NRRL B-50979), Bacillus thuringiensis EE417 (NRRL B-50979) or Bacillus cereus EE444 (NRRL B-50977).

[001326] Embodiment 131 is a fusion protein according to any one of Embodiments 4, 8 and 12-15 or a recombinant member of the Bacillus cereus family according to Embodiment 16 or 130, wherein the targeting sequence comprises:

an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, where 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 20-35 of SEQ ID NO: 1, where 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 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 63%;

an amino acid sequence having at least about 62% identity with amino acids 20-35 of SEQ ID NO: 1, where 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 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%;

an amino acid sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 72%;

an amino acid sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%;

an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%; or

an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 90%.

[001327] Embodiment 132 is a fusion protein according to any one of Embodiments 4, 8 and 12-15 or a recombinant member of the Bacillus cereus family according to 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, where 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.

[001328] Embodiment 133 is a fusion protein or recombinant member of the Bacillus cereus family of Embodiment 131, wherein the targeting sequence consists of an amino acid sequence.

[001329] Embodiment 134 is a fusion protein according to any one of embodiments 4, 8 and 12-15 or a recombinant member of the Bacillus cereus family according to embodiment 16 or 130, wherein the fusion protein comprises an exosporium protein or an exosporium protein fragment containing the 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.

[001330] Embodiment 135 is a fusion protein according to Embodiment 1 or a recombinant member of the Bacillus cereus family according to Embodiment 16 or 130, wherein the fusion protein comprises an exosporium protein having 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.

[001331] Embodiment 136 is a fusion protein according to any of embodiments 1-15 and 131-135 or a recombinant member of the Bacillus cereus family according to any of embodiments 16-29, 48-63 and 99-135, wherein the targeting sequence, protein the exospore or exospore protein fragment contain the amino acid sequence GXT at their C-terminus, where X is any amino acid.

[001332] Embodiment 137 is a fusion protein according to any of embodiments 1-15 and 131-136 or a recombinant member of the Bacillus cereus family according to any of embodiments 16-29, 48-63 and 99-136, wherein the targeting sequence, protein the exosporium or exosporium protein fragment contains an alanine residue at the position of the targeting sequence which corresponds to amino acid 20 of SEQ ID NO: 1.

[001333] Embodiment 138 is a fusion protein according to any of embodiments 1-15 and 131-137 or a recombinant member of the Bacillus cereus family according to any of embodiments 16-29, 48-63 and 99-137, wherein the targeting sequence, protein exosporium or exosporium protein fragment additionally contain 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, which corresponds to amino acid 20 of SEQ ID NO: 1.

[001334] Embodiment 139 is a fusion protein according to any of embodiments 1-15 and 131-138 or a recombinant member of the Bacillus cereus family according to any of embodiments 16-29, 48-63 and 99-138, wherein the fusion protein, except furthermore, contains an amino acid linker between the targeting sequence, the exospore protein or exospore protein fragment and the protein or peptide of interest.

[001335] Embodiment 140 is a fusion protein or recombinant member of the Bacillus cereus family of Embodiment 139, wherein the linker includes a polyalanine linker, a polyglycine linker, or a linker containing a mixture of both alanine and glycine residues.

[001336] Embodiment 141 is a fusion protein or recombinant member of the Bacillus cereus family of Embodiment 139 or 140, wherein the linker contains a protease recognition site.

[001337] Embodiment 142 is a fusion protein comprising at least one protein or peptide of interest and a spore coat protein, wherein the spore coat protein comprises a CotB/H protein, a spore X protein, or a CotY protein, wherein the CotY protein comprises the amino acid sequence, having at least 80% identity with SEQ ID NO: 258 or 259.

[001338] Embodiment 143 is a fusion protein according to embodiment 142, wherein the spore coat protein comprises a CotB/H protein.

[001339] Embodiment 144 is a fusion protein according to embodiment 142, wherein the spore coat protein comprises X spore protein.

[001340] Embodiment 145 is a fusion protein according to embodiment 142, wherein the spore coat protein comprises a CotY protein, wherein the CotY protein contains an amino acid sequence having at least 80% identity with SEQ ID NO: 258 or 259.

[001341] Embodiment 146 is a fusion protein according to any of embodiments 142-145, 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% identity with SEQ ID NO: 255, 257, 258 or 259.

[001342] Embodiment 147 is a recombinant spore-forming bacterium that expresses a fusion protein according to any of embodiments 142-146.

[001343] Embodiment 148 is a recombinant spore-forming bacterium according to embodiment 147, wherein the recombinant spore-forming bacterium comprises an endophytic bacterial strain, a plant growth promoting bacterial strain, or a bacterial strain that is both endophytic and plant growth promoting.

[001344] Embodiment 149 is 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 spore surface of the bacterium, wherein the spore coat protein includes CotB protein, protein CotC, CgeA protein, CotB/H protein, CotG protein, X spore coat protein, or CotY protein; and wherein the recombinant spore-forming bacterium comprises an endophytic bacterial strain, a plant growth promoting bacterial strain, or a bacterial strain that is both endophytic and plant growth promoting.

[001345] Embodiment 150 is a plant seed coated with a recombinant spore-forming bacterium, wherein the recombinant spore-forming bacterium expresses a fusion protein comprising at least one protein of interest or a peptide and a spore coat protein that targets the fusion protein to the spore surface of the bacterium, wherein the protein the spore envelope includes CotB protein, CotC protein, CgeA protein, CotB/H protein, Cot G protein, X spore protein, or CotY protein.

[001346] Embodiment 151 is a recombinant spore-forming bacterium according to embodiment 147, 148, or 149, or a plant seed according to embodiment 150, wherein the recombinant spore-forming bacterium comprises a bacterium of the genus Bacillus, Lysinibacillus , Virginibacillus , Clostridia , or Paenibacillus .

[001347] Embodiment 152 is a recombinant bacterium of the genus Bacillus according to 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 protein a spore coat protein that targets the fusion protein to the spore surface of the bacterium, wherein the spore coat protein includes CotB protein, CotC protein, CgeA protein, CotB/H protein, Cot G protein, X spore coat protein, or CotY protein.

[001348] Embodiment 153 is a recombinant bacterium of the genus Bacillus according to embodiment 152, wherein the recombinant bacterium comprises an endophytic bacterial strain, a plant growth promoting bacterial strain, or a bacterial strain that is both endophytic and plant growth promoting.

[001349] Embodiment 154 is a plant seed coated with a recombinant spore-forming bacterium according to Embodiment 152 or 153.

[001350] Embodiment 155 is a plant seed according to embodiment 150, 151, or 154, wherein the recombinant spore-forming bacterium comprises an endophytic bacterial strain, a plant growth promoting bacterial strain, or a bacterial strain that is both endophytic and plant growth promoting.

[001351] Embodiment 156 is a recombinant spore-forming bacterium according to Embodiment 148, 149, or 153, or a seed according to Embodiment 155, wherein the endophytic bacterial strain, the plant growth-promoting bacterial strain, or the bacterial strain that is both endophytic and growth-promoting plants, includes Bacillus megaterium EE385 (NRRL B-50980), Bacillus sp. EE387 (NRRL B-50981), Bacillus circulans EE388 (NRRL B-50982), Bacillus subtilis EE405 (NRRL B-50978), Lysinibacillus fusiformis 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).

[001352] Embodiment 157 is a recombinant spore-forming bacterium or seed according to Embodiment 156, wherein the endophytic bacterial strain comprises Bacillus subtilis EE405 (NRRL B-50978) or Bacillus megaterium EE385 (NRRL B-50980).

[001353] Embodiment 158 is a recombinant spore-forming bacterium according to Embodiment 148, 149, or 153, or a seed according to Embodiment 155, wherein the endophytic bacterial strain, the plant growth-promoting bacterial strain, or the bacterial strain that is both endophytic and growth-promoting plants, includes Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus 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 EE-B00377 (NRRL B-67119), Bacillus pseudomycoides EE-B00366 (NRRL B-67120), Bacillus mycoides EE- B00363 (NRRL B-67121), Bacillus mycoides BT155 (NRRL #B-50921), Bacillus mycoides EE118 (NRRL #B-50918), Bacillus mycoides EE141 (NRRL #B-50921), Bacillus mycoides BT46-3 (NRRL #B -50922), represented a member of the Bacillus cereus EE128 family (NRRL no. B-50917), Bacillus thuringiensis BT013A (NRRL no. B-50924), or a member of the Bacillus cereus EE349 family (NRRL no. B-50928).

[001354] Embodiment 159 is a recombinant spore-forming bacterium according to any one of Embodiments 149, 151, 152, 153, and 156-158, or a seed according to Embodiment 150, 151, and 154-158, 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% identity with any of SEQ ID NOs: 252-259.

[001355] Embodiment 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 spore surface of the bacterium, wherein the recombinant spore-forming bacterium is not a recombinant member of the Bacillus cereus family , and the protein that targets the fusion protein to the bacterial spore surface 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.

[001356] Embodiment 161 is a recombinant spore-forming bacterium according to Embodiment 160, wherein the protein that targets the fusion protein to the bacterial spore surface 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.

[001357] Embodiment 162 is a recombinant spore-forming bacterium according to embodiment 160 or 161, wherein the recombinant spore-forming bacterium comprises an endophytic bacterial strain, a plant growth promoting bacterial strain, or a bacterial strain that is both endophytic and plant growth promoting.

[001358] Embodiment 163 is a recombinant spore-forming bacterium according to Embodiment 162, wherein the endophytic bacterial strain, plant growth promoting bacterial strain, or bacterial strain that is both endophytic and plant growth promoting, comprises Bacillus megaterium EE385 (NRRL B- 50980), Bacillus sp. EE387 (NRRL B-50981), Bacillus circulans EE388 (NRRL B-50982), Bacillus subtilis EE405 (NRRL B-50978), Lysinibacillus fusiformis 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).

[001359] Embodiment 164 is a recombinant spore-forming bacterium according to Embodiment 163, wherein the endophytic bacterial strain comprises Bacillus sp. EE387 (NRRL B-50981).

[001360] Embodiment 165 is a fusion protein according to any of embodiments 142-146, a recombinant spore-forming bacterium according to any of embodiments 147-149, 151-153 and 156-164, or a seed according to any of embodiments 150, 151 and 154-159, wherein the fusion protein further comprises an amino acid linker between the spore coat protein and the protein or peptide of interest.

[001361] Embodiment 166 is a fusion protein, recombinant spore-forming bacterium or seed according to embodiment 165, wherein the linker comprises a polyalanine linker, a polyglycine linker, or a linker containing a mixture of both alanine and glycine residues.

[001362] Embodiment 167 is a fusion protein, recombinant spore-forming bacterium or seed according to embodiment 165 or 166, wherein the linker contains a protease recognition site.

[001363] Embodiment 168 is a fusion protein according to any one of embodiments 1-3 and 142-146, a recombinant member of the Bacillus cereus family according to any one of embodiments 16-29, 48-63 and 99-141, a recombinant spore-forming bacterium according to any of embodiments 147-149, 151-153, and 156-167, or the plant seed of any of embodiments 150, 151, and 154-167, wherein the protein or peptide of interest comprises a plant growth promoting protein or peptide, a protein or peptide that protects the plant from a pathogen, a protein or peptide that increases the plant's resistance to stress, a plant-binding protein or peptide, an enzyme that catalyzes the production of nitric oxide, or a nucleic acid-binding protein or peptide.

[001364] Embodiment 169 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 168, wherein the protein or peptide of interest comprises a plant growth promoting protein or peptide.

[001365] Embodiment 170 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or a plant seed according to embodiment 169, wherein the plant growth promoting protein or peptide comprises a peptide hormone, a non-hormonal peptide, an enzyme involved in the production or activation of the stimulatory plant growth compound or enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source.

[001366] Embodiment 171 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 170, wherein the plant growth promoting protein or peptide comprises a peptide hormone.

[001367] Embodiment 172 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 171, wherein the peptide hormone comprises phytosulfokine, clavata 3 (CLV3), systemin, ZmlGF, or SCR/SP11.

[001368] Embodiment 173 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or a bacterium seed according to Embodiment 172, wherein the phytosulfokine comprises phytosulfokine-α.

[001369] Embodiment 174 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 170, wherein the plant growth promoting protein or peptide comprises a non-hormonal peptide.

[001370] Embodiment 175 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 174, wherein the non-peptide hormone comprises RKN 16D10, Hg-Syv46, eNOD40 peptide, melittin, mastoparan, Mas7, RHPP, POLARIS or kunitz trypsin inhibitor (KTI).

[001371] Embodiment 176 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 170, wherein the plant growth promoting protein or peptide comprises an enzyme involved in the production or activation of the plant growth promoting compound.

[001372] Embodiment 177 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 176, wherein the plant growth promoting compound comprises a compound produced by bacteria or fungi in the rhizosphere.

[001373] Embodiment 178 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 177, wherein the compound produced by bacteria or fungi in the rhizosphere comprises 2,3-butanediol.

[001374] Embodiment 179 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 176, wherein the plant growth stimulating compound comprises plant growth hormone.

[001375] Embodiment 180 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 179, wherein the plant growth hormone comprises cytokinin or a cytokinin derivative, ethylene, auxin or an auxin derivative, gibberellic acid or a giberralic acid derivative. acid, abscisic acid or abscisic acid derivative, jasmonic acid or jasmonic acid derivative.

[001376] Embodiment 181 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 180, wherein the plant growth hormone comprises cytokinin or a cytokinin derivative, and the cytokinin or cytokinin derivative comprises kinetin, cis-zeatin, trans-zeatin, 6-benzylaminopurine, dihydroxyzeatin, N6-(D2-isopentenyl)adenine, ribosylzeatin, N6-(D2-isopentenyl)adenosine, 2-methylthio-cis-ribosylzeatin, cis-ribosylzeatin, trans-ribosylzeatin, 2-methylthio- trans-ribosylzeatin, ribosylzeatin-5-monophosphate, N6-methylaminopurine, N6-dimethylaminopurine, 2'-deoxyzeatin riboside, 4-hydroxy-3-methyl-trans-2-butenylaminopurine, ortho-topoline, meta-topolin, benzyladenine, ortho-methyltopolin, meta-methyltopoline or a combination thereof; or, when the plant growth hormone comprises an auxin or an auxin derivative, the auxin or auxin derivative may include an active auxin, an inactive auxin, a conjugated auxin, a naturally occurring auxin, a synthetic auxin, or a combination thereof.

[001377] Embodiment 182 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 180 or 181, wherein the plant growth hormone comprises auxin or an auxin derivative and the auxin or auxin derivative comprises indole-3- acetic acid, indole-3-pyruvic acid, indole-3-acetaldoxime, indole-3-acetamide, indole-3-acetonitrile, indole-3-ethanol, indole-3-pyruvate, indole-3-acetaldoxime, indole-3- butyric acid, phenylacetic acid, 4-chloroindole-3-acetic acid, glucose-conjugated auxin, or a combination thereof.

[001378] Embodiment 183 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to any one of embodiments 176-182, wherein the plant growth promoting protein or peptide comprises an enzyme involved in the production or activation of the plant growth promoting compound, and the enzyme involved in the production or activation of the plant growth-promoting compound includes acetoin reductase, indole-3-acetamide hydrolase, tryptophan monooxygenase, acetolactate synthetase, α-acetolactate decarboxylase, pyruvate decarboxylase, diacetyl reductase, butanediol dehydrogenase, aminotransferase (e.g., tryptophan aminotransferase, inoxidyl caraminotransferase), -3-pyruvate decarboxylase, indole-3-acetaldehyde dehydrogenase, tryptophan side chain oxidase, nitrile hydrolase, nitrilase, peptidase, protease, adenosine phosphate isopentenyl transferase, phosphatase, adenosine kinase, adenine phosphoribosyl transfer erase, CYP735A, 5'-ribonucleotide phosphohydrolase, adenosine nucleosidase, zeatin-cis-trans isomerase, zeatin-O-glucosyltransferase, β-glucosidase, cis-hydroxylase, cis-hydroxylase CK, N-glucosyltransferase CK, 2,5-ribonucleotide phosphohydrolase, adenosine nucleosidase, purine nucleoside phosphorylase, zeatin reductase, hydroxylamine reductase, 2-oxoglutarate dioxygenase, gibberellic 2B/3B hydrolase, gibberallin-3-oxidase, gibberallin-20-oxidase, chitosanase, chitinase, β-1,3-glucanase, β-1,4-glucanase, β- 1,6-glucanase, aminocyclopropane-1-carboxylic acid deaminase, or an enzyme involved in the production of the nod factor.

[001379] Embodiment 184 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 183, wherein the enzyme involved in the production or activation of the plant growth-promoting compound includes an enzyme involved in the production of the nod factor, and the enzyme involved in the production of the nod factor includes nodA, nodB or nodI; or wherein the enzyme involved in the production or activation of the plant growth stimulating compound comprises an aminotransferase and the aminotransferase includes tryptophan aminotransferase.

[001380] Embodiment 185 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 183, wherein the enzyme involved in the production or activation of the plant growth-promoting compound comprises a protease or peptidase that degrades proteins, peptides, proproteins or preproproteins to form a biologically active peptide.

[001381] Embodiment 186 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 183, wherein the protease or peptidase comprises subtilisin, acid protease, alkaline protease, proteinase, endopeptidase, exopeptidase, thermolysin, papain , pepsin, trypsin, pronase, carboxylase, serine protease, glutamine protease, aspartate protease, cysteine protease, threonine protease or metalloproteinase.

[001382] Embodiment 187 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 185 or 186, wherein the biologically active peptide comprises RKN 16D10 or RHPP.

[001383] Embodiment 188 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to any of embodiments 185-187, wherein the protease or peptidase cleaves proteins in the protein-rich material.

[001384] Embodiment 189 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 188, wherein the protein-rich material is soy flour or yeast extract.

[001385] Embodiment 190 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 183, wherein the enzyme involved in the production or activation of the plant growth-promoting compound comprises chitosanase, wherein the chitosanase comprises the 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.

[001386] Embodiment 191 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 170, wherein the plant growth promoting protein or peptide comprises an enzyme that degrades or modifies a nutrient source of bacteria, fungi, or plants .

[001387] Embodiment 192 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 191, wherein the enzyme that degrades or modifies the nutrient source of bacteria, fungi, or plants includes cellulase, lipase, lignin oxidase , protease, glycoside hydrolase, phosphatase, nitrogenase, nuclease, amidase, nitrate reductase, nitrite reductase, amylase, ammonia oxidase, ligninase, glucosidase, phospholipase, phytase, pectinase, glucanase, sulfatase, urease, xylanase, or siderophore.

[001388] Embodiment 193 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 192, wherein the phospholipase includes phospholipase A1, phospholipase A2, phospholipase C, phospholipase D, or lysophospholipase.

[001389] Embodiment 194 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 193, wherein the phospholipase comprises phospholipase C and phospholipase C comprises an amino acid sequence having at least 85%, at least at least 95%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 312.

[001390] Embodiment 195 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 192, wherein the enzyme that degrades or modifies the nutrient source of bacteria, fungi or plants comprises cellulase and the cellulase includes endo-cellulase , exocellulase or β-glucosidase.

[001391] Embodiment 196 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 195, wherein the cellulase includes endocellulase and the endocellulase includes endoglucanase.

[001392] Embodiment 197 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 195 or 196, wherein the cellulase includes endocellulase and the endocellulase includes Bacillus subtilis endoglucanase, Bacillus thuringiensis endoglucanase, Bacillus cereus endoglucanase, or Bacillus clausii endoglucanase.

[001393] Embodiment 198 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 197, wherein the cellulase comprises a Bacillus subtilis endoglucanase and the Bacillus subtilis endoglucanase contains an amino acid sequence having at least 85% at least 95%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 311.

[001394] Embodiment 199 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 195, wherein the cellulase includes exocellulase and the exocellulase includes Trichoderma reesei exocellulase .

[001395] Embodiment 200 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 195, wherein the cellulase comprises β-glucosidase and the β-glucosidase comprises Bacillus subtilis β-glucosidase, Bacillus β-glucosidase thuringiensis , Bacillus cereus β-glucosidase, or Bacillus clausii β-glucosidase.

[001396] Embodiment 201 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 192, wherein the enzyme that degrades or modifies a nutrient source of bacteria, fungi, or plants comprises a lipase, and the lipase comprises a Bacillus subtilis lipase, a Bacillus thuringiensis lipase, a Bacillus cereus lipase, or a Bacillus clausii lipase.

[001397] Embodiment 202 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 192, wherein the enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source comprises lignin oxidase, and the lignin oxidase comprises lignin peroxidase, laccase, glyoxal oxidase, ligninase or manganese peroxidase.

[001398] Embodiment 203 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 192, wherein an enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source comprises a protease, and the protease comprises subtilisin, acid protease, alkaline protease, proteinase, peptidase, endopeptidase, exopeptidase, thermolysin, papain, pepsin, trypsin, pronase, carboxylase, serine protease, glutamine protease, aspartate protease, cysteine protease, threonine protease or metalloproteinase.

[001399] Embodiment 204 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 192, wherein the enzyme that degrades or modifies a nutrient source of bacteria, fungi, or plants comprises a phosphatase, and the phosphatase comprises phosphoric acid monoester hydrolase, phosphomonoesterase, phosphoric acid diester hydrolase, phosphodiesterase, triphosphoric acid monoester hydrolase, phosphorylanhydride hydrolase, pyrophosphatase, phytase, trimetaphosphatase or triphosphatase.

[001400] Embodiment 205 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 204, wherein the enzyme that degrades or modifies a nutrient source of bacteria, fungi, or plants comprises a phosphomonoesterase, and the phosphomonoesterase comprises PhoA4; or wherein the enzyme that degrades or modifies a bacterial, fungal or plant nutrient source includes a phytase, and the phytase includes Bacillus subtilis EE148 phytase or Bacillus thuringiensis BT013A phytase.

[001401] Embodiment 206 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 192, wherein the enzyme that degrades or modifies a nutrient source of bacteria, fungi, or plants comprises nitrogenase, and the nitrogenase comprises nitrogenase of the Nif family.

[001402] Embodiment 207 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 206, wherein the Nif family nitrogenase comprises NifBDEHKNXV Paenibacillus massiliensis .

[001403] Embodiment 208 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 168, wherein the protein or peptide of interest comprises a protein or peptide that protects the plant from a pathogen.

[001404] Embodiment 209 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 208, wherein the protein or peptide that protects the plant from a pathogen includes a protein or peptide that enhances the plant's immune system.

[001405] Embodiment 210 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 209, wherein the plant immune system enhancing protein includes harpin, harpin-like protein, α-elastin, β- elastin, systemin, phenylalanine ammonia lyase, elicitin, defensin, cryptogain, flagellin protein or flagellin peptide.

[001406] Embodiment 211 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 210, wherein the flagellin peptide comprises flg22.

[001407] Embodiment 212 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 208, wherein the protein or peptide that protects the plant from a pathogen has antibacterial activity, antifungal activity, or both antibacterial and and antifungal activity.

[001408] Embodiment 213 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 212, wherein the protein or peptide that protects the plant from a pathogen includes bacteriocin, lysozyme, peptide lysozyme, siderophore, avidin , streptavidin, nonribosomal active peptide, conalbumin, albumin, lactoferrin, lactoferrin peptide or TasA.

[001409] Embodiment 214 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 213, wherein the lysozyme peptide comprises LysM or wherein the lactoferrin peptide comprises LfcinB.

[001410] Embodiment 215 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or a plant seed of embodiment 208, wherein the protein or peptide that protects the plant from a pathogen has insecticidal activity, helminthicidal activity, inhibits insect predation, or worms, or a combination of these effects.

[001411] Embodiment 216 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 215, wherein the protein or peptide that protects the plant from a pathogen includes an insecticidal bacterial toxin, an endotoxin, a Cry toxin, a protein or a peptide that is a protease, cysteine protease or chitinase inhibitor.

[001412] Embodiment 217 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 216, wherein the protein or peptide that protects the plant from a pathogen comprises an insecticidal bacterial toxin, and the insecticidal bacterial toxin comprises an insecticidal toxin VIP; where the protein or peptide that protects the plant from a pathogen includes a protein or peptide that is a protease inhibitor, and the protein or peptide that is a protease inhibitor includes a trypsin inhibitor or an arrowhead protease inhibitor; or where the protein or peptide that protects the plant from the pathogen includes a Cry toxin and the Cry toxin includes a Cry toxin from Bacillus thuringiensis .

[001413] Embodiment 218 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 217, wherein the Cry toxin comprises a Cry toxin from Bacillus thuringiensis and the Cry toxin from Bacillus thuringiensis comprises a Cry5B protein or a Cry21A protein .

[001414] Embodiment 219 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 208, wherein the protein that protects the plant from a pathogen includes an enzyme.

[001415] Embodiment 220 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 219, wherein the enzyme comprises a protease or lactonase.

[001416] Embodiment 221 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 220, wherein the protease or lactonase is specific for a bacterial signaling molecule.

[001417] Embodiment 222 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 221, wherein the bacterial signal molecule comprises a homoserine lactone bacterial signal molecule.

[001418] Embodiment 223 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or plant seed according to any one of embodiments 220-222, wherein the enzyme comprises lactonase and the lactonase comprises 1,4-lactonase, 2-pyrone- 4,6-dicarboxylate lactonase, 3-oxoadipat-enol lactonase, actinomycin lactonase, deoxylimonate A-ring lactonase, gluconolactonase, L-rhamnono-1,4-lactonase, limonin-D-ring lactonase, steroid lactonase, triacetate lactonase, or xylono -1,4-lactonase.

[001419] Embodiment 224 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 219, wherein the enzyme is specific to a cellular component of a bacterium or fungus.

[001420] Embodiment 225 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 224, wherein the enzyme comprises β-1,3-glucanase, β-1,4-glucanase, β-1 ,6-glucanase, chitosanase, chitinase, chitosanase-like enzyme, lyticase, peptidase, protease, protease, mutanolysin, stafolysin, or lysozyme.

[001421] Embodiment 226 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 225, wherein the enzyme comprises chitosanase, wherein the chitosanase contains an amino acid sequence having at least 85%, at least 95 %, at least 98%, at least 99% or 100% identity with SEQ ID NO: 313.

[001422] Embodiment 227 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 225, wherein the protease comprises an alkaline protease, an acidic protease, or a neutral protease.

[001423] Embodiment 228 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or a plant seed according to any one of embodiments 208-227, wherein the protein or peptide that protects the plant from a pathogen protects the plant from a bacterial pathogen, a fungal pathogen, worm pathogen, or insect pathogen.

[001424] Embodiment 229 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 228, wherein the bacterial pathogen includes an α-class proteobacterium, a β-class proteobacterium, a γ-class proteobacterium, or a combination thereof; or where the bacterial pathogen includes Agrobacterium tumefaciens , Pantoea stewartii , Erwinia carotovora , Ralstonia solanacearum , Pseudomonas syringae , Pseudomonas aeruginosa , Xanthomonas campestris , or a combination thereof.

[001425] Embodiment 230 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 228, wherein the protein or peptide that protects the plant from a pathogen protects the plant from predation by a worm pathogen or insect pathogen .

[001426] Embodiment 231 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or a plant seed according to embodiment 228 or 230, wherein the worm or insect pathogen includes caterpillar, epsilon bollworm, corn borer, fall armyworm , butterfly cutworm, Japanese beetle, small corn stem borer, corn weevil, sprout fly larva, caterpillar releasing web, corn stem moth, southern corn rootworm, southern potato wireworm, stem grinder, sugar cane beetle, grub grub, cabbage scoop , cotton weevil, yellow striped caterpillar, red-breasted leech, bed bug, aphid, cutworm, Mexican bean weevil, soybean cutworm, soybean stem grinder, or a combination thereof.

[001427] Embodiment 232 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 168, wherein the protein or peptide of interest comprises at least one protein or peptide that enhances plant stress tolerance .

[001428] Embodiment 233 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 232, wherein the protein or peptide that enhances plant stress tolerance includes an enzyme that degrades the stress-causing compound.

[001429] Embodiment 234 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or seed according to embodiment 233, wherein the stress-causing compound comprises aminocyclopropane-1-carboxylic acid (ACC), reactive oxygen species, nitric oxide, oxylipin , a phenolic compound, or a combination thereof.

[001430] Embodiment 235 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 233 or 234, wherein the enzyme that degrades the stress-causing compound includes superoxide dismutase, oxidase, catalase, aminocyclopropane-1 deaminase -carboxylic acid, peroxidase, antioxidant enzyme or antioxidant peptide.

[001431] Embodiment 236 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 235, wherein the enzyme that degrades the stress-causing compound includes superoxide dismutase.

[001432] Embodiment 237 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 235 or 236, wherein the superoxide dismutase comprises superoxide dismutase 1 (SODA1) or superoxide dismutase 2 (SODA2).

[001433] Embodiment 238 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 237, wherein the superoxide dismutase comprises an amino acid sequence having at least 85%, at least 95%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 155 or 156.

[001434] Embodiment 239 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 232, wherein the protein or peptide that enhances plant stress resistance includes a protein or peptide that protects the plant from stressful impact of the external environment.

[001435] Embodiment 240 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 239, wherein environmental stress includes drought, flood, heat, frost, salt, heavy metals, low pH , high pH values, or a combination thereof.

[001436] Embodiment 241 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 239 or 240, wherein the protein or peptide that protects the plant from environmental stress includes a protein that induces microcrystal formation ice, prolinase, phenylalanine ammonia lyase, isochorismate synthase, isochorismate pyruvate lyase, or choline dehydrogenase.

[001437] Embodiment 242 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 168, wherein the fusion protein comprises at least one plant-binding protein or peptide.

[001438] Embodiment 243 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 242, wherein the plant-binding protein or peptide comprises adhesin, flagellin, omptin, lectin, expansin, a biofilm structural protein, pilus protein, curl protein, intimin, invasin, agglutinin, afimbria protein.

[001439] Embodiment 244 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 243, wherein the plant-binding protein comprises adhesin and the adhesin comprises ricadhesin; or wherein the plant-binding protein comprises a biofilm structural protein and the biofilm structural protein comprises TasA or YuaB.

[001440] Embodiment 245 is a fusion protein according to embodiment 4, or a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 168, wherein the protein or peptide of interest comprises an enzyme that catalyzes the production of nitric oxide .

[001441] Embodiment 246 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 245, wherein the enzyme that catalyzes nitric oxide production comprises nitric oxide synthase or arginase.

[001442] Embodiment 247 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 245, wherein the enzyme that catalyzes nitric oxide production comprises nitric oxide synthase.

[001443] Embodiment 248 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 247, wherein the nitric oxide synthase comprises Bacillus thuringiensis nitric oxide synthase or Bacillus subtilis nitric oxide synthase.

[001444] Embodiment 249 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 248, wherein the nitric oxide synthase comprises nitric oxide synthase from Bacillus thuringiensis BT013A or Bacillus subtilis 168.

[001445] Embodiment 250 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or plant seed according to any one of embodiments 245-249, 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.

[001446] Embodiment 251 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or plant seed according to any of embodiments 245-250, wherein the fusion protein comprises SEQ ID NO: 262 or 263.

[001447] Embodiment 252 is a fusion protein according to embodiment 4, or a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 168, wherein the protein or peptide of interest comprises a nucleic acid binding protein or peptide.

[001448] Embodiment 253 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 252, wherein the nucleic acid binding protein or peptide comprises an RNA binding protein or peptide or a DNA binding protein or peptide.

[001449] Embodiment 254 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 253, wherein the nucleic acid binding protein or peptide comprises an RNA binding protein or peptide.

[001450] Embodiment 255 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 254, wherein the RNA-binding protein or peptide comprises a non-specific RNA-binding protein or peptide.

[001451] Embodiment 256 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 254, wherein the RNA-binding protein or peptide comprises a specific RNA-binding protein or peptide.

[001452] Embodiment 257 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 254, wherein the RNA binding protein or peptide comprises an Hfq protein.

[001453] Embodiment 258 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or plant seed according to embodiment 257, wherein the Hfq protein comprises Bacillus thuringiensis Hqf protein.

[001454] Embodiment 259 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 253, wherein the nucleic acid binding protein or peptide comprises a DNA binding protein or peptide.

[001455] Embodiment 260 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 259, wherein the DNA binding protein or peptide comprises a small acid soluble spore protein (SASP).

[001456] Embodiment 261 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or a plant seed according to embodiment 260, wherein the SASP comprises an SASP encoded by an SspA gene, an SspB gene, an SspC gene, an SspD gene, an SspE gene, a SspF, SspG gene, SspH gene, SspI gene, SspJ gene, SspK gene, SspL gene, SspM gene, SspN gene, SspO gene, or SspP gene.

[001457] Embodiment 262 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 261, wherein the SASP includes SASPα, SASPβ, or SASPγ.

[001458] Embodiment 263 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or a plant seed according to any one of embodiments 260-262, wherein the SASP comprises a Bacillus thuringiensis SASP.

[001459] Embodiment 264 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or plant seed according to any one of embodiments 252-263, wherein the nucleic acid binding protein or peptide 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 any of SEQ ID NOs: 264-266.

[001460] Embodiment 265 is a fusion protein, a recombinant member of the Bacillus cereus family, or a recombinant spore-forming bacterium, or plant seed according to any of embodiments 252-264, wherein the fusion protein comprises SEQ ID NOS: 267, 268, or 269.

[001461] Embodiment 266 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to any of embodiments 252-254 and 259, wherein the nucleic acid binding protein or peptide comprises a nuclease having an inactivated active site.

[001462] Embodiment 267 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to any of embodiments 252-266, further comprising a nucleic acid molecule linked to a nucleic acid binding protein or peptdom.

[001463] Embodiment 268 is a fusion protein, a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or plant seed according to embodiment 267, wherein the nucleic acid molecule comprises a modulating RNA molecule; RNA-i molecule; miRNA; an aptamer or a DNA molecule that encodes a modulating RNA molecule, an RNA-i molecule, a microRNA or an aptamer.

[001464] Embodiment 269 is a fusion protein according to Embodiment 4 or a recombinant member of the Bacillus cereus family or plant seed according to any of Embodiments 16-29, 48-63, 99-141 and 168-268, wherein the targeting sequence comprises SEQ ID NO: 96.

[001465] Embodiment 270 is a recombinant member of the Bacillus cereus family that co-expresses at least one fusion protein containing a plant growth promoting protein or peptide, a protein or peptide that protects a plant from a pathogen, a protein or peptide that enhances plant resistance stress, an enzyme that catalyses the production of nitric oxide, or a nucleic acid binding protein or peptide according to any one of embodiments 168-241 and 245-269, and at least one fusion protein comprising a plant-binding protein or peptide according to any one of the embodiments implementation 242-244 and 269.

[001466] Embodiment 271 is a recombinant member of the Bacillus cereus family according to any of embodiments 16-141 and 168-270, wherein the recombinant member of the Bacillus cereus family includes Bacillus anthracis , Bacillus cereus , Bacillus thuringiensis , Bacillus mycoides , Bacillus pseudomycoides , Bacillus samanii , Bacillus gaemokensis, Bacillus weihenstephensis , Bacillus toyoiensis , or a combination thereof.

[001467] Embodiment 272 is a recombinant member of the Bacillus cereus family according to embodiment 271, wherein the recombinant member of the Bacillus cereus family comprises Bacillus thuringiensis or Bacillus mycoides .

[001468] Embodiment 273 is a recombinant member of the Bacillus cereus family according to embodiment 271 or 272, wherein the recombinant member of the Bacillus cereus family comprises a plant growth promoting bacterial strain.

[001469] Embodiment 274 is a recombinant member of the Bacillus cereus family of Embodiment 273, wherein the plant growth promoting bacterial strain produces an insecticidal toxin, produces a fungicidal compound, produces a nematocidal compound, produces a bactericidal compound, is resistant to one or more antibiotics, contains one or several freely replicating plasmids, binds to plant roots, colonizes plant roots, forms biofilms, solubilizes nutrients, secretes organic acids, or has a combination of these effects.

[001470] Embodiment 275 is a recombinant member of the Bacillus cereus family of Embodiment 274, wherein the insecticidal toxin comprises Cry toxin; where the fungicidal compound includes β-1,3-glucanase, chitosanase, lyticase, or a combination thereof; or where the nematocidal compound includes a Cry toxin.

[001471] Embodiment 276 is a recombinant member of the Bacillus cereus family according to any one of Embodiments 273-275, wherein the plant growth-promoting bacterial strain comprises Bacillus mycoides BT155 (NRRL No. B-50921), Bacillus mycoides EE118 (NRRL No. B-50918) , Bacillus mycoides EE141 (NRRL No. B-50921), Bacillus mycoides BT46-3 (NRRL No. B-50922), member of the family Bacillus cereus EE128 (NRRL No. B-50917), Bacillus thuringiensis BT013A (NRRL No. B-50924), member family Bacillus cereus EE349 (NRRL No. B-50928); member of the family Bacillus cereus EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120) or Bacillus mycoides EE-B00363 (NRRL B-67121).

[001472] Embodiment 277 is a recombinant member of the Bacillus cereus family of Embodiment 276, wherein the plant growth-promoting bacterial strain comprises Bacillus mycoides BT155 (NRRL No. B-50921), Bacillus mycoides EE141 (NRRL No. B-50921), or Bacillus thuringiensis BT013A (NRRL No. B-50924).

[001473] Embodiment 278 is a recombinant member of the Bacillus cereus family according to any of embodiments 16-141 and 168-275, wherein the recombinant member of the Bacillus cereus family comprises an endophytic bacterial strain.

[001474] Embodiment 279 is a recombinant Bacillus cereus of Embodiment 278, wherein the endophytic bacterial strain comprises Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus EE439 (NRRL B-50979), Bacillus thuringiensis EE417 (NRRL B-50979) Bacillus cereus EE444 (NRRL B-50977) or Bacillus thuringiensis EE319 (NRRL B-50983) B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120) or Bacillus mycoides EE-B00363 (NRRL B-67121).

[001475] Embodiment 280 is a recombinant member of the Bacillus cereus family of embodiment 279, wherein the endophytic bacterial strain comprises a member of the Bacillus cereus family 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 EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120) or Bacillus mycoides EE-B00363 (NRRL B-67121).

[001476] Embodiment 281 is a recombinant member of the Bacillus cereus family according to any of embodiments 16-141 and 186-275, wherein the recombinant member of the Bacillus cereus family comprises a bacterial strain that is capable of degrading the herbicide or pesticide.

[001477] Embodiment 282 is a recombinant Bacillus cereus of Embodiment 281, wherein the bacterial strain that is capable of degrading the herbicide or pesticide comprises Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120); or Bacillus mycoides EE-B00363 (NRRL B-67121).

[001478] Embodiment 283 is a recombinant member of the Bacillus cereus family according to any of embodiments 125, 128, 281, and 282, wherein the bacterial strain that is capable of degrading the herbicide or pesticide degrades a sulfonylurea herbicide, an aryltriazine herbicide, dicamba, 2,4-D, phenoxy herbicide, pyrethrin, pyrethroid, or a combination thereof.

[001479] Embodiment 284 is a recombinant member of the Bacillus cereus family of Embodiment 283, wherein the sulfonylurea herbicide comprises sulfentrazone.

[001480] Embodiment 285 is a recombinant member of the Bacillus cereus family of Embodiment 282 or 283, wherein the bacterial strain that is capable of degrading the pesticide degrades pyrethrin.

[001481] Embodiment 286 is a recombinant member of the Bacillus cereus family according to any of embodiments 16-141 and 186-275, wherein the recombinant member of the Bacillus cereus family comprises a probiotic bacterial strain.

[001482] Embodiment 287 is a recombinant Bacillus cereus of Embodiment 286, wherein the probiotic bacterial strain comprises Bacillus cereus EE349 NRRL No. B-50928), Bacillus cereus EE439 (NRRL B-50979), Bacillus thuringiensis EE417 (NRRL B-50979) or Bacillus cereus EE444 (NRRL B-50977).

[001483] Embodiment 288 is a recombinant member of the Bacillus cereus family according to embodiments 16-141 and 168-287, wherein the recombinant member of the Bacillus cereus family contains an inactivating mutation in its BclA gene.

[001484] Embodiment 289 is a recombinant member of the Bacillus cereus family of Embodiment 288, wherein the inactivating mutation in the BclA gene comprises a knockout of the BclA gene.

[001485] Embodiment 290 is a recombinant member of the Bacillus cereus family according to 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, an exospore protein or a fragment of an exosporium protein, in a fusion protein or a portion thereof.

[001486] Embodiment 291 is a recombinant member of the Bacillus cereus family according to any one of Embodiments 17-27, wherein the modulator protein is expressed under the control of a native promoter or a portion thereof.

[001487] Embodiment 292 is a recombinant member of the Bacillus cereus family according to any of embodiments 16-29, 48-63, 99-141, and 168-291, wherein the fusion or modulator protein is expressed under the control of a highly expressed sporulation promoter.

[001488] Embodiment 293 is a recombinant member of the Bacillus cereus family of embodiment 292, wherein the highly expressed sporulation promoter comprises a sigma-K sporulation-specific polymerase promoter sequence.

[001489] Embodiment 294 is a recombinant member of the Bacillus cereus family according to any of embodiments 290-293, 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 any of the nucleic acid sequences of SEQ ID NO: 157-231.

[001490] Embodiment 295 is a recombinant member of the Bacillus cereus family according to any of embodiments 290-293, wherein the promoter comprises a nucleic acid sequence having at least 80%, at least 90%, at least 95%, at least 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.

[001491] Embodiment 296 is a recombinant member of the Bacillus cereus family according to any one of Embodiments 293-295, wherein the sequence or sequences of the sporulation-specific polymerase sigma-K promoter has 100% identity with the corresponding nucleotides of any of SEQ ID NOs: 157-231 .

[001492] Embodiment 297 is a biologically pure bacterial culture, wherein the bacteria in the bacterial culture are:

(a) a member of the family Bacillus cereus EE439 (NRRL B-50979);

(b) Bacillus thuringiensis EE417 (NRRL B-50979);

(c) Bacillus cereus EE444 (NRRL B-50977);

(d) Bacillus thuringiensis EE319 (NRRL B-50983);

(e) Bacillus thuringiensis EE-B00184 (NRRL B-67122);

(f) a member of the family Bacillus cereus EE-B00377 (NRRL B-67119);

(g) Bacillus pseudomycoides EE-B00366 (NRRL B-67120); or

(h) Bacillus mycoides EE-B00363 (NRRL B-67121).

[001493] Embodiment 298 is a biologically pure bacterial culture according to embodiment 297, wherein the bacteria in the bacterial culture is a member of the Bacillus cereus EE439 family (NRRL B-50979) and the bacteria have a 16S ribosomal RNA sequence having at least 98% , at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 277.

[001494] Embodiment 299 is a biologically pure bacterial culture according to Embodiment 297, wherein the bacteria in the bacterial culture are Bacillus thuringiensis EE417 (NRRL B-50979) and the bacteria have a 16S ribosomal RNA sequence having at least 98% at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 278.

[001495] Embodiment 300 is a biologically pure bacterial culture according to Embodiment 297, wherein the bacteria in the bacterial culture are Bacillus cereus EE444 (NRRL B-50977) and the bacteria have a 16S ribosomal RNA sequence having at least 98% at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 279.

[001496] Embodiment 301 is a biologically pure bacterial culture according to Embodiment 297, wherein the bacteria in the bacterial culture are Bacillus thuringiensis EE319 (NRRL B-50983) and the bacteria have a 16S ribosomal RNA sequence having at least 98% at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 280.

[001497] Embodiment 302 is a biologically pure bacterial culture according to embodiment 297, wherein the bacteria in the bacterial culture are Bacillus thuringiensis EE-B00184 (NRRL B-67122) and the bacteria have a 16S ribosomal RNA sequence having at least 98% , at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 301.

[001498] Embodiment 303 is a biologically pure bacterial culture according to embodiment 297, wherein the bacteria in the bacterial culture are family representativeBacillus cereus EE-B00377 (NRRL B-67119) and the bacteria have a 16S ribosomal RNA sequence having at least 98%, at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 304.

[001499] Embodiment 304 is a biologically pure bacterial culture according to embodiment 297, wherein the bacteria in the bacterial culture are Bacillus pseudomycoides EE-B00366 (NRRL B-67120) and the bacteria have a 16S ribosomal RNA sequence having at least 98% , at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 303.

[001500] Embodiment 305 is a biologically pure bacterial culture according to embodiment 297, wherein the bacteria in the bacterial culture are Bacillus mycoides EE-B00363 (NRRL B-67121) and the bacteria have a 16S ribosomal RNA sequence having at least 98% , at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 302.

[001501] Embodiment 306 is a biologically pure bacterial culture wherein the bacteria in the bacterial culture are bacterial mutants according to any one of embodiments 297-305 comprising one or more mutations wherein the bacteria are endophytic.

[001502] Embodiment 307 is a biologically pure bacterial culture wherein the bacteria in the bacterial culture are:

(a) Bacillus megaterium EE385 (NRRL B-50980);

(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) Bacillus pumilus EE-B00143 (NRRL B-67123).

[001503] Embodiment 308 is a biologically pure bacterial culture according to embodiment 307, wherein the bacteria in the bacterial culture are Bacillus megaterium EE385 (NRRL B-50980) and the bacteria have a 16S ribosomal RNA sequence having at least 98% at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 281.

[001504] Embodiment 309 is a biologically pure bacterial culture according to embodiment 307, wherein the bacteria in the bacterial culture is Bacillus sp. EE387 (NRRL B-50981) and bacteria have a 16S ribosomal RNA sequence having at least 98%, at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 282.

[001505] Embodiment 310 is a biologically pure bacterial culture according to embodiment 307, wherein the bacteria in the bacterial culture are Bacillus circulans EE388 (NRRL B-50982) and the bacteria have a 16S ribosomal RNA sequence having at least 98% at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 283.

[001506] Embodiment 311 is a biologically pure bacterial culture according to embodiment 307, wherein the bacteria in the bacterial culture are Bacillus subtilis EE405 (NRRL B-50978) and the bacteria have a 16S ribosomal RNA sequence having at least 98% at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 284.

[001507] Embodiment 312 is a biologically pure bacterial culture according to embodiment 307, wherein the bacteria in the bacterial culture are Lysinibacillus fusiformis EE442 (NRRL B-50975) and the bacteria have a 16S ribosomal RNA sequence having at least 98% at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 285.

[001508] Embodiment 313 is a biologically pure bacterial culture according to embodiment 307, wherein the bacteria in the bacterial culture are Lysinibacillus sphaericus EE443 (NRRL B-50976) and the bacteria have a 16S ribosomal RNA sequence having at least 98% at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 286.

[001509] Embodiment 314 is a biologically pure bacterial culture according to embodiment 307, wherein the bacteria in the bacterial culture are Bacillus pumilus EE-B00143 (NRRL B-67123) and the bacteria have a 16S ribosomal RNA sequence having at least 98% , at least 99% or 100% sequence identity with the sequence of SEQ ID NO: 305.

[001510] Embodiment 315 is a biologically pure bacterial culture, wherein the bacteria in the bacterial culture are mutants according to any of embodiments 307-314 containing one or more mutations, wherein the bacteria are endophytic.

[001511] Embodiment 316 is a biologically pure bacterial culture wherein the bacteria in the bacterial culture are mutants according to any of embodiments 307-315 containing one or more mutations, wherein the bacteria are probiotic.

[001512] Embodiment 317 is an inoculum for application to plants, plant seeds, plant growth media, or an area surrounding a plant or plant seed, wherein the inoculum contains an effective amount of a biologically pure bacterial culture according to any one of Embodiments 297-316, and agriculturally acceptable carrier.

[001513] Embodiment 318 is an inoculum according to embodiment 317, wherein the inoculum contains an effective amount of a mixture containing at least two biologically pure bacterial cultures according to any one of embodiments 297-316.

[001514] Embodiment 319 is an inoculum according to embodiment 317 or 318, wherein the inoculum further comprises an effective amount of rhizobacteria.

[001515] Embodiment 320 is an inoculum according to embodiment 319, wherein the rhizobacteria are a biologically pure bacterial culture of a rhizobacteria strain.

[001516] Embodiment 321 is an inoculum according to embodiment 319 or 320, wherein the rhizobacteria include bacteria of the genus Bradyrhizobium , bacteria of the genus Rhizobium , or a combination thereof.

[001517] Embodiment 322 is an inoculum according to Embodiment 321, wherein the bacteria of the genus Bradyrhizobium include Bradyrhizobium japonicum .

[001518] Embodiment 323 is an inoculum according to embodiment 322, wherein the bacteria of the genus Rhizobium include Rhizobium phaseoli , Rhizobium leguminosarum , or a combination thereof.

[001519] Embodiment 324 is a plant seed coated with: (i) an enzyme that catalyses the production of nitric oxide; (ii) superoxide dismutase; or (iii) a recombinant microorganism that expresses an enzyme that catalyzes the production of nitric oxide, or superoxide dismutase, wherein the expression of the enzyme that catalyzes the production of nitric oxide, or superoxide dismutase, is increased compared to the expression of the enzyme that catalyzes the production of nitric oxide, or superoxide dismutase, in the microorganism wild type under the same conditions.

[001520] Embodiment 325 is a plant seed according to Embodiment 324, wherein the plant seed is coated with an enzyme that catalyses the production of nitric oxide.

[001521] Embodiment 326 is a plant seed according to embodiment 324 or 325, wherein the plant seed is coated with a recombinant microorganism that expresses an enzyme that catalyses the production of nitric oxide.

[001522] Embodiment 327 is a plant seed according to any of embodiments 324-326, wherein the enzyme that catalyzes nitric oxide production comprises nitric oxide synthase or arginase.

[001523] Embodiment 328 is the seed of the plant according to Embodiment 327, wherein the enzyme that catalyzes nitric oxide production includes nitric oxide synthase.

[001524] Embodiment 329 is the seed of the plant according to Embodiment 328, wherein the nitric oxide synthase comprises nitric oxide synthase from Bacillus thuringiensis BT013A or Bacillus subtilis 168.

[001525] Embodiment 330 is a seed of a plant according to 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.

[001526] Embodiment 331 is a plant seed according to any one of embodiments 324-330, wherein the plant seed is coated with superoxide dismutase.

[001527] Embodiment 332 is a plant seed according to any of embodiments 324-331, wherein the plant seed is coated with a recombinant microorganism that expresses superoxide dismutase.

[001528] Embodiment 333 is a plant seed according to any one of embodiments 324-332, wherein the superoxide dismutase comprises superoxide dismutase 1 (SODA1) or superoxide dismutase 2 (SODA2).

[001529] Embodiment 334 is a plant seed according to 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.

[001530] Embodiment 335 is a plant seed according to any one of embodiments 324-334, wherein the recombinant microorganism includes a Bacillus species, Escherechia coli , an Aspergillus species, or a Sacchromyces species.

[001531] Embodiment 336 is the seed of the plant of Embodiment 335, wherein the Bacillus species includes a member of the Bacillus cereus family, Bacillus subtilis, Bacillus licheniformis, or Bacillus megaterium .

[001532] Embodiment 337 is the seed of a plant according to Embodiment 335, wherein the Aspergillus species includes Aspergillus niger or where the Sacchromyces species includes Sacchromyces cerevisiae .

[001533] Embodiment 338 is a formulation comprising a recombinant member of the Bacillus cereus family according to any of embodiments 16-141 and 168-296 or a recombinant spore-forming bacterium according to any of embodiments 147-149, 152-154 and 156-268 and is acceptable in terms of agriculture carrier.

[001534] Embodiment 339 is a formulation containing exosporium fragments derived from spores of a recombinant member of the Bacillus cereus family according to any of Embodiments 100-124, 130-141, and 168-296, and an agriculturally acceptable carrier.

[001535] Embodiment 340 is the seed of a plant according to any one of embodiments 150, 151, and 154-268, wherein the seed is coated with a formulation containing a recombinant spore-forming bacterium and an agriculturally acceptable carrier.

[001536] Embodiment 341 is a plant seed according to any one of embodiments 324-337, wherein the seed is coated with a formulation containing an enzyme or recombinant microorganism and an agriculturally acceptable carrier.

[001537] Embodiment 342 is a formulation according to Embodiment 338 or 339, a plant seed according to Embodiment 340 or 341, or an inoculum according to any of Embodiments 317-323, wherein the agriculturally acceptable carrier comprises a dispersing agent, a surfactant active agent, additive, water, thickener, anticaking agent, sludge breaker, composting compound, granular composition, diatomaceous earth, oil, colorant, stabilizer, preservative, polymer, coating, or a combination thereof.

[001538] Embodiment 343 is a formulation, plant seed or inoculum according to Embodiment 342, wherein the agriculturally acceptable carrier includes an additive, and the additive includes oil, gum, resin, clay, polyoxyethylene glycol, terpene, viscous organic compound, complex fatty acid ester, sulfated alcohol, alkylsulfonate, petroleum sulfonate, alcohol sulfate, sodium alkyl butanediamate, sodium thiobutanedioate polyester, acetonitrile benzene derivative, proteinaceous material, or a combination thereof; the agriculturally acceptable carrier comprises a thickener, and the thickener comprises long chain polyethylene glycol alkylsulfonate, polyoxyethylene oleate, or a combination thereof; the agriculturally acceptable carrier includes a surfactant, and the surfactant includes a heavy petroleum oil, a heavy petroleum distillate, a polyol fatty acid ester, a polyethoxylated fatty acid ester, an arylalkylpolyoxyethylene glycol, an alkylamine acetate, an alkylarylsulfonate, a polyhydric alcohol, an alkyl phosphate or a combination of them; or the agriculturally acceptable carrier comprises an anti-caking agent, and the anti-caking agent comprises sodium salt, calcium carbonate, diatomaceous earth, or a combination thereof.

[001539] Embodiment 344 is a formulation, plant seed, or inoculum according to Embodiment 343, wherein the additive comprises a protein material, and the protein material includes a dairy product, wheat flour, soy flour, blood, albumin, gelatin, alfalfa flour, yeast extract, or their combination; or the anticaking agent comprises a sodium salt, and the sodium salt includes monomethylnaphthalenesulfonate sodium, dimethylnaphthalenesulfonate sodium, sodium sulfite, sodium sulfate, or a combination thereof.

[001540] Embodiment 345 is a formulation, a plant seed according to any of Embodiments 338-344, or an inoculum according to any of Embodiments 317-323 and 342-344, wherein the agriculturally acceptable carrier includes vermiculite, charcoal, filter press mud for carbonization in sugar production, rice hulls, carboxymethyl cellulose, peat, perlite, fine sand, calcium carbonate, flour, alum, starch, talc, polyvinylpyrrolidone, or a combination thereof.

[001541] Embodiment 346 is a composition, a plant seed according to any of embodiments 338-345, or an inoculum according to any of embodiments 317-323 and 342-345, wherein the composition or inoculum comprises a seed coating composition, a liquid application composition onto plants or plant growth media, or a solid formulation for application to plants or plant growth media.

[001542] Embodiment 347 is the formulation, plant seed or inoculum of embodiment 346, wherein the seed coating formulation comprises an aqueous or oily solution for application to the seeds, or a powder or granular formulation for application to the seeds.

[001543] Embodiment 348 is the formulation, plant seed, or inoculum of embodiment 346, wherein the liquid formulation for application to the plants or plant growth medium comprises a concentrated formulation or a ready-to-use formulation.

[001544] Embodiment 349 is the formulation, plant seed or inoculum of embodiment 346, wherein the solid formulation for application to the plants or plant growth medium comprises a granular formulation or a powdered agent.

[001545] Embodiment 350 is a composition according to any of embodiments 338 and 342-349, a plant seed according to embodiment 340 or 341, or an inoculum according to any of embodiments 317-323 and 342-349, wherein the composition or inoculum further comprises an agrochemical comprising a fertilizer, a micronutrient material, an insecticide, a herbicide, a plant growth modifier, a fungicide, an insecticide, a molluscicide, an algicide, a bacterial inoculum, a fungal inoculum, or a combination thereof.

[001546] Embodiment 351 is a composition, plant seed or inoculum according to embodiment 350, wherein the composition or inoculum comprises a bacterial inoculum and the bacterial inoculum comprises a plant growth-promoting bacterial strain.

[001547] Embodiment 352 is a composition, plant seed or inoculum according to Embodiment 351, wherein the plant growth promoting bacterial strain produces an insecticidal toxin, produces a fungicidal compound, produces a nematocidal compound, produces a bactericidal compound, is resistant to one or more antibiotics, contains one or more freely replicating plasmids, binds to plant roots, colonizes plant roots, forms biofilms, solubilizes nutrients, secretes organic acids, or has a combination of these effects.

[001548] Embodiment 353 is a formulation, plant seed or inoculum according to Embodiment 352, wherein the insecticidal toxin comprises Cry toxin; where the fungicidal compound includes β-1,3-glucanase, chitosanase, lyticase, or a combination thereof; or where the nematocidal compound includes a Cry toxin.

[001549] Embodiment 354 is a formulation, plant seed, or inoculum according to any one of Embodiments 351-353, wherein the plant growth promoting bacterial strain comprises Bacillus aryabhattai CAP53 (NRRL No. B-50819), Bacillus aryabhattai CAP56 (NRRL No. B-50817 ), Bacillus flexus BT054 (NRRL No. B-50816), Paracoccus kondratievae NC35 (NRRL No. B-50820), Bacillus mycoides BT155 (NRRL No. B-50921), Enterobacter cloacae CAP12 (NRRL No. B-50822), Bacillus nealsonii BOBA57 ( NRRL No. NRRL B-50821), Bacillus mycoides EE118 (NRRL No. B-50918), Bacillus subtilis EE148 (NRRL No. B-50927), Alcaligenes faecalis EE107 (NRRL No. B-50920), Bacillus mycoides EE141 (NRRL No. B-50916 ), Bacillus mycoides BT46-3 (NRRL No. B-50922), Bacillus cereus EE128 (NRRL No. B-50917), Bacillus thuringiensis BT013A (NRRL No. B-50924), Paenibacillus massiliensis BT23 (NRRL No. B-50923), member of the family Bacillus cereus EE349 (NRRL No. B-50928), Bacillus subtilis EE218 (NRRL No. B-50926), Bacillus megaterium EE281 (NRRL No. B-50925), Bacillus cereus EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120), Bacillus mycoides EE-B00363 (NRRL B-67121), Bacillus pumilus EE-B00143 (NRRL B-67123) or Bacillus thuringiensis EE-B00184 (NRRL B-67122), or their combination.

[001550] Embodiment 355 is the formulation, plant seed, or inoculum of Embodiment 354, wherein the plant growth-promoting bacterial strain comprises Paracoccus kondratievae NC35 (NRRL No. B-50820), Bacillus aryabhattai CAP53 (NRRL No. B-50819), or Bacillus megaterium EE281 (NRRL No. B-50925), and the composition further comprises a recombinant member of the Bacillus cereus family according to embodiment 276 or 277.

[001551] Embodiment 356 is a plant seed coated with a recombinant Bacillus cereus according to any of Embodiments 16-141 and 168-296, exospore fragments derived from spores of a recombinant Bacillus cereus according to any of Embodiments 100-124, 130-141 and 168-296, recombinant spore-forming bacterium according to any of embodiments 147-149, 151-153 and 156-268, biologically pure bacterial culture according to any of embodiments 297-316, inoculum according to any of embodiments 317-323 and 342-355, or formulations according to any of embodiments 338, 339, and 342-355.

[001552] Embodiment 357 is a method for promoting plant growth, comprising:

introducing into the plant growth medium a recombinant member of the Bacillus cereus family according to any of embodiments 16-141 and 168-296 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342-355; or

applying to a plant, plant seed, or area surrounding a plant or plant seed, a recombinant member of the Bacillus cereus family according to any of embodiments 16-141 and 168-296 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342- 355;

wherein the recombinant member of the Bacillus cereus family expresses a fusion protein containing a plant growth promoting protein or peptide.

[001553] Embodiment 358 is a method for promoting plant growth, comprising:

adding to the plant growth medium a recombinant spore-forming bacterium according to any one of embodiments 147-149, 151-153, and 156-268, or a composition comprising a recombinant spore-forming bacterium according to any one of embodiments 338 and 342-355; or

applying to a plant, plant seed, or area surrounding a plant or plant seed, a recombinant spore-forming bacterium according to any one of embodiments 147-149, 151-153, and 156-268 or a formulation comprising a recombinant spore-forming bacterium according to any one of embodiments 338 and 342- 355;

wherein the recombinant spore-forming bacterium expresses a fusion protein containing a plant growth promoting protein or peptide.

[001554] Embodiment 359 is a method of protecting a plant from a pathogen or increasing plant stress tolerance, comprising:

adding to the plant growth medium a recombinant member of the Bacillus cereus family according to any of embodiments 16-141 and 168-296 or a formulation containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342-355, or

applying to a plant, plant seed, or area surrounding a plant or plant seed, a recombinant member of the Bacillus cereus family according to any of embodiments 16-141 and 168-296 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342- 355;

where the recombinant member of the Bacillus cereus family expresses a fusion protein containing a protein or peptide that protects the plant from a pathogen, or a protein or peptide that increases the resistance of the plant to stress.

[001555] Embodiment 360 is a method of protecting a plant from a pathogen or increasing plant stress tolerance, comprising:

adding to the plant growth medium a recombinant spore-forming bacterium according to any one of embodiments 147-149, 151-153, and 156-268 or a composition comprising a recombinant spore-forming bacterium according to any one of embodiments 338 and 342-355; or

applying to a plant, plant seed, or area surrounding a plant or plant seed, a recombinant spore-forming bacterium according to any one of embodiments 147-149, 151-153, and 156-268 or a formulation comprising a recombinant spore-forming bacterium according to any one of embodiments 338 and 342- 355;

wherein the recombinant spore-forming bacterium expresses a fusion protein containing a protein or peptide that protects the plant from a pathogen, or a protein or peptide that enhances the plant's resistance to stress.

[001556] Embodiment 361 is the method of embodiment 359 or 360, wherein the method is a method of protecting a plant from a pathogen, and plants grown in a plant growth medium containing a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium are less sensitive to pathogen infection compared to plants grown under the same conditions in an identical plant growth medium that does not contain a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium.

[001557] Embodiment 362 is the method of Embodiment 359 or 360, wherein the method is a method of increasing plant stress tolerance and plants grown in plant growth media containing a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium are less sensitive to stresses compared to plants grown under the same conditions in an identical plant growth medium. which does not contain a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium.

[001558] Embodiment 363 is a method for immobilizing spores of a recombinant member of the Bacillus cereus family on a plant, comprising:

introducing into the plant growth medium a recombinant member of the Bacillus cereus family according to any of embodiments 16-141 and 168-296 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342-355; or

applying to a plant, plant seed, or area surrounding a plant or plant seed, a recombinant member of the Bacillus cereus family according to any of embodiments 16-141 and 168-296 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342- 355;

wherein the recombinant member of the Bacillus cereus family expresses a fusion protein containing a plant-binding protein or peptide.

[001559] Embodiment 364 is a method for immobilizing a spore of a recombinant spore-forming bacterium on a plant, comprising:

adding to the plant growth medium a recombinant spore-forming bacterium according to any one of embodiments 147-149, 151-153, and 156-268 or a composition comprising a recombinant spore-forming bacterium according to any one of embodiments 338 and 342-355; or

applying to a plant, plant seed, or area surrounding a plant or plant seed, a recombinant spore-forming bacterium according to any one of embodiments 147-149, 151-153, and 156-268 or a formulation comprising a recombinant spore-forming bacterium according to any one of embodiments 338 and 342- 355;

wherein the recombinant spore-forming bacterium expresses a fusion protein containing a plant-binding peptide.

[001560] Embodiment 365 is a method for promoting germination of a plant seed, comprising:

introducing into the plant growth medium a recombinant member of the Bacillus cereus family according to any of embodiments 16-141 and 168-296 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342-355; or

applying to a plant, plant seed, or area surrounding a plant or plant seed, a recombinant member of the Bacillus cereus family according to any of embodiments 16-141 and 168-296 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342- 355;

where the recombinant member of the Bacillus cereus family expresses a fusion protein containing superoxide dismutase or an enzyme that catalyzes the production of nitric oxide.

[001561] Embodiment 366 is a method for promoting germination of a plant seed, comprising:

adding to the plant growth medium a recombinant spore-forming bacterium according to any one of embodiments 147-149, 151-153, and 156-268 or a composition comprising a recombinant spore-forming bacterium according to any one of embodiments 338 and 342-355; or

applying to a plant, plant seed, or area surrounding a plant or plant seed, a recombinant spore-forming bacterium according to any one of embodiments 147-149, 151-153, and 156-268 or a formulation comprising a recombinant spore-forming bacterium according to any one of embodiments 338 and 342- 355;

wherein the recombinant spore-forming bacterium expresses a fusion protein containing superoxide dismutase, or an enzyme that catalyses the production of nitric oxide.

[001562] Embodiment 367 is a method according to any one of embodiments 357-360 and 363-366, wherein a plant growth promoting protein or peptide, a protein or peptide that protects the plant from a pathogen, a protein or peptide that increases the plant's resistance to stress effects, a plant-binding protein or peptide, or superoxide dismutase, or an enzyme that catalyzes the production of nitric oxide is physically associated with the exospore of a recombinant member of the Bacillus cereus family or with the spore envelope of a recombinant spore-forming bacterium.

[001563] Embodiment 368 is a method according to any one of embodiments 365-367, wherein the method comprises applying a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or formulation to a plant seed.

[001564] Embodiment 369 is a method according to any one of Embodiments 365-371, further comprising adding L-arginine to a plant growth medium, applying to a seed of a plant, a plant, or an area surrounding a plant, or a plant seed.

[001565] Embodiment 370 is the method of Embodiment 369, wherein the method comprises applying L-arginine to the aerial part of the plant.

[001566] Embodiment 371 is the method of Embodiment 369, wherein the method comprises applying L-arginine to a plant seed.

[001567] Embodiment 372 is a method according to any one of embodiments 365-371, wherein seeds in a plant growth medium containing a recombinant Bacillus cereus or a recombinant spore-forming bacterium, or seeds coated with a recombinant Bacillus cereus or a recombinant spore-forming bacterium, have an increased germination rate or have a longer main root after germination compared to seeds germinated under the same conditions in an identical plant growth medium that does not contain a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium, or seeds on which no recombinant member of the Bacillus cereus family or recombinant spore-forming bacterium grown under the same conditions is applied.

[001568] Embodiment 373 is a method for delivering nucleic acids to a plant, comprising:

introducing into the plant growth medium a recombinant member of the Bacillus cereus family according to any of embodiments 16-141 and 168-296 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342-355; or

applying to the plant, plant seed, or area surrounding the plant or plant seed, a recombinant member of the Bacillus cereus family according to embodiments 16-141 and 168-296 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342-355;

wherein the recombinant member of the Bacillus cereus family expresses a fusion protein comprising a nucleic acid-binding protein, and wherein the nucleic acid-binding protein or peptide is linked to the nucleic acid molecule.

[001569] Embodiment 374 is the method of 373 wherein the recombinant member of the Bacillus cereus family comprises an endophytic bacterial strain.

[001570] Embodiment 375 is the method of Embodiment 374, wherein the endophytic bacterial strain comprises Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus 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 EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120) or Bacillus mycoides EE-B00363 (NRRL B-67121).

[001571] Embodiment 376 is the method of Embodiment 375, wherein the endophytic bacterial strain comprises Bacillus cereus 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 EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120) or Bacillus mycoides EE-B00363 (NRRL B-67121).

[001572] Embodiment 377 is a method for delivering nucleic acids to a plant, comprising:

adding to the plant growth medium a recombinant spore-forming bacterium according to any one of embodiments 147-149, 151-153, and 156-268 or a composition comprising a recombinant spore-forming bacterium according to any one of embodiments 338 and 342-355; or

applying to a plant, plant seed, or area surrounding a plant or plant seed, a recombinant spore-forming bacterium according to any one of embodiments 147-149, 151-153, and 156-268 or a formulation comprising a recombinant spore-forming bacterium according to any one of embodiments 338 and 342- 355;

wherein the recombinant spore-forming bacterium expresses a fusion protein comprising a nucleic acid-binding protein, and wherein the nucleic acid-binding protein or peptide is linked to the nucleic acid molecule.

[001573] Embodiment 378 is the method of Embodiment 377, wherein the recombinant spore-forming bacterium comprises an endophytic bacterial strain.

[001574] Embodiment 379 is the method of Embodiment 378, wherein the endophytic bacterial strain comprises Bacillus megaterium EE385 (NRRL B-50980), Bacillus sp. EE387 (NRRL B-50981), Bacillus circulans EE388 (NRRL B-50982), Bacillus subtilis EE405 (NRRL B-50978), Lysinibacillus fusiformis EE442 (NRRL B-50975), or Lysinibacillus sphaericus EE443 (NRRL B-50976), or Bacillus pumilus EE-B00143 (NRRL B-67123).

[001575] Embodiment 380 is a method of delivering a nucleic acid molecule to an animal, insect, or worm, comprising feeding the animal, insect, or worm a plant modified to contain a level of the nucleic acid molecule that exceeds the level of the nucleic acid molecule in the same plant, which has not been modified grown under the same conditions.

[001576] Embodiment 381 is a method for delivering a nucleic acid molecule to an animal, insect, or worm, comprising feeding the animal, insect, or worm:

a recombinant member of the Bacillus cereus family expressing a fusion protein comprising a protein or peptide of interest and a targeting sequence, exospore protein or exospore protein fragment, which targets the fusion protein into the exospore of the recombinant member of the Bacillus cereus family; or

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 bacterial spore surface;

wherein the protein or peptide of interest comprises a nucleic acid-binding protein or peptide and a nucleic acid molecule linked to the nucleic acid-binding protein or peptide.

[001577] Embodiment 382 is the method according to embodiment 380 or 381, wherein the worm is a nematode.

[001578] Embodiment 383 is a method for delivering a nucleic acid molecule to a fungus or protozoan, comprising contacting the fungus or protozoan with:

a recombinant member of the Bacillus cereus family expressing a fusion protein comprising a protein or peptide of interest and a targeting sequence, exospore protein or exospore protein fragment, which targets the fusion protein into the exospore of the recombinant member of the Bacillus cereus family; or

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 bacterial spore surface;

wherein the protein or peptide of interest comprises a nucleic acid-binding protein or peptide and a nucleic acid molecule is linked to the nucleic acid-binding protein or peptide.

[001579] Embodiment 384 is a method according to any one of embodiments 373-379 and 381-383, wherein the nucleic acid binding protein or peptide is physically linked to the exospore of a recombinant member of the Bacillus cereus family or to the spore envelope of a recombinant spore-forming bacterium.

[001580] Embodiment 385 is a method according to any one of embodiments 373-384, wherein the nucleic acid molecule comprises a modulating RNA molecule; RNA-i molecule; miRNA; aptamer; or a DNA molecule that encodes a modulating RNA molecule, an RNA-i molecule, a microRNA or an aptamer.

[001581] Embodiment 386 is a method according to any of embodiments 381-385, wherein the recombinant Bacillus cereus includes the recombinant Bacillus cereus according to any of embodiments 16-29, 48-63, 100-141, and 186-296 .

[001582] Embodiment 387 is a method according to any one of embodiments 381-386, wherein the fusion protein comprises a fusion protein according to any one of embodiments 252-266.

[001583] Embodiment 388 is a method according to any one of embodiments 381-385, wherein the spore coat protein comprises CotB protein, CotC protein, CgeA protein, CotB/H protein, CotG protein, X spore coat protein, or CotY protein.

[001584] Embodiment 389 is the 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% identity with any of SEQ ID NOs: 252-259.

[001585] Embodiment 390 is a method for promoting plant growth, comprising:

adding to the plant growth medium a recombinant member of the Bacillus cereus family according to any of embodiments 64-70, 74-77 and 87-99 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342-355;

or applying to the plant, plant seed, or area surrounding the plant or plant seed, a recombinant member of the Bacillus cereus family according to any one of embodiments 64-70, 74-77, and 87-99 or a formulation containing a recombinant member of the Bacillus cereus family according to any one of the embodiments implementation 338 and 342-355;

where the recombinant member of the Bacillus cereus family expresses an enzyme involved in nutrient solubilization, a protease, BclA protein, BclB protein, CotE protein, CotO protein, an ExsY protein, ExsFA/BxpB protein, CotY protein, ExsFB protein, ExsJ protein, ExsH protein, protein YjcA, YjcB protein, BclC protein, BxpA protein, BclE protein, BetA/BAS3290 protein, ExsA protein, ExsK protein, ExsB protein, YabG protein, or Tgl protein, where expression of an enzyme involved in nutrient solubilization, protease, BclA protein, BclB protein, CotE protein, CotO protein, ExsY protein, ExsFA/BxpB protein, CotY protein, ExsFB protein, ExsJ protein, ExsH protein, YjcA protein, YjcB protein, BclC protein, BxpA protein, BclE protein, BetA/BAS3290 protein, ExsA, ExsK protein, ExsB protein, YabG protein, or Tgl protein is increased compared to the expression of an enzyme involved in nutrient solubilization, protease, BclA protein, BclB protein, CotE protein, CotO protein, ExsY protein, ExsFA/BxpB protein, CotY protein , ExsFB protein, ExsJ protein, ExsH protein, YjcA protein, YjcB protein, BclC protein, BxpA protein, BclE protein, BetA/BAS3290 protein, ExsA protein, ExsK protein, ExsB protein, YabG protein, or Tgl protein in a wild-type Bacillus cereus under the same conditions.

[001586] Embodiment 391 is a method for improving plant stress tolerance, comprising:

introducing into the plant growth medium a recombinant member of the Bacillus cereus family according to any of embodiments 64, 65 and 78-83 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342-355;

or applying to the plant, plant seed, or area surrounding the plant or plant seed, a recombinant member of the Bacillus cereus family according to any of embodiments 64, 65 and 78-83 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342 -355;

wherein the recombinant member of the Bacillus cereus family expresses superoxide dismutase or arginase, wherein the expression of superoxide dismutase or arginase is increased compared to the expression of superoxide dismutase or arginase in a wild-type member of the Bacillus cereus family under the same conditions.

[001587] Embodiment 392 is a method of protecting a plant from a pathogen, comprising:

adding to the plant growth medium a recombinant member of the Bacillus cereus family according to any of embodiments 64, 65 and 74-77 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342-355;

or applying to the plant, plant seed, or area surrounding the plant or plant seed, a recombinant member of the Bacillus cereus family according to any of embodiments 64, 65, and 74-77, or a composition containing a recombinant member of the Bacillus cereus family, according to any one of embodiments 338 and 342 -355;

wherein the recombinant member of the Bacillus cereus family expresses a protease, wherein the expression of the protease is increased compared to the expression of the protease in a wild-type member of the Bacillus cereus family under the same conditions.

[001588] Embodiment 393 is a method of slowing spore germination of a member of the Bacillus cereus family, comprising modifying a member of the Bacillus cereus family to express inosinuridine hydrolase or alanine racemase, wherein the expression of inosinuridine hydrolase or alanine racemase is increased compared to the expression of inosinuridine hydrolase or alanine racemase in a wild-type member of the Bacillus cereus family in the same conditions.

[001589] Embodiment 394 is a method according to any one of embodiments 357-379 and 381-393, further comprising inactivating the recombinant Bacillus cereus or recombinant spore-forming bacterium prior to being introduced into a plant growth medium or applied to a plant, plant seed, or area surrounding a plant or plant seed.

[001590] Embodiment 395 is the method of Embodiment 394, wherein the inactivation comprises exposing a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium to a heat treatment; exposure to gamma radiation; X-ray exposure, UV-A exposure; exposure to UV-B; treatments with glutaraldehyde, formaldehyde, hydrogen peroxide, acetic acid, bleach, chloroform or phenol, or a combination thereof.

[001591] Embodiment 396 is the method of embodiment 394 or 395, wherein the inactivation comprises modifying a recombinant member of the Bacillus cereus family or a recombinant spore-forming bacterium to express a spore outgrowth protease or non-specific endonuclease, wherein expression of the spore outgrowth protease or non-specific endonuclease is increased compared to expression of a spore germinating protease or a non-specific endonuclease in a wild-type member of the Bacillus cereus family under the same conditions, and wherein the recombinant spore-forming bacterium comprises a recombinant bacterium of the genus Bacillus .

[001592] Embodiment 397 is a method for removing exospores from spores of a recombinant member of the Bacillus cereus family, comprising:

exposing a suspension containing spores of a recombinant member of the Bacillus cereus family according to any one of embodiments 100-124 to centrifugation or filtration to obtain exospore fragments that are separated from the spores;

where the exosporium fragments contain a fusion protein.

[001593] Embodiment 398 is the method of Embodiment 397, wherein the method includes subjecting the suspension containing spores to centrifugation, and further comprising collecting the supernatant, wherein the supernatant contains exospore fragments and is substantially free of spores.

[001594] Embodiment 399 is the method of Embodiment 397, wherein the method includes subjecting the spore-containing suspension to filtration, and further comprising collecting the filtrate, wherein the filtrate contains exospore fragments and is substantially free of spores.

[001595] Embodiment 400 is a method for promoting plant growth, comprising:

adding exosporium fragments or the composition of Embodiment 339 to the plant growth medium; or

applying fragments of the exospore or composition according to embodiment 339 to the plant, plant seed, or an area surrounding the plant or plant seed;

wherein the exospore fragments are derived from spores of a recombinant member of the Bacillus cereus family according to any one of embodiments 100-124 and contain a fusion protein, and the fusion protein contains a plant growth promoting protein or peptide.

[001596] Embodiment 401 is a method of protecting a plant from a pathogen or increasing plant stress tolerance, comprising:

adding exosporium fragments or the composition of Embodiment 339 to the plant growth medium; or

applying fragments of the exospore or composition according to embodiment 339 to the plant, plant seed, or an area surrounding the plant or plant seed;

wherein the exospore fragments are derived from spores of a recombinant member of the Bacillus cereus family according to any of embodiments 100-124 and contain a fusion protein, and the fusion protein contains a protein or peptide that protects the plant from a pathogen or a protein or peptide that increases the plant's resistance to stress.

[001597] Embodiment 402 is the method of Embodiment 401, wherein the method is a method of protecting a plant from a pathogen, and wherein the fusion protein comprises a protein or peptide that protects the plant from the pathogen.

[001598] Embodiment 403 is a method of protecting a plant from a pathogen according to embodiment 402, wherein plants grown in a plant growth medium containing exosporium fragments are less susceptible to pathogen infection compared to plants grown under the same conditions in an identical a plant growth medium that does not contain exospore fragments.

[001599] Embodiment 404 is the method of Embodiment 401, wherein the method is a method for enhancing plant stress tolerance, and wherein the fusion protein comprises a protein or peptide that enhances plant stress tolerance.

[001600] Embodiment 405 is a method for improving plant stress tolerance according to embodiment 404, wherein plants grown in a plant growth medium containing exosporium fragments are less stress sensitive than plants grown under the same conditions. in an identical plant growth medium that does not contain exospore fragments.

[001601] Embodiment 406 is a method for immobilizing exospore fragments on a plant, comprising:

adding exosporium fragments or the composition of Embodiment 339 to the plant growth medium; or

applying fragments of the exospore or composition according to embodiment 339 to the plant, plant seed, or an area surrounding the plant or plant seed;

wherein the exospore fragments are derived from spores of a recombinant member of the Bacillus cereus family according to any one of embodiments 100-124 and comprises a fusion protein, and the fusion protein comprises a plant-binding protein or peptide.

[001602] Embodiment 407 is a method according to any of embodiments 363, 364, and 406, wherein the plant-binding protein or peptide selectively targets and retains a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or exospore fragments in a plant.

[001603] Embodiment 408 is the method of embodiment 407 wherein the plant-binding protein or peptide selectively targets and retains a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or exosporium fragments in plant roots, root substructures, an aerial part of a plant, or an aerial substructure plant parts.

[001604] Embodiment 409 is a method for promoting germination of a plant seed, comprising:

adding exosporium fragments or the composition of Embodiment 339 to the plant growth medium; or

applying exosporium fragments or the composition according to embodiment 339 to a plant seed or an area surrounding a plant or plant seed;

wherein the exospore fragments are derived from spores of a recombinant member of the Bacillus cereus family according to any one of embodiments 100-124 and contain a fusion protein, and the fusion protein contains superoxide dismutase or an enzyme that catalyzes the production of nitric oxide.

[001605] Embodiment 410 is the method of Embodiment 409, wherein the method comprises applying exospore fragments to a plant seed.

[001606] Embodiment 411 is the method of Embodiment 409 or 410, further comprising adding L-arginine to the plant growth medium, applying to the plant seed, or an area surrounding the plant seed.

[001607] Embodiment 412 is the method of Embodiment 411, wherein the method comprises applying L-arginine to a plant seed.

[001608] Embodiment 413 is a method according to any one of embodiments 409-412, wherein the seeds in the plant growth medium containing exosporium fragments, or the seeds to which the exosporium fragments are applied, have an increased germination rate or have a longer main root after germination compared to the same seeds germinated under the same conditions in an identical plant growth medium that does not contain exospore fragments, or the same seeds germinated under the same conditions to which exospore fragments were not applied.

[001609] Embodiment 414 is a method for delivering nucleic acids to a plant, comprising:

adding exosporium fragments or the composition of Embodiment 339 to the plant growth medium; or

applying fragments of the exospore or composition according to embodiment 339 to the plant, plant seed, or an area surrounding the plant or plant seed;

wherein the exospore fragments are derived from spores of a recombinant member of the Bacillus cereus family according to any of embodiments 100-124 and contain a fusion protein, and the fusion protein comprises a nucleic acid binding protein or peptide, and wherein the nucleic acid binding protein or peptide is linked to a nucleic acid molecule.

[001610] Embodiment 415 is a method for delivering a nucleic acid molecule to an animal, insect, or worm, comprising feeding the animal, insect, or worm exosporium fragments, wherein the exospore fragments are derived from a recombinant member of the Bacillus cereus family according to any of embodiments 100-124 and contain a fusion a protein, wherein the fusion protein comprises a nucleic acid-binding protein or peptide, and wherein the nucleic acid-binding protein or peptide is linked to the nucleic acid molecule.

[001611] Embodiment 416 is the method of Embodiment 415, wherein the worm is a nematode.

[001612] Embodiment 417 is a method of delivering a nucleic acid molecule to a fungus or protozoan, comprising contacting the fungus or protozoan with exospore fragments, wherein the exospore fragments are derived from a recombinant member of the Bacillus cereus family according to any one of embodiments 100-124 and contain a fused a protein, wherein the fusion protein comprises a nucleic acid-binding protein or peptide, and wherein the nucleic acid-binding protein or peptide is linked to the nucleic acid molecule.

[001613] Embodiment 418 is a method according to any one of embodiments 414-417, wherein the nucleic acid molecule comprises a modulating RNA molecule; RNA-i molecule; miRNA; an aptamer or a DNA molecule that encodes a modulating RNA molecule, an RNA-i molecule, a microRNA or an aptamer.

[001614] Embodiment 419 is a method according to any of embodiments 414-418, wherein the fusion protein comprises a fusion protein according to any of embodiments 253-266.

[001615] Embodiment 420 is a method for promoting the germination of a plant seed, comprising adding to, or applying to the plant, plant seed, or area surrounding the plant or plant seed: (i) an enzyme that catalyzes the production of nitric oxide; (ii) superoxide dismutase; or (iii) a recombinant microorganism that expresses an enzyme that catalyzes the production of nitric oxide or superoxide dismutase, wherein the expression of the enzyme that catalyzes the production of nitric oxide or superoxide dismutase is increased compared to the expression of the enzyme that catalyzes the production of nitric oxide or superoxide dismutase in the wild-type microorganism. type under the same conditions.

[001616] Embodiment 421 is the method of Embodiment 420, wherein the method comprises applying an enzyme or microorganism to a plant seed.

[001617] Embodiment 422 is the method of embodiment 420 or 421, further comprising applying L-arginine to a plant growth medium, to a plant seed, or to an area surrounding the plant seed.

[001618] Embodiment 423 is the method of Embodiment 422, wherein the method comprises applying L-arginine to a plant seed.

[001619] Embodiment 424 is a method according to any one of embodiments 420-423, wherein the seeds in the plant growth medium containing the enzyme or microorganism, or the seeds coated with the enzyme or microorganism, have an increased level of germination or a longer main root after germination compared to seeds germinated under the same conditions in an identical plant growth medium that does not contain the enzyme or microorganism, or seeds not coated with the enzyme or microorganism germinated under the same conditions.

[001620] Embodiment 425 is a method according to any one of embodiments 420-424, wherein the enzyme that catalyzes nitric oxide production comprises nitric oxide synthase or arginase.

[001621] Embodiment 426 is the method of Embodiment 425, wherein the enzyme that catalyzes nitric oxide production comprises nitric oxide synthase.

[001622] Embodiment 427 is the method of Embodiment 426, wherein the nitric oxide synthase comprises nitric oxide synthase from Bacillus thuringiensis BT013A or Bacillus subtilis 168.

[001623] Embodiment 428 is a method according to 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%, of at least 99% or 100% sequence identity with SEQ ID NO: 260 or 261.

[001624] Embodiment 429 is a method according to any one of embodiments 420-438, wherein the superoxide dismutase comprises superoxide dismutase 1 (SODA1) or superoxide dismutase 2 (SODA2).

[001625] Embodiment 430 is the 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.

[001626] Embodiment 431 is a method according to any one of embodiments 420-430, wherein the recombinant microorganism comprises a Bacillus species, Escherechia coli , an Aspergillus species, or a Sacchromyces species.

[001627] Embodiment 432 is the method of Embodiment 431, wherein the Bacillus species includes a member of the family Bacillus cereus , Bacillus subtilis, Bacillus licheniformis, or Bacillus megaterium .

[001628] Embodiment 433 is the method of Embodiment 431 wherein the Aspergillus species includes Aspergillus niger or where the Sacchromyces species includes Sacchromyces cerevisiae .

[001629] Embodiment 434 is a method according to any one of embodiments 420-433, wherein the enzyme or recombinant microorganism is introduced into the plant growth medium or applied to the plant, plant seed, or area surrounding the plant or plant seed, in a composition containing the enzyme or a recombinant microorganism and an agriculturally acceptable carrier.

[001630] Embodiment 435 is a method for delivering enzymes to a plant, comprising:

adding to the plant growth medium a recombinant member of the Bacillus cereus family according to embodiments 16-141 and 168-296 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342-355; or

applying to the plant, plant seed, or area surrounding the plant or plant seed, a recombinant member of the Bacillus cereus family according to embodiments 16-141 and 168-296 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342-355;

where the protein or peptide of interest includes an enzyme.

[001631] Embodiment 436 is a method for delivering enzymes to a plant, comprising:

adding to the plant growth medium a recombinant spore-forming bacterium according to any of embodiments 147-149, 151-153, and 156-268 or a composition comprising a recombinant spore-forming bacterium according to any of embodiments 338 and 342-355; or

applying to a plant, plant seed, or area surrounding a plant or plant seed, a recombinant spore-forming bacterium according to any one of embodiments 147-149, 151-153, and 156-268 or a composition comprising a recombinant spore-forming bacterium according to any one of embodiments 338 and 342- 355;

where the protein or peptide of interest includes an enzyme.

[001632] Embodiment 437 is a method for delivering enzymes to a plant according to embodiment 435 or 436, wherein the enzyme is physically associated with the exospore of a recombinant member of the Bacillus cereus family or with the spore envelope of the recombinant spore-forming bacterium.

[001633] Embodiment 438 is a method for delivering enzymes to a plant, comprising:

adding exosporium fragments or the composition of Embodiment 339 to the plant growth medium; or

applying fragments of the exospore or composition according to embodiment 339 to the plant, plant seed, or an area surrounding the plant or plant seed;

wherein the exospore fragments are derived from spores of a recombinant member of the Bacillus cereus family according to any one of embodiments 100-124 and contain a fusion protein and the protein or peptide of interest comprises an enzyme.

[001634] Embodiment 439 is the method of Embodiment 438, wherein the method further comprises treating the plant with a penetration enhancer, surfactant, detergent, oil, or combination thereof.

[001635] Embodiment 440 is a method according to any one of embodiments 435-441, wherein the enzyme is useful for biomass degradation, cellulosic material digestion, aiding digestion in the digestive system of a target animal that can be plant fed, or for biofuel production.

[001636] Embodiment 441 is the method of embodiment 440, wherein the enzyme comprises a non-specific protease, metalloproteinase, cellulase, xylanase, phosphatase, endoglucanase, exoglucanase, β-glucosidase, amylase, pectinase, xylosidase, lipase, phospholipase, or a combination thereof.

[001637] Embodiment 442 is a method according to any one of embodiments 434-441, wherein the plant is a crop selected from corn, alfalfa, wheat, pasture, forage, soybean, millet, jicama, sweet sorghum, sugarcane, or their combinations.

[001638] Embodiment 443 is a method for changing a property of a plant, comprising:

adding to the plant growth medium a recombinant member of the Bacillus cereus family according to embodiments 16-141 and 168-296 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342-355; or

applying to the plant, plant seed, or area surrounding the plant or plant seed, a recombinant member of the Bacillus cereus family according to embodiments 16-141 and 168-296 or a composition containing a recombinant member of the Bacillus cereus family according to any of embodiments 338 and 342-355;

wherein the protein or peptide of interest comprises a plant signaling molecule or an enzyme that influences plant composition.

[001639] Embodiment 444 is a method for changing a property of a plant, comprising:

adding to the plant growth medium a recombinant spore-forming bacterium according to any one of embodiments 147-149, 151-153, and 156-268 or a composition comprising a recombinant spore-forming bacterium according to any one of embodiments 338 and 342-355; or

applying to a plant, plant seed, or area surrounding a plant or plant seed, a recombinant spore-forming bacterium according to any one of embodiments 147-149, 151-153, and 156-268 or a composition comprising a recombinant spore-forming bacterium according to any one of embodiments 338 and 342- 355;

wherein the protein or peptide of interest comprises a plant signaling molecule or an enzyme that influences plant composition.

[001640] Embodiment 445 is the method of embodiment 443 or 444, wherein the protein or peptide of interest is physically linked to the exospore of a recombinant member of the Bacillus cereus family or to the spore envelope of a recombinant spore-forming bacterium.

[001641] Embodiment 446 is a method for changing a property of a plant, comprising:

adding exosporium fragments or the composition of Embodiment 339 to the plant growth medium; or

applying fragments of the exospore or composition according to embodiment 339 to the plant, plant seed, or an area surrounding the plant or plant seed;

wherein the exospore fragments are derived from spores of a recombinant member of the Bacillus cereus family according to any one of embodiments 100-124 and contain a fusion protein, and the protein or peptide of interest comprises a plant signal molecule or an enzyme that affects plant composition.

[001642] Embodiment 447 is the method of Embodiment 446, wherein the method further comprises treating the plant with a penetration enhancer, surfactant, detergent, oil, or combination thereof.

[001643] Embodiment 448 is a method according to any one of embodiments 435-447, wherein the recombinant Bacillus cereus , recombinant spore-forming bacterium, or exospore fragments are applied to the foliar portion of the plant prior to harvest.

[001644] Embodiment 449 is a method according to any one of embodiments 443-450, wherein the enzyme comprises an endoglucanase, a protease, a phospholipase, an aminocyclopropane-1-carboxylic acid deaminase, a lipase, or a combination thereof.

[001645] Embodiment 450 is a method according to any one of embodiments 443-448, wherein the plant signaling molecule comprises a flagellin peptide, cryptogain, harpin, a harpin-like protein, an enzyme that degrades or modifies a nutrient source of bacteria, fungi, or plants, or a combination of them.

[001646] Embodiment 451 is a method according to any one of embodiments 443-450, wherein the plant is selected from grape, sugar cane, millet, sweet sorghum, cereal, cereal, rapeseed, canola, salt, sunflower, marijuana, or a combination thereof .

[001647] Embodiment 452 is a method according to any one of embodiments 443-451, wherein the property of the plant to be changed includes a metabolic change.

[001648] Embodiment 453 is a method according to embodiment 452, wherein the metabolic change comprises increasing macronutrient or micronutrient uptake or tissue content of the plant by increasing its root system, increasing protein content, increasing nitrogen uptake, modified oil content, modified sugar or sucrose content , an increased content of a medical compound, an increased content of cannabinoids, or a combination of both.

[001649] Embodiment 454 is a method according to any of embodiments 357-379, 390-396, 400-414, and 420-453, wherein the plant growth medium is supplemented with an enzyme substrate or cofactor.

[001650] Embodiment 455 is the method of Embodiment 454 wherein the substrate or cofactor comprises tryptophan, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, indole, trimetaphosphate, ferrodoxin, acetoin, diacetyl, pyruvate, acetolactate, pectin, cellulose, methylcellulose, starch, chitin , pectin, protein flour, cellulose derivative, phosphate, acetoin, chitosan, inactive indole-3-acetic acid derivative, inactive gibberellic acid derivative, xylan, arabinoxylan, fat, wax, oil, phytic acid, lignin, humic acid, choline, derivative choline, proline, proline-rich protein, proline-rich flour, phenylalanine, chorismate, L-arginine, NADH, NADPH, ATP, GTP, cytochrome C, cytochrome p450, or a combination thereof.

[001651] Embodiment 456 is the method of Embodiment 455, wherein the adenosine triphosphate comprises adenosine 3-triphosphate.

[001652] Embodiment 457 is a method according to any of embodiments 357-379, 390-396, 400-414, and 420-456, comprising coating seeds with a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or exosporium fragments, or a formulation containing a recombinant a member of the Bacillus cereus family, a recombinant spore-forming bacterium or exospore fragments, before sowing.

[001653] Embodiment 458 is a method according to any one of embodiments 357-379, 390-396, 400-414, and 420-457, comprising applying a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or exosporium fragments, or a formulation containing the recombinant a representative of the Bacillus cereus family, a recombinant spore-forming bacterium or exosporium fragments, on the aerial part of the plant.

[001654] Embodiment 459 is a method according to any one of embodiments 357-379, 390-396, 400-414, and 420-458, wherein adding a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium, or exosporium fragments to the plant growth medium comprises introducing into the medium a liquid or solid composition containing a recombinant member of the Bacillus cereus family, a recombinant spore-forming bacterium or exospore fragments.

[001655] Embodiment 460 is the method of embodiment 459, wherein the method comprises applying the composition to the plant growth medium before, at the same time as, or after sowing the seeds, seedlings, cuttings, bulbs, or plants into the plant growth medium.

[001656] Embodiment 461 is a method according to any one of embodiments 357-379, 390-396, 400-414, and 420-460, further comprising adding at least one agrochemical to the plant growth medium or applying at least one agrochemical on plants or seeds.

[001657] Embodiment 462 is the method of Embodiment 461, wherein the agrochemical comprises a fertilizer, a micronutrient material, an insecticide, a herbicide, a fungicide, a molluscicide, an algicide, a plant growth modifier, a bacterial inoculum, a fungal inoculum, or a combination thereof.

[001658] Embodiment 463 is the formulation, plant seed, or inoculum of Embodiment 350, or the method of Embodiment 462, wherein the agrochemical comprises a fertilizer and the fertilizer comprises a liquid fertilizer; where the agrochemical includes a micronutrient material, and the microfertilizer material includes boric acid, borate, boric frit, copper sulfate, copper frit, copper chelate, sodium tetraborate decahydrate, ferrous sulfate, iron oxide, ammonium ferrous sulfate, iron frit, iron chelate, manganese sulfate, manganese oxide, manganese chelate, manganese chloride, manganese frit, sodium molybdate, molybdic acid, zinc sulfate, zinc oxide, zinc carbonate, zinc frit, zinc phosphate, zinc chelate, or a combination thereof; where the agrochemical includes an insecticide, and the insecticide includes an organophosphorus compound, a carbamate, a pyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate, fluoride, sulfur, a haloaromatic substituted urea, a hydrocarbon ester, a biobased insecticide, or a combination thereof; where the agrochemical includes a herbicide and the herbicide includes a chlorophenoxy compound, a nitrophenol compound, a nitrocresol compound, a dipyridyl compound, acetamide, an aliphatic acid, an anilide, a benzamide, a benzoic acid, a benzoic acid derivative, anisic acid, anisic acid derivative, benzonitrile, benzothiadiazinone dioxide, thiocarbamate, carbamate , carbanylate, chlorpyridinyl, cyclohexenone derivative, dinitroaminobenzene derivative, fluoronitrotoluidine compound, isoxazolidinone, nicotinic acid, isopropylamine, isopropylamine derivative, oxadiazolinone, phosphate, phthalate, picolinic acid compound, triazine, triazole, uracil, urea derivative, endothall, sodium chlorate, or a combination thereof ; wherein the agrochemical contains a fungicide and the fungicide contains a substituted benzene, thiocarbamate, ethylene bis-dithiocarbamate, thiophthalidamide, a copper compound, an organomercury compound, an organotin compound, a cadmium compound, anilazine, benomyl, cyclohexamide, dodine, etridiasol, iprodione, metlaxyl, thiamimefon, triforin, or their combination; wherein the agrochemical comprises a fungal inoculum and the fungal inoculum includes a fungal inoculum of the Glomeraceae family, a fungal inoculum of the Claroidoglomeraceae family, a fungal inoculum of the Gigasporaceae family, a fungal inoculum of the Acaulosporaceae family, a fungal inoculum of the Sacculosporaceae family, a fungal inoculum of the Entrophosporaceae family, a fungal inoculum of the Pacidsporaceae family, a fungal inoculum of the Diversisporaceae family, fungal inoculum of the Paraglomeraceae family, fungal inoculum of the Archaeosporaceae family, fungal inoculum of the Geosiphonaceae family, fungal inoculum of the Ambisporaceae family, fungal inoculum of the Scutellosporaceae family, fungal inoculum of the Dentiscultataceae family, fungal inoculum of the Racocetraceae family, mushroom inoculum of the Basidiomycota division, fungal inoculum of the Ascomycota division, fungal inoculum of the Zygomycota division their combination; или где агрохимикат включает бактериальный инокулят и бактериальный инокулят включает бактериальный инокулят рода Rhizobium , бактериальный инокулят рода Bradyrhizobium , бактериальный инокулят рода Mesorhizobium , бактериальный инокулят рода Azorhizobium , бактериальный инокулят рода Allorhizobium , бактериальный инокулят рода Sinorhizobium , бактериальный инокулят рода Kluyvera , бактериальный инокулят рода Azotobacter , bacterial inoculum of the genus Pseudomonas , bacterial inoculum of the genus Azospirillium , bacterial inoculum of the genus Bacillus , bacterial inoculum of the genus Streptomyces , bacterial inoculum of the genus Paenibacillus , bacterial inoculum of the genus Paracoccus , bacterial inoculum of the genus Enterobacter , bacterial inoculum of the genus Alcaligenes , bacterial genus inoculum of the genus Mycobacter , a bacterial inoculum of the genus Gliocladium , a bacterial inoculum of the genus Glomus , a bacterial inoculum of the genus Klebsiella , or a combination thereof.

[001659] Embodiment 464 is the formulation, plant seed or inoculum of Embodiment 350, or the method of Embodiment 462, wherein the agrochemical comprises a fungicide, and the fungicide comprises aldimorph, ampropylphos, ampropylphos potassium, andoprim, anilazine, azoconazole, azoxystrobin, benalaxyl, benodanil, benomyl, benzamacryl, benzamacryl-isobutyl, bialaphos, binapacryl, biphenyl, bitertanol, blasticidin-S, boscalid, bromuconazole, bupirimate, butiobate, calcium polysulfide, capsimycin, captafol, captan, carbendazim, carvone, quinomethionate, chlobentiazone, chlorebphenazole, chlorone , chloropicrin, chlorothalonil, chlosolinate, closilacone, kufraneb, cymoxanil, cyproconazole, cyproconazole, cyprofuram, debacarb, dichlorophen, diclobutrazol, diclofluanid, diclomesin, dicloran, diethophencarb, dimethirimol, dimethomorph, dimoxystrobin, diniconazolite, dinocapazole, diphenylimino-M , dithalymphos, dithianon, dodemorph, dodin, drazoxolone, edifenphos, epoxiconazole, ethaconazole, etirimol, etridiazol, famoxadone, fenapanil, fenarimol, fenbuconazole, fenfuram, fenitropan, fenpiclonil, fenpropidine, fenpropimorph, fentin acetate, fentin hydroxide, ferbam, ferimzone, fluazinam, flemetover, fluoromide, fluquinconazole, flurprimidol, flusilazole, flutosulfamide, flutosulfamide, folpet, fosetyl aluminum, fosetyl sodium, phthalide, fuberidazole, furalaxyl, furametpyr, furcarbonyl, furconazole, furconazole cis, furmecyclox, guazatin, hexachlorobenzene, hexaconazole, hymexazole, imazalil, imibenconazole, iminoctadine, iminoctadine albesilate, iminocotadin acetate, triacetate iprobenfos (IBP), iprodione, irumamycin, isoprothiolane, isovaledione, casugamycin, kresoxim-methyl, copper preparations such as: copper hydroxide, copper naphthenate, copper oxychloride, copper sulfate, copper oxide, copper oxin and Bordeaux mixture, mancuprum, mancozeb , maneb, meferimzone, mepanipyrim, mepronil, metalaxyl, metconazole, metasulfocarb, metfuroxam, metiram, metomeclam, metsulfax, mildiomycin, myclobutanil, myclo zoline, nickel dimethyldithiocarbamate, nitrotal-isopropyl, nuarimol, ofuras, oxadixyl, oxamocarb, oxolinic acid, oxycarboxy, oxyfentiin, paclobutrazol, pefurazoate, penconazole, pencycuron, fosdifen, pimaricin, piperalin, polyoxin, polyoxorim, probenazole, prochloraz, procymidone, propamocarb, propanosine-sodium, propiconazole, propineb, prothiocinazole, pyrazophos, pyrifenox, pyrimethanil, pyroquilon, piroxifur, quinoconazole, quintozene (PCNB), sulfur and sulfur preparations, tebuconazole, tecloftalam, teknazen, tetsiklasis, tetraconazole, thiabendazole, ticifen, thifluzamide, thiophanate- methyl, thioximide, tolclofos-methyl, tolylfluanid, triadimefon, triadimenol, triazbutyl, triazoxide, trichlamide, tricyclazole, tridemorph, trifloxystrobin, triflumizole, triforin, uniconazole, validamycin A, vincozoline, viniconazole, zarilamide, zineb, ziram, and also Dagger G, OK-8705, OK-8801, a-(1,1-dimethylethyl)-(3-(2-phenoxyethyl)-1H-1,2,4-triazole-1-ethanol, a-(2,4-dichlorophenyl) -[3-fluoro-3-propyl-1H-1,2,4-triazole-1-ethanol, a -(2,4-dichlorophenyl)-[3-methoxy-a-methyl-1 H-1,2,4-triazol-1-ethanol, a-(5-methyl-1,3-dioxan-5-yl) -[3-[[4-(trifluoromethyl)-phenyl]methylene]-1H-1,2,4-triazole-1-ethanol, (5RS,6RS)-6-hydroxy-2,2,7,7-tetramethyl -5-(1 H-1,2,4-triazol-1-yl)-3-octanone, (E)-a-(methoxyimino)-N-methyl-2-phenoxyphenylacetamide, 1-isopropyl{2-methyl- 1-[[[1-(4-methylphenyl)ethyl]amino]carbonyl]propyl}carbamate, 1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone -O-(phenylmethyl)oxime, 1-(2-methyl-1-naphthalenyl)-1H-pyrrole-2,5-dione, 1-(3,5-dichlorophenyl)-3-(2-propenyl)-2, 5-pyrrolidinedione, 1-[(diiodomethyl)sulfonyl]-4-methylbenzene, 1-[[2-(2,4-dichlorophenyl)-1,3-dioxolan-2-yl]methyl]-1H-imidazole, 1- [[2-(4-chlorophenyl)-3-phenyloxiranyl]methyl]-1H-1,2,4-triazole, 1-[1-[2-[(2,4-dichlorophenyl)-methoxy]phenyl]ethenyl] -1H-imidazole, 1-methyl-5-nonyl-2-(phenylmethyl)-3-pyrrolidinol, 2',6'-dibromo-2-methyl-4'-trifluoromethoxy-4'-trifluoromethyl-1, 3-thiazolecarboxanilide , 2,2-dichloro-N-[1-(4-chlorophenyl)ethyl]-1-ethyl-3-methylcyclopropanecarboxamide, 2,6-dichloro-5-(methylthio)-4-pyr imidinylthiocyanate, 2,6-dichloro-N-(4-trifluoromethylbenzyl)benzamide, 2,6-dichloro-N-[[4-(trifluoromethyl)phenyl]methyl]benzamide, 2-(2,3,3-triiodo-2 -propenyl)-2H-tetrazole, 2-[(1-methylethyl)-sulfonyl]-5-(trichloromethyl)-1,3,4-thiadiazole, 2-[[6-deoxy-4-O-(4-O -methyl-(3-D-glycopyranosyl)-a-D-glucopyranosyl]amino]-4-methoxy-1H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile, 2-aminobutane, 2-bromo-2-(bromomethyl )pentandinitrile, 2-chloro-N-(2,3-dihydro-1,1,3-trimethyl-1H-inden-4-yl)-3-pyridinecarboxamide, 2-chloro-N-(2,6-dimethylphenyl) -N-(isothiocyanatemethyl)acetamide, 2-phenylphenol (OPP), 3,4-dichloro-1-[4-(difluoromethoxy)phenyl]pyrrole-2,5-dione, 3,5-dichloro-N-[cyano[ (1-methyl-2-propynyl)oxy]methyl]benzamide, 3-(1,1-dimethylpropyl-1-oxo-1H-indene-2-carbonitrile, 3-[2-(4-chlorophenyl)-5-ethoxy -3-isoxazolidinyl]pyridine, 4-chloro-2-cyano-N,N-dimethyl-5-(4-methylphenyl)-1H-imidazole-1-sulfonamide, 4-methyltetrazolo[1,5-a]quinazoline-5 (4H)-one, 8-(1,1-dimethylethyl)-N-ethyl-N-propyl-1,4-dioxaspiro[4,5]decan-2-methanamine, 8-hydroxyquinoline sulfate, 9H-xanthene -2-[(phenylamino)carbonyl]-9-carboxylic hydrazide, bis-(1-methylethyl)-3-methyl-4-[(3-methylbenzoyl)oxy]-2,5-thiophene dicarboxylate, cis-1-(4 -chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)cycloheptanol, cis-4-[3-[4-(1,1-dimethylpropyl)phenyl-2-methylpropyl]-2,6 -dimethylmorpholine hydrochloride, ethyl [(4-chlorophenyl)-azo]cyanoacetate, potassium bicarbonate, methanetetrathiol sodium salt, methyl 1-(2,3-dihydro-2,2-dimethylinden-1-yl)-1H-imidazol-5 -carboxylate, methyl N-(2,6-dimethylphenyl)-N-(5-isoxazolylcarbonyl)-DL-alaninate, methyl-N-(chloroacetyl)-N-(2,6-dimethylphenyl)-DL-alaninate, N- (2,3-dichloro-4-hydroxyphenyl)-1-methylcyclohexanecarboxamide, N-(2,6-dimethylphenyl)-2-methoxy-N-(tetrahydro-2-oxo-3-furanyl)acetamide, N-(2, 6-dimethylphenyl)-2-methoxy-N-(tetrahydro-2-oxo-3-thienyl)acetamide, N-(2-chloro-4-nitrophenyl)-4-methyl-3-nitrobenzenesulfonamide, N-(4-cyclohexylphenyl) )-1,4,5,6-tetrahydro-2-pyrimidinamine, N-(4-hexylphenyl)-1,4,5,6-tetrahydro-2-pyrimidinamine, N-(5-chloro-2-methylphenyl)- 2-methoxy-N-(2-oxo-3-oxazolidinyl)acetamide, N-(6- methoxy)-3-pyridinyl)cyclopropanecarboxamide, N-[2,2,2-trichloro-1-[(chloroacetyl)amino]ethyl]benzamide, N-[3-chloro-4,5-bis(2-propynyloxy)phenyl ]-N'-methoxymethanenimidamide, N-formyl-N-hydroxy-DL-alanine-sodium salt, O,O-diethyl [2-(dipropylamino)-2-oxoethyl]ethylphosphoramidothioate, O-methyl S-phenylphenylpropylphosphoramidothioate, S-methyl -1,2,3-benzothiadiazol-7-carbothioate and spiro[2H]-1-benzopyran-2,1'(3'H)-isobenzofuran]-3'-one, N-trichloromethyl)thio-4-cyclohexane- l,2-dicarboximide, tetramethylthioperoxydicarboxylic diamide, methyl-N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate, 4-(2,2-difluoro-1,3-benzodioxol-4-yl) -1-H-pyrrole-3-carbonitrile or a combination thereof.

[001660] Embodiment 465 is the formulation, plant seed, or inoculum of Embodiment 350, or the method of Embodiment 462, wherein the agrochemical comprises a bacterial inoculum of the genus Bacillus and the bacterial inoculum of the genus Bacillus comprises Bacillus argri, Bacillus aizawai, Bacillus albolactis, Bacillus amyloliquefaciens Bacillus cereus Bacillus coagulans Bacillus endoparasiticus Bacillus endorhythmos Bacillus kurstaki Bacillus lacticola Bacillus lactimorbus Bacillus lactis Bacillus laterosporus Bacillus lentimorbus Bacillus licheniformis Bacillus megaterium Bacillus medusa popillae, Bacillus pumilus, Bacillus siamensis, Bacillus sphearicus, Bacillus spp., Bacillus subtilis, Bacillus thuringiensis, Bacillus unifagellatu, or a combination thereof.

[001661] Embodiment 466 is the formulation, plant seed or inoculum of Embodiment 350 or the method of Embodiment 462, wherein the agrochemical comprises a herbicide and the herbicide comprises 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor , ametrine, atrazine, aminopyralid, benefin, bensulfuron, bensulide, bentazone, bromacyl, bromxinil, butylate, carfentrazone, chlorimuron, chlorsulfuron, cletodim, clomazone, clopyralid, cloransulam, cycloate, DCPA, desmedifam, dicambu, dichlobenil, diclofop, diflufene diclosulam, , Dimethenamid, Diquat, Diuron, DSMA, Endothal, EPTC, Ethalfluralin, Ethofumesate, Fenoxaprop, Fluazifop-P, Flucarbazone, Flufanecet, Flumetsulam, Flumiclorac, Flumioxazine, Fluometuron, Fluroxypyr, Fomesafen, Foramsulfuron, Glufosinate, Glyphosinsulphate, Iphosinubenacet, , imazamox, imazapic, imazakquin, imazethapyr, isoxaben, isoxaflutol, lactofen, linuron, MCPA, MCPB, mesotrione, metolachlor-s, metribuzin, metsulfuron, molinate, MSM A, napropamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxydiazone, oxyfluorfen, paraquat, pelargonic acid, pendimethalin, phenmedifam, picloram, primisulfuron, prodiamine, prometrin, pronamide, propanil, prosulfuron, pyrazon, pyritiobac, quinclorac, quizalofop, setoxytimsulmuron , siduron, simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, tebacil, thiazopyr, thifensulfuron, thiobencarb, tralkoxydim, triallat, triasulfuron, tribenuron, triclopyr, trifluralin, triflusulfuron, or a combination thereof.

[001662] Embodiment 467 is the formulation, plant seed, or inoculum of Embodiment 350, or the method of Embodiment 463, wherein the formulation or inoculum comprises a herbicide and a bacterial strain that is capable of degrading the herbicide.

[001663] Embodiment 468 is a formulation, plant seed, inoculum, or method according to Embodiment 467, wherein the bacterial strain that is capable of degrading the herbicide comprises Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus EE- B00377 (NRRL B-67119), Bacillus pseudomycoides EE-B00366 (NRRL B-67120) or Bacillus mycoides EE-B00363 (NRRL B-67121), or a combination thereof.

[001664] Embodiment 469 is a formulation, plant seed, inoculum, or method according to any 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.

[001665] Embodiment 470 is a formulation, plant seed, inoculum, or method according to Embodiment 469, wherein the sulfonylurea compound comprises sulfentrazone.

[001666] Embodiment 471 is a formulation, plant seed or inoculum according to any of Embodiments 350 and 463-470, or a method according to any of Embodiments 461-470, wherein the fertilizer comprises 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, calcium ammonium nitrate, calcium sulfate, calcined magnesite, calcined limestone, calcium oxide, calcium nitrate, dolomite limestone, slaked lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, potassium magnesium sulfate, potassium sulfate, sodium nitrates, magnesian limestone, magnesia, urea, urea-formaldehydes, ammonium urea nitrate, sulfur-coated urea, polymer-coated urea, isobutylidene diurea, K ₂ SO ₄ -2MgSO ₄ , kainite, sylvinite, k iserite, Epsom salt, elemental sulfur, lime clay, crushed oyster shells, fishmeal, cake, fish current, blood meal, phosphate ore, superphosphates, slag, bone meal, wood ash, manure, bat guano, peat moss, compost, glauconite sand, cotton seed meal, feather meal, crab meal, fish emulsion, humic acid, or a combination thereof.

[001667] Embodiment 472 is a method of delivering beneficial bacteria to an animal, comprising feeding the animal a plant modified to contain a level of endophytic and probiotic strain of bacteria greater than the level of endophytic and probiotic strain of bacteria in the same plant that has not been modified, grown under the same conditions.

[001668] Embodiment 473 is the method of Embodiment 472, wherein the plant fed to the animal comprises a plant grown in a plant growth medium containing an endophytic and probiotic strain of bacteria, or a formulation comprising an endophytic and probiotic strain of bacteria, a plant, on which has been coated with an endophytic and probiotic strain of bacteria, a plant grown from a seed that has been coated with an endophytic and probiotic strain of bacteria, a plant grown in an area that has been coated with an endophytic and a probiotic strain of bacteria, or a seed grown in an area that has been coated with an endophytic and a probiotic strain of bacteria.

[001669] Embodiment 474 is a method according to embodiment 472 or 473, wherein the endophytic and probiotic bacterial strain comprises a Bacillus or Lysinibacillus species .

[001670] Embodiment 475 is the method of Embodiment 474, wherein the Bacillus species includes Bacillus subtilis , Bacillus firmus , Bacillus amyloliquefaciens , Bacillus cereus , Bacillus toyocerin, Bacillus megaterium, Bacillus pumilus, Bacillus licheniformis, or a combination thereof.

[001671] Embodiment 476 is a method according to any one of embodiments 472-475, wherein the endophytic and probiotic bacterial strain comprises Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus EE439 (NRRL B-50979), Bacillus thuringiensis EE417 (NRRL B-50979), Bacillus cereus EE444 (NRRL B-50977), Bacillus megaterium EE385 (NRRL B-50980), Bacillus sp. EE387 (NRRL B-50981), Bacillus circulans EE388 (NRRL B-50982), Bacillus subtilis EE405 (NRRL B-50978), Lysinibacillus fusiformis EE442 (NRRL B-50975), or Lysinibacillus sphaericus EE443 (NRRL B-50976), Bacillus pumilus EE-B00143 (NRRL B-67123), or a combination thereof.

[001672] Embodiment 477 is a method of delivering proteins or peptides to an animal, comprising feeding the animal a recombinant member of the Bacillus cereus family expressing a fusion protein containing a protein or peptide of interest and a targeting sequence, an exosporium protein, or an exosporium protein fragment that targets the fusion protein to exospores of a recombinant member of the Bacillus cereus family, or feeding the animal with exospore fragments derived from a recombinant member of the Bacillus cereus family expressing a fusion protein containing a protein or peptide of interest and a targeting sequence, exospore protein or exospore protein fragment, which targets the fusion protein into the exospore of the recombinant member of the Bacillus cereus family.

[001673] Embodiment 478 is the method of Embodiment 477, wherein the recombinant Bacillus cereus includes the recombinant Bacillus cereus according to any of Embodiments 16-29, 48-63, 125-141, and 186-296, or wherein the exospore fragments include exosporium fragments derived from a member of the Bacillus cereus family according to any one of embodiments 100-124.

[001674] Embodiment 479 is a method according to embodiment 477 or 478, wherein the recombinant member of the Bacillus cereus family comprises an endophytic bacterial strain.

[001675] Embodiment 480 is the method of Embodiment 479, wherein the endophytic bacterial strain comprises Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus 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 EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120) or Bacillus mycoides EE-B00363 (NRRL B-67121).

[001676] Embodiment 481 is the method of Embodiment 480 wherein the endophytic bacterial strain comprises Bacillus cereus 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 EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120) or Bacillus mycoides EE-B00363 (NRRL B-67121).

[001677] Embodiment 482 is a method according to any one of embodiments 477-479, wherein the recombinant member of the Bacillus cereus family comprises a probiotic bacterial strain.

[001678] Embodiment 483 is the method of Embodiment 482, wherein the probiotic bacterial strain comprises a member of the familyBacillus cereus EE349 (NRRL #B-50928), member of the familyBacillus cereus EE439 (NRRL B-50979),Bacillus thuringiensis EE417 (NRRL B-50979),Bacillus cereus EE444 (NRRL B-50977),Bacillus thuringiensis BT013A (NRRL #B-50924) or a combination of them.

[001679] Embodiment 484 is a method according to any one of embodiments 479-481, wherein the recombinant member of the Bacillus cereus family is in a plant fed to the animal.

[001680] Embodiment 485 is a method according to embodiment 477 or 478, wherein the recombinant member of the Bacillus cereus family comprises a bacterial strain that is capable of colonizing the plant's phylloplana.

[001681] Embodiment 486 is the method of Embodiment 485, wherein the bacterial strain that is capable of colonizing the phylloplana of a plant comprisesBacillus mycoidesBT155 (NRRL #B-50921),Bacillus mycoides EE118 (NRRL #B-50918),Bacillus mycoides EE141 (NRRL #B-50916),Bacillus mycoides BT46-3 (NRRL #B-50922), member of the familyBacillus cereus EE218 (NRRL #B-50926),Bacillus thuringiensis BT013A (NRRL #B-50924), family memberBacillus cereus EE-B00377 (NRRL B-67119),Bacillus pseudomycoides EE-B00366 (NRRL B-67120) orBacillus mycoides EE-B00363 (NRRL B-67121).

[001682] Embodiment 487 is a method according to any one of embodiments 485 or 486, wherein the recombinant member of the Bacillus cereus family is on the phylloplane of the plant fed to the animal.

[001683] Embodiment 488 is a vaccine comprising a pharmaceutically acceptable carrier and a recombinant member of the Bacillus cereus family that expresses the fusion protein of Embodiment 12.

[001684] Embodiment 489 is a vaccine comprising a pharmaceutically acceptable carrier and exospore fragments, wherein the exospore fragments are derived from a recombinant member of the Bacillus cereus family according to any of embodiments 100-124 and contain a fusion protein and the fusion protein contains an antigen.

[001685] Embodiment 490 is a vaccine comprising a pharmaceutically acceptable carrier and a recombinant member of the Bacillus cereus family according to any one of Embodiments 17-27, wherein the fusion protein contains an antigen.

[001686] Embodiment 491 is a vaccine according to any one of embodiments 488-490, wherein the vaccine is suitable for intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, topical, oral, intranasal, intradermal, transepithelial administration, or by inhalation.

[001687] Embodiment 492 is a vaccine according to any one of embodiments 488-490, wherein the vaccine is suitable for oral or transepithelial administration.

[001688] Embodiment 493 is a method of inducing an immunogenic response in an individual, the method comprising administering to the individual a vaccine according to any of embodiments 488-492.

[001689] Embodiment 494 is a method for reducing environmental pollutants, comprising exposing the polluted environment to spores of a recombinant member of the Bacillus cereus family that express the fusion protein of Embodiment 13.

[001690] Embodiment 495 is a method for reducing pollutants in the environment, comprising exposing the polluted environment to exospore fragments, wherein the exospore fragments are derived from a recombinant member of the Bacillus cereus family according to any one of embodiments 100-124 and contain a fusion protein, and wherein the fusion protein contains a reclamation enzyme.

[001691] Embodiment 496 is a method for reducing environmental contaminants, comprising exposing the polluted environment to spores of a recombinant member of the Bacillus cereus family according to any one of Embodiments 17-27, wherein the fusion protein contains a remediation enzyme.

[001692] Embodiment 497 is a method according to any one of embodiments 494-496, wherein the contaminated environment includes a gas, liquid, semi-liquid, gel, film, semi-solid, or solid.

[001693] Embodiment 498 is a method according to embodiment 497 wherein the solid includes surface soil, subsoil, compost, crop residue, leaves, mulch, felled trees, biofilm, mud layer, humus, sludge, sand, slag, sediment, sewage, waste rock, nuclear waste, ammunition and shells, hospital waste, car parts for pressing, metal trim, insulation waste, food waste, asbestos, batteries, metalworking scrap, landfill waste, woodworking waste, textile production waste , cullet, leather waste, rubber waste, plastic waste, electronic component waste, agricultural waste, photographic waste, ceramic waste, pharmaceutical waste, wax, spent catalysts, or combinations thereof

[001694] Embodiment 499 is a method according to Embodiment 497, wherein the liquid includes drinking water, groundwater, surface water, saline solutions, reservoirs, lagoons, aquatic ecosystems, industrial wastewater, acid mine water, spent automotive fluid, spent electrolytic baths, degreasers, dry cleaning solutions, machine coolants, drilling fluid waste, cutting fluid waste, hydraulic fracturing fluid waste, lubricant waste, paint, domestic waste water, oily effluent, pulp mill effluent, water treatment systems , a sewage treatment system, a sewer, a settling pond, a sewage tank, a lake, a river, or a combination thereof.

[001695] Embodiment 500 is a method according to embodiment 497, wherein the gas includes air, exhaust gas such as emissions from power plants, incinerators, crematoria, or refineries, process effluent, landfill gas, natural gas, propane, or a combination thereof.

[001696] Embodiment 501 is a method according to any one of embodiments 494-500, wherein the contaminant comprises a chemical warfare agent including Sarin (GB; o-isopropylmethylphosphonofluoridate); soman (GD; o-pinacolylmethylphosphonofluoridate); cyclosarin (GF; o-cyclohexylmethylphosphonofluoridate); VX (O-ethyl-S-[2-(diisopropylamino)ethyl]methylphosphonothioate); tabun (GA; N,N-dimethylethylphosphoroamidocyanidate), DFP (diisopropylphosphorofluoridate), or mustard gas.

[001697] Embodiment 502 is a method according to any one of embodiments 494-500, wherein the contaminant comprises an inorganic compound including arsenic, antimony, barium, beryllium, cadmium, chromium, copper, iron, lead, manganese, mercury, nickel, selenium , silver, tin, thallium, uranium, zinc, or a combination thereof.

[001698] Embodiment 503 is a method according to any one of embodiments 494-500, wherein the contaminant includes an organic compound containing 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.

[001699] Embodiment 504 is a method according to any one of embodiments 494-500, wherein the contaminant includes crude oil, refined oil, fuel oil, diesel fuel, gasoline, hydraulic oil, and kerosene, or a volatile component thereof.

[001700] Embodiment 505 is a method according to any one of embodiments 494-500, wherein the contaminant includes an explosive, fertilizer, pesticide, insecticide, or herbicide.

[001701] Embodiment 506 is a method according to any one of embodiments 494-500, wherein 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 dioxygenase, a laccase, lignin peroxidase, manganese peroxidase, peroxidase, dehalogenase, catalase, amylase, reductase, oxidase, amidase, ligninase, xylanase, pectinase, xylosidase, endoglucanase, exoglucanase, glucosidase, biofilm inhibiting peptide, herbicide degrading enzyme, pesticide degrading enzyme, or a combination thereof.

[001702] Embodiment 507 is the method of Embodiment 506 wherein the pesticide degrading enzyme comprises pyrethrinase.

[001703] Embodiment 508 is the method of embodiment 506, wherein the enzyme comprises a herbicide-degrading enzyme or a pesticide-degrading enzyme, and the recombinant member of the Bacillus cereus family includes a bacterial strain that is capable of degrading the herbicide or pesticide.

[001704] Embodiment 509 is the method of Embodiment 508, wherein the bacterial strain that is capable of degrading the herbicide or pesticide comprises Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus EE-B00377 (NRRL B- 67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120) or Bacillus mycoides EE-B00363 (NRRL B-67121).

[001705] Embodiment 510 is a method according to any one of embodiments 494-509, wherein recombinant Bacillus cereus spores or exosporium fragments are brought into contact with a contaminated environment by incorporating the spores or exospore fragments into a stream containing the contaminant, contacting a stream containing a pollutant with an immobilizing material containing spores or exospore fragments, including spores or exospore fragments in granules to be mixed with a polluted environment, spraying spores or exospore fragments onto or into a polluted environment, injecting spores or exospore fragments into a polluted environment environment, or watering a polluted environment with spores or fragments of exospores.

[001706] Embodiment 511 is a method for phytoremediation of contaminated soil, the method comprising:

introducing spores of a recombinant member of the Bacillus cereus family into contaminated soil; or

applying spores of a recombinant member of the Bacillus cereus family to a plant planted in contaminated soil, or a plant seed to be planted in contaminated soil, or an area of contaminated soil surrounding the plant or plant seed;

where spores of a recombinant member of the Bacillus cereus family express a fusion protein containing at least one protein or peptide of interest and a targeting sequence, an exosporium protein or an exosporium protein fragment, which targets the fusion protein into the exospore of the spores of the recombinant member of the Bacillus cereus family, where the fusion protein contains a fusion the protein of Embodiment 13, and wherein the recombinant member of the Bacillus cereus family comprises an endophytic bacterial strain or a root colonizing bacterial strain.

[001707] Embodiment 512 is a method for phytoremediation of contaminated soil, a method comprising expression of a remediation enzyme in spores of a member of the Bacillus cereus family, wherein expression of the remediation enzyme in the recombinant member of the Bacillus cereus family is increased compared to expression of the remediation enzyme in spores of a member of the wild Bacillus cereus family type.

[001708] Embodiment 513 is a method for phytoremediation of contaminated soil, the method comprising:

introducing a recombinant spore-forming bacterium into contaminated soil; or

application of a 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 the plant or plant seed, where the recombinant spore-forming bacterium expresses a fusion protein containing at least one protein or peptide of interest and a spore coat protein that targets the fusion protein to the spore surface of the bacterium, wherein the spore coat protein includes CotB protein, CotC protein, CgeA protein, CotB/H protein, Cot G protein, X spore coat protein, or CotY protein; the recombinant spore-forming bacterium includes an endophytic bacterial strain or a root colonizing bacterial strain; and the protein or peptide of interest comprises a reclamation enzyme.

[001709] Embodiment 514 is the method of Embodiment 513, wherein the recombinant spore-forming bacterium comprises an endophytic bacterial strain.

[001710] Embodiment 515 is a method for phytoremediation of contaminated soil, the method comprising applying exospore fragments to contaminated soil, or applying exospore 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, wherein the exospore fragments are derived from spores of a recombinant member of the Bacillus cereus family according to any one of embodiments 100-124 and contain a fusion protein, wherein the fusion protein contains a reclamation enzyme.

[001711] Embodiment 516 is a method for phytoremediation of contaminated soil, comprising applying spores of a recombinant member of the Bacillus cereus family according to any of embodiments 17-27 to the contaminated soil or applying spores of a recombinant member of the Bacillus cereus family according to any of embodiments 17-27 to a plant planted in contaminated soil, or a plant seed for planting in contaminated soil, or an area of contaminated soil surrounding the plant or plant seed, where the fusion protein contains a reclamation enzyme.

[001712] Embodiment 517 is a method according to any one of embodiments 511-516, wherein spores or exospore fragments are applied to a plant or plant seed, and the plant or plant grown from the plant seed is tolerant of a given contaminant to be reclaimed from the contaminated soil.

[001713] Embodiment 518 is a method according to any one of embodiments 494-517, wherein the spores of the recombinant member of the Bacillus cereus family comprise an endophytic bacterial strain.

[001714] Embodiment 519 is the method of Embodiment 518, wherein the endophytic bacterial strain comprises Bacillus cereus EE349 (NRRL No. B-50928), Bacillus cereus 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 EE-B00377 (NRRL B-67119) ; Bacillus pseudomycoides EE-B00366 (NRRL B-67120); or Bacillus mycoides EE-B00363 (NRRL B-67121).

[001715] Embodiment 520 is a method for reducing environmental contaminants, comprising exposing a polluted environment to spores of a strain of a member of the Bacillus cereus family that are capable of degrading a herbicide or pesticide; where the pollutants in the environment include a herbicide, a pesticide, or a combination thereof; and where a member of the Bacillus cereus family that is capable of degrading the herbicide or pesticide includes a member of the Bacillus cereus family EE349 (NRRL No. B-50928), a member of the Bacillus cereus family EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120); Bacillus mycoides EE-B00363 (NRRL B-67121) or a combination thereof.

[001716] Embodiment 521 is the method of embodiment 520, wherein the strain of a Bacillus cereus that is capable of degrading the herbicide or pesticide comprises a recombinant Bacillus cereus that expresses a fusion protein containing at least one protein or peptide of interest and a targeting sequence, an exospore protein or an exospore protein fragment, which targets the fusion protein to the exospore of a recombinant member of the Bacillus cereus family.

[001717] Embodiment 522 is the method of embodiment 521, wherein the protein or peptide of interest comprises a herbicide-degrading enzyme, a pesticide-degrading enzyme, or a combination thereof.

[001718] Embodiment 523 is a method of treating a fracturing fluid to break an emulsion or gel in the fluid, the method comprising adding spores of a recombinant member of the Bacillus cereus family to the fracturing fluid, wherein the recombinant member of the Bacillus cereus family expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, an exospore protein or an exospore protein fragment, that targets the fusion protein to the exospore of a recombinant member of the Bacillus cereus family, and the protein or peptide of interest comprises an enzyme suitable for breaking an emulsion or gel.

[001719] Embodiment 524 is the method of Embodiment 523, wherein the recombinant Bacillus cereus includes the recombinant Bacillus cereus according to any of Embodiments 16-29, 48-63, 125-141, and 168-296.

[001720] Embodiment 525 is a method of treating a fracturing fluid to break an emulsion or gel in the fluid, the method comprising adding exospore fragments to the fracturing fluid, wherein the exospore fragments are from a recombinant member of the Bacillus cereus family according to any of Embodiments 100-124 and contain a fusion protein, and where the fusion protein contains an enzyme suitable for breaking the emulsion or gel.

[001721] Embodiment 526 is a method according to any one of Embodiments 523-525, wherein the fusion protein includes the fusion protein according to Embodiment 14.

[001722] Embodiment 527 is a method according to any of embodiments 523-526, wherein the enzyme comprises hemicellulase, amylase, pectinase, carbohydrate hydrolyase, cellulase, agarase, polygalacturonase, endoglucanase, or a combination thereof.

[001723] Embodiment 528 is a method according to any one of embodiments 523-527, wherein the emulsion or gel comprises a polymer, gum arabic, agar, xanthan gum, cellulose, carboxymethylcellulose, carboxymethylhydroxyethylcellulose, hydroxyethylmethylcellulose, guar, a guar derivative, or a combination thereof.

[001724] Embodiment 529 is a method according to any one of embodiments 523-528, wherein spores are injected into a well that is in contact with a subterranean hydrocarbon containing formation.

[001725] Embodiment 530 is a surface disinfection method comprising exposing the surface to a recombinant member of the Bacillus cereus family that expresses the fusion protein of Embodiment 15.

[001726] Embodiment 531 is a surface disinfection method comprising exposing a surface to exospore fragments, wherein the exospore fragments are derived from a recombinant member of the Bacillus cereus family according to any one of embodiments 100-124 and comprise a fusion protein, and wherein the fusion protein comprises an antibacterial protein or peptide.

[001727] Embodiment 532 is a surface disinfection method comprising exposing the surface to a recombinant member of the Bacillus cereus family according to any one of Embodiments 17-27, wherein the fusion protein comprises an antibacterial protein or peptide.

[001728] Embodiment 533 is a method according to any one of embodiments 530-532, wherein the antibacterial protein or peptide minimizes or prevents the formation of, or binding to, a surface by viral agents, bacteria, amoebae, parasites, or molds.

[001729] Embodiment 534 is a method according to any one of embodiments 530-533, wherein the antibacterial protein or peptide comprises a protease, nuclease, antimicrobial peptide, LysM, LfcinB, lysostaphin, albumin, defensin, bacteriocin, lipopeptide, innate immune system peptide, lysozyme, lyticase, or a combination thereof.

[001730] Embodiment 535 is the use of fusion proteins containing the enzyme as the protein or peptide of interest according to any of Embodiments 13 or 15, or a recombinant member of the Bacillus cereus family expressing the fusion protein containing the enzyme as the protein or peptide of interest according to any one of embodiments 13 or 15, for treating or degumming a lubricant, oil or fat; processing hides into leather; production of biofuels, biodiesel or bioethanol; processing or conversion of sugars; starch processing; paper or linen processing; processing animal or fungal by-products or isolating amino acids; directed splitting of technical waste; food and feed additives; dietary supplements; animal feed; industrial cleaning; grain processing; cosmetic production; deodorization; food or beverage processing; brewing optimization or brewing additives; detergent additives or fabric or yarn processing.

[001731] Embodiment 536 is the use of exospore fragments to process or degumming a lubricant, oil, or fat; processing hides into leather; production of biofuels, biodiesel or bioethanol; processing or conversion of sugars; starch processing; paper or linen processing; processing animal or fungal by-products or isolating amino acids; directed splitting of technical waste; food and feed additives; dietary supplements; animal feed; industrial cleaning; grain processing; cosmetic production; deodorization; food or beverage processing; brewing optimization or brewing additives; detergent additives or fabric or yarn processing; wherein the exospore fragments are derived from a recombinant member of the Bacillus cereus family according to any one of embodiments 100-124 and contain a fusion protein, and wherein the fusion protein contains an enzyme.

[001732] Embodiment 537 is the use of a recombinant member of the Bacillus cereus family according to any of Embodiments 17-27 for treating or degumming a lubricant, oil, or fat; processing hides into leather; production of biofuels, biodiesel or bioethanol; processing or conversion of sugars; starch processing; paper or linen processing; processing animal or fungal by-products or isolating amino acids; directed splitting of technical waste; food and feed additives; dietary supplements; animal feed; industrial cleaning; grain processing; cosmetic production; deodorization; food or beverage processing; brewing optimization or brewing additives; detergent additives or fabric or yarn processing; where the fusion protein contains the enzyme.

[001733] Embodiment 538 is a use according to any of embodiments 535-537 wherein the enzyme comprises β-lactamase, protease, lipase, phospholipase, cellulase, endoglucanase, exogluconase, pectinase, ligninase, amylase, polygalacturonase, glucosidase, galactosidase, hydrolyase carbohydrates, cell wall hydrolase, nuclease, hemicellulase, xylanase, mannase, laccase, lactase, esterase, phytase, phosphatase, invertase, glucose oxidase, catalase, lyticase, acetolactate decarboxylase or urease.

[001734] Embodiment 539 is the use according to Embodiment 538, wherein the amylase comprises α-amylase, β-amylase, or glucoamylase; or where the esterase includes pectin methylesterase.

[001736] Embodiment 541 is the method of Embodiment 540, wherein the targeting sequence comprises:

an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, where 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 20-35 of SEQ ID NO: 1, where 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 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 63%;

an amino acid sequence having at least about 62% identity with amino acids 20-35 of SEQ ID NO: 1, where 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 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%;

an amino acid sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 72%;

an amino acid sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%;

an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 81%; or

an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 90%.

[001737] Embodiment 542 is the method of Embodiment 540, where 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, where 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.

[001738] Embodiment 543 is the method of Embodiment 541, wherein the targeting sequence consists of an amino acid sequence.

[001739] Embodiment 544 is the method of embodiment 540, wherein the exosporium protein or exosporium protein fragment comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to 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.

[001740] Embodiment 545 is a method according to any one of embodiments 477-544, wherein the targeting sequence, exosporium protein, or exosporium protein fragment contains the amino acid sequence GXT at its C-terminus, where X is any amino acid.

[001741] Embodiment 546 is a method according to any of embodiments 477-545, wherein the targeting sequence, exosporium protein, or exosporium protein fragment contains an alanine residue at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1.

[001742] Embodiment 547 is a method according to any one of embodiments 477-546, wherein the targeting sequence, exospore protein, or exosporium protein fragment further comprises a methionine, serine, or threonine residue at the amino acid position immediately preceding the first amino acid of the targeting sequence, exospore protein, or exosporium protein fragment, or at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1.

[001743] Embodiment 548 is a method according to any one of embodiments 477-547, wherein the fusion protein further comprises an amino acid linker between the targeting sequence, exosporium protein or exosporium protein fragment, and the protein or peptide of interest.

[001744] Embodiment 549 is the method of Embodiment 548, wherein the linker comprises a polyalanine linker, a polyglycine linker, or a linker. containing a mixture of both alanine and glycine residues.

[001745] Embodiment 550 is the method according to embodiment 548 or 549, wherein the linker contains a protease recognition site.

[001746] Embodiment 551 is a method according to any one of embodiments 447-550, wherein the fusion protein is expressed under the control of a sporulation promoter native to the targeting sequence, an exosporium protein or an exospore protein fragment of the fusion protein, or a portion thereof.

[001747] Embodiment 552 is a method according to any one of embodiments 447-551, wherein the fusion protein is expressed under the control of a highly expressed sporulation promoter.

[001748] Embodiment 553 is the method of any embodiment 552 wherein the highly expressed sporulation promoter comprises a sigma-K sporulation-specific polymerase promoter sequence.

[001749] Embodiment 554 is a method according to 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 any of the nucleic acid sequences of SEQ ID NO: 157-231.

[001750] Embodiment 555 is the method according to embodiment 553 or 554, the sporulation-specific polymerase sigma-K promoter sequence or sequences or sequences have 100% identity with the corresponding nucleotides of any of SEQ ID NOs: 157-231.

[001751] Embodiment 556 is a method for delivering proteins or peptides to an animal, comprising feeding the animal a recombinant spore-forming bacterium according to any one of Embodiments 147-149, 151-153, and 156-167.

[001752] Embodiment 557 is the method of Embodiment 556, wherein the recombinant spore-forming bacterium comprises an endophytic and a probiotic strain of bacteria.

[001753] Embodiment 558 is the method of Embodiment 557, wherein the endophytic and probiotic bacterial strain comprises Bacillus megaterium EE385 (NRRL B-50980), Bacillus sp. EE387 (NRRL B-50981), Bacillus circulans EE388 (NRRL B-50982), Bacillus subtilis EE405 (NRRL B-50978), Lysinibacillus fusiformis EE442 (NRRL B-50975) or Lysinibacillus sphaericus EE443 (NRRL B-50976), Bacillus pumilus EE-B00143 (NRRL B-67123), or a combination thereof.

[001754] Embodiment 559 is a method according to any one of embodiments 556-558, wherein the recombinant spore-forming bacterium is contained in a plant fed to the animal.

[001755] Embodiment 560 is a method according to any of embodiments 472-487 and 540-559 wherein the plant is processed before feeding to the animal.

[001756] Embodiment 561 is the method of Embodiments 472-487 and 540-560, wherein the method further comprises:

adding an endophytic bacterial strain or a composition comprising an endophytic bacterial strain to the plant growth medium; or

applying an endophytic bacterial strain or a composition comprising an endophytic bacterial strain to a plant, a plant seed, or an area surrounding the plant or plant seed;

where the plant fed to the animal includes a plant grown in a plant growth medium containing an endophytic and a probiotic strain of bacteria or a composition containing an endophytic and a probiotic strain of bacteria, a plant on which an endophytic and a probiotic strain of bacteria is applied, a plant grown from the seed of a plant , on which the endophytic and probiotic strain of bacteria is applied, a plant grown in the area on which the endophytic and probiotic strain of bacteria is applied, or a seed grown in the area on which the endophytic and probiotic strain of bacteria is applied.

[001757] Embodiment 562 is a method according to any of embodiments 477-487 and 540-561, wherein the protein or peptide of interest comprises an enzyme.

[001758] Embodiment 563 is the method of Embodiment 562 wherein the enzyme comprises xylanase, xylosidase, phytase, phosphatase, protease, cellulase, endoglucanase, exogluconase, glucanase, amylase, lipase, phospholipase, glycosylase, galactanase, α-galactosidase, β -glucosidase, amylase, pectinase, biotinase, polygalacturonase, ligninase, or a combination thereof.

[001759] Embodiment 564 is the method of Embodiment 563, wherein the lipase comprises phospholipase A1, phospholipase A2, phospholipase C, phospholipase D, lysophospholipase, or a combination thereof.

[001760] Embodiment 565 is the method of Embodiment 563 wherein the amylase comprises α-amylase or β-amylase.

[001761] Embodiment 566 is the method of Embodiment 563 wherein the enzyme comprises a xylanase or phytase.

[001762] Embodiment 567 is a method according to any one of embodiments 472-487 and 540-566, wherein the animal includes a mammal, bird, fish, or crustacean.

[001763] Embodiment 568 is the method of Embodiment 567, wherein the mammal includes sheep, goat, cow, pig, deer, alpaca, bison, camel, donkey, horse, mule, llama, rabbit, dog, or cat; where the bird includes chicken, turkey, duck, goose, quail or pheasant; where the fish includes salmon, trout, tilapia, tuna, catfish or carp; or where the crustacean includes small shrimp, large shrimp, lobster, crab or crayfish.

[001764] In view of the foregoing, it can be seen that several objectives of the invention are achieved and other advantageous results are provided.

[001765] Since various changes can be made to the products, compositions and methods described above without deviating from the scope of the invention, it is understood that all material contained in the description described above and shown in the accompanying drawings should be interpreted as illustrative, but not limiting.

Claims (635)

1. A recombinant member of the Bacillus cereus family that expresses a fusion protein containing: (A) the protein or peptide of interest, and (B) a targeting sequence, exosporium protein or exosporium protein fragment that targets the fusion protein into the exospores of a recombinant member of the Bacillus cereus family, wherein the targeting sequence, exosporium protein or exosporium protein fragment comprises: (1) a targeting sequence containing an amino acid sequence with at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 54%; (2) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 1; (3) a targeting sequence containing amino acids 20-35 of SEQ ID NO: 1; (4) a targeting sequence containing SEQ ID NO: 1; (5) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 2; (6) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 1; (7) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 1; (8) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 1; (9) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 1; (10) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 1; (11) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 3; (12) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 3; (13) a targeting sequence containing SEQ ID NO: 3; (14) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 4; (15) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 3; (16) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 3; (17) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 3; (18) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 3; (19) a targeting sequence containing amino acids 1-38 of SEQ ID NO: 5; (20) a targeting sequence containing amino acids 23-38 of SEQ ID NO: 5; (21) a targeting sequence containing SEQ ID NO: 5; (22) exosporium protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 6; (23) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 5; (24) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 5; (25) a targeting sequence containing amino acids 8-38 of SEQ ID NO: 5; (26) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 5; (27) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 5; (28) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 5; (29) a targeting sequence containing amino acids 1-28 of SEQ ID NO: 7; (30) a targeting sequence containing amino acids 13-28 of SEQ ID NO: 7; (31) a targeting sequence containing SEQ ID NO: 7; (32) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 8; (33) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 7; (34) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 7; (35) a targeting sequence containing amino acids 8-28 of SEQ ID NO: 7; (36) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 7; (37) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 9; (38) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 9; (39) a targeting sequence containing SEQ ID NO: 9; (40) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 10; (41) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 9; (42) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 9; (43) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 9; (44) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 11; (45) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 11; (46) a targeting sequence containing SEQ ID NO: 11; (47) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 12; (48) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 11; (49) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 11; (50) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 11; (51) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 11; (52) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 11; (53) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 13; (54) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 13; (55) a targeting sequence containing SEQ ID NO: 13; (56) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 14; (57) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 13; (58) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 13; (59) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 13; (60) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 13; (61) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 13; (62) a targeting sequence containing amino acids 1-43 of SEQ ID NO: 15; (63) a targeting sequence containing amino acids 28-43 of SEQ ID NO: 15; (64) a targeting sequence containing SEQ ID NO: 15; (65) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 16; (66) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 15; (67) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 15; (68) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 15; (69) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 15; (70) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 15; (71) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 15; (72) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 15; (73) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 17; (74) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 17; (75) a targeting sequence containing SEQ ID NO: 17; (76) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 18; (77) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 17; (78) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 17; (79) a targeting sequence containing amino acids 8-27 of SEQ ID NO: 17; (80) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 17; (81) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 19; (82) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 19; (83) a targeting sequence containing SEQ ID NO: 19; (84) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 20; (85) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 19; (86) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 19; (87) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 19; (88) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 19; (89) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 19; (90) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 21; (91) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 21; (92) a targeting sequence containing SEQ ID NO: 21; (93) exosporium protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 22; (94) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 21; (95) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 21; (96) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 21; (97) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 21; (98) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 21; (99) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 23; (100) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 23; (101) a targeting sequence comprising SEQ ID NO: 23; (102) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 24; (103) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 23; (104) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 23; (105) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 23; (106) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 25; (107) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 25; (108) a targeting sequence comprising SEQ ID NO: 25; (109) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 26; (110) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 25; (111) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 25; (112) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 25; (113) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 27; (114) a targeting sequence containing amino acids 15-30 of SEQ ID NO: 27; (115) a targeting sequence comprising SEQ ID NO: 27; (116) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 28; (117) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 27; (118) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 27; (119) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 27; (120) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 27; (121) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 29; (122) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 29; (123) a targeting sequence comprising SEQ ID NO: 29; (124) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 30; (125) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 29; (126) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 29; (127) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 29; (128) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 29; (129) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 29; (130) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 31; (131) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 31; (132) a targeting sequence containing SEQ ID NO: 31; (133) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 32; (134) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 31; (135) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 31; (136) a targeting sequence containing amino acids 8-24 of SEQ ID NO: 31; (137) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 33; (138) a targeting sequence comprising SEQ ID NO: 33; (139) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 34; (140) a targeting sequence containing amino acids 1-16 of SEQ ID NO: 35; (141) a targeting sequence containing SEQ ID NO: 35; (142) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 36; (143) a targeting sequence containing amino acids 1-29 of SEQ ID NO: 43; (144) a targeting sequence containing amino acids 14-29 of SEQ ID NO: 43; (145) a targeting sequence containing SEQ ID NO: 43; (146) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 44; (147) a targeting sequence containing amino acids 2-29 of SEQ ID NO: 43; (148) a targeting sequence containing amino acids 5-29 of SEQ ID NO: 43; (149) a targeting sequence containing amino acids 8-29 of SEQ ID NO: 43; (150) a targeting sequence containing amino acids 10-29 of SEQ ID NO: 43; (151) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 45; (152) a targeting sequence containing amino acids 20-35 of SEQ ID NO: 45; (153) a targeting sequence containing SEQ ID NO: 45; (154) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 46; (155) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 45; (156) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 45; (157) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 45; (158) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 45; (159) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 45; (160) a targeting sequence containing amino acids 1-43 of SEQ ID NO: 47; (161) a targeting sequence containing amino acids 28-43 of SEQ ID NO: 47; (162) a targeting sequence containing SEQ ID NO: 47; (163) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 48; (164) a targeting sequence containing amino acids 2-43 of SEQ ID NO: 47; (165) a targeting sequence containing amino acids 5-43 of SEQ ID NO: 47; (166) a targeting sequence containing amino acids 8-43 of SEQ ID NO: 47; (167) a targeting sequence containing amino acids 10-43 of SEQ ID NO: 47; (168) a targeting sequence containing amino acids 15-43 of SEQ ID NO: 47; (169) a targeting sequence containing amino acids 20-43 of SEQ ID NO: 47; (170) a targeting sequence containing amino acids 25-43 of SEQ ID NO: 47; (171) a targeting sequence containing amino acids 1-32 of SEQ ID NO: 49; (172) a targeting sequence containing amino acids 17-32 of SEQ ID NO: 49; (173) a targeting sequence containing SEQ ID NO: 49; (174) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 50; (175) a targeting sequence containing amino acids 2-32 of SEQ ID NO: 49; (176) a targeting sequence containing amino acids 5-32 of SEQ ID NO: 49; (177) a targeting sequence containing amino acids 8-32 of SEQ ID NO: 49; (178) a targeting sequence containing amino acids 10-32 of SEQ ID NO: 49; (179) a targeting sequence containing amino acids 15-32 of SEQ ID NO: 49; (180) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 51; (181) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 51; (182) a targeting sequence comprising SEQ ID NO: 51; (183) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 52; (184) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 51; (185) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 51; (186) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 51; (187) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 51; (188) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 51; (189) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 53; (190) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 53; (191) a targeting sequence containing SEQ ID NO: 53; (192) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 54; (193) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 53; (194) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 53; (195) a targeting sequence containing amino acids 8-33 of SEQ ID NO: 53; (196) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 53; (197) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 53; (198) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 55; (199) a targeting sequence containing amino acids 15-30 of SEQ ID NO: 55; (200) a targeting sequence comprising SEQ ID NO: 55; (201) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 56; (202) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 55; (203) a targeting sequence containing amino acids 5-30 of SEQ ID NO: 55; (204) a targeting sequence containing amino acids 8-30 of SEQ ID NO: 55; (205) a targeting sequence containing amino acids 10-30 of SEQ ID NO: 55; (206) a targeting sequence containing amino acids 1-130 of SEQ ID NO: 57; (207) a targeting sequence containing amino acids 115-130 of SEQ ID NO: 57; (208) a targeting sequence containing SEQ ID NO: 57; (209) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 58; (210) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 57; (211) a targeting sequence containing amino acids 5-130 of SEQ ID NO: 57; (212) a targeting sequence containing amino acids 8-130 of SEQ ID NO: 57; (213) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 57; (214) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 57; (215) a targeting sequence containing amino acids 40-130 of SEQ ID NO: 57; (216) a targeting sequence containing amino acids 50-130 of SEQ ID NO: 57; (217) a targeting sequence containing amino acids 60-130 of SEQ ID NO: 57; (218) a targeting sequence containing amino acids 70-130 of SEQ ID NO: 57; (219) a targeting sequence containing amino acids 80-130 of SEQ ID NO: 57; (220) a targeting sequence containing amino acids 90-130 of SEQ ID NO: 57; (221) a targeting sequence containing amino acids 100-130 of SEQ ID NO: 57; (222) a targeting sequence containing amino acids 110-130 of SEQ ID NO: 57; (223) exospore protein containing an amino acid sequence with 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 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 containing SEQ ID NO: 103; (232) a targeting sequence comprising SEQ ID NO: 104; (233) a targeting sequence containing SEQ ID NO: 105; (234) a targeting sequence comprising SEQ ID NO: 106; (235) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 108; (236) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 109; (237) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 110; (238) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 111; (239) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 112; (240) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 113; (241) an exosporium protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 114; (242) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 115; (243) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 116; (244) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 117; (245) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 118; (246) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 119; (247) exosporium protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 120; (248) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 121; (249) a targeting sequence containing amino acids 22-31 of SEQ ID NO: 1; (250) a targeting sequence containing amino acids 22-33 of SEQ ID NO: 1; (251) a targeting sequence containing amino acids 20-31 of SEQ ID NO: 1; (252) a targeting sequence containing amino acids 14-23 of SEQ ID NO: 3; (253) a targeting sequence containing amino acids 14-25 of SEQ ID NO: 3; (254) a targeting sequence containing amino acids 12-23 of SEQ ID NO: 3; (255) a targeting sequence containing amino acids 1-30 of SEQ ID NO: 59; (256) a targeting sequence comprising SEQ ID NO: 59; (257) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 60; (258) a targeting sequence containing amino acids 2-30 of SEQ ID NO: 59; (259) a targeting sequence containing amino acids 4-30 of SEQ ID NO: 59; (260) a targeting sequence containing amino acids 6-30 of SEQ ID NO: 59; (261) a targeting sequence containing amino acids 1-33 of SEQ ID NO: 61; (262) a targeting sequence containing amino acids 18-33 of SEQ ID NO: 61; (263) a targeting sequence containing SEQ ID NO: 61; (264) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 62; (265) a targeting sequence containing amino acids 2-33 of SEQ ID NO: 61; (266) a targeting sequence containing amino acids 5-33 of SEQ ID NO: 61; (267) a targeting sequence containing amino acids 10-33 of SEQ ID NO: 61; (268) a targeting sequence containing amino acids 15-33 of SEQ ID NO: 61; (269) a targeting sequence containing amino acids 1-35 of SEQ ID NO: 63; (270) a targeting sequence comprising SEQ ID NO: 63; (271) an exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 64; (272) a targeting sequence containing amino acids 2-35 of SEQ ID NO: 63; (273) a targeting sequence containing amino acids 5-35 of SEQ ID NO: 63; (274) a targeting sequence containing amino acids 8-35 of SEQ ID NO: 63; (275) a targeting sequence containing amino acids 10-35 of SEQ ID NO: 63; (276) a targeting sequence containing amino acids 15-35 of SEQ ID NO: 63; (277) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 65; (278) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 65; (279) a targeting sequence comprising SEQ ID NO: 65; (280) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 66; (281) a targeting sequence comprising SEQ ID NO: 107; (282) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 65; (283) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 65; (284) a targeting sequence containing amino acids 1-27 of SEQ ID NO: 67; (285) a targeting sequence containing amino acids 12-27 of SEQ ID NO: 67; (286) a targeting sequence containing SEQ ID NO: 67; (287) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 68; (288) a targeting sequence containing amino acids 2-27 of SEQ ID NO: 67; (289) a targeting sequence containing amino acids 5-27 of SEQ ID NO: 67; (290) a targeting sequence containing amino acids 10-27 of SEQ ID NO: 67; (291) a targeting sequence containing amino acids 1-38 of SEQ ID NO: 69; (292) a targeting sequence containing amino acids 23-38 of SEQ ID NO: 69; (293) a targeting sequence containing SEQ ID NO: 69; (294) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 70; (295) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 69; (296) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 69; (297) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 69; (298) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 69; (299) exosporium protein containing SEQ ID NO: 72; (300) a targeting sequence comprising SEQ ID NO: 73; (301) exospore protein containing an amino acid sequence with at least 95% identity with SEQ ID NO: 74; (302) a targeting sequence containing amino acids 1-42 of SEQ ID NO: 75; (303) a targeting sequence containing amino acids 27-42 of SEQ ID NO: 75; (304) a targeting sequence comprising SEQ ID NO: 75; (305) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 76; (306) a targeting sequence containing amino acids 2-42 of SEQ ID NO: 75; (307) a targeting sequence containing amino acids 5-42 of SEQ ID NO: 75; (308) a targeting sequence containing amino acids 10-42 of SEQ ID NO: 75; (309) a targeting sequence containing amino acids 15-42 of SEQ ID NO: 75; (310) a targeting sequence containing amino acids 20-42 of SEQ ID NO: 75; (311) a targeting sequence containing amino acids 25-42 of SEQ ID NO: 75; (312) a targeting sequence containing amino acids 1-24 of SEQ ID NO: 77; (313) a targeting sequence containing amino acids 9-24 of SEQ ID NO: 77; (314) a targeting sequence comprising SEQ ID NO: 77; (315) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 78; (316) a targeting sequence containing amino acids 2-24 of SEQ ID NO: 77; (317) a targeting sequence containing amino acids 5-24 of SEQ ID NO: 77; (318) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 80; (319) a targeting sequence containing amino acids 1-38 of SEQ ID NO: 81; (320) a targeting sequence containing amino acids 23-38 of SEQ ID NO: 81; (321) a targeting sequence comprising SEQ ID NO: 81; (322) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 82; (323) a targeting sequence containing amino acids 2-38 of SEQ ID NO: 81; (324) a targeting sequence containing amino acids 5-38 of SEQ ID NO: 81; (325) a targeting sequence containing amino acids 10-38 of SEQ ID NO: 81; (326) a targeting sequence containing amino acids 15-38 of SEQ ID NO: 81; (327) a targeting sequence containing amino acids 20-38 of SEQ ID NO: 81; (328) a targeting sequence containing amino acids 1-34 of SEQ ID NO: 83; (329) a targeting sequence comprising SEQ ID NO: 83; (330) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 84; (331) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 86; (332) a targeting sequence containing amino acids 1-28 of SEQ ID NO: 87; (333) a targeting sequence containing amino acids 13-28 of SEQ ID NO: 87; (334) a targeting sequence comprising SEQ ID NO: 87; (335) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 88; (336) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 87; (337) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 87; (338) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 87; (339) a targeting sequence containing amino acids 1-28 of SEQ ID NO: 89; (340) a targeting sequence comprising SEQ ID NO: 89; (341) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 90; (342) a targeting sequence containing amino acids 2-28 of SEQ ID NO: 89; (343) a targeting sequence containing amino acids 5-28 of SEQ ID NO: 89; (344) a targeting sequence containing amino acids 10-28 of SEQ ID NO: 89; (345) a targeting sequence containing amino acids 1-93 of SEQ ID NO: 91; (346) a targeting sequence comprising SEQ ID NO: 91; (347) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 92; (348) a targeting sequence containing amino acids 2-93 of SEQ ID NO: 91; (349) a targeting sequence containing amino acids 10-93 of SEQ ID NO: 91; (350) a targeting sequence containing amino acids 20-93 of SEQ ID NO: 91; (351) a targeting sequence containing amino acids 30-93 of SEQ ID NO: 91; (352) a targeting sequence containing amino acids 40-93 of SEQ ID NO: 91; (353) a targeting sequence containing amino acids 50-93 of SEQ ID NO: 91; (354) a targeting sequence containing amino acids 60-93 of SEQ ID NO: 91; (355) a targeting sequence containing amino acids 1-130 of SEQ ID NO: 93; (356) a targeting sequence comprising SEQ ID NO: 93; (357) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 94; (358) a targeting sequence containing amino acids 2-130 of SEQ ID NO: 93; (359) a targeting sequence containing amino acids 10-130 of SEQ ID NO: 93; (360) a targeting sequence containing amino acids 20-130 of SEQ ID NO: 93; (361) a targeting sequence containing amino acids 30-130 of SEQ ID NO: 93; (362) exospore protein containing an amino acid sequence with at least 85% identity with SEQ ID NO: 122; (363) a targeting sequence containing amino acids 20-33 of SEQ ID NO: 1; (364) a targeting sequence containing amino acids 21-33 of SEQ ID NO: 1; (365) a targeting sequence containing amino acids 23-31 of SEQ ID NO: 1; (366) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 96; (367) a targeting sequence containing amino acids 1-13 of SEQ ID NO: 96; (368) a targeting sequence containing amino acids 12-25 of SEQ ID NO: 3; (369) a targeting sequence containing amino acids 13-25 of SEQ ID NO: 3; (370) a targeting sequence containing amino acids 15-23 of SEQ ID NO: 3; (371) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 97; (372) a targeting sequence containing amino acids 1-13 of SEQ ID NO: 97; (373) a targeting sequence containing amino acids 23-36 of SEQ ID NO: 5; (374) a targeting sequence containing amino acids 23-34 of SEQ ID NO: 5; (375) a targeting sequence containing amino acids 24-36 of SEQ ID NO: 5; (376) a targeting sequence containing amino acids 26-34 of SEQ ID NO: 5; (377) a targeting sequence containing amino acids 13-26 of SEQ ID NO: 7; (378) a targeting sequence containing amino acids 13-24 of SEQ ID NO: 7; (379) a targeting sequence containing amino acids 14-26 of SEQ ID NO: 7; (380) a targeting sequence containing amino acids 16-24 of SEQ ID NO: 7; (381) a targeting sequence containing amino acids 9-22 of SEQ ID NO: 9; (382) a targeting sequence containing amino acids 9-20 of SEQ ID NO: 9; (383) a targeting sequence containing amino acids 10-22 of SEQ ID NO: 9; (384) a targeting sequence containing amino acids 12-22 of SEQ ID NO: 9; (385) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 105; (386) a targeting sequence containing amino acids 1-13 of SEQ ID NO: 105; (387) a targeting sequence containing amino acids 18-31 of SEQ ID NO: 11; (388) a targeting sequence containing amino acids 18-29 of SEQ ID NO: 11; (389) a targeting sequence containing amino acids 19-31 of SEQ ID NO: 11; (390) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 98; (391) a targeting sequence containing amino acids 1-13 of SEQ ID NO: 98; (392) a targeting sequence containing amino acids 18-31 of SEQ ID NO: 13; (393) a targeting sequence containing amino acids 18-29 of SEQ ID NO: 13; (394) a targeting sequence containing amino acids 19-31 of SEQ ID NO: 13; (395) a targeting sequence containing amino acids 21-29 of SEQ ID NO: 13; (396) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 99; (397) a targeting sequence containing amino acids 1-13 of SEQ ID NO: 99; (398) a targeting sequence containing amino acids 28-41 of SEQ ID NO: 15; (399) a targeting sequence containing amino acids 28-39 of SEQ ID NO: 15; (400) a targeting sequence containing amino acids 29-41 of SEQ ID NO: 15; (401) a targeting sequence containing amino acids 31-39 of SEQ ID NO: 15; (402) a targeting sequence containing amino acids 12-25 of SEQ ID NO: 17; (403) a targeting sequence containing amino acids 13-25 of SEQ ID NO: 17; (404) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 100; (405) a targeting sequence containing amino acids 18-31 of SEQ ID NO: 19; (406) a targeting sequence containing amino acids 18-29 of SEQ ID NO: 19; (407) a targeting sequence containing amino acids 19-31 of SEQ ID NO: 19; (408) a targeting sequence containing amino acids 21-29 of SEQ ID NO: 19; (409) a targeting sequence containing amino acids 18-31 of SEQ ID NO: 21; (410) a targeting sequence containing amino acids 18-29 of SEQ ID NO: 21; (411) a targeting sequence containing amino acids 19-31 of SEQ ID NO: 21; (412) a targeting sequence containing amino acids 21-29 of SEQ ID NO: 21; (413) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 101; (414) a targeting sequence containing amino acids 1-13 of SEQ ID NO: 101; (415) a targeting sequence containing amino acids 9-22 of SEQ ID NO: 23; (416) a targeting sequence containing amino acids 9-20 of SEQ ID NO: 23; (417) a targeting sequence containing amino acids 10-22 of SEQ ID NO: 23; (418) a targeting sequence containing amino acids 12-20 of SEQ ID NO: 23; (419) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 102; (420) a targeting sequence containing amino acids 1-13 of SEQ ID NO: 102; (421) a targeting sequence containing amino acids 9-22 of SEQ ID NO: 25; (422) a targeting sequence containing amino acids 9-20 of SEQ ID NO: 25; (423) a targeting sequence containing amino acids 10-22 of SEQ ID NO: 25; (424) a targeting sequence containing amino acids 12-20 of SEQ ID NO: 25; (425) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 103; (426) a targeting sequence containing amino acids 1-13 of SEQ ID NO: 103; (427) a targeting sequence containing amino acids 15-28 of SEQ ID NO: 27; (428) a targeting sequence containing amino acids 15-26 of SEQ ID NO: 27; (429) a targeting sequence containing amino acids 16-28 of SEQ ID NO: 27; (430) a targeting sequence containing amino acids 18-26 of SEQ ID NO: 27; (431) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 104; (432) a targeting sequence containing amino acids 1-13 of SEQ ID NO: 104; (433) a targeting sequence containing amino acids 1-13 of SEQ ID NO: 33; (434) a targeting sequence containing amino acids 1-11 of SEQ ID NO: 33; (435) a targeting sequence containing amino acids 3-11 of SEQ ID NO: 33; (436) a targeting sequence containing amino acids 1-14 of SEQ ID NO: 35; (437) a targeting sequence containing amino acids 1-12 of SEQ ID NO: 35; (438) a targeting sequence containing amino acids 2-14 of SEQ ID NO: 35; (439) a targeting sequence containing amino acids 14-27 of SEQ ID NO: 43; (440) a targeting sequence containing amino acids 14-25 of SEQ ID NO: 43; (441) a targeting sequence containing amino acids 15-27 of SEQ ID NO: 43; (442) a targeting sequence containing amino acids 20-33 of SEQ ID NO: 45; (443) a targeting sequence containing amino acids 20-31 of SEQ ID NO: 45; (444) a targeting sequence containing amino acids 21-33 of SEQ ID NO: 45; (445) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 106; (446) a targeting sequence containing amino acids 1-13 of SEQ ID NO: 106; (447) a targeting sequence containing amino acids 28-41 of SEQ ID NO: 47; (448) a targeting sequence containing amino acids 28-39 of SEQ ID NO: 47; (449) a targeting sequence containing amino acids 18-31 of SEQ ID NO: 53; (450) a targeting sequence containing amino acids 18-29 of SEQ ID NO: 53; (451) a targeting sequence containing amino acids 19-31 of SEQ ID NO: 53; (452) a targeting sequence containing amino acids 18-31 of SEQ ID NO: 61; (453) a targeting sequence containing amino acids 18-29 of SEQ ID NO: 61; (454) a targeting sequence containing amino acids 19-31 of SEQ ID NO: 61; (455) a targeting sequence containing amino acids 9-22 of SEQ ID NO: 65; (456) a targeting sequence containing amino acids 9-20 of SEQ ID NO: 65; (457) a targeting sequence containing amino acids 10-22 of SEQ ID NO: 65; (458) a targeting sequence containing amino acids 1-15 of SEQ ID NO: 107; (459) a targeting sequence containing amino acids 1-13 of SEQ ID NO: 107; (460) a targeting sequence containing amino acids 12-25 of SEQ ID NO: 67; (461) a targeting sequence containing amino acids 12-23 of SEQ ID NO: 67; (462) a targeting sequence containing amino acids 13-25 of SEQ ID NO: 67; (463) a targeting sequence containing amino acids 15-23 of SEQ ID NO: 67; (464) a targeting sequence containing amino acids 23-36 of SEQ ID NO: 69; (465) a targeting sequence containing amino acids 23-34 of SEQ ID NO: 69; (466) a targeting sequence containing amino acids 24-36 of SEQ ID NO: 69; (467) a targeting sequence containing amino acids 26-34 of SEQ ID NO: 69; (468) a targeting sequence containing amino acids 27-40 of SEQ ID NO: 75; (469) a targeting sequence containing amino acids 27-38 of SEQ ID NO: 75; (470) a targeting sequence containing amino acids 9-22 of SEQ ID NO: 77; (471) a targeting sequence containing amino acids 9-20 of SEQ ID NO: 77; (472) a targeting sequence containing amino acids 10-22 of SEQ ID NO: 77; (473) a targeting sequence containing amino acids 12-20 of SEQ ID NO: 77; (474) a targeting sequence containing amino acids 23-36 of SEQ ID NO: 81; (475) a targeting sequence containing amino acids 23-34 of SEQ ID NO: 81; (476) a targeting sequence containing amino acids 24-36 of SEQ ID NO: 81; (477) a targeting sequence containing amino acids 26-34 of SEQ ID NO: 81; (478) a targeting sequence containing amino acids 13-26 of SEQ ID NO: 87; (479) a targeting sequence containing amino acids 13-24 of SEQ ID NO: 87; or (480) a targeting sequence containing amino acids 14-26 of SEQ ID NO: 87; and where is the recombinant member of the Bacillus cereus family: (i) contains a mutation in the ExsY gene, where the ExsY gene encodes a protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 123; (ii) contains a mutation in the CotE gene, where the CotE gene encodes a protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 149; (iii) expresses an ExsY protein, wherein the expression of the ExsY protein is increased compared to the expression of the ExsY protein in a wild-type member of the Bacillus cereus family under the same conditions, where the ExsY protein contains a carboxy-terminal tag containing a globular protein, and where the ExsY protein contains an amino acid sequence having at least 85% identity with SEQ ID NO: 123; (iv) expresses a BclB protein, wherein the expression of the BclB protein is increased compared to the expression of the BclB protein in a wild-type member of the Bacillus cereus family under the same conditions, and wherein the BclB protein contains an amino acid sequence having at least 85% identity with SEQ ID NO: 143 or 144; (v) expresses a YjcB protein, wherein the expression of the YjcB protein is increased compared to the expression of the YjcB protein in a wild-type member of the Bacillus cereus family under the same conditions, and wherein the YjcB protein contains an amino acid sequence having at least 85% identity with SEQ ID NO: 134 or 135; (vi) contains a mutation in the CotY gene, where the CotY gene encodes a protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 125; (vii) contains a mutation in the ExsA gene, where the ExsA gene encodes a protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 150; and/or (viii) contains a mutation in the CotO gene, where the CotO gene encodes a protein containing an amino acid sequence having at least 85% identity with SEQ ID NO: 126; and where the mutation 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 and/or the mutation in the CotO gene provides spores of members of the Bacillus cereus family having exospores, which is easier to remove from the spore compared to wild-type spore exospore. 2. The recombinant member of the Bacillus cereus family according to claim 1, wherein the recombinant member of the Bacillus cereus family contains a mutation in the CotE gene. 3. A recombinant member of the Bacillus cereus family according to claim 2, wherein the mutation in the CotE gene partially or completely inhibits the ability of CotE to attach exospores to the spore. 4. A recombinant member of the Bacillus cereus family according to claim 2, wherein the mutation in the CotE gene includes a knockout of the CotE gene or a dominant negative form of the CotE gene. 5. The recombinant member of the Bacillus cereus family according to claim 1, wherein the recombinant member of the Bacillus cereus family expresses the ExsY protein, where the expression of the ExsY protein is increased compared to the expression of the ExsY protein in the wild-type member of the Bacillus cereus family under the same conditions, and where the ExsY protein contains C -end tag containing a globular protein. 6. A recombinant member of the Bacillus cereus family according to claim 5, wherein the globular protein has a molecular weight of 25 to 100 kDa. 7. A recombinant member of the Bacillus cereus family according to claim 5, wherein the globular protein comprises green fluorescent protein (GFP) or a variant thereof. 8. A recombinant member of the Bacillus cereus family according to claim 5, wherein expression of an ExsY protein containing a carboxy-terminal tag containing a globular protein inhibits the binding of the ExsY protein to its targets in the exospore. 9. The recombinant member of the Bacillus cereus family according to claim 1, wherein the recombinant member of the Bacillus cereus family expresses the BclB protein, wherein the expression of the BclB protein is increased compared to the expression of the BclB protein in the wild-type member of the Bacillus cereus family under the same conditions. 10. The recombinant member of the Bacillus cereus family according to claim 9, wherein the expression of the BclB protein results in the formation of a fragile exospore. 11. The recombinant member of the Bacillus cereus family according to claim 1, wherein the recombinant member of the Bacillus cereus family expresses the YjcB protein, wherein the expression of the YjcB protein is increased compared to the expression of the YjcB protein in the wild type member of the Bacillus cereus family under the same conditions. 12. A recombinant member of the Bacillus cereus family according to claim 11, wherein the expression of the YjcB protein causes the formation of the exospore in fragments instead of a complete structure. 13. The recombinant member of the Bacillus cereus family according to claim 1, wherein the recombinant member of the Bacillus cereus family contains a mutation in the ExsY gene. 14. The recombinant member of the Bacillus cereus family according to claim 13, wherein the mutation in the ExsY gene includes a knockout of the ExsY gene. 15. A recombinant member of the Bacillus cereus family according to claim 13, wherein the mutation in the ExsY gene partially or completely inhibits the ability of ExsY to fully form an exospore or bind an exospore to a spore. 16. The recombinant member of the Bacillus cereus family according to claim 1, wherein the recombinant member of the Bacillus cereus family contains a mutation in the CotY gene. 17. A recombinant member of the Bacillus cereus family according to claim 16, wherein the mutation in the CotY gene includes knockout of the CotY gene. 18. A recombinant member of the Bacillus cereus family according to claim 17, wherein the mutation in the CotY gene results in the formation of a fragile exospore. 19. The recombinant member of the Bacillus cereus family according to claim 1, wherein the recombinant member of the Bacillus cereus family contains a mutation in the ExsA gene. 20. A recombinant member of the Bacillus cereus family according to claim 19, wherein the mutation in the ExsA gene includes a knockout of the ExsA gene. 21. A recombinant member of the Bacillus cereus family according to claim 19, wherein the mutation in the ExsA gene results in the formation of a fragile exospore. 22. The recombinant member of the Bacillus cereus family according to claim 1, wherein the recombinant member of the Bacillus cereus family contains a mutation in the CotO gene. 23. A recombinant member of the Bacillus cereus family according to claim 22, wherein the mutation in the CotO gene includes a knockout of the CotO gene or a dominant negative form of the CotO gene. 24. A recombinant member of the Bacillus cereus family according to claim 22, wherein the mutation in the CotO gene results in the formation of exosporium in stripes. 25. A recombinant member of the Bacillus cereus family according to claim 1, wherein the targeting sequence contains an amino acid sequence with at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 63 %. 26. A recombinant member of the Bacillus cereus family according to claim 1, wherein the targeting sequence contains an amino acid sequence with at least about 62% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 72 %. 27. A recombinant member of the Bacillus cereus family according to claim 1, wherein the targeting sequence contains an amino acid sequence with at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, where the identity with amino acids 25-35 is at least about 90 %. 28. A recombinant representative of the Bacillus cereus family according to claim 1, where the targeting sequence or exospore protein contains: (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, where 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. 29. A recombinant member of the Bacillus cereus family according to claim 1, wherein the targeting sequence contains SEQ ID NO: 96. 30. A recombinant member of the Bacillus cereus family according to claim 1, wherein the exosporium protein or exosporium protein fragment comprises an amino acid sequence with at least 95% identity with 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, 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. 31. The recombinant member of the Bacillus cereus family of claim 1, wherein the targeting sequence, exosporium protein, or exosporium protein fragment comprises the GXT amino acid sequence at its C-terminus, where X is any amino acid. 32. The recombinant member of the Bacillus cereus family according to claim 1, wherein the targeting sequence, exosporium protein, or exosporium protein fragment 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 exosporium protein fragment, or at position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1. 33. The recombinant member of the Bacillus cereus family of claim 1, wherein the fusion protein further comprises an amino acid linker between the targeting sequence, the exosporium protein or exosporium protein fragment, and the protein or peptide of interest. 34. A recombinant member of the Bacillus cereus family according to claim 33, wherein the linker comprises a polyalanine linker, a polyglycine linker, or a linker containing a mixture of both alanine and glycine residues. 35. The recombinant member of the Bacillus cereus family according to claim 1, wherein the protein or peptide of interest comprises a plant growth stimulating protein or peptide, a protein or peptide that protects the plant from a pathogen, a protein or peptide that increases plant stress resistance, binds to plant protein or peptide, an enzyme that catalyzes the production of nitric oxide, or a nucleic acid-binding protein or peptide. 36. The recombinant member of the Bacillus cereus family of claim 35, wherein the protein or peptide of interest comprises a plant growth promoting protein or peptide. 37. The recombinant member of the Bacillus cereus family of claim 36, wherein the plant growth promoting protein or peptide comprises a peptide hormone or a non-hormonal peptide. 38. A recombinant member of the Bacillus cereus family according to claim 37, where the peptide hormone includes phytosulfokine, clavata 3 (CLV3), systemin, ZmlGF or SCR/SP11, or where the non-hormonal peptide includes RKN 16D10, Hg-Syv46, eNOD40 peptide, melittin, mastoparan, Mas7, RHPP, POLARIS, or kunitz trypsin inhibitor (KTI). 39. The recombinant member of the Bacillus cereus family of claim 1, wherein the protein or peptide of interest comprises an enzyme involved in the production or activation of a plant growth stimulating compound. 40. The recombinant member of the Bacillus cereus family according to claim 39, wherein the plant growth stimulating compound comprises 2,3-butanediol or a plant growth hormone, wherein the plant growth hormone comprises a cytokinin or a cytokinin derivative, ethylene, an auxin or an auxin derivative, gibberellic acid or a gibberellic acid derivative. acid, abscisic acid or an abscisic acid derivative, or jasmonic acid or a jasmonic acid derivative. 41. The recombinant member of the Bacillus cereus family of claim 39, wherein the enzyme involved in the production or activation of the plant growth stimulating compound comprises acetoin reductase, indole-3-acetamide hydrolase, tryptophan monooxygenase, acetolactate synthetase, α-acetolactate decarboxylase, pyruvate decarboxylase, diacetyl reductase, butanediol dehydrogenase, aminotransferase, триптофандекарбоксилазу, аминоксидазу, индол-3-пируватдекарбоксилазу, индол-3-ацетальдегиддегидрогеназу, оксидазу боковой цепи триптофана, нитрилгидролазу, нитрилазу, пептидазу, протеазу, аденозинфосфатизопентенилтрансферазу, фосфатазу, аденозинкиназу, аденинфосфорибозилтрансферазу, CYP735A, 5'-рибонуклеотидфосфогидролазу, аденозиннуклеозидазу, зеатин-цис- trans-isomerase, zeatin-O-glucosyltransferase, β-glucosidase, cis-hydroxylase, cis-hydroxylase CK, N-glucosyltransferase CK, 2,5-ribonucleotide phosphohydrolase, adenosine nucleosidase, purine nucleoside phosphorylase, zeatin reductase, hydroxylamine reductase, 2-oxoglutarate dioc cigenase, gibberellic 2B/3B hydrolase, gibberallin-3-oxidase, gibberallin-20-oxidase, chitosanase, chitinase, β-1,3-glucanase, β-1,4-glucanase, β-1,6-glucanase, aminocyclopropane deaminase -1-carboxylic acid or an enzyme involved in the production of the factor nod. 42. The recombinant member of the Bacillus cereus family of claim 41, wherein the enzyme involved in the production or activation of the plant growth stimulating compound comprises a protease or peptidase that cleaves proteins, peptides, proproteins, or preproproteins to form a biologically active peptide, wherein the protease or peptidase comprises subtilisin, acid protease, alkaline protease, proteinase, endopeptidase, exopeptidase, thermolysin, papain, pepsin, trypsin, pronase, carboxylase, serine protease, glutamine protease, aspartate protease, cysteine protease, threonine protease or metalloproteinase. 43. The recombinant member of the Bacillus cereus family of claim 1, wherein the protein or peptide of interest comprises an enzyme. 44. The recombinant member of the Bacillus cereus family of claim 43, wherein the enzyme includes an enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source. 45. A recombinant member of the Bacillus cereus family according to claim 43, where the enzyme includes cellulase, lipase, lignin oxidase, protease, glycoside hydrolase, phosphatase, nitrogenase, nuclease, amidase, nitrate reductase, nitrite reductase, amylase, ammonia oxidase, ligninase, glucosidase, phospholipase, phytase, pectinase, glucanase, sulfatase, urease, xylanase or chitosanase. 46. The recombinant member of the Bacillus cereus family according to claim 45, wherein the enzyme includes a phospholipase and the phospholipase includes phospholipase A1, phospholipase A2, phospholipase C, phospholipase D, or lysophospholipase. 47. The recombinant member of the Bacillus cereus family according to claim 46, wherein the phospholipase includes phospholipase C and phospholipase C includes an amino acid sequence with at least 85% identity with SEQ ID NO: 312. 48. The recombinant member of the Bacillus cereus family according to claim 46, wherein the phospholipase includes phospholipase C and phospholipase C includes an amino acid sequence with at least 95% identity with SEQ ID NO: 312. 49. The recombinant member of the Bacillus cereus family according to claim 46, wherein the phospholipase includes phospholipase C and phospholipase C includes an amino acid sequence with at least 98% identity with SEQ ID NO: 312. 50. The recombinant member of the Bacillus cereus family according to claim 46, wherein the phospholipase includes phospholipase C and phospholipase C includes an amino acid sequence with 100% identity with SEQ ID NO: 312. 51. The recombinant member of the Bacillus cereus family according to claim 45, wherein the enzyme includes cellulase and the cellulase includes endo-cellulase, exo-cellulase, or β-glucosidase. 52. The recombinant member of the Bacillus cereus family according to claim 51, wherein the cellulase includes endo-cellulase and the endo-cellulase includes endoglucanase. 53. The recombinant member of the Bacillus cereus family according to claim 51, wherein the cellulase includes endo-cellulase and the endo-cellulase includes Bacillus subtilis endo-cellulase, Bacillus thuringiensis endo-cellulase, Bacillus cereus endo-cellulase, or Bacillus clausii endo-cellulase . 54. The recombinant member of the Bacillus cereus family according to claim 53, wherein the cellulase comprises Bacillus subtilis endocellulase and the Bacillus subtilis endocellulase comprises an amino acid sequence with at least 85% identity with SEQ ID NO: 311. 55. The recombinant member of the Bacillus cereus family according to claim 53, wherein the cellulase comprises Bacillus subtilis endocellulase and the Bacillus subtilis endocellulase comprises an amino acid sequence with at least 95% identity with SEQ ID NO: 311. 56. The recombinant member of the Bacillus cereus family according to claim 53, wherein the cellulase comprises Bacillus subtilis endocellulase and the Bacillus subtilis endocellulase comprises an amino acid sequence with at least 98% identity with SEQ ID NO: 311. 57. The recombinant member of the Bacillus cereus family according to claim 53, wherein the cellulase comprises Bacillus subtilis endo-cellulase and the Bacillus subtilis endo-cellulase comprises an amino acid sequence with 100% identity with SEQ ID NO: 311. 58. The recombinant member of the Bacillus cereus family of claim 45, wherein the enzyme comprises a lipase and the lipase includes a Bacillus subtilis lipase, a Bacillus thuringiensis lipase, a Bacillus cereus lipase, or a Bacillus clausii lipase. 59. The recombinant member of the Bacillus cereus family according to claim 46, wherein the enzyme includes lignin oxidase and the lignin oxidase includes lignin peroxidase, laccase, glyoxal oxidase, ligninase, or manganese peroxidase. 60. A recombinant member of the Bacillus cereus family according to claim 45, where the enzyme includes a protease and the protease includes subtilisin, acid protease, alkaline protease, proteinase, peptidase, endopeptidase, exopeptidase, thermolysin, papain, pepsin, trypsin, pronase, carboxylase, serine protease, glutamine protease, aspartate protease, cysteine protease, threonine protease or metalloproteinase. 61. The recombinant member of the Bacillus cereus family according to claim 45, wherein the enzyme comprises a phosphatase and the phosphatase comprises phosphoric acid monoester hydrolase, phosphomonoesterase, phosphoric acid diester hydrolase, phosphodiesterase, triphosphoric acid monoester hydrolase, phosphorylanhydride hydrolase, pyrophosphatase, phytase, trimetaphosphatase, or triphosphatase . 62. The recombinant member of the Bacillus cereus family of claim 61, wherein the enzyme comprises a phosphomonoesterase and the phosphomonoesterase comprises PhoA4, or wherein the enzyme comprises a phytase and the phytase includes Bacillus subtilis EE148 phytase or Bacillus thuringiensis BT013A phytase. 63. The recombinant member of the Bacillus cereus family according to claim 45, wherein the enzyme includes a nitrogenase and the nitrogenase includes a nitrogenase of the Nif family. 64. A recombinant member of the Bacillus cereus family according to claim 45, wherein the enzyme includes chitosanase, where the chitosanase contains an amino acid sequence with at least 85% identity with SEQ ID NO: 313. 65. A recombinant member of the Bacillus cereus family according to claim 45, wherein the enzyme includes chitosanase, where the chitosanase contains an amino acid sequence with at least 95% identity with SEQ ID NO: 313. 66. A recombinant member of the Bacillus cereus family according to claim 45, wherein the enzyme comprises a chitosanase, wherein the chitosanase contains an amino acid sequence with at least 98% identity with SEQ ID NO: 313. 67. A recombinant member of the Bacillus cereus family according to claim 45, wherein the enzyme includes chitosanase, where the chitosanase contains an amino acid sequence with 100% identity with SEQ ID NO: 313. 68. A recombinant representative of the Bacillus cereus family according to claim 43, characterized in that the enzyme includes aminocyclopropane-1-carboxylic acid deaminase. 69. The recombinant member of the Bacillus cereus family of claim 35, wherein the protein or peptide of interest includes a protein or peptide that protects the plant from a pathogen. 70. A recombinant member of the Bacillus cereus family according to claim 69, wherein the protein or peptide that protects the plant from the pathogen includes a protein or peptide that enhances the immune system of plants. 71. A recombinant member of the Bacillus cereus family according to claim 70, wherein the protein or peptide that enhances the plant immune system includes harpin, harpin-like protein, α-elastin, β-elastin, systemin, phenylalanine ammonia lyase, elicitin, defensin, cryptogein, flagellin protein, or peptide flagellin. 72. The recombinant member of the Bacillus cereus family according to claim 69, wherein the protein or peptide that protects the plant from the pathogen has antibacterial activity, antifungal activity, or both antibacterial and antifungal activity, or where the protein or peptide that protects the plant from the pathogen has insecticidal activity, helminticidal activity, inhibits predation by insects or worms, or a combination thereof. 73. A recombinant member of the Bacillus cereus family according to claim 72, where the protein or peptide that protects the plant from the pathogen includes bacteriocin, lysozyme, lysozyme peptide, avidin, streptavidin, nonribosomal active peptide, conalbumin, albumin, lactoferrin, lactoferrin peptide, TasA, insecticidal bacterial toxin, endotoxin, Cry toxin, protease-inhibiting protein or peptide, cysteine protease or chitinase. 74. A recombinant member of the Bacillus cereus family according to claim 73, where the protein or peptide that protects the plant from the pathogen includes an insecticidal bacterial toxin and an insecticidal bacterial toxin includes a VIP insecticidal toxin; where the protein or peptide that protects the plant from the pathogen includes a protease-inhibiting protein or peptide and the protease-inhibiting protein or peptide includes a trypsin inhibitor or an arrowhead protease inhibitor; or where the protein or peptide that protects the plant from the pathogen includes a Cry toxin and the Cry toxin includes a Cry toxin from Bacillus thuringiensis . 75. The recombinant member of the Bacillus cereus family according to claim 74, wherein the Cry toxin includes a Cry toxin from Bacillus thuringiensis and the Cry toxin, including a Cry toxin from Bacillus thuringiensis , includes a Cry5B or Cry21A protein. 76. A recombinant member of the Bacillus cereus family according to claim 69, wherein the protein or peptide that protects the plant from the pathogen includes an enzyme. 77. A recombinant member of the Bacillus cereus family according to claim 76, wherein the enzyme comprises a protease or lactonase, where the protease or lactonase is specific for the bacterial signaling molecule homoserine. 78. A recombinant member of the Bacillus cereus family according to claim 76, where the enzyme is specific to the cellular component of a bacterium or fungus, where the enzyme includes β-1,3-glucanase, β-1,4-glucanase, β-1,6-glucanase, chitosanase, chitinase, chitosanase-like enzyme, lyticase, peptidase, proteinase, protease, mutanolysin, stafolysin, or lysozyme. 79. The recombinant member of the Bacillus cereus family according to claim 35, wherein the protein or peptide of interest comprises at least one protein or peptide that enhances plant stress resistance. 80. A recombinant member of the Bacillus cereus family according to claim 79, wherein the protein or peptide that enhances the plant's resistance to stress includes an enzyme that degrades the stress-causing compound, where the stress-causing compound includes aminocyclopropane-1-carboxylic acid (ACC), active oxygen species, nitric oxide, oxylipin, phenol, or a combination thereof. 81. A recombinant member of the Bacillus cereus family according to claim 80, wherein the protein or peptide that enhances plant resistance to stress includes an enzyme that degrades a stress-causing compound, and the enzyme includes superoxide dismutase, oxidase, catalase, aminocyclopropane-1-carboxylic acid deaminase , peroxidase, antioxidant enzyme or antioxidant peptide. 82. The recombinant member of the Bacillus cereus family of claim 81, wherein the enzyme that degrades the stress-causing compound includes superoxide dismutase, where the superoxide dismutase includes superoxide dismutase 1 (SODA1) or superoxide dismutase 2 (SODA2). 83. A recombinant member of the Bacillus cereus family according to claim 82, wherein the superoxide dismutase comprises an amino acid sequence with at least 85% identity with SEQ ID NO: 155 or 156. 84. The recombinant member of the Bacillus cereus family of claim 82, wherein the superoxide dismutase comprises an amino acid sequence with at least 95% identity with SEQ ID NO: 155 or 156. 85. The recombinant member of the Bacillus cereus family of claim 82, wherein the superoxide dismutase comprises an amino acid sequence with at least 98% identity with SEQ ID NO: 155 or 156. 86. A recombinant member of the Bacillus cereus family according to claim 82, wherein the superoxide dismutase comprises an amino acid sequence with 100% identity with SEQ ID NO: 155 or 156. 87. A recombinant representative of the Bacillus cereus family according to claim 79, where the protein or peptide that increases the plant's resistance to stress includes a protein or peptide that protects the plant from environmental stress, where environmental stress includes drought, flood, heat , freezing, salt, heavy metals, low pH, high pH, or a combination. 88. A recombinant member of the Bacillus cereus family according to claim 87, wherein the protein or peptide that protects the plant from environmental stressors includes a protein that induces the formation of ice microcrystals, prolinase, phenylalanine ammonia lyase, isochorismate synthase, isochorismate pyruvate lyase, or choline dehydrogenase. 89. The recombinant member of the Bacillus cereus family of claim 35, wherein the fusion protein comprises at least one plant-binding protein or peptide. 90. A recombinant member of the Bacillus cereus family according to claim 89, wherein the plant-binding protein or peptide comprises adhesin, ricadhesin, flagellin, omptin, lectin, expansin, biofilm structural protein, TasA, YuaB, pilus protein, curl protein, intimin, invasin, agglutinin, afimbria protein. 91. The recombinant member of the Bacillus cereus family of claim 35, wherein the protein or peptide of interest includes an enzyme that catalyzes the production of nitric oxide. 92. The recombinant member of the Bacillus cereus family of claim 91, wherein the enzyme that catalyzes the production of nitric oxide comprises nitric oxide synthase or arginase. 93. The recombinant member of the Bacillus cereus family of claim 92, wherein the enzyme that catalyses the production of nitric oxide comprises nitric oxide synthase, wherein the nitric oxide synthase comprises nitric oxide synthase from Bacillus thuringiensis or nitric oxide synthase from Bacillus subtilis . 94. The recombinant member of the Bacillus cereus family of claim 93, wherein the nitric oxide synthase comprises nitric oxide synthase from Bacillus thuringiensis BT013A or Bacillus subtilis 168. 95. The recombinant member of the Bacillus cereus family of claim 92, wherein the nitric oxide synthase comprises an amino acid sequence with at least 85% identity with SEQ ID NO: 260 or 261. 96. The recombinant member of the Bacillus cereus family of claim 92, wherein the nitric oxide synthase comprises an amino acid sequence with at least 95% identity with SEQ ID NO: 260 or 261. 97. The recombinant member of the Bacillus cereus family of claim 92, wherein the nitric oxide synthase comprises an amino acid sequence with at least 98% identity with SEQ ID NO: 260 or 261. 98. A recombinant member of the Bacillus cereus family according to claim 92, wherein the nitric oxide synthase comprises an amino acid sequence with 100% identity with SEQ ID NO: 260 or 261. 99. The recombinant member of the Bacillus cereus family of claim 35, wherein the protein or peptide of interest comprises a nucleic acid binding protein or peptide. 100. The recombinant member of the Bacillus cereus family of claim 99, wherein the nucleic acid binding protein or peptide comprises an Hfq protein or a small acid soluble spore protein (SASP). 101. The recombinant member of the Bacillus cereus family of claim 102, wherein the SASP includes SASPα, SASPβ, or SASPγ. 102. The recombinant member of the Bacillus cereus family of claim 99, wherein the nucleic acid binding protein or peptide comprises an amino acid sequence with at least 85% identity to any of SEQ ID NOs: 264-266. 103. The recombinant member of the Bacillus cereus family of claim 99, wherein the nucleic acid binding protein or peptide comprises an amino acid sequence with at least 95% identity to any of SEQ ID NOs: 264-266. 104. The recombinant member of the Bacillus cereus family of claim 99, wherein the nucleic acid binding protein or peptide comprises an amino acid sequence with at least 98% identity to any of SEQ ID NOs: 264-266. 105. The recombinant member of the Bacillus cereus family of claim 99, wherein the nucleic acid binding protein or peptide comprises an amino acid sequence with 100% identity to any of SEQ ID NOs: 264-266. 106. The recombinant member of the Bacillus cereus family according to claim 99, where the fusion protein contains SEQ ID NO: 267, 268 or 269. 107. The recombinant member of the Bacillus cereus family of claim 99, wherein the nucleic acid binding protein or peptide comprises a nuclease having an inactivated active site. 108. A recombinant member of the Bacillus cereus family according to claim 99, further comprising a nucleic acid molecule associated with a nucleic acid-binding protein or peptide, where the nucleic acid molecule includes a modulating RNA molecule; RNA-i molecule; miRNA; aptamer; or a DNA molecule that encodes a modulating RNA molecule, an RNA-i molecule, a microRNA or an aptamer. 109. A recombinant member of the Bacillus cereus family according to claim 1, characterized in that the recombinant member of the Bacillus cereus family includes Bacillus anthracis , Bacillus cereus , Bacillus thuringiensis , Bacillus mycoides , Bacillus pseudomycoides , Bacillus samanii, Bacillus gaemokensis, Bacillus weihenstephensis , Bacillus toyoiensis or their combination. 110. A recombinant member of the Bacillus cereus family according to claim 109, characterized in that the recombinant member of the Bacillus cereus family includes Bacillus mycoides BT155 (NRRL No. B-50921), Bacillus mycoides EE118 (NRRL No. B-50918), Bacillus mycoides EE141 ( NRRL No. B-50921), Bacillus mycoides BT46-3 (NRRL No. B-50922), member of the family Bacillus cereus EE128 (NRRL No. B-50917), Bacillus thuringiensis BT013A (NRRL No. B-50924), member of the family Bacillus cereus EE349 (NRRL No. B-50928); member of the family Bacillus cereus EE-B00377 (NRRL B-67119); Bacillus pseudomycoides EE-B00366 (NRRL B-67120); Bacillus mycoides EE-B00363 (NRRL B-67121); member of the family Bacillus cereus EE439 (NRRL B-50979); Bacillus thuringiensis EE417 (NRRL B-50979); Bacillus cereus EE444 (NRRL B-50977); Bacillus thuringiensis EE319 (NRRL B-50983); or Bacillus thuringiensis EE-B00184 (NRRL B-67122). 111. A recombinant member of the Bacillus cereus family according to claim 1, characterized in that the recombinant member of the Bacillus cereus family additionally includes an inactivating mutation in its BclA gene. 112. The recombinant member of the Bacillus cereus family according to claim 111, wherein the inactivating mutation in the BclA gene includes a knockout of the BclA gene. 113. Composition containing exosporium fragments derived from spores of a recombinant member of the Bacillus cereus family according to any one of claims 1 to 112, and an agriculturally acceptable carrier, wherein the composition stimulates plant growth, protects the plant from a pathogen and/or increases plant resistance to stressful conditions. 114. Plant seed coated with exospore fragments derived from spores of a recombinant member of the Bacillus cereus family according to any one of claims 1-112. 115. A plant seed according to claim 114, characterized in that the plant seed is covered with exospore fragments. 116. A method for stimulating plant growth, comprising introducing exosporium fragments into a plant growth medium or applying exosporium fragments to a plant, plant seed or area surrounding a plant or plant seed, wherein the exosporium fragments are derived from spores of a recombinant member of the Bacillus cereus family according to any one of claims 1 to 112 and contain a fusion protein and the fusion protein comprises a plant growth stimulating protein or peptide. 117. A method of protecting a plant from a pathogen or increasing plant resistance to stress, including introducing exosporium fragments into the plant growth medium, wherein the exosporium fragments are derived from spores of a recombinant member of the Bacillus cereus family according to any one of claims 1 to 112 and contain a fusion protein and the fusion protein contains a protein or peptide that protects the plant from a pathogen, or a protein or peptide that increases the plant's resistance to stress. 118. A method for immobilizing exospore fragments on a plant, including introducing exosporium fragments into a plant growth medium or applying exosporium fragments to a plant, plant seed or area surrounding a plant or plant seed, wherein the exospore fragments are derived from spores of a recombinant member of the Bacillus cereus family according to any one of claims 1 to 112 and contain a fusion protein and the fusion protein comprises a plant-binding protein or peptide. 119. A method for stimulating the germination of a plant seed, comprising introducing exosporium fragments into a plant growth medium or applying exospore fragments to a plant seed or an area surrounding a plant or plant seed, wherein the exospore fragments are derived from spores of a recombinant member of the Bacillus cereus family according to any one of claims 1 to 112 and contain a fusion protein and the fusion protein contains superoxide dismutase or an enzyme that catalyses the production of nitric oxide. 120. A method for delivering nucleic acids to a plant, comprising introducing exosporium fragments into a plant growth medium or applying exosporium fragments to a plant, plant seed or area surrounding a plant or plant seed, wherein the exospore fragments are derived from spores of a recombinant member of the Bacillus cereus family according to any one of claims 1 to 112 and contain a fusion protein and the fusion protein comprises a nucleic acid binding protein or peptide, and wherein the nucleic acid binding protein or peptide is linked to a nucleic acid molecule. 121. A method for protecting a plant from a pathogen or increasing plant resistance to stress, including applying exosporium fragments to a plant, plant seed or area surrounding a plant or plant seed, wherein the exosporium fragments are derived from spores of a recombinant member of the Bacillus cereus family according to any one of claims 1 to 112 and contain a fusion protein and the fusion protein contains a protein or peptide that protects the plant from a pathogen, or a protein or peptide that increases the plant's resistance to stress.

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