WO2007050103A2 - Recombinant bacteria without selection marker - Google Patents

Abstract

The present invention provides recombinant bacteria strains, such as Bacillus or Clostridium strains, lacking one or more genes commonly used for selecting recombinants (i.e., selection marker genes). More particularly, the present invention provides recombinant bacteria strains, containing one or more additional copies of its own gene or genes and/or one or more foreign genes artificially inserted in the host chromosomal DNA or extra-chromosomal DNA (e.g., a plasmid) without any selection marker gene or genes, such as an antibiotic resistance gene of any foreign origin. This allows for the introduction of the resultant recombinant Bacillus or Clostridium strain into the environment or into animals or humans without any contaminating selection marker gene. The recombinant strain, while lacking detrimental genes such as selection marker genes, may contain one or more desirable genes such as genes encoding insecticidal proteins or antigens for immunizing animals and humans. The present invention also provides methods for producing the recombinant bacterial strains.

Description

RECOMBINANT BACTERIA WITHOUT SELECTION MARKER

CROSS REFERENCE TO RELATED APPLICATIONS

[ooi] This application claims priority to 60/631 ,305 filed on November 29, 2004, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[002] The present invention relates to one or more recombinant Bacillus or Clostridium bacteria ("recombinant strain") comprising one or more additional copies of its own gene or genes and/or one or more foreign genes artificially inserted in the host chromosomal DNA or extra-chromosomal DNA without the presence of one or more selection marker genes of any foreign origin. The present invention also comprises methods for producing said recombinant bacteria. The invention also relates to any products produced by the recombinant strain during fermentation. The recombinant strain can produce a new foreign protein (i.e., an "exogenous protein") from a foreign gene inserted into the recombinant strain and/or an additional amount of its own protein from an extra copy of the gene added to the recombinant strain. The invention further relates to the use of said recombinant strain and its products to control insects and/or the use of said recombinant strain for animal immunizations.

BACKGROUND OF THE INVENTION

[003] Bacillus and Clostridium are rod-shaped, gram-positive bacteria some of which are known for their insect pathogenicity. For example, bacillus thuringiensis (Bt) is well known for its use as an insecticide in spray-on formulations and in transgenic plants. The commercial spray-on formulations of Bt insecticides contain spores and one or more insecticidal proteins that are produced by Bt. Many tons of live spores are applied as pesticides every year without significant adverse environmental impacts.

[004] The excellent safety record of Bt's commercial usage is due to its high specificity to target insects. Bt insecticidal proteins are inactive in or safe to animals and non-target insects. This high specificity, while beneficial, has certain drawbacks which can limit the use of Bt insecticides in commercial applications. For example, a commercial formulation based on the Bt subspecies kurstaki is highly active against lepidopteron insects but is inactive against most non-lepidopteran insects such as coleopteran insects. If one wants to use Bt-based insecticides to control both lepidopteran and coleopteran insects at the same time, a formulation with a mixture of two Bt strains is needed. While this strain mixing is feasible, it doubles the production costs and dilutes the concentration of each strain's products. Producing Bt by fermentation is costly. The fermentation process takes 48 hours or longer, and the post-fermentation recovery process incurs additional costs. [005] In similar fashion, some Clostridium species also are known to produce insecticidal proteins. For example, one strain of Clostridium produces a mosquitocidal protein similar to those produced by Bt. Another Clostridium strain, known as C. brevifaciens, is active against lepidopteran insects. In order to control lepidopteran and dipteran insects (e.g., mosquitoes) with Clostridium, one would need to mix the two strains with the same attending drawbacks discussed above for mixing Bt strains. Thus, there is a need to produce bacterial-based insecticidal products having broad insecticidal specificity without the disadvantages of mixing strains. Further, there is a need to do so without relying on the use of selection markers to select for recombinant strains since the application of recombinant strains containing selection markers could potentially have environmentally deleterious effects. For example, the application of recombinant strains carrying antibiotic-resistance genes could introduce the antibiotic-resistant trait to bacteria in the environment, thereby conferring unwanted antibiotic resistance to naturally-occurring bacteria resident in areas where the application of insecticide formulations containing recombinant strains occurs.

[006] The present invention overcomes the disadvantages inherent in an insecticide formulation containing a mixture of two strains of recombinant bacteria by providing formulations that do not require two separate strains of recombinant bacteria. The methods of the present invention accomplish this by providing for the selection of recombinant strains that do not require selection marker genes. The present invention also provides for the recombinant strains themselves and formulations of the same, which are intended to be applied as insecticidal products having similar safety profiles as wild-type strains but having a broader range of insecticidal activity and without introducing undesirable selection markers to the environment.

SUMMARY OF THE INVENTION

[007] The present invention provides methods that overcome the difficulties of making recombinant strains without selection marker genes, i.e., the invention provides methods for finding or selecting a desired recombinant strain that acquires a new gene placed in its chromosomal and/or extra- chromosomal DNA without a selection marker gene of any foreign origin present in the recombinant strain.

[008] The above shortcomings associated with the mixing of two strains of Bacillus or two strains of Clostridium insecticidal bacteria can be overcome by placing or cloning an additional insecticidal gene in the host strain. Such recombinant strains then produce an additional insecticidal protein encoded by the gene newly placed in the host. The added gene can encode a foreign protein having an insect specificity, which is not found in the host or one of the genes the host already contains. [009] The invention relates to one or more recombinant Bacillus or Clostridium bacteria ("recombinant strain") comprising one or more additional copies of its own gene or genes and/or one or more foreign genes artificially inserted in the host chromosomal DNA or extra-chromosomal DNA without the presence of one or more selection marker genes of any foreign origin and methods for producing said recombinant bacteria. A recombinant strain lacking a selection marker gene is advantageous as it will not contaminate the environment with any undesired selection marker gene, such as an antibiotic resistance gene (e.g., a vancomycin-resistance gene), when released. The invention also relates to any products produced by the recombinant strain during fermentation. The recombinant strain can produce a new foreign protein (i.e., an "exogenous protein") from the gene introduced to the recombinant strain and/or an additional amount of its own protein from an extra copy of the gene added to the recombinant strain. The invention further relates to the use of said recombinant strain and its products to control insects and/or the use of said recombinant strain for animal immunizations.

[oio] The integration of a foreign gene or genes in a Bt host may be made by utilizing Bt's own DNA recombination system. Alternatively, added recombination factors may be employed, for example by the introduction into the Bt host of plasmids coding for said recombination factors. The present invention employs a two-step integration process. In one embodiment, at least one selection marker gene, such as an antibiotic-resistance gene, is integrated at one or more selected sites of chromosomal DNA or extra-chromosomal DNA to produce a recombinant Bt selectable by one or more selection markers. Then, the one or more integrated selection marker genes is replaced with one or more desired genes, such as a heterologous insecticidal crystal protein gene of foreign origin, by a double recombination event. In this embodiment, there remains a requirement for screening many transformants due to the lack of a selection marker (i.e., negative selection or screening) but it is easier than direct, single-step integration of the desired gene without using any selection marker gene.

[Oil] In another embodiment, a host organism gene (i.e., a "target gene") is used to select a transformant. In this instance, a functional or non-functional DNA fragment is first inserted into the target gene of the host in a way that disrupts the target gene. Second, the inserted DNA fragment is replaced with a desired gene in a way that restores the target gene's activity thereby supplying a detectable activity that was disrupted by the first insertion event. In this specific embodiment, the target gene function is used as a selection marker. Examples of target genes include, without limitation, a phospholipase C gene for which a color indicator is available, an alpha-amylase gene which is needed for an organism to grow in a medium requiring alpha-amylase activity, and a sporulation regulation gene such as spoOA. When spoOA is disrupted, Bt fails to produce spores but can grow vegetatively. The vegetative cells are killed by heat (e.g., 80 ⁰C) but spores are not. By treating transformed cells with heat, only those capable of producing spores can survive, and therefore this can be used as a basis for selection.

[012] In another embodiment, the methods of the present invention provide for a recombinant strain having no selection marker gene of any foreign origin and having one or more useful traits such as a wide activity spectrum and increased productivity of the host's own insecticidal genes. There is no need to limit the utility of such recombinant strains to insecticide use. Bacillus and Clostridium can produce a variety of proteins including antigens and enzymes (see, e.g., International Patent Application No. PCT/US2005/25788, herein incorporated by reference in its entirety).

[013] In one embodiment, an antigen-expressing recombinant strain made using the methods of the present invention can be used safeiy as a vaccine, since the vaccine has no detrimental selection markers. There are several possible ways in which an antigen can be expressed including on the surface of a spore or as part of crystals that Bt produces during sporulation (see International Patent Application No. PCT/US2005/25788, previously incorporated by reference in its entirety).

[014] In the embodiments of the invention, the recombinant strain has no selection marker that can contaminate the environment, cause adverse effects in humans who would be interested in using the invention, or elicit harmful effects in animals, for example, through a vaccination process. The methods of the present invention would exclude any potential environmentally-detrimental genes from the final recombinant organism (such as antibiotic resistance genes).

[Ois] The present invention also provides for the use of bacteria producing insecticidal toxins other than those in Bacillus and Clostridium genera. Any insect-pathogenic bacteria species or strain is envisioned for use as a host and/or donor of a gene in order to produce a recombinant strain for insect control.

[016] Another aspect of the present invention includes recombinant bacteria that are generated by the methods of the present invention and therefore include a recombined endogenous gene, a recombined endogenous gene copy and a DNA insert. In preferred embodiments, the recombinant bacteria lack exogenous antibiotic resistance markers and more preferably lack any exogenous selectable marker. In various embodiments, the recombinant bacteria may be a gram-positive bacteria, a bacteria of the Bacillus or Clostridium genera, or a strain of Bt. The DNA insert in preferred embodiments is a foreign gene, but may be any DNA sequence. The foreign gene preferably includes a promoter that is operable in the recombinant bacteria which is preferably operably linked to a protein encoding sequence. The protein encoding sequence may be screenable and even selectable, but in preferred embodiments, the protein encoding sequence is not selectable and more preferably is neither selectable nor screenable (i.e., the DNA insert lacks any readily detectable phenotype). In certain variations, the DNA insert may further include a transcription termination site downstream of the protein encoding sequence. In preferred embodiments, the protein encoding sequence will encode an insecticidal gene. In other embodiments, the protein encoding sequence will encode an antigen or an enzyme.

[017] In certain embodiments, the recombined endogenous gene and the recombined endogenous gene copy will be identical and in other embodiments, they will be different. The recombined endogenous gene may include one or more mutations that may be screenable and/or selectable. The recombined endogenous gene copy may also include one or more mutations that may be screenable and/or selectable.

[018] In yet another aspect of the present invention, the recombinant bacteria above containing the DNA insert are generated by a two step process. The first step involves introducing a first recombination vector that includes a marker gene and a first endogenous gene copy and isolating the intermediate bacteria that have a first recombined endogenous gene, a first recombined endogenous gene copy and the marker gene between the endogenous gene and the endogenous gene copy. The second step involves introducing a second recombination vector that includes the DNA insert between a second endogenous gene copy and a third endogenous gene copy, and isolating the recombinant bacteria that have the DNA insert between a second recombined endogenous gene and a second recombined endogenous gene copy. In preferred embodiments the first endogenous gene copy includes a region that is sufficiently homologous to a region of an endogenous gene to allow recombination. In certain embodiments, the marker gene is selectable or screenable and the first step of isolating involves selecting or screening for the marker gene. In some embodiments, the second endogenous gene copy has a region that is sufficiently homologous to the first recombined endogenous gene to allow recombination and the third endogenous gene copy is sufficiently homologous to the first recombined gene copy to allow recombination. In preferred embodiments, the first recombined endogenous gene and the first recombined endogenous gene copy are selectably or screenably different and the second isolating step involves selecting or screening for recombinant bacteria that lack that difference, preferably the screenable or selectable difference will be a lack of function in one of the genes and the recombinant bacteria will be isolated by screening or selection based upon the lack of the function (i.e., both the endogenous gene and the endogenous gene copy in the recombinant bacteria lack the function).

[019] In another embodiment, the first recombined endogenous gene and the first recombined endogenous gene copy will be screenably or selectably identical and the recombinant bacteria will be isolated by selecting for a screenable or selectable difference in the second recombined endogenous gene. In preferred embodiments, both the first recombined gene and the first recombined gene copy will lack a function that is selectable or screenable. In preferred embodiments, the screen or selection will be for restoration or gain of the function in the second recombined endogenous gene.

[020] In other embodiments, the marker gene is selectable or screenable. In preferred embodiments, the marker gene is an antibiotic resistance gene, a specific metabolic enzyme gene or enzyme genes in a pathway that utilizes a special nutrient substitute or an enzyme that catalyzes a chemical compound to form a distinctive color.

[02i] The aspects and embodiments of the DNA insert of the foregoing methods include all aspects and embodiments of the DNA insert in the recombinant bacteria discussed above.

BRIEF DESCRIPTION OF THE DRAWINGS

[022] FIGURE 1 is a plasmid map of pSCO1.

[023] FIGURE 2 is a plasmid map of p8DA.

[024] FIGURE 3 is a plasmid map of pSCO2.

[025] FIGURE 4 is a plasmid map of pSCO3.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

[026] The present invention allows for the production of a recombinant bacterial strain by a two-step process comprising inserting a foreign gene such as an antibiotic resistance gene, or a non-functional DNA fragment, in the chromosomal or extra-chromosomal DNA of a host strain and then replacing the inserted gene or DNA fragment with a desired gene from which a useful protein can be expressed. As a result, there is no selection marker gene remaining in the final recombinant strain. This process relies on the host strain's natural capacity for recombining homologous DNA sequences.

[027] A recombinant strain lacking a selection marker gene is advantageous as it will not contaminate the environment with any undesired selection marker gene, such as an antibiotic resistance gene, when released. The present invention also provides products produced by the recombinant strain during fermentation. The recombinant strain can produce a new foreign protein (i.e., an "exogenous protein") from the gene introduced to the recombinant strain and/or an additional amount of its own protein from an extra copy of the gene added to the recombinant strain.

[028] The present invention also contemplates the creation of recombinant strains useful in the vaccination of humans and animals.

[029] There are a number of known methods of cloning a gene into a host Bacillus or Clostridium strain, especially in Bt. For example, Karamata and Piot (U.S. Patent No. 4,797,279, herein incorporated by reference in its entirety) invented a method of using cell conjugation to transfer a plasmid from one Bt strain specific to lepidopteran insects to another strain specific to coleopteran insects. The resulting conjugate contains double specificities active both against lepidopteran and coleopteran insects. Similarly, Gonzares, Jr., et al., (U.S. Patent No. 5,080,897, herein incorporated by reference in its entirety) invented another conjugation method to transfer a plasmid-coded insecticidal gene from one Bt strain to another. These methods can produce a recombinant Bt strain with improved insecticidal specificity but they suffer from a number of problems. For example, the methods depend on transmissible native plasmid. If a gene encoding a desired trait is on a non- transmissible plasmid, this method does not work. Also, this method cannot increase the copy number of one gene on one plasmid.

[030] A synthetic plasmid can be used to place or to clone a new gene in a Bt host as disclosed by Baum ef al. (Appl. Environ. Microbiol., 56(11):3420-3428, 1990) and Gamel and Piot (Gene. 120(1): 17-26, 1992). In both cases, the synthetic plasmids contain an antibiotic resistance marker, which can be transferred to other bacteria when the recombinant host Bt strain is released into the open environment. The self-replicating, extra-chromosomal DNA can be transferred to another Bt strain or even to a non-Bt strain by mating. The possibility of such gene transfers is not an environmentally-desirable outcome. Another problem, which may occur with the synthetic plasmid method, is instability of the plasmid in the host cell. According to a telephonic communication between Dr. Piot and the Applicants, the synthetic plasmid disappeared rapidly when the host recombinant Bt strain was grown in a commercial-scale fermentation tank.

[03i] To overcome plasmid instability and gene mobilization problems, Kalman et al. demonstrated that placing a heterologous Bt crystal protein gene in the chromosome of a Bt host strain (which contains a number of resident crystal insecticidal protein genes) resulted in the recombinant Bt strain producing a new crystal protein from the cloned gene, in addition to the proteins being produced by the native host genes (Appl. Environ. Microbiol. 61 (8): 3063-3068, 1995). Similarly, Adams et al., disclosed in U.S. Patent No. 5,955,367 (herein incorporated by reference in its entirety), a method to insert a Bt insecticidal crystal protein gene in the chromosome of a Bt host strain. These reports clearly indicate that Bt is capable of obtaining a new trait by DNA recombination. However, in both cases, there was a selection marker gene that was also inserted in the chromosome in order to select and identify the transformants. It is clear that the difficulty of producing, finding, and selecting a useful recombinant strain without a selection marker gene exists. The lack of reports of cloning a gene without any selection marker suggests that the selection of such a recombinant Bt is not feasible without an extraordinary effort of screening millions or even trillions of transformed cells.

[032] The present invention provides methods that overcome the difficulties of making recombinant strains without selection marker genes, i.e., the invention provides methods for finding or selecting a desired recombinant strain that acquires a new gene placed in its chromosomal and/or extra- chromosomal DNA without a selection marker gene of any foreign origin present in the recombinant strain.

II. General Techniques

[033] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture ( J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and CC. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); and Short Protocols in Molecular Biology (Wiley and Sons, 1999); all of which are incorporated by reference for the needed techniques. Furthermore, procedures employing commercially available assay kits and reagents will typically be used according to manufacturer-defined protocols unless otherwise noted.

III. Definitions

[034] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried- out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

[035] As defined herein, "Bt" or "Bacillus thuringiensis" is a rod-shaped, spore-forming, gram positive bacterium that belongs to the genus Bacillus and produces, during sporulation, one or more proteinaceous parasporal crystalline inclusion bodies that are often, but not always, insecticidak

[036] A "strain" as defined herein is a population of cells entirely derived from a single cell.

[037] A "foreign gene" as defined herein is a gene or a fragment of DNA, which does not naturally occur in the host bacterial cell.

[038] A "host strain" as defined herein is a bacterial strain that receives one or more foreign genes and expresses said one or more foreign genes.

[039] "Integration" as defined herein is a process, or a result of a process, of inserting a fragment of DNA into chromosomal or extra-chromosomal DNA of a host strain.

[040] A "transformant" as defined herein is a resulting bacterial strain created by transformation or by placing a foreign gene in a host bacterial strain.

[04i] The term "exogenous" as used herein means derived from outside the Bt host strain and the term "exogenous proteins" includes proteins, peptides, and polypeptides. Conversely, the term "endogenous" means derived from within the Bt host strain. The term "heterologous" as used herein means derived from a different genetic source. The term "homologous" as used herein means similar in structure and evolutionary origin.

[042] "Polynucleotide" and "nucleic acid" refer to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs. It will be Understood that, where required by context, when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C)In which "U" replaces "T."

[043] The term "upstream" refers to the region or DNA extending in a 5' direction and the term "downstream" refers to an area on the same strand of DNA, that is located past the gene if one moves along the strand in a 5'-3' direction (the normal direction of transcription and leading strand replication).

[044] A "gene" is a defined hereditary unit that occupies a specific location on a chromosome, determines a particular characteristic in an organism by directing the formation of a specific protein, and is capable of replicating itself at each cell division. The term "reading frame" refers to a contiguous, non-overlapping set of triplet codons in RNA or DNA that begin from a specific nucleotide. A "codon" is defined as the basic unit of the genetic code, comprising three-nucleotide sequences of messenger ribonucleic acid (mRNA), each of which is translated into one amino acid in protein synthesis.

[045] A "recombinant" as defined herein is an adjective that signifies a condition of a bacterial strain, which acquired, by an act of man, one or more additional copies of its own gene or one or more foreign genes incorporated into the chromosome and/or extra-chromosomal DNA by recombination.

[046] "Selection marker" as defined herein is a gene or gene product that can be used to select a recombinant strain. Selection marker and "selectable marker" are used interchangeably herein.

[047] A "cloning vector" is defined as a DNA molecule originating from a virus, a plasmid, or the cell of a higher organism into which another DNA fragment of appropriate size can be integrated without loss of the vector's capacity for self-replication. Vectors introduce foreign DNA into host cells, where it can be reproduced. Vectors are often recombinant molecules containing DNA sequences from several sources. The DNA introduced with the vector is replicated whenever the cell divides.

[048] "Improved" as defined herein is an adjective meaning at least 1.2 times higher than the original state in the relevant measurable biological activity or product quantity that is to be enhanced.

[049] "Activity spectrum" as defined herein is a target or substrate specificity of any biologically active agent (e.g. insect control agent) that has beneficial biological activity against multiple target species. [050] A "promoter" is an array of nucleic acid control sequences that direct transcription of an associated polynucleotide, which may be a heterologous or a native polynucleotide. A promoter includes nucleic acid sequences near the start site of transcription, such as an RNA polymerase binding site. An "expression cassette" refers to a series of polynucleotide elements that permit transcription of a gene in a host cell. Typically, the expression cassette includes a promoter and a heterologous or native polynucleotide sequence that is transcribed.

[05i] A "linker" is a double-stranded oligonucleotide containing a number of restriction endonuclease recognition sites. A "restriction endonuclease recognition site" or a "restriction site" is a specific nucleotide sequence at which a particular restriction enzyme cleaves the DNA.

[052] A "restriction enzyme" or "restriction endonuclease" is a protein that recognizes specific, short nucleotide sequences and cleaves DNA at those sites.

[053] • The term "operably linked" refers to a functional relationship between two parts in which the activity of one part (e.g., the ability to regulate transcription) results in an action on the other part (e.g., transcription of the sequence). Thus, a polynucleotide is "operably linked to a promoter" when there is a functional linkage between a polynucleotide expression control sequence (such as a promoter or other transcription regulation sequences) and a second polynucleotide sequence (e.g., a native or a heterologous polynucleotide), where the expression control sequence directs transcription of the polynucleotide.

[054] The "polymerase chain reaction" or "PCR" is method for amplifying a DNA base sequence using a heat-stable polymerase and two primers, one complementary to the plus strand at one end of the sequence to be amplified and the other complementary to the minus strand at the other end. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence. PCR also can be used to detect the existence of the defined sequence in a DNA sample.

[055] The terms "primer", "oligonucleotide", and "oligonucleotide primer" can be used interchangeably and are defined as a short polynucleotide chain. "Polymerase" is defined as an enzyme that catalyzes the synthesis of nucleic acids on preexisting nucleic acid templates.

[056] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Amino acids may be referred to herein by either their commonly known three letter symbols or by Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes, i.e., the one-letter symbols recommended by the IUPAC-IUB.

[057] An "antibody" is a protein produced in response to an antigen (often a virus or bacterium). It is able to combine with and neutralize the antigen. An "antigen" is a substance that causes an immune system response.

[058] "High stringency conditions" may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.015 M sodium citrate/0.1% sodium dodecyl sulfate at 50-68 ⁰C; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (v/v) formamide with 0.1 % bovine serum albumin/0.1 % Ficoll/0.1 % polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mWI sodium chloride, 75 mM sodium citrate at 42 ⁰C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 Dg/ml), 0.1 % SDS, and 10% dextran sulfate at 42 ⁰C, with washes at 42 ⁰C. in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55 ⁰C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55 °C.

[059] The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. Stringent conditions are sequence- dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 ⁰C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.05 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 ⁰C for short probes (e.g., 10 to 50 nucleotides) and at least about 60 ⁰C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of DNA duplex destabilizing agents such as formamide.

[060] The terms "identical" or percent "identity", in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Preferably, the percent identity exists over a region of the sequence that is at least about 25 amino acids in length, more preferably over a region that is 50 or 100 amino acids in length. This definition also refers to the complement of a test sequence, provided that the test sequence has a designated or substantial identity to a reference sequence. Preferably, the percent identity exists over a region of the sequence that is at least about 25 nucleotides in length, more preferably over a region that is 50 or 100 nucleotides in length.

[061] The phrase "substantially identical," in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 60%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions.

[062] By "DNA" is meant a polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in double-stranded or single-stranded form, either relaxed or supercoiled. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes single- and double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. The term captures molecules that include the four bases adenine, guanine, thymine, or cytosine, as well as molecules that include base analogs which are known in the art.

[063] A "recombined endogenous gene" and a "recombined endogenous gene copy" refer to endogenous genes within the bacteria of interest that have undergone recombination with a foreign nucleic acid that has at least a region within it that has sufficient homology to recombine with the endogenous gene. The recombination may occur via an endogenous recombination mechanism, i.e., using the bacteria's own recombination proteins or via an exogenous recombination mechanism, i.e., using proteins provided exogenously through a plasmid or other method available to those of skill in the art. Such recombination may include homologous recombination and site-specific recombination such as through the cre-lox recombination machinery. As one of skill in the art will appreciate, the recombined endogenous gene may be identical to the original endogenous gene or may include various changes such as point mutations, insertions, and deletions. The recombined endogenous gene will be recognizable by one of skill in the art in that it will contain at least a portion in the 5' upstream region or the 3' downstream region that is still identical to the original endogenous gene.

IV. Methods and Uses of the Present Invention

A. Methods of the Present Invention

[064] In one embodiment, the first insertion of a foreign gene or nonfunctional DNA fragment into the chromosome or extra-chromosomal DNA is made to a desired host that can be released into the environment. The first integration can be made with a circular or non-circular (i.e., linear) DNA. In the case of a circular DNA, it may contain a replication of origin that does not work in the host strain. The integration of circular DNA occurs at a single point driven by the host strain's natural capability for recombining homologous sequences. Therefore, the DNA that is inserted into the host strain must be long enough to support homologous recombination (e.g., from 10 base pairs to several thousand base pairs and the DNA sequence must be homologous to the target site of the host DNA). Finding a specific target site is made by probing the host DNA by a number of methods known in the art including PCR amplification and sequencing of the target DNA from the host strain by utilizing available knowledge about the host's genomic sequence (e.g., Bacillus subtilis DNA sequence). Another example of a method to find the target DNA sequence is to clone, identify, and sequence the target DNA fragment or fragments from the host strain.

[065] The DNA fragment that contains the DNA sequence homologous to the target DNA sequence of the host strain and a piece of a foreign gene or DNA or DNA fragment that goes into the host DNA can be introduced into the host strain by any method known in the art including, but not limited to, electroporation, protoplasting of cells, transduction, chemical transformation and regeneration. In this first integration step, the foreign gene or DNA or DNA fragment that is integrated in the host cell can be a selection marker or any DNA fragment, which may encode one or more functional or nonfunctional proteins. In one embodiment, a selection marker is used at this first integration step. In one example, an antibody-resistance gene, such as a tetracycline resistance gene from pBC16.1, can be used. In this case, a medium containing a selective concentration of tetracycline can be used to identify the recombinant host. Since no replication origin exists in the integration DNA that is introduced to the host, only those recombinant strains that have acquired the tetracycline-resistance selection marker gene can grow on a tetracycline-containing medium. In another embodiment of the first integration step, one can place a fragment of DNA at a target site of a host strain in a way that disrupts the function of a gene that exists at the target site. The disrupted gene in the target site can be restored during the second integration step described below, and is used for selecting the recombinant host resulting from the second integration step. [066] The methods of the present invention involve a second integration step in a recombinant strain after the first integration step. The major objective of the second integration step is to replace the DNA fragment that was inserted in the host DNA during the first integration step with a gene that encodes a protein of a desired trait, such as an insecticidal crystal protein, from any source including the host strain itself, and other heterologous bacterial strains. The DNA sequence that is introduced to the host strain can be a circular or non-circular DNA; in either form it is not capable of replicating in the host strain. Like the integration sequence used in the first step, the integration sequence of the second step contains one or more fragments of DNA that are homologous to the target DNA sequence of the host strain. The integration sequence must be organized in such a way that removes the foreign gene (e.g., an antibiotic-resistance gene such as a tetracycline-resistance gene) or nonfunctional DNA fragment that has been integrated in the host DNA by the first integration step. Also, the second integration step needs to restore the gene function of the integration target site of the host strain when the second integration takes place. In one embodiment, all unnecessary foreign DNA sequences are removed from the host strain by the second integration step. In another embodiment, the restored target gene of the host strain is used to select the recombinant host. Such target genes include, without limitation, the alpha-amylase gene and spoOA gene as described in the examples of the present invention, infra. In the case of alpha-amylase, the recombinant host can grow on a medium containing starch as a sole carbon source.

[067] Those host cells whose amylase gene is disrupted during the first integration process and failed to acquire the integration sequence during the second integration stage cannot grow on the starch medium. The spoOA gene also can be used as a selection marker between disrupted and restored conditions. The desired recombinant strain that is selected for has the restored, functionaf spoOA allowing it to produce mature spores. On the other hand, those that failed to sporulate due to the failure of restoring the functional spoOA during the second integration step cannot survive heat treatment (e.g., 80° C for 20 min). Bacillusi 'Clostridium spores are highly heat stable.

[068] In one embodiment, the integration sequence that replaces the gene or DNA fragments inserted into the host DNA during the first integration step can be a foreign gene from a bacterial strain different from the host strain. For example, a coleoptera-specific gene, cryδDa, can be introduced to a Bt subspecies kurstaki strain called HD73. As is well known in the art, HD73 is available from multiple sources including the USDA, ARS Northern Regional Research Center (NRRL), and the Ohio State University Bacillus Genetic Stock Center (BGSC). The HD73 strain is active against lepidopteran insects but is not active against coleopteran insects due to the lack of coleopteran-active protein(s) in its crystal. However, the resulting recombinant Bt HD73 strain is active against lepidopteran insects and coleopteran insects. As can be appreciated by the skilled artisan, such a Bt strain having wide-spectrum insecticidal activity is highly desirable in controlling a complex insect infestation.

[069] In another embodiment, the integration sequence that replaces the gene or DNA fragments inserted into the host Bt DNA during the first integration step can be a gene from the host strain. For example, an additional copy of a resident gene isolated from extra-chromosomal DNA (i.e., plasmid) can be placed in the chromosome. The resulting recombinant host strain produces an increased amount of the gene product in comparison with the original, non-recombinant host strain.

[070] The fermentation products of the recombinant bacterial strains of the invention, containing spores and insecticidal proteins, can be formulated into a pest-control agent for example, a water or solvent dispersible powder, water or solvent dispersible granules, water or solvent emulsifiable liquid concentrate, dusting powder, encapsulated granules, and other formulations and mixtures known in the art. In order to formulate the fermentation products of the recombinant strain, inactive ingredients are added, such as a dispersing agent, emulsifying agent, wetting agent, sticker, UV blocker, UV and light absorber, granulating agent, encapsulation agent, or other additives known to those skilled in the art to facilitate handling and application for particular target pests.

[07i] In yet another embodiment of the present invention, a gene is inserted that encodes an antigen that is derived from a pathogen of human or animal disease. By so doing, the inserted antigen gene can be directed to synthesize and transport the gene product to the surface of bacterial spores. Such recombinant spores can then be delivered to animals or humans by spraying properly formulated recombinant spores for nasal intake, or by injection, or by oral ingestion, or any other route of administration known in the art in order to immunize the animals and humans.

[072] Since the recombinant bacterial strain does not contain any foreign selection marker, its environmental release as a pest control agent or its introduction to animals, including humans, for immunization and other purposes are highly desirable since environmental exposure (e.g., exposure to animals, humans, and endogenous microorganisms such as bacteria living in soil) to undesirable genetic elements is eliminated by the methods disclosed herein.

[073] This invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)). [074] For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). Sizes are derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated or established from gel electrophoresis, from mass spectrometry, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.

[075] Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984).

[076] The sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al., Gene 16:21-26 (1981).

[077] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection. One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. MoI. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1 , more preferably less than about 0.01 , and most preferably less than about 0.001.

[078] When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Alternatively, when one includes such conservative substitutions in the comparison, a percent "similarity" can be noted, as opposed to a percent "identity". Means for making this adjustment are well known to those of skill in the art. The scoring of conservative substitutions can be calculated according to, e.g., the algorithm of Meyers & Millers, Computer Applic. Biol. Sci. 4:11-17 (1988), e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).

[079] Also included within the definition of target proteins of the present invention are amino acid sequence variants of wild-type target proteins. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site-specific mutagenesis of nucleotides in the DNA encoding the target protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Variant target protein fragments having up to about 100-150 amino acid residues may be prepared by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the target protein amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics.

[080] Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to about 20 amino acids, although considerably longer insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases, deletions may be much longer.

[08i] Substitutions, deletions, and insertions or any combinations thereof may be used to arrive at a final derivative. Generally, these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger characteristics may be tolerated in certain circumstances.

[082] The following six groups each contain amino acids that are conservative substitutions for one another (see, e.g., Creighton, Proteins (1984)):

[083] 1) Alanine (A), Serine (S), Threonine (T);

[084] 2) Aspartic acid (D)₁ Glutamic acid (E); [085] 3) Asparagine (N), Glutamine (Q);

[086] 4) Arginine (R), Lysine (K);

[087] 5) lsoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[088] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

B. Uses of the Present Invention

[089] The present invention relates to one or more recombinant Bacillus or Clostridium bacteria ("recombinant strain") comprising one or more additional copies of its own gene or genes and/or one or more foreign genes artificially inserted in the host chromosomal DNA or extra-chromosomal DNA without the presence of one or more selection marker genes of any foreign origin. The present invention also comprises methods for producing said recombinant bacteria. The invention also relates to any products produced by the recombinant strain during fermentation. The recombinant strain can produce a new foreign protein (i.e., an "exogenous protein") from a foreign gene inserted into the recombinant strain and/or an additional amount of its own protein from an extra copy of the gene added to the recombinant strain. The invention further relates to the use of said recombinant strain and its products to control insects and/or the use of said recombinant strain for animal immunizations.

[090] The methods of the present invention provide for the selection of recombinant strains that do not require selection marker genes. The present invention also provides for the recombinant strains themselves and formulations of the same, which are intended to be applied as insecticidal products having similar safety profiles as wild-type strains but having a broader range of insecticidal activity and without introducing undesirable selection markers to the environment.

[09i] More particularly, the invention relates to one or more recombinant Bacillus or Clostridium bacteria ("recombinant strain") comprising one or more additional copies of its own gene or genes and/or one or more foreign genes artificially inserted in the host chromosomal DNA or extra- chromosomal DNA without the presence of one or more selection marker genes of any foreign origin and methods for producing said recombinant bacteria. A recombinant strain lacking a selection marker gene is advantageous as it will not contaminate the environment with any undesired selection marker gene, such as an antibiotic resistance gene (e.g., a vancomycin-resistance gene), when released. Therefore, such a strain will be useful in the control of deleterious insects but will not contribute to antibiotic resistance in bacteria living in areas where the recombinant bacterial insecticidal product is applied. [092] The invention also relates to any products produced by the recombinant strain during fermentation. The recombinant strain can produce a new foreign protein (i.e., an "exogenous protein") from the gene introduced to the recombinant strain and/or an additional amount of its own protein from an extra copy of the gene added to the recombinant strain. The invention further relates to the use of said recombinant strain and its products to control insects and/or the use of said recombinant strain for animal immunizations.

[093] The present invention provides for a recombinant strain having no selection marker gene of any foreign origin and having one or more useful traits such as a wide activity spectrum and increased productivity of the host's own insecticidal genes. There is no need to limit the utility of such recombinant strains to insecticide use. Bacillus and Clostridium can produce a variety of proteins including antigens and enzymes.

[094] The host for the expression vector can be any Bacillus thuringiensis strain including those Bt strains used to make commercial insecticides. Genomically similarly structured Bacillus strains can also be used. Examples of Baccillus thuringiensis strains and strains similarly genomically structured include, but are not limited to, B. albus, B. anthracis, B. brevis, B. cereus, B. coagulans, B. colistinus, B. larvae, B. lentimorbus, B. licehnfiformis, B. megaterium, B. mycoides, B. polymyxa, B. popilHae (aka: Paenibacillus popilliae), B. radicicola, B. sphaericus, B. stearothermophilus, B. subtilis, B. theromoacidurans and B. thuringiensis. With respect to B. thuringiensis the strains include, but are not limited to, any subspecies including Bacillus thuringiensis subsp. aizawai, Bacillus thuringiensis subsp. galleriae, Bacillus thuringiensis subsp. entomocidus, Bacillus thuringiensis subsp. tenebrionis, Bacillus thuringiensis subsp. thuringiensis, Bacillus thuringiensis subsp. alesti, Bacillus thuringiensis subsp. canadiensis, Bacillus thuringiensis subsp. darmstadiensis, Bacillus thuringiensis subsp. dendrolimus, Bacillus thuringiensis subsp. finitimus, Bacillus thuringiensis subsp. kenyae, Bacillus thuringiensis subsp. morrisoni, Bacillus thuringiensis subsp. subtoxicus, Bacillus thuringiensis subsp. toumanoffi, Bacillus thuringiensis subsp. pondicheήensis, Bacillus thuringiensis subsp. shandogiensis, Bacillus thuringiensis subsp. sotto, Bacillus thuringiensis subsp. nigeriae, Bacillus thuringiensis subsp. yunnanensis, Bacillus thuringiensis subsp. dakota, Bacillus thuringiensis subsp. indiana, Bacillus thuringiensis subsp. tohokuensis, Bacillus thuringiensis subsp. kumamotoensis, Bacillus thuringiensis subsp. tochigiensis, Bacillus thuringiensis subsp. thompsoni, Bacillus thuringiensis subsp. wuhanensis, Bacillus thuringiensis subsp. kyushuensis, Bacillus thuringiensis subsp. ostriniae, Bacillus thuringiensis subsp. tolworthi, Bacillus thuringiensis subsp. Pakistani, Bacillus thuringiensis subsp. japonensis, Bacillus thuringiensis subsp. colmeri, Bacillus thuringiensis subsp. pondicheriensis, Bacillus thuringiensis subsp. shandongiensis, Bacillus thuringiensis subsp. neoleonensis, Bacillus thuringiensis subsp. coreanensis, Bacillus thuringiensis subsp. silo, Bacillus thuringiensis subsp. mexcanensis, Bacillus thuringiensis subsp. israelensis, Bacillus thuringiensis subsp. berliner, Bacillus thuringiensis subsp. cameroun, Bacillus thuήngiensis subsp. onghei, Bacillus thuringiensis subsp. fukuokaensis, Bacillus thuringiensis subsp. higo, Bacillus thuringiensis subsp. israelensis, Bacillus thuringiensis subsp. japonensis Buibui, Bacillus thuringiensis subsp. jegathesan, Bacillus thuringiensis subsp. kenyae, Bacillus thuringiensis subsp. kunthala, Bacillus thuringiensis subsp. medellin, Bacillus thuringiensis subsp. roskildiensis, Bacillus thuringiensis subsp. san diego, Bacillus thuringiensis subsp. shanghai, Bacillus thuringiensis subsp. sotto, Bacillus thuringiensis subsp. tenebήonis, and Bacillus thuringiensis subsp. thompsoni.

[095] lnsecticidal protein genes occur naturally in Bt as well as some other Bacillus species, such as Bacillus popilliae. lnsecticidal proteins, also called delta-endotoxins, or crystal protein, form crystals visible by phase contrast microscopy, lnsecticidal protein genes (also called crystal protein genes) used in the present invention may include, but are not limited to, the following list (see also at the website biols.susx.ac.uk/home/Neil_C/rickmore/Bt/toxins2.html where sequences to the following proteins can be obtained either directly or via links to other websites): Cry1Aa1, Cry1Aa2, Cry1Aa3, Cry1Aa4, Cry1Aa5, Cry1Aa6, Cry1Aa7, CryiAaδ, Cry1Aa9, Cry1Aa10, Cry1Aa11, Cry1Aa12, Cry1Aa13, Cry1Aa14, Cry1Ab1, Cry1Ab2, Cry1Ab3, Cry1Ab4, Cry1Ab5, Cry1Ab6, Cry1Ab7, CryiAbδ, Cry1Ab9, Cry1Ab10, Cry1Ab11, Cry1Ab12, Cry1Ab13, Cry1Ab14, Cry1Ab15, Cry1Ab16, Cry 1 Ad, Cry1Ac2, Cry1Ac3, Cry1Ac4, Cry1Ac5, Cry1Ac6, Cry1Ac7, CryiAcδ, Cry1Ac9, Cry 1 AdO, Cry1Ac11, Cry1Ac12, Cry1Ac13, Cry1Ac14, Cry1Ac15, Cry1Ad1, Cry1Ad2, Cry1Ae1, Cry1Af1, Cry1Ag1, Cry1Ah1, Cry1Ai1, Cry1Ba1, Cry1Ba2, Cry1Ba3, Cry1Ba4, Cry1Bb1, Cry1Bc1, Cry1Bd1, Cry1Bd2, Cry1Be1, Cry1Be2, Cry1Bf1, Cry1Bf2, Cry1Bg1, Cry1Ca1, Cry1Ca2, Cry1Ca3, Cry1Ca4, Cry1Ca5, Cry1Ca6, Cry1Ca7, CryiCaδ, Cry1Ca9, Cry1Ca10, Cry1Cb1, Cry1Cb2, Cry1Da1, Cry1Da2, Cry1Db1, Cry1Db2, Cry1Ea1, Cry1Ea2, Cry1Ea3, Cry1Ea4, Cry1Ea5, Cry1Ea6, Cry1Eb1, Cry1Fa1, Cry1Fa2, Cry1Fb1, Cry1Fb2, Cry1Fb3, Cry1Fb4, Cry1Fb5, Cry1Ga1, Cry1Ga2, Cry1Gb1, Cry1Gb2, CrylGc, Cry1Ha1, Cry1Hb1, Cry1la1, Cry1la2, Cry1la3, Cry1la4, Cry1la5, Cry1la6, Cry1la7, Cryilaδ, Cry1la9, Cry1la10, Cry1la11, Cry1lb1, Cryild, Cry1lc2, Cry1ld1, Cry1le1, Cry1lf1, CryUal, CryUbi, CryUd, CryUc2, CryUdi, Cry1Ka1, Cry2Aa1, Cry2Aa2, Cry2Aa3, Cry2Aa4, Cry2Aa5, Cry2Aa6, Cry2Aa7, Cry2Aa8, Cry2Aa9, Cry2Aa10, Cry2Aa11, Cry2Ab1, Cry2Ab2, Cry2Ab3, Cry2Ab4, Cry2Ab5, Cry2Ab6, Cry2Ac1, Cry2Ac2, Cry2Ac3, Cry2Ad1, Cry2Ae1, Cry3Aa1, Cry3Aa2, Cry3Aa3, Cry3Aa4, Cry3Aa5, Cry3Aa6, Cry3Aa7, Cry3Ba1, Cry3Ba2, Cry3Bb1, Cry3Bb2, Cry3Bb3, Cry3Ca1, Cry4Aa1, Cry4Aa2, Cry4Aa3, Cry4Ba1, Cry4Ba2, Cry4Ba3, Cry4Ba4, Cry4Ba5, Cry5Aa1, Cry5Ab1, CryδAd, Cry5Ba1, Cry6Aa1, Cry6Aa2, Cry6Ba1, Cry7Aa1, Cry7Ab1, Cry7Ab2, CryδAal, CryδBai, Cry8Bb1, CryδBd, CryδCal, CryδCa2, CryδDai, CryδDa2, Cry8Da3, CryδEal, Cry9Aa1, Cry9Aa2, Cry9Ba1, Cry9Ca1, Cry9Ca2, Cry9Da1, Cry9Da2, Cry9Ea1, Cry9Ea2, Cry9Eb1, Cry9Ed, Cry10Aa1, Cry10Aa2, Cry10Aa3, Cry11Aa1, Cry11Aa2, Cry11Aa3, Cry11Ba1, Cry11Bb1, Cry12Aa1, Cry13Aa1, Cry14Aa1, Cry15Aa1, Cry16Aa1, Cry17Aa1, Cry1δAa1, Cry18Ba1, Cry18Ca1, Cry19Aa1, Cry19Ba1, Cry20Aa1, Cry21Aa1, Cry21Aa2, Cry21Ba1, Cry22Aa1, Cry22Aa2, Cry22Ab1, Cry22Ab2, Cry22Ba1 , Cry23Aa1 , Cry24Aa1 , Cry25Aa1 , Cry26Aa1 , Cry27Aa1 , Cry28Aa1 , Cry28Aa2, Cry29Aa1 , Cry30Aa1 , Cry30Ba1 , , Cry31Aa1 , Cry31Aa2, Cry32Aa1 , Cry32Ba1 , Cry32Ca1 , Cry32Da1 , Cry33Aa1 , Cry34Aa1 , Cry34Aa2, Cry34Ab1 , Cry34Ac1 , Cry34Ac2, Cry34Ba1 , Cry35Aa1 , Cry35Aa2, Cry35Ab1 , Cry35Ab2, Cry35Ac1 , Cry35Ba1 , Cry36Aa1 , Cry37Aa1 , Cry38Aa1 , Cry39Aa1 , Cry40Aa1, Cry40Ba1, Cry41Aa1, Cry41Ab1, Cry42Aa1, Cry43Aa1, Cry43Ba1, Cry44Aa, Cry45Aa, Cry46Aa, Cry47Aa, Cyt1Aa1 , Cyt1Aa2, Cyt1Aa3, Cyt1Aa4, Cyt1Aa5, Cyt1Ab1 , Cyt1 Ba1 , Cyt2Aa1, Cyt2Aa2, Cyt2Ba1 , Cyt2Ba2, Cyt2Ba3, Cyt2Ba4, Cyt2Ba5, Cyt2Ba6, Cyt2Ba7, Cyt2Ba8, Cyt2Ba9, Cyt2Bb1 , Cyt2Bc1 , Cyt2Ca1, Vip3A(a) and Vip3A(b). Gene sequences for the above-mentioned genes may be found in any public source including public databases such as those maintained by the National Center for Biotechnology Information, university databases, publications including those from various scientific journals and others well known to those of skill in the art. •

[096] Recombinant or engineered insecticidal protein genes can also be used in the methods of the present invention. For example, a hybrid insecticidal protein gene made by fusing the N-terminal coding region of one insecticidal protein gene with the C-terminal coding region of another insecticidal protein gene can be used. In another example, the insecticidal protein gene is engineered to encode a number of amino acid changes.

[097] The present invention is also useful for one or more hosts that belong to genera similar to Bacillus, such as Clostridium including, but not limited to, C. acetobutylicum, C. bifermentans, C. botulinum, C. brevifaciens, C. butyricum, C. chauvoei, C. dissolvens, C. fallax, C. histolyticum, C. nigrificans, C. novyi, C. paterurianum, C. perfringens, C. putrificum, C. septicum, C. sporogenes, C. tetani, C. thermohyfrosulfuricum, C. thermosaccharolyticum and C. welchii. Some of these Bacillus and Clostridium strains are well known insect pathogens exerting their effects by producing insecticidal proteins.

[098] The present invention also provides for the use of any insect pathogens other than those in Bacillus and Clostridium genera. For example, a Serratia species is known to produce an insect- active protein (Hurst et al., J Bacteriol. 2004 186:5116-5128, 2004). Any insect-pathogenic bacteria species or strain is envisioned for use as a host and/or donor of a gene in order to produce a recombinant strain for insect control.

[099] The double crossover recombination step described in the present invention is useful to remove an unwanted gene or genes from the host strain (see Example 1 , infra). Patel et al. report {Antimicrobial Agents and Chemotherapy, 44: 705-709, 02000) that B. popilliae used in a commercial biopesticide contains a vancomycin-resistance gene. Obviously, it is highly desirable to remove such a gene from any insecticide formulation before it is released into the environment and allowed to be propagated to other bacterial species and strains thereby spreading vancomycin resistance.

[oioo] The methods of the present invention also provide an antigen-expressing recombinant strain, which can be used safely as a vaccine, since the vaccine has no detrimental selection markers. There are several possible ways in which an antigen can be expressed including on the surface of a spore or as part of crystals that Bt produces during sporulation (see, e.g., International Patent Application No. PCT/US2005/25788, previously incorporated by reference in its entirety).

[oioi] The disease-associated antigens include, but are not limited to, toxins, virulence factors, cancer antigens, such as tumor-associated or specific antigens expressed on cancer cells, antigens associated with autoimmunity disorders, antigens associated with inflammatory conditions, antigens associated with allergic reactions, antigens associated with infectious agents, and autoantigens that play a role in¹ induction of autoimmune diseases.

[0102] Among the tumor-specific antigens that may be used in the methods of the invention are: bullous pemphigoid antigen 2, prostate mucin antigen (PMA) (Beckett and Wright (1995) Int. J. Cancer 62: 703-710), tumor associated Thomsen-Friedenreich antigen (Dahlenborg et al. (1997) Int. J. Cancer 70: 63-71), prostate-specific antigen (PSA) (Dannull and Belldegrun (1997) Br. J. Urol. 1 : 97-103), EpCam/KSA antigen, luminal epithelial antigen (LEA.135) of breast carcinoma and bladder transitional cell carcinoma (TCC) (Jones et al. (1997) Anticancer Res. 17: 685-687), cancer- associated serum antigen (CASA) and cancer antigen 125 (CA 125) (Kierkegaard et al. (1995) Gynecol. Oncol. 59: 251-254), the epithelial glycoprotein 40 (EGP40) (Kievit et al. (1997) Int. J. Cancer 71 : 237-245), squamous cell carcinoma antigen (SCC) (Lozza et al. (1997) Anticancer Res. 17: 525-529), cathepsin E (Mota et al. (1997) Am. J. Pathol. 150: 1223-1229), tyrosinase in melanoma (Fishman et al. (1997) Cancer 79: 1461-1464), cell nuclear antigen (PCNA) of cerebral cavemomas (Notelet et al. (1997) Surg Neurol. 47: 364-370), DF3/MUC1 breast cancer antigen (Apostolopoulos et al. (1996) Immunol. Cell. Biol. 74: 457-464; Pandey et al. (1995) Cancer Res. 55: 4000-4003), carcinoembryonic antigen (Paone et al. (1996) J. Cancer Res. Clin. Oncol. 122: 499-503; Schlom et al. (1996) Breast Cancer Res. Treat. 38: 27-39), tumor-associated antigen CA 19-9 (Tolliver and O'Brien (1997) South Med. J. 90: 89-90; Tsuruta et al. (1997) Urol. Int. 58: 20-24), human melanoma antigens MART-1/Melan-A27-35 and gp100 (Kawakami and Rosenberg (1997) Int. Rev. Immunol. 14: 173-192; Zajac et al. (1997) Int. J. Cancer 71 : 491-496), the T and Tn pancarcinoma (CA) glycopeptide epitopes (Springer (1995) Crit. Rev. Oncog. 6: 57-85), a 35 kD tumor-associated autoantigen in papillary thyroid carcinoma (Lucas et al. (1996) Anticancer Res. 16: 2493-2496), KH-1 adenocarcinoma antigen (Deshpande and Danishefsky (1997) Nature 387: 164-166), the A60 mycobacterial antigen (Maes et al. (1996) J. Cancer Res. Clin. Oncol. 122: 296-300), heat shock proteins (HSPs) (Blachere and Srivastava (1995) Semin. Cancer Biol. 6: 349-355), and MAGE, tyrosinase, melan-A and gp75 and mutant oncogene products (e.g., p53, ras, and HER-2/neu (Bueler and Mulligan (1996) MoI. Med. 2: 545-555; Lewis and Houghton (1995) Semin. Cancer Biol. 6: 321- 327; Theobald et al. (1995) Proc. Nat'l. Acad. Sci. USA 92: 11993-11997).

[0103] Viral antigens that can be used with the present invention include, but are not limited to, hepatitis B capsid protein, hepatitis C capsid protein, hepatitis A capsid protein, Norwalk diarrheal virus capsid protein, influenza A virus N2 neuraminidase (Kilbourne et al. (1995} Vaccine 13: 1799- 1803); Dengue virus envelope (E) and premembrane (prM) antigens (Feighny et al. (1994) Am. J. Trop. Med. Hyg. 50: 322-328; Putnak et al. (1996) Am. J. Trop. Med. Hyg. 55: 504-10); HIV antigens Gag, Pol, Vif and Nef (Vogt et al. (1995) Vaccine 13: 202-208); HIV antigens gp120 and gp160 (Achour et al. (1995) Cell. MoI. Biol. 41:395-400; Hone et al. (1994) Dev. Biol. Stand. 82: 159-162); gp41 epitope of human immunodeficiency virus (Eckhart et al. (1996) J. Gen. Virol. 77:2001-2008); rotavirus antigen VP4 (Mattion et al. (1995) J. Virol. 69:5132-5137); the rotavirus protein VP7 or VP7sc (Emslie et al. (1995) J. Virol. 69: 1747-1754; Xu et al. (1995) J. Gen. Virol. 76: 1971-1980; Chen et al. (1998) Journal of Virology VoI 72:7; pp 5757-5761); herpes simplex virus (HSV) glycoproteins gB, gC, gD, gE, gG, gH, and gl (Fleck et al. (1994) Med. Microbiol. Immunol. (Berl) 183: 87-94 (Mattion, 1995); Ghiasi et al. (1995) Invest. Ophthalmol. Vis. Sci. 36: 1352-1360; McLean et al. (1994) J. Infect. Dis. 170: 1100-1109); immediate-early protein ICP47 of herpes simplex virus-type 1 (HSV-1) (Banks et al. (1994) Virology 200:236-245); immediate-early (IE) proteins ICP27, ICPO, and ICP4 of herpes simplex virus (Manickan et al. (1995) J. Virol. 69: 4711-4716); influenza virus nucleoprotein and hemagglutinin (Deck et al. (1997) Vaccine 15: 71-78; Fu et al. (1997) J. Virol. 71 : 2715-2721); B19 parvovirus capsid proteins VP1 (Kawase et al. (1995) Virology 211: 359-366) or VP2 (Brown et al. (1994) Virology 198: 477-488); Hepatitis B virus core and e antigen (Schodel et al. (1996) I 'ntervirology 39: 104-106); hepatitis B surface antigen (Shiau and Murray (1997) J. Med. Virol. 51: 159-166); hepatitis B surface antigen fused to the core antigen of the virus (Id.); Hepatitis B virus core-preS2 particles (Nemeckova et al. (1996) Acta Virol. 40: 273-279); HBV preS2-S protein (Kutinova et al. (1996) Vaccine 14: 1045-1052); VZV glycoprotein I (Kutinova et al. (1996) Vaccine 14: 1045-1052); rabies virus glycoproteins (Xiang et al. (1994) Virology 199: 132-140; Xuan et al. (1995) Virus Res. 36: 151-161) or ribonucleocapsid (Hooper et al. (1994) Proc. Nat'l. Acad. Sci. USA 91 : 10908-10912); human cytomegalovirus (HCMV) glycoprotein B (UL55) (Britt et al. (1995) J. Infect. Dis. 171: 18-25); the hepatitis C virus (HCV) nucleocapsid protein in a secreted or a nonsecreted form, or as a fusion protein with the middle (pre-S2 and S) or major (S) surface antigens of hepatitis B virus (HBV) (Inchauspe et al. (1997) DNA Cell Biol. 16: 185-195; Major et al. (1995) J. Virol. 69: 5798- 5805); the hepatitis C virus antigens: the core protein (pC); E1 (pE1) and E2 (pE2) alone or as fusion proteins (Saito et al. (1997) Gastroenterology 112: 1321-1330); the gene encoding respiratory syncytial virus fusion protein (PFP-2) (Falsey and Walsh (1996) Vaccine 14: 1214-1218; Piedra et al. (1996) Pediatr. Infect. Dis. J. 15: 23-31); the VP6 and VP7 genes of rotaviruses (Choi et al. (1997) Virology 232: 129-138; Jin et al. (1996) Arch. Virol. 141 : 2057-2076); the E1 , E2, E3, E4, E5, E6 and E7 proteins of human papillomavirus (Brown et al. (1994) Virology 201 : 46-54; Dillner et al. (1995) Cancer Detect. Prev. 19:381-393; Krul et al. (1996) Cancer Immunol. Immunother. 43: 44-48; Nakagawa et al. (1997) J. Infect. Dis. 175: 927-931); a human T-lymphotropic virus type I gag protein (Porter et al. (1995) J. Med. Virol. 45: 469-474); Epstein-Barr virus (EBV) gp340 (Mackett et al. (1996) J. Med. Virol. 50:263-271); the Epstein-Barr virus (EBV) latent membrane protein LMP2 (Lee et al. (1996) Eur. J. Immunol. 26: 1875-1883); Epstein-Barr virus nuclear antigens 1 and 2 (Chen and Cooper (1996) J. Virol. 70: 4849-4853; Khanna et al. (1995) Virology ' 214: 633-637); the measles virus nucleoprotein (N) (Fooks et al. (1995) Virology 210: 456-465); and cytomegalovirus glycoprotein gB (Marshall et al. (1994) J. Med. Virol. 43: 77-83) or glycoprotein gH (Rasmussen et al. (1994) J. Infect. Dis. 170: 673-677).

10104] Examples of medical conditions and/or diseases where down-regulation or decreased immune response is desirable include, but are not limited to, allergy, asthma, autoimmune diseases (e.g., rheumatoid arthritis, SLE, diabetes mellitus, myasthenia gravis, reactive arthritis, ankylosing spondylitis, and multiple sclerosis), septic shock, organ transplantation, and inflammatory conditions, including IBD, psoriasis, pancreatitis, and various immunodeficiencies.

[0105]_' Autoimmune diseases and inflammatory conditions are often characterized by an accumulation of inflammatory cells, such as lymphocytes, macrophages, and neutrophils, at the sites of inflammation. Altered cytokine production levels are often observed, with increased levels of cytokine production. Several autoimmune diseases, including diabetes and rheumatoid arthritis, are linked to certain MHC haplotypes. Other autoimmune-type disorders, such as reactive arthritis, have been shown to be triggered by bacteria such as Yersinia and Shigella, and evidence suggests that several other autoimmune diseases, such as diabetes, multiple sclerosis, rheumatoid arthritis, may also be initiated by viral or bacterial infections in genetically susceptible individuals. Examples of antigens for use in recombinant strains and methods of the present invention to treat autoimmune diseases, inflammatory conditions, and other immunodeficiency-associated conditions are provided in Punnonen et al. (1999) WO 99/41369; Punnonen et al. (1999) WO 99/41383; Punnonen et al. (1999) WO 99/41368; and Punnonen et al. (1999) WO 99/41402), each of which is incorporated herein by reference for all purposes.

[0i06j For treatment or prevention of such diseases or conditions, recombinant strains comprising one or more polypeptides, proteins, peptides, or nucleic acids capable of reducing or suppressing an immune response (e.g., antigens specific for or associated with a disease), such as T cell proliferation or activation, can be administered according to the methods described herein. [0107] For example, in another aspect, the invention provides recombinant strains and vaccines for treating allergies, and prophylactic and therapeutic treatment methods utilizing such strains and vaccines.

[0108] Examples of allergies that can be treated using the methods and recombinant strains of the invention include, but are not limited to, allergies against house dust mite, grass pollen, birch pollen, ragweed pollen, hazel pollen, cockroach, rice, olive tree pollen, fungi, mustard, bee venom. Antigens of interest include those of animals, including the mite (e.g., Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis), such as the allergens der p1 (Scobie et al. (1994) Biochem. Soc. Trans. 22: 448S; Yssel et al. (1992) J. Immunol. 148: 738-745), der p2 (Chua et al. (1996) CHn. Exp. Allergy 26: 829-837), der p3 (Smith and Thomas (1996) Clin. Exp. Allergy 26: 571- 579), der p5, der p V (Lin et al. (1994) J. Allergy Clin. Immunol. 94: 989-996), der p6 (Bennett and Thomas (1996) Clin. Exp. Allergy 26: 1150-1154), der p 7 (Shen et al. (1995) Clin. Exp. Allergy 25: 416-422), der f2 (Yuuki et al. (1997) Int. Arch. Allergy Immunol. 112: 44-48), der f3 (Nishiyama et al. (1995) FEBS Lett. 377: 62-66), der f7 (Shen et al. (1995) CHn. Exp. Allergy 25: 1000-1006); Eur m 1 and Eur m 2; Mag 3 (Fujikawa et al. (1996) MoI. Immunol. 33: 311-319). Also of interest as antigens for use with the invention are the house dust mite allergens Tyr p2 (Eriksson et al. (1998) Eur. J. Biochem. 251 : 443-447), Lep d1 (Schmidt et al. (1995) FEBS Lett 370: 11-14), and glutathione S- transferase (O'Neill et al. (1995) Immunol Lett. 48: 103-107); the 25,589 Da, 219 amino acid polypeptide with homology with glutathione S-transferases (O'Neill et al. (1994) Biochim. Biophys. Acta. 1219: 521-528); BIo t 5 (Arruda et al. (1995) Int. Arch. Allergy Immunol. 107: 456-457); bee venom phospholipase A2 (Carballido et al. (1994) J. Allergy CHn. Immunol. 93: 758-767; Jutel et al. (1995) J. Immunol. 154: 4187-4194); bovine dermal/dander antigens BDA 11 (Rautiainen et al. (1995) J. Invest. Dermatol. 105: 660-663) and BDA20 (Mantyjarvi et al. (1996) J. Allergy Clin. Immunol. 97: 1297-1303); the major horse allergen Equ d (Gregoire et al. (1996) J. Biol. Chem. 271 : 32951- 32959); Jumper ant M. pilosula allergen Myr p I and its homologous allergenic polypeptides Myr p2 (Donovan et al. (1996) Biochem. MoI. Biol. Int. 39: 877-885); 1-13, 14, 16 kD allergens of the mite Blomia tropicalis (Caraballo et al. (1996) J. Allergy CHn. Immunol. 98: 573-579); the cockroach allergens BIa g Bd90K (Helm et al. (1996) J. Allergy CHn. Immunol. 98: 172-80) and BIa g 2 (Arruda et al. (1995) J. Biol. Chem. 270: 19563-19568); the cockroach Cr-Pl allergens (Wu et al. (1996) J. Biol. Chem. 271 : 17937-17943); fire ant venom allergen, Sol i 2 (Schmidt et al. (1996) J. Allergy CHn. Immunol. 98: 82-88); the insect Chironomus thummi major allergen Chi t 1-9 (Kipp et al. (1996) Int. Arch. Allergy Immunol. 110: 348-353); dog allergen Can f 1 or cat allergen FeI d 1 (Ingram et al. (1995) J. Allergy CHn. Immunol. 96: 449-456); albumin, derived, for example, from horse, dog or cat (Goubran Botros et al. (1996) Immunology 88: 340-347); deer allergens with the molecular mass of 22 kD, 25 kD or 60 kD (Spitzauer et al. (1997) CHn. Exp. Allergy 27: 196-200); and the 20 kd major allergen of cow (Ylonen et al. (1994) J. Allergy CHn. Immunol. 93: 851-858). [0109] Therapeutic and prophylactic agents and vaccines against food allergens and treatment methods for food allergies can also be developed using recombinant strains and the methods of the present invention. Suitable antigens for development of such vaccines include, for example, profilin (Rihs et al. (1994) Int. Arch. Allergy Immunol. 105: 190-194); rice allergenic cDNAs belonging to the alpha-amylase/trypsin inhibitor gene family (Alvarez et al. (1995) Biochim Biophys Acta 1251 : 201- 204); the main olive allergen, Ole e I (Lombardero et al. (1994) CHn Exp Allergy 24: 765-770); Sin a 1 , the major allergen from mustard (Gonzalez De La Pena et al. (1996) Eur J Biochem. 237: 827-832); parvalbumin, the major allergen of salmon (Lindstrom et al. (1996) Scand. J. Immunol. 44: 335-344); apple allergens, such as the major allergen MaI d 1 (Vanek-Krebitz et al. (1995) Biochem. Biophys. Res. Commun. 214: 538-551); and peanut allergens, such as Ara h I (Burks et al. (1995) J. CHn. Invest. 96: 1715-1721).

[oiioj Methods for administering vaccine products comprising recombinant strains of the present invention include those known to those having ordinary skill in the art. Suitable routes of administration or "delivery systems" include parenteral delivery and enteral delivery, such as, for example, oral, transdermal, transmucosal, intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracapsular, intraspinal, intrasternal, intrapulmonary, intranasal, vaginal, rectal, intraocular, and intrathecal, buccal (e.g., sublingual), respiratory, topical, ingestion, and local delivery, such as by aerosol or transdermal^, and the like. Methods for administering proteins, polypeptides, peptides, nucleic acids, and other molecules of interest to mucosal tissue via pulmonary inhalation, nasal, oral, vaginal, and/or rectal delivery are provided. The methods comprise preparing and administering to a subject a composition comprising a recombinant strain of the present invention. Such composition may include a carrier or excipient. In one embodiment of the invention, a polypeptide, protein, peptide, nucleic acid, or other molecule of interest is displayed on the surface of the spore. In another embodiment, the polypeptide, protein, or peptide of interest is expressed by the vegetative cells resulting from the germination and/or vegetative reproduction of a spore. In yet another embodiment, the spore displays a polypeptide, protein, or peptide with DNA binding capabilities that is bound to a DNA molecule encoding an antigen or immunomodulatory molecule or that is an antigen or immunomodulatory molecule.

[oiii] These methods of inoculation and/or immunization can also be utilized for herd animals in a field, such as cattle grazing over an extended area, or for fish in their native aquatic habitats. Subject animals also include wild animals. For example, subjects include American buffalo (bison), which often carry the disease brucellosis, which can infect humans and causes spontaneous abortions in cattle. In another embodiment, rabies vaccinations or therapeutic or prophylactic agents comprising spore systems of the invention are administered to a variety of wild animal populations in a particular area by distributing spores from an overflying plane. Thus, the present invention provides a relatively inexpensive means for vaccinating or treating wild populations against a variety of illnesses and diseases. Diseases and illnesses that are potential targets of this vaccination approach include those caused by cholera (e.g., enterotoxins from V. cholerae), Japanese encephalitis, tick-borne encephalitis, Venezuelan Equine encephalitis, enterotoxins produced by Staphylococcus and Streptococcus species, and enterotoxigenic strains of E. coli (e.g., heat-labile toxin from E. coli), and salmonella toxin, shigella toxin and Campylobacter toxin, dengue fever, and hantavirus.

[0112] Distribution of the vaccine or other prophylactic or therapeutic agent comprising a recombinant strain of the present invention to fish in the aquaculture or aquarium trades can be accomplished by injection or immersion techniques. Immersion, or dipping, is an inoculation or vaccination method well known to one of skill in the art (see e.g., Vinitnantharat et al. (1999) Adv. Vet. Med. 41 :539-550). A dip treatment involves dipping whole fish in a dilution of the inoculant or vaccine whereupon the inoculant or vaccine is absorbed by the gills. Intraperitoneal injection is another vaccination method well known to one of skill in the art. Injection involves anesthetizing and injecting the fish intraperitoneal^ (Vinitnantharat et al. (1999) Adv. Vet. Med. 41:539-550). Diseases of cultivated fish that may be treated using a spore system of the invention include, but are not limited to, infectious pancreatic necrosis (IPN), infectious hematopoietic necrosis (IHN), Vibriosis (Vibrio anguillarum), cold-water vibriosis {Vibrio salmonicida), Vibrio ordalii, winter ulcer (Vibrio viscosus), Vibrio wodanis, yersiniosis (Yersinia ruckeri) or Enteric Red Mouth, Bacterial Kidney Disease, Fumnculosis (Aeromonas salmonicida subsp. salmonicida), Saddleback, Gafkemia, Dollfustrema vaneyi, Cryptobia bullocki, Cryptobia salmositica, Listeria monocytogenes, Photobacterium damsela subsp. piscicida, and Microcotyl sebastis. Fish species of interest include, but are not limited to, salmonids, including Rainbow Trout (Onchorhycus mykiss), salmon (Salmo salar), Coho salmon (Oncorhynchus kisutch), Steelhed (Oncorhynchus mykiss), rockfish (Sebastis schlegeli), catfish (lctalurus punctatus), yellowtail, Pseudobagrus fulvidraco, Gilt-head Sea Bream, Red Drum, European Sea Bass fish, striped bass, white bass, yellow perch, whitefish, sturgeon, largemouth bass, Northern pike, walieye, black crappie, fathead minnows, and Golden Shiner minnows. Invertebrates of interest include, but are not limited to, oysters, shrimp, crab, and lobsters.

[0113] Delivery by pulmonary inhalation, nasal delivery, gill delivery, or respiratory delivery provides a promising route for absorption of polypeptides and other molecules of interest having poor oral bioavailability due to inefficient transport across the gastrointestinal epithelium or high levels of first- pass hepatic clearance. By "nasal delivery" is intended that the polypeptide is administered to the subject through the nose. By "pulmonary inhalation" is intended that the polypeptide or other substance of interest is administered to the subject through the airways in the nose or mouth so as to result in delivery of the polypeptide or other substance to the lung tissues and into the interior of the lung. Both nasal delivery and pulmonary inhalation can result in delivery of the polypeptide or other substance to the lung tissues and into the interior of the lung, also referred to herein as "pulmonary delivery." By "respiratory delivery" is intended that the polypeptide or other substance is administered to the subject through the respiratory system of the subject so as to result in delivery of the polypeptide or other substance to the organs and tissues of the respiratory system of the subject organism. The organs and tissues of the respiratory system of a subject organism include, but are not limited to, the lungs, nose, or gills. Potential advantages of these delivery routes for polypeptides and other molecules of interest include a greater extent of absorption due to an absorptive surface area of approximately 140 m.sup.2 and high volume of blood flowing through the lungs (5000 ml/min in the human lung) (Hollinger (1985), pp. 1-20, in Respiratory Pharmacology and Toxicology (Saunders, Pa.)). Further potential benefits of administration via pulmonary inhalation include lack of some forms of peptidase and/or protease activity when compared with the gastrointestinal tract and lack of first- pass hepatic metabolism of absorbed compounds. Interest in this delivery route has increased in recent years since a number of potential peptide-, polypeptide-, or protein-containing pharmaceuticals or drugs are absorbed more efficiently from the lung than from the gastrointestinal tract (Patton and Plate (1992) Adv. Drug Del. Rev. 8:179-196; Niven (1993) Pharm. Technol. 17:72-82). In fish, respiratory delivery of vaccines is the primary mode of vaccination due to the technical difficulties associated with injection of each fish and the destruction of most vaccines in the digestive tract of the fish.

[0114] Successful respiratory delivery of peptides, polypeptides, or proteins is dependent upon a number of factors but delivery can be readily optimized by varying such factors in routine experimentation by one of skill in the art. The extent of absorption within the respiratory tissues varies with size and structure of the polypeptide, peptide, or protein and the delivery device used. Recombinant strains, alone or in combination with other suitable components, can be made into aerosol formulations (e.g., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Delivery devices include nebulizers, metered-dose inhalers, powder inhalers, and dipping bags. Preparation of compositions, including those comprising spore systems, as an aqueous liquid aerosol, a nonaqueous suspension aerosol, or dry powder aerosol for pulmonary administration using these respective delivery devices can influence polypeptide stability, and hence bioavailability as well as biological activity following delivery. See Wall (1995) Drug Delivery 2:1-20; Krishnamurthy (March 1999) BioPharm., pp. 34-38). The enhanced stability of the recombinant strains of the present invention is therefore of value in administration by respiratory delivery. In addition, the Bt spore is between 1 and 1.5 uM in size which is the optimal size range for deep lung delivery, further enhancing its efficacy as a respiratory delivery vehicle. [0115] The following examples are provided to show that the methods of the present invention may be used to produce recombinant spores having a variety of uses. Those skilled in the art will recognize that while specific embodiments have been illustrated and described, they are not intended to limit the invention.

V. EXAMPLES

[0116] Materials used in the examples:

[0117] Bacterial Strains and Plasmids:

[0118] Bt SDS-502 strain: obtained from SDS-Biotech KK, Tukuba, Japan

[0119] Bt kurstaki HD73 strain: obtained from USDA, ARS, NRRL, Peoria, Illinois

[0120] E. coli TG1 : obtained from Invitrogen, Carlsbad, California

[0121] E. coli-Bt Shuttle Vector: obtained from Dr. Asano, Hokkaido University, Sapporo, Japan

[0122] pBluescript KS(+); obtained from Stratagene, La JoIIa, California

[0123] pBC16.1 : obtained from Bacillus Genetic Stock Center, Ohio State University, Columbus, Ohio

[0124] pUC19: obtained from New England Biolabs, Bevery, Massachusetts

[0125] tetR: The genetic sequence can be obtained, for example, from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov)

[0126] phospholipase C: The genetic sequence can be obtained, for example, from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov)

[0127] Bt genomic DNA samples are prepared from SDS-502 and HD73 strains using Qiagen QiaPrep Spin Miniprep kit (cat# 27106) with some modifications. Fresh Bacillus cells grown in LB for 3 hr at 30 ⁰C are harvested by centrifugation. The cells are suspended in P1 in the kit with 5 mg/ml lysozyme and incubated at 37 ⁰C for 30 min. To this suspension, an equal amount of 4% SDS is added instead of P2 in the kit. After this step, the instructions included in the kit are followed. [0128] EXAMPLE 1

[0129] The tetracycline resistance (tetR) gene is cloned from pBC16.1 and inserted into the phospholipase C gene site in the Bt host chromosome. Second, the inserted tetR gene is replaced with the Bt cryδDa (insecticidal crystal protein) gene from SDS-502.

[0130] PCR primers:

[0131] TefC-P/?osΛ/:CAGGAATTCCTTAAGGAACAGCAATAAGAAGTTAATTTTG <SEQ ID NO: 1>

[0132] 7efC-PήosΛ/-R/C:CAAAATTAACTTCTTATTGCTGTTCCTTAAGGAATTCCTG <SEQ ID

NO:2>

[0133] PΛos3P-R/C:ATATACTGCAGGAAGCTGAAGCAGCTCCG {Pst\) <SEQ ID NO:3>

[0134] TeWAMTTAGTGCACGTTCAACAAACGGGCC (ApalA) <SEQ ID NO:4>

[0135] P/7OS3-R/C: GAAGCTGAAGCAGCTCCG <SEQ ID NO:5>

[0136] 8DMATATACCATGGGTCCAAATAATCAAAATGAATATG (Λ/CO/I) <SEQ ID NO:6>

[0137] 8DC-R/C:TTATAGGATCCTTACTCTTCTTCTAACACGAGTTCTAC (BamH\) <SEQ ID NO:7>

10138] P/?osΛ/:AGCAATAAGAAGTTAATTTTG <SEQ ID NO:8>

[0139] SΛuΛRGATCCTTTTTTTATAACAGGAATTCG <SEQ ID NO:9>

[0140] S/?i/Λ/O-R/C:CGAATTGGAGCTCCACCGCGGTG <SEQ ID NO: 10>

[0141] Insertion of tetR in Bt

An approximately 1.2 kb Phospholipase C gene (Phos fragment: ATG deleted protein coding region plus flanking 3' sequence) is amplified by PCR from Bt HD73 genomic DNA with primers TetC-PhosN and Phos3P-R/C, and an approximately 1.6 kb tetracycline resistance gene (TetR fragment) is amplified by PCR from pBC16-1 with primers TetNA and TetC-PhosN-R/C. These PCR amplified fragments, Phos (X12952, Henner et al., Sequence of the Bt phosphatidylinositol specific phsopholipase C, Nucleic Acids Res. 16, 10383, 1988) and TetR (Palva et al., Nucleotide sequence of the tetracycline resistance gene of pBC16 from Bacillus cereus., Nucleic Acids Res. 18, 1635, 1990), are purified by gel electrophoresis and are assembled by thermal cycle reaction with Taq polymerase utilizing the TetC-PhosN sequence overlap (15 cycles at 45 ⁰C annealing without primers). The assembled -2.8 kb TetR-Phos is then rescued by PCR reaction (25 cycles) with primers TetNA and Phos3P-R/C and digested with ApaL\ and Pst\. The digested fragment is purified by gel electrophoresis. In order to clone this ApaL\ and Psfl-cut TetR-Phos in pUC19, pUC19 is cut with Apal_1 and Pstl producing 4 bands, 270, 500, 670 and 1250 bp. The 670 bp band that contains the E. coli ori is purified by gel electrophoresis. The ~2.8 kb TetR-Phos is ligated with the 670-bp ApaLI-Pstl E. coli ori fragment of pUC19 and cloned in E. coli TG1 with tetδ selection. Several colonies are picked, and restriction sites and the Phospholipase sequence are checked by mapping and PCR amplification. This pUC19 based plasmid called pSCO1 (FIG. 1) is amplified in E. coli GM2163 to produce a methylation minus pSCO1 preparation used to transform competent Bt HD73 cells by electroporation. About 500 ng of pSCO1 are used to transform 106 competent Bt cells suspended in 100 μl 0.5 M sucrose containing 5 mM HEPS, pH 7. Electroporation is conducted at 1.25 kV, 3 microFaradays without any parallel resistance (infinite resistance). The electroporated cells are placed on LBtetiO to find tetracycline resistant colonies. The insertion of pSCO1 in the Bt chromosome by single crossover recombination is confirmed by PCR with Phosδ and Phos3-R/C.

[0142] Cloning Cry8D in pBluescript with cry1 Ca promoter and cry1 Ac terminator:

[0143] A Bacillus-E. coli shuttle vector containing cryi la (cryV), cryiCa promoter and cryiAc terminator is obtained from Dr. S. Asano, Hokkaido University, Sapporo, Japan (Curr. Microbiol. 32 (1996), 195-200) and cut with Apal and Notl producing -2.7 kb and ~6 kb fragments. The 2.7 kb fragment is purified by gel electrophoresis and is cloned into pBluescript KS(+) between Apa\ and Λ/ofl. The resultant Bluescript plasmid is called pBScryl la. The 3.6 kb cryδDa coding region is amplified by PCR from the Bt SDS-502 strain with primers 8DN and 8DC-R/C, digested with Nott and Bamti\ and purified by gel electrophoresis. The pBScrylla plasmid is cut with Notl and BamH\ producing -2.1 kb and -3.6 kb fragments. The 3.6 kb fragment is purified by gel electrophoresis. The 3.6 kb pBScryl la fragment is ligated with the Not\/BamH\-cut 3.6 kb cryδDa gene and cloned in E. coli TG1 to produce the plasmid called p8DA (FIG. 2).

[0144] Cloning the phospholipase C gene in pδDA:

[0145] A 1.2 kb fragment of non-functional phospholipase C gene consisting of ATG-deleted coding region and flanking 3' sequence is amplified by PCR from Bt HD73 genomic DNA with primers phosN and Phos3-R/C. This PCR amplified phospholipase fragment is cloned in pδDA that has been cut open with Apa\ and treated with the Klenow fragment of E. coli DNA polymerase. A clone having the phospholipase gene in the same orientation as that of the cry8Da gene is selected. This new plasmid is called p8DA-1 P. The p8DA-1P plasmid is cut with Not\ and treated with Klenow. Another copy of the 1.2 kb PCR amplified the phospholipase fragment is cloned in the Λ/ofl-cut p8DA-1 P. A clone having the phospholipase gene in the same orientation as that of the cryδDa gene is selected. Now p8DA has two copies of the phospholipase fragment, one at the Apa\ site and the other at the Λ/ofl site. This new plasmid is called p8DA-2P.

[0146] Replacing the tetracycline gene with cryδDa by double-crossing integration:

[0147] Approximately 6.6-kb phos-cry8Da-phos fragment is amplified by PCR using TaKaRa Ex Taq™ polymerase from p8DA-2P with primers ShuAP and ShuNO-R/C and is electroporated into competent Bt HD73 cells that contain the tetracycline resistance gene in the chromosome. About 1 μg of DNA was used in competent Bt cells suspended in 100 μl of 0.5 M sucrose containing 5 mM HEPS, pH 7. The electroporated cells are plated on 400 20x20-cm LB-agar plates (no selection) at a cell density of about 3000 per plate and incubate overnight at 30 ⁰C. At this cell density, individual colonies are well ' isolated. The colonies are transferred to new LBtetiO plates with sheets of nitrocellulose membrane to determine which colonies are sensitive to tetracycline. A few tetracycline- sensitive colonies are picked and the insertion of cryδDa and removal of tetracycline resistance gene is confirmed by colony PCR using primers PhosN and 8DC-R/C. Alternatively, transformed cells containing the cryδDa gene can be screened by PCR or antiserum made against the CryδDa protein. In the case of PCR screening, cells are harvested from copied plates made by nitrocellulose membrane transfer, the genomic DNA samples are prepared from cells obtained from each plate, and each DNA sample is analyzed by PCR for cryδDa. In order to have a reasonable cell count in one DNA preparation, plates are divided by 8 (each containing about 250 colonies). From those samples with cryδDa positive, the PCR screening is repeated with subdivided colonies from those positive plate areas. For antiserum screening, the cells on each plate subdivision are allowed to grow until sporulation in LB (the Applicants have found that B. subtilis does not sporulate on LB). Crystals and spores are collected and analyzed for cryδDa. Colonies on the positive plates are subdivided and screened for cryδDa expression until individual colonies with cryδDa are found.

[0148] Additional examples related to Example 1 :

[0149] Example 1, described supra, utilizes an antibiotic resistance gene that is inserted into the host during the first step. When that first inserted gene is replaced with a desired gene to be introduced to the host, the resulting recombinant host with the desired gene can be identified for the lack of the first inserted gene. Any other selection or identification markers such as color markers like GFP and beta- galactosidase genes can be used in place of the antibiotic resistance marker gene. As with an antibiotic-resistance gene, the color marker gene or beta-galactosidase gene, or DNA fragment or any other gene or DNA sequence known to those of skill in the art that is readily adaptable for selection purposes that has been inserted in the host DNA in the first step is removed from the host when the second step integration takes place.

[0150] EXAMPLE 2:

[0151] This example utilizes a gene in the host as a selection marker. First, a functional (e.g., antibiotic resistance gene) or a non-functional DNA sequence is inserted by single cross over reaction into a host target gene (e.g., the sporulation gene or amylase gene) in such a way as to disrupt the function of the target gene, and then the disrupted gene is restored by inserting the disrupted part of the target site gene along with a desired gene to be introduced to the host. The restored target gene can be used to select the recombinant cells that have the inserted desired gene.

[0152] Primers:

[0153] PANE: ATATAGAATTCGTAGCTGCTCAAGATGATATGG (EcoRI) <SEQ ID NO: 11 >

[0154] PACP-R/C: ATATACTGCAGTTAGGACGAAATAGAATCAATATTCC(PStI) <SEQ ID NO:12>

[0155] Spo3-R/C: GATTATGATGTCAAACCCTCG <SEQ ID NO:13>

[0156] Insertion of tetR into the spoOA gene of the host Bt:

[0157] A 630 bp fragment of N-terminal and C-terminal truncated spoOA (protein A) gene is amplified by PCR from Bt HD73 genomic DNA with primers PANE and PACP-R/C. The PCR amplified fragment is cut with EcoRI and Pst\ and purified by gel electrophoresis. The pSCO1 plasmid is cut with EcoRI and Pst\ to generate -2.3, -0.8, -0.4 kb fragment. The 2.3 kb EcoM-Psti fragment of pSCO1 is purified by gel electrophoresis and ligated with the 630 bp fragment of the EcoR\/Pst\ cut spoOA gene. This ligated DNA is cloned in E. coli TG1 using tet5 selection. Several colonies are picked, and restriction sites and spoOA sequence are checked by mapping and PCR amplification. This pSCO1 based plasmid, called pSCO2 (FIG. 3), is amplified in E. coli GM2163 to produce a methylation minus preparation used to transform competent Bt HD73 cells by electroporation. About 500 ng of pSCO2 are used in 106 competent Bt cells suspended in 100 μl of 0.5 M sucrose containing 5 mM HEPS, pH 7. The electroporated cells are placed on LBtetiO to find tetracycline-resistant colonies. The insertion of pSCO2 in the Bt chromosome by single crossover recombination is confirmed by PCR. These recombinant Bt hosts fail to produce spores as a result of the tetracycline resistance gene inserted into the spoOA gene. The loss of sporulation function of the host Bt is confirmed by examination under a microscope.

[0158] Cloning the spoOA gene in p8DA:

[0159] A 1.05 kb fragment containing the spoOA (protein A) coding region, without the translation start codon and its 3" flanking region, is amplified by PCR from Bt HD73 genomic DNA with primers PAN and Spo3-R/C and cloned in >4pal-cut, Klenow-treated pδDA in E. coli TG1. A clone having the spoOA gene in the same orientation as that of the cryδDa gene is found by restriction enzyme mapping and PCR. This new plasmid is called p8DA-1S. The p8DA-1S plasmid is cut with Not\ and treated with Klenow. Another copy of the 1.05 kb PCR amplified spoOA gene fragment is cloned in the /Vofl-cut p8DA-1S. A clone having the spoOA gene in the same orientation as that of the cryδDa gene is selected. The p8DA vector plasmid now has two copies of the spoOA gene, one at the Apa\ site and the other at the Λ/ofl site. This new plasmid is called p8DA-2S.

[0160] Replacing the spoOA gene with cryδDa by double-crossing integration:

[0161] Approximately 6.2-kb spoOA-cry8Da-spoOA fragment is amplified by PCR using TaKaRa Ex Taq™ polymerase from p8DA-2S with primers ShuAP and ShuNO-R/C and is electroporated into competent Bt HD1 cells whose spoOA has been disrupted. About 1 μg of DNA is used in competent Bt cells suspended in 100 μl 0.5 M sucrose containing 5 mM HEPS, pH 7. The electroporated Bt cells are plated on LB-agar plates (no selection) and incubated overnight at 30 ⁰C. The cells are collected from the plates, suspended in 10 ml LB liquid medium, and incubated at 30 ⁰C for 72 hr with shaking. Those cells which fail to sporulate are killed at 80 ⁰C for 15 min. The heat-treated Bt culture is diluted with 100 ml of LB liquid medium and incubated at 30 ⁰C for 48 hr with shaking. At this point, the existence of spores is confirmed under the microscope. From this 100-ml culture, a 10-ιml aliquot is removed, treated at 80 ⁰C for 15 min and plated on LB-agar plates with serial dilutions. The plates are incubated overnight at 30 ⁰C to produce a number of well-isolated colonies. A few colonies are picked and checked for the cryδDa insertion by PCR and sporulation by microscopic observation.

[0162] EXAMPLE 3:

[0163] In this example, alpha-amylase gene is used as the target site where a desired gene is inserted without any foreign selection marker.

[0164] Primers: [0165] AmN: TTTGGGTGAATTCAATCAAAAGGG (EcoRI) <SEQ ID NO:14>

[0166] AmCP-R/C:ATATACTGCAGTTAAGGATATCCCTCTGACAGAG (Pstl) <SEQ ID NO:15>

[0167] Am3-R/C:CTACTGGTGTATACTCAG <SEQ I D NO: 16>

[0168] Insertion of tetR into the alpha-amylase gene of the host Bt:

[0169] An 880 bp fragment of N-terminal and C-terminal truncated alpha-amylase (amy) gene is amplified by PCR from Bt HD73 genomic DNA with primers AmN and AmCP-R/C. The PCR amplified fragment is cut with EcoRI and Pst\ and purified by gel electrophoresis. The pSCO1 plasmid is cut with EcoR\ and Pst\ to generate -2.3, -0.8, -0.4 kb fragments. The 2.3 kb EcoRI-Psfl fragment of pSCO1 is purified by gel electrophoresis and ligated with the 880 bp fragment of the EcoRUPstl cut amy gene. The ligated DNA is cloned in E. coli TG1 using tet5 selection. Several colonies are picked, and restriction sites and amy sequence are checked by mapping and PCR amplification. The pSCO1- based plasmid, called pSCO3 (FIG. 4), is amplified in E. coli GM2163 to produce a methylation minus DNA preparation used to transform competent Bt HD73 cells by electroporation. About 500 ng of pSCO3 are used in 106 competent Bt cells suspended in 100 μl 0.5 M sucrose containing 5 mM HEPS, pH 7. The electroporated cells are placed on LBteti O to find tetracycline-resistant colonies. The insertion of pSCO3 in the Bt chromosome by single crossover recombination is confirmed by PCR. These recombinant Bt hosts fail to produce spores as a result of the tetracycline resistance gene's insertion into amy.

[0170] Cloning the amy gene in p8DA:

[0171] A 1.6 kb fragment containing the amy coding region without approximately 90 codons at the N-terminal up to the naturally occurring EcoRI site and its 3' flanking region is amplified by PCR from Bt HD73 genomic DNA with primers AmN and Am3-R/C and cloned in /\pal-cut, Klenow-treated p8DA in E. coli TG1. A clone having the spoOA gene in the same orientation as that of the cryδDa gene is found by restriction enzyme mapping and PCR. This new plasmid is called p8DA-1A. The p8DA-1A plasmid is cut with Λ/ofl and treated with Klenow. Another copy of the 1.6 kb PCR amplified amy gene fragment is cloned in the Λ/ofl-cut p8DA-1A. A clone having the amy gene in the same orientation as that of the cryδDa gene is selected. Now the pδDA vector plasmid has two copies of the amy gene, one at the Apa\ site and the other at the Not\ site. This new plasmid is called p8DA-2A.

[0172] Replacing the amy gene with cryδDa by double-crossing integration: [0173] Approximately 7.4-kb amy-cry8Da-amy fragment is amplified by PCR using TaKaRa Ex Taq™ polymerase from p8DA-2S with primers ShuAP and ShuNO-R/C and is electroporated into competent Bt HD73 cells whose amy has been disrupted. About 1 μg of DNA is used in 106 competent Bt cells and suspended in 100 μl 0.5 M sucrose containing 5 mM HEPS, pH 7. The electroporated Bt cells are plated on starch-ammonium sulfate-agarose plates and incubated overnight at 30 °C. The cells are collected from the plates, suspended in 1 ml water and plated on the starch plates. The plates are incubated overnight at 30 ⁰C to produce a number of well-isolated colonies. A few colonies are picked and checked for the cry8Da insertion by PCR.

[0174] Alternative means to produce double-crossing insertion sequence:

[0175] Plasmids, p8DA-2P, p8DA-2S, p8DA-2A, are linearized by digestion with ApaU and used to insert the cryδDa gene at phospholipase C (p8DA-2P), spoOA(p8DA-2S) and alpha-amylase (p8DA- 2A) sites.

[0176] Although the foregoing invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced without departing from the spirit and scope of the invention. Therefore, the description should not be construed as limiting the scope of the invention, which is delineated by the appended claims.

[0177] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference.

Additional Sequences

[0178] Phospholipase C from Bacillus cereus.

MKKKVLALAAAITLVAPLQSVAFAHENDGGSKIKIVHRWSAEDKHKEGVNSHLWIVNRAIDIMSRNTTLVKQDRV AQLNEWRTELENGIYAADYENPYYDNSTFASHFYDPDNGKTYIPFAKQAKETGAKYFKLAGESYKNKDMKQAFFY LGLSLHYLGDVNQPMHAANFTNLSYPQGFHSKYENFVDTIKDNYKVTDGNGYWNWKGTNPEDWIHGAΆVVAKQDY SGIVNDNTKDWFVKAAVSQEYADKWRAEVTPMTGKRLMDAQRVTAGYIQLWFDTYGDR <SEQ ID NO:17>

Atggaggatttcagatgaaaaagaaagtacttgctttagcagcagctattacattagtagctcctttacaaagcg ttgcatttgctcatgaaaacgatgggggaagtaaaataaaaatagttcaccgctggtctgctgaagataaacata aagaaggcgtaaattctcatttatggattgtaaaccgtgcgattgatattatgtctcgcaatacaacacttgtaa aacaagatcgagttgcacaattaaatgaatggcgtacggagttagagaacggtatttatgctgctgactatgaaa atccttattatgataatagtacatttgcttcacatttctatgatccagacaatggaaaaacatatattccatttg caaagcaggcaaaagaaactggagctaaatattttaaattagctggtgaatcatataaaaataaagatatgaaac aagcattcttctatttaggattatctcttcattatttaggagatgtaaaccaaccgatgcatgcagcaaacttta caaatctttcgtatccacaaggattccattctaaatagaaaactttgtagatacgataaaagataattataaagt aacggatggaaatggatattggaattggaaaggtacaaatccagaagattggatccatggagcggcagtagtggc gaaacaagattactctggaattgtaaatgataatacgaaagattggttcgtaaaagcagctgtgtcacaagaata tgcagataaatggcgtgctgaagttacaccgatgacaggtaagcgattaatggatgcccaacgtgttactgctgg atacattcagctttggtttgatacgtacggagatcgttaa <SEQ ID NO: 18>

[0179] TetR

MNTSYSQSNLRHNQILIWLCILSFFSVLNEMVLNVSLPDIANDFNKPPASTNWVNTAFMLTFSIGTAVYGKLSDQ LGIKRLLLFGIIINCFGSVIGFVGHSFSLLIMARFIQGAGAAAFPALVMVVVARYIPKENRGKAFGLIGSIVAMG

IVSVLS-FLIFVKHIRKVTDPFVDPGLGKNIPFMIGVLCGGIIFGTVAGFVSMVPYMMKDVHQLSTAEIGSVIIFP

LKQQEAGAGMSLLNFTSFLSEGTGIAIVGGLLSIPLLDQRLLPMEVDQSTYLYSNLLLLFSGIIVISWLVTLNVY KHSQRDF <SEQ ID NO: 19>

Upstream promoter TetR tattgttgta taagtgatga aatactgaat ttaaaactta gtttatatgt ggtaaaatgt tttaatcaag tttaggagga attaattatg aagtgtaatg aatgtaacag ggttcaatta aaagagggaa gcgtatcatt aaccctataa actacgtctg ccctcattat tggagggtga a <SEQ ID NO: 20>

coding TetR atgtgaata catcctattc acaatcgaat ttacgacaca accaaatttt aatttggctt tgcattttat ctttttttag cgtattaaat gaaatggttt tgaacgtctc attacctgat attgcaaatg attttaataa accacctgcg agtacaaact gggtgaacac agcctttatg ttaacctttt ccattggaac agctgtatat ggaaagctat ctgatcaatt aggcatcaaa aggttactcc tatttggaat tataataaat tgtttcgggt cggtaattgg gtttgttggc cattctttct tttccttact tattatggct cgttttattc aaggggctgg tgcagctgca tttccagcac tcgtaatggt tgtagttgcg cgctatattc caaaggaaaa taggggtaaa gcatttggtc ttattggatc gatagtagcc atgggagaag gagtcggtcc agcgattggt ggaatgatag cccattatat tcattggtcc tatcttctac tcattcctat gataacaatt atcactgttc cgtttcttat gaaattatta aagaaagaag taaggataaa aggtcatttt gatatcaaag gaattatact aatgtctgta ggcattgtat tttttatgtt gtttacaaca tcatatagca tttcttttct tatcgttagc gtgctgtcat tcctgatatt tgtaaaacat atcaggaaag taacagatcc ttttgttgat cccggattag ggaaaaatat accttttatg attggagttc tttgtggggg aattatattt ggaacagtag cagggtttgt ctctatggtt ccttatatga tgaaagatgt tcaccagcta agtactgccg aaatcggaag tgtaattatt ttccctggaa caatgagtgt cattattttc ggctacattg gtgggatact tgttgataga agaggtcctt tatacgtgtt aaacatcgga gttacatttc tttctgttag ctttttaact gcttcctttc ttttagaaac aacatcatgg ttcatgacaa ttataatcgt atttgtttta ggtgggcttt cgttcaccaa aacagttata tcaacaattg tttcaagtag cttgaaacag caggaagctg gtgctggaat gagtttgctt aactttacca gctttttatc agagggaaca ggtattgcaa ttgtaggtgg tttattatcc atacccttac ttgatcaaag gttgttacct atggaagttg atcagtcaac ttatctgtat agtaatttgt tattactttt ttcaggaatc attgtcatta gttggctggt taccttgaat gtatataaac attctcaaag ggatttctaa <SEQ ID NO: 21>

Down stream of termination TetR atcgttaagg gatcaacttt gggagagagt tcaaaattga tccttttttt ataacaggaa ttcaaatctt tttgttccat taaa <SEQ ID NO: 22>

Claims

We claim:

1. A recombinant bacteria comprising a recombined endogenous gene, a recombined endogenous gene copy, and a DNA insert, wherein the DNA insert is between the recombined endogenous gene and the recombined endogenous gene copy, and wherein the recombinant bacteria lacks an exogenous selection marker.

2. The recombinant bacteria of claim 1 wherein the bacteria is used in a biologically active agent that is released into the open environment.

3. The recombinant bacteria of claim 1 wherein the bacteria is a gram-positive bacteria.

4. The recombinant bacteria of claim 1 wherein the bacteria is an insect pathogenic bacteria.

5. The recombinant bacteria of claim 1 wherein the bacteria is a Bacillus bacteria or a Clostridium bacteria.

6. The recombinant bacteria of claim 1 wherein the bacteria is a Bacillus species such as B. albus, B. anthracis, B. brevis, B. cereus, B. coagulans, B. colistinus, B. larvae, B. lentimorbus, B. licehnfiformis, B. megaterium, B. mycoides, B. polymyxa, B. popilliae (aka: Paenibacillus popilliae), B. radicicola, B. sphaericus, B. stearothermophilus, B. subtilis, B. theromoacidurans and B. thuringiensis or a Clostridium species such as C. acetobutylicum, C. bifermentans, C. botulinum, C. brevifaciens, C. butyricum, C. chauvoei, C. dissolvens, C. fallax, C. histolyticum, C. nigrificans, C. novyi, C. paterurianum, C. perfringens, C. putrificum, C. septicum, C. sporogenes, C. tetani, C. thermohyfrosulfuricum, C. thermosaccharolyticum and C. welchii..

7. The recombinant bacteria of claim 1 wherein the recombined endogenous gene and the recombined endogenous gene copy are identical.

8. The recombinant bacteria of claim 1 wherein the recombined endogenous gene and the recombined endogenous gene copy are different.

9. The recombinant bacteria of claim 1 wherein the recombined endogenous gene is a selectable gene or a screenable gene.

10. The recombinant bacteria of claim 1 wherein the recombined endogenous gene is neither a selectable gene nor a screenable gene.

11. The recombinant bacteria of claim 1 wherein the recombined endogenous gene copy comprises a first mutation.

12. The recombinant bacteria of claim 11 wherein the first mutation is a selectable mutation or a screenable mutation.

13. The recombinant bacteria of claim 11 wherein the first mutation is a truncation mutation, an internal mutation, or a chimeric mutation.

14. The recombinant bacteria of claim 11 wherein the recombined endogenous gene comprises a second mutation.

15. The recombinant bacteria of claim 14 wherein the first mutation and the second mutation are different.

16. The recombinant bacteria of claim 1 wherein the recombined endogenous gene is selected from the group consisting of phospholipase C, amylase and sporulation genes.

17. The recombinant bacteria of claim 1 wherein the DNA insert is a foreign gene encoding a protein selected from the group consisting of insecticidal proteins, antigenic proteins, and enzymes.

18. The recombinant bacteria of claim 17 wherein the foreign gene comprises a promoter operable in the recombinant bacteria operably linked to a protein coding sequence.

19. The recombinant bacteria of claim 18 wherein the protein coding sequence does not encode a protein that is used to identify and select the recombinant bacteria.

20. The recombinant bacteria of claim 19 wherein the protein coding sequence does not encode a screenable protein.

21. The recombinant bacteria of claim 19 wherein the protein coding sequence encodes a protein selected from the group consisting of Cry1Aa1, Cry1Aa2, Cry1Aa3, Cry1Aa4, Cry1Aa5, Cry1Aa6, Cry1Aa7, Cry1Aa8, Cry1Aa9, Cry1Aa10, Cry1Aa11, Cry1Aa12, Cry1Aa13, Cry1Aa14, Cry1Ab1, Cry1Ab2, Cry1Ab3, Cry1Ab4, Cry1Ab5, Cry1Ab6, Cry1Ab7, CryiAbδ, Cry1Ab9, Cry1Ab10, Cry1Ab11, Cry1Ab12, Cry1Ab13, Cry1Ab14, Cry1Ab15, Cry1Ab16, Cry1Ac1, Cry1Ac2, Cry1Ac3, Cry1Ac4, Cry1Ac5, Cry1Ac6, Cry1Ac7, CryiAcδ, Cry1Ac9, CryiAdO, Cry1Ac11, Cry1Ac12, Cry1Ac13, Cry1Ac14, Cry1Ac15, Cry1Ad1, Cry1Ad2, Cry1Ae1, CryiAfi, Cry1Ag1, Cry1Ah1, Cry1Ai1, Cry1Ba1, Cry1Ba2, Cry1Ba3, Cry1Ba4, Cry1Bb1, Cry1Bc1, Cry1Bd1, Cry1Bd2, Cry1Be1, Cry1Be2, Cry1Bf1, Cry1Bf2, Cry1Bg1, Cry1Ca1, Cry1Ca2, Cry1Ca3, Cry1Ca4, Cry1Ca5, Cry1Ca6, Cry1Ca7, Cry1Ca8, Cry1Ca9, Cry1Ca10, Cry1Cb1, Cry1Cb2, Cry1Da1, Cry1Da2, Cry1Db1, Cry1Db2, Cry1Ea1, Cry1Ea2, Cry1Ea3, Cry1Ea4, Cry1Ea5, Cry1Ea6, Cry1Eb1, Cry1Fa1, Cry1Fa2, Cry1Fb1, Cry1Fb2, Cry1Fb3, Cry1Fb4, Cry1Fb5, Cry1Ga1, Cry1Ga2, Cry1Gb1, Cry1Gb2, CrylGc, Cry1Ha1, Cry1Hb1, Cry1la1, Cry1la2, Cry1la3, Cry1la4, Cry1la5, Cry1la6, Cry1la7, Cryilaδ, Cry1la9, Cry1la10, CryllaH, Cry1lb1, Crylld, Cry1lc2, Cry1ld1, Cry1le1, Cry1lf1, CryUal, CryUbi, CryUd, CryUc2, CryUdi, Cry1Ka1, Cry2Aa1, Cry2Aa2, Cry2Aa3, Cry2Aa4, Cry2Aa5, Cry2Aa6, Cry2Aa7, Ctγ2Aa8, Cry2Aa9, Cry2Aa10, Cry2Aa11, Cry2Ab1, Cry2Ab2, Cry2Ab3, Cry2Ab4, Cry2Ab5, Cry2Ab6, Cry2Ac1, Cry2Ac2, Cry2Ac3, Cry2Ad1, Cry2Ae1, Cry3Aa1, Cry3Aa2, Cry3Aa3, Cry3Aa4, Cry3Aa5, Cry3Aa6, Cry3Aa7, Cry3Ba1, Cry3Ba2, Cry3Bb1, Cry3Bb2, Cry3Bb3, Cry3Ca1, Cry4Aa1, Cry4Aa2, Cry4Aa3, Cry4Ba1, Cry4Ba2, Cry4Ba3, Cry4Ba4, Cry4Ba5, Cry5Aa1, Cry5Ab1, CryδAd, Cry5Ba1, Cry6Aa1, Cry6Aa2, Cry6Ba1, Cry7Aa1, Cry7Ab1, Cry7Ab2, CryδAal, CryδBai, CryδBbi, CryδBd, CryδCal, CryδCa2, CryδDai, CryδDa2, CryδDa3, CryδEal, Cry9Aa1, Cry9Aa2, Cry9Ba1, Cry9Ca1, Cry9Ca2, Cry9Da1, Cry9Da2, Cry9Ea1, Cry9Ea2, Cry9Eb1, Cry9Ec1, Cry10Aa1, Cry10Aa2, Cry10Aa3, Cry11Aa1, Cry11Aa2, Cry11Aa3, Cry11Ba1, Cry11Bb1, Cry12Aa1, Cry13Aa1, Cry14Aa1, Cry15Aa1, Cry16Aa1, Cry17Aa1, Cry1δAa1, Cry1δBa1, Cry1δCa1, Cry19Aa1, Cry19Ba1, Cry20Aa1, Cry21Aa1, Cry21Aa2, Cry21Ba1, Cry22Aa1, Cry22Aa2, Cry22Ab1, Cry22Ab2, Cry22Ba1, Cry23Aa1, Cry24Aa1 , Cry25Aa1 , Cry26Aa1, Cry27Aa1, Cry28Aa1, Cry2δAa2, Cry29Aa1, Cry30Aa1, Cry30Ba1, , Cry31Aa1, Cry31Aa2, Cry32Aa1, Cry32Ba1, Cry32Ca1, Cry32Da1, Cry33Aa1, Cry34Aa^'i, Cry34Aa2, Cry34Ab1, Cry34Ac1, Cry34Ac2, Cry34Ba1, Cry35Aa1, Cry35Aa2, Cry35Ab1, Cry35Ab2, Cry35Ac1, Cry35Ba1, Cry36Aa1, Cry37Aa1, Cry33Aa1, Cry39Aa1, Cry40Aa1, Cry40Ba1, Cry41Aa1, Cry41Ab1, Cry42Aa1, Cry43Aa1, Cry43Ba1, Cry44Aa, Cry45Aa, Cry46Aa, Cry47Aa, Cyt1Aa1, Cyt1Aa2, Cyt1Aa3, Cyt1Aa4, Cyt1Aa5, Cyt1Ab1, Cyt1Ba1, Cyt2Aa1, Cyt2Aa2, Cyt2Ba1, Cyt2Ba2, Cyt2Ba3, Cyt2Ba4, Cyt2Ba5, Cyt2Ba6, Cyt2Ba7, Cyt2Baδ, Cyt2Ba9, Cyt2Bb1, Cyt2Bc1, Cyt2Ca1, Vip3A(a) and Vip3A(b).

22. A method of integrating a DNA insert into a recombinant bacteria comprising:

a) introducing a first recombination vector comprising a first endogenous gene copy and a marker gene into a bacteria;

b) isolating an intermediate bacteria comprising a first recombined endogenous gene, a first recombined endogenous gene copy and the marker gene wherein the marker gene is between the first recombined endogenous gene and the first recombined endogenous gene copy;

c) introducing a second recombination vector comprising a second endogenous gene copy, a third endogenous gene copy, and the DNA insert into the intermediate bacteria, wherein the DNA insert is between the second endogenous gene copy and the third endogenous gene copy; and

d) isolating a recombinant bacteria comprising a second recombined endogenous gene, a second recombined endogenous gene copy and the DNA insert, wherein the DNA insert is between the second recombined endogenous gene and the second recombined endogenous gene copy and wherein said recombinant bacteria does not have an exogenous antibiotic resistance marker.

23. The method of claim 22 wherein the first endogenous gene copy comprises a region that has sufficient homology to a region within an endogenous gene within the bacteria to permit recombination between the first endogenous gene copy and the endogenous gene.

24. The method of claim 22 wherein the second endogenous gene copy comprises a region that has sufficient homology to a region within the first recombined endogenous gene to permit a first recombination between the second endogenous gene copy and the first recombined endogenous gene and wherein the third endogenous gene copy comprises a region that has sufficient homology to a region within the first recombined endogenous gene copy to permit a second recombination between the third endogenous gene copy and the first recombined endogenous gene copy.

25. The method of claim 22 wherein marker gene is a selectable marker or a screenable marker.

26. The method of claim 25 wherein the first isolating step is performed by selecting or screening for the marker gene in the intermediate bacteria.

27. The method of claim 22 wherein the marker gene is selected from the group consisting of an antibiotic resistance gene, a gene encoding a specific metabolic enzyme that utilizes a special nutrient substitute, a gene encoding an enzyme that catalyzes a chemical compound to form a distinctive color, a gene encoding a fluorescent protein, and a gene that encodes a protein with specific affinity for another molecule.

28. The method of claim 22 wherein the first recombined endogenous gene and the first recombined endogenous gene copy have a difference in function that is screenable or selectable, and wherein the second isolating step is performed by selecting or screening for the recombinant bacteria that lack the difference.

29. The method of claim 28 wherein the difference comprises a mutation in the first recombined gene copy.

30. The method of claim 28 wherein the difference comprises a truncation in the first recombinant gene copy.

31. The method of claim 24 wherein the first recombined endogenous gene and the first recombined endogenous gene copy both lack a function that is screenable or selectable, and wherein the second isolating step is performed by selecting or screening for the recombinant bacteria that have the function.

32. The method of claim 31 wherein the function is performed by the second recombined endogenous gene.

33. The method of claim 22 wherein the DNA insert is a foreign gene.

34. The recombinant bacteria of claim 33 wherein the foreign gene comprises a promoter operable in the recombinant bacteria operably linked to a protein coding sequence.

35. The method of claim 34 wherein the protein coding sequence encodes a selectable protein or a screenable protein.

36. The method of claim 34 wherein the protein coding sequence does not encode a selectable protein or a screenable protein.

37. The method of claim 34 wherein the protein coding sequence encodes a protein selected from the group consisting of insecticidal proteins, antigenic proteins, and enzymes.