WO1994025611A1 - Integrative dna segment comprising gene encoding insecticidal protein - Google Patents

Abstract

This invention relates to a method of increasing the insecticidal spectrum and/or toxicity of Bacillus thuringiensis strains whereby an exogenous crystal toxin encoding DNA sequence is stably integrated into the chromosomal DNA of a host B.t. wherein the exogenous DNA sequence is expressed and further wherein crystal toxin encoding DNA sequences on resident plasmids may be expressed. The invention further relates to the B.t. host including said exogenous DNA sequences, methods of preparing said host, insecticidal compositions containing said host and methods of use.

Description

INTEGRATIVE DNA SEGMENT COMPRISING GENE ENCODING INSECTICIDAL PROTEIN

This invention relates to the field of microbial insecticides, and more particularly, to the construction of hybrid microbes possessing greater insect toxicities and broader insect host ranges. This invention is useful in the protection of plants from insect infestation.

In recent years there has been considerable interest in Bacillus thuringiensis (B.t.) and their use in the biological control of insect infestation of plants. These insect pathogens from a toxic crystalline protein during sporulation, called the parasporal crystal.

B.t. strains with different insect host spectra are classified into different serotypes or subspecies based on their flagellar antigens. Most B.t. strains are active against larvae of certain members of the lepidopteran order including caterpillars of butterflies and moths but some also show toxicity against members of the dipteran or coleopteran order including mosquito larvae and beetle larvae respectively. Toxic activity has not yet been demonstrated for several crystal-producing strains.

The crystalline inclusions of B.t. dissolve in the midgut of the larvae, releasing one or more insecticidal crystal proteins, or δ-endotoxins, of 27 to 140Kd. Most crystal proteins are pro- toxins that are proteolytically converted to smaller, toxic polypeptides in the insect midgut. In general, it is well known in the art to refer to the crystal proteins as Cry and die gene encoding said protein as cry.

Over 42 B.t. cry genes have been characterized. A classification scheme for cry genes is published by Hofte and Whitely, 1989, Microbiol. Rev., 53:242, and genes are divided into four classes and several subclasses, by the structural similarities and insecticidal spectra of the encoded proteins. The four major classes are those encompassing the Lepidoptera- specific (I), Lepidoptera- and Diptera-specific (II), Coleoptera-specific (IE), and Diptera- specific (IN) genes.

The Lepidoptera-specific genes (cryl) encode 130 to 140Kd molecular weight proteins which accumulate in bipyramidal crystalline inclusions during the sporulation of B.t. The cryl genes can be distinguished from other cry genes by sequence homology (>50% amino acid identity). Three of these genes, cryΙA(a), cryIA(b), and cryΙA(c), show more than 80% amino acid identity and have therefore been considered as a separate subgroup. The more recently identified cry-EB, crylC, and ciylD genes differ from each other and from the crylA genes. The CrylA, CrylB, and CrylC proteins in crystal preparations have been distinguished in 29 strains of 11 serotypes by using 35 monoclonal antibodies as shown by Hofte et al. (Microbiol. Rev. 53:242-255 (1989)).

The Lepidoptera- and Diptera-specific class includes genes which encode 65Kd proteins which form cuboidal inclusions. The first crylLA gene was cloned from B.t. subsp. kurstaki HD-263 and expressed in Bacillus megaterium. Cells producing the CryllA protein were toxic for the lepidopteran species Heliothis virescens and Lvmantria dispar as well as for larvae of the dipteran Aedes aegvpti.

The Coleoptera-specific class encode gene products which are active on Coleoptera species and the proteins are about 70Kda. At least three Coleoptera-specific B.t. strains have been described: B.t. tenebrionis, B.t. san diego. and B.t. EG2158. The strains produce rhomboidal crystals containing one major protein.

The Diptera-specific class of crylV genes is composed of a rather heterogeneous group of Diptera-specific crystal protein genes. The cytA and four other genes were isolated from the same 72Mdalton (Md) plasmid present in strains of B.t. israelensis. The crylVA, crylNB, crylVC, and cryIND genes encode proteins of 135, 128, 78 and 72Kd, respectively. These proteins assemble together with the 26Kd cytA gene product, in ovoid crystal complexes. A crystal complex with the same or a similar protein composition has also been observed in the B.t. morrisoni PG-14 strain. Toxicity tests with preparations of cry IV class crystal proteins, derived either from B.t. israelensis or from recombinant E. coli or Bacillus are, to various extents, toxic against larvae of some mosquito species.

Since the original classification by Hofte and Whitely a number of novel cry genes have been cloned and their nucleotide sequences determined. A different classification system has been published by Yamamoto and Powell, 1993 "Bacillus thuringiensis Crystal Proteins pps 3-42 in Advanced Engineered Pesticides", ed. Leo Kim.

Commercial preparations of B.t. are commonly used on many agricultural crops, shade trees and ornamentals to control various insect species and these preparations are applied with the same equipment used for application of chemical insecticides. Methods for assessing spray coverage are well known to those skilled in the art.

The compositions comprising the hybrid bacterium may be applied as a spray, dust or bait, alone, or in conjunction with parasites, predators or other control procedures such as chemical insecticides radiation-induced sterilization, chemosterilants, pheromones, etc. Stressors may enhance the pathogenicity or activate chronic infections with the hybrid bacterium of the invention.

Known methods for transformation of B.t. include protoplast fusion, protoplast transfection, transduction, electroporation and conjugation-like processes.

Electroporation is frequently used for transforming B.t. due to its simplicity, speed and efficiency. A B.t. shuttle vector was also developed in 1989. The utility of this shuttle vector was first demonstrated by moving a B.t. crystal toxin gene into a crystal minus (Cry) B.t. strain called cryB with the resulting transformant expressing the 130Kd crystal protein.

Various cloning and expression vectors derived from native B.t. plasmids have been recently developed, for example, shuttle vectors incorporating the B.t. israelensis 3.65Mdalton plasmid and pBR322; plasmids from B.t. kurstaki HD263. HD73, HD1; plasmids from B.t. israelensis and E. coli vectors. One such shuttle vector was used to move a coleopteran-active toxin gene into a B.t. israelensis strain, widening the spectrum of insecticidal activity to include both Diptera and Coleoptera.

A serious problem associated with the use of this technology is the instability or incompatibility between the native and exogenous plasmid. Frequently, one or more native B.t. plasmids are unable to coexist with an exogenous plasmid(s) introduced into the bacterium, and the native plasmid is rapidly lost through segregation. The difficulty arises when the native plasmids contain one or more cry genes to be preserved in the transgenic B.t. strain. This problem can be overcome by eliminating the portion of the B.t. plasmid vector causing the incompatibility between the recombinant (or exogenous) and native plasmids.

Methods of stably introducing exogenous DNA into bacteria have been investigated. A B.t.- based, native-compatible vector has been used to transfer a coleopteran-active crylllA gene into a B.t. kurstaki strain and the resulting transgenic strain exhibited high levels of Coleopteran activity while still retaining its wild-type Lepidopteran activity. Bacteria may also be transformed with plasmids incapable of autonomous replication and carrying a selectable marker if the plasmid carries a segment of DNA that is homologous to a portion of the host chromosome. In the example above, a plasmid has been shown to interact with the host by a single crossover mechanism that involves plasmid and homologous host sequences. The result is an integrated plasmid flanked by direct repeats of the homologous DNA segment. Such insertions are mutagenic if both ends of the duplicated segment are contained within a single transcription unit.

Delecluse et al. applied homologous recombination to inactivate the cytA insecticidal protein- encoding gene in B.t. israelensis. (Delecluse et al., J. Bacteriol. 173:3374-3381 (1991)). An integrational vector containing partial cytA sequences was constructed and homologously recombined with the cytA gene present on the 72Mdalton resident plasmid of B.t. israelensis. In addition, Calogero et al. constructed a B. subtilis integrational vector carrying the entire CryΙA(c) coding region of B.t. kurstaki HD73 (Calogero, S., et al., Appl. Env. Microbial. 55:446-453 (1989)). The plasmid vector was found to express the HD73 cryΙA(c) gene. PCT/US91/05930 (WO/93/03619) describes a recombinant B.t. bacterium with a shuttle vector carrying an insecticidal gene and a method of preparing the recombinant bacterium.

However, neither the general methods described above nor the specific references offer any suggestion that the problem of B.t. stability could be solved by chromosomal integration.

The present invention is concerned with the stable maintenance and expression of crystal genes wherein the cry genes are integrated into the chromosome of B.t. strains. Additionally the invention concerns the construction of B.t. strains with broader host ranges and/or new specificities. Specifically, the invention concerns the construction of B.t. strains with high spodoptera activity and retained potency against Lepidopterous insects.

This invention relates to a DNA segment comprising one or more insecticide-encoding DNA sequences capable of being replicated and expressed in B.t. and a DNA sequence which directs insertion via homologous recombination of the DNA segment into chromosomal B.t. DNA.

This invention also relates to a DNA segment which comprises one or more insecticide- encoding DNA sequences capable of being replicated and expressed in B.t. and a DNA sequence capable of randomly integrating into the B.t. genome.

The invention also includes a hybrid vector which comprises a vector such as a plasmid or a shuttle vector and the DNA segment of this invention operatively linked thereto.

Also part of this invention is a hybrid B.t. host having integrated in its chromosome at least one of the insecticide-encoding DNA sequence of said DNA segment.

This invention also relates to a method of preparing the DNA segment of the invention comprising the steps of: obtaining a DNA sequence homologous to a chromosomal B.t. DNA sequence; and operatively linking thereto at least one insecticide-encoding DNA sequence so that when B.t. is transformed with the DNA segment or a hybrid vector having the DNA sequence operatively linked thereto the insecticide-encoding DNA sequence is expressed.

Still another method of preparing a transformed B.t. host is provided herein that comprises a) obtaining a DNA sequence capable of randomly integrating into the B.t. chromosome DNA; b) operatively linking to said DNA sequence one or more insecticide-encoding DNA sequences capable of being replicated and expressed in B.t.; c) obtaining a DNA segment; d) transforming a Bacillus thuringiensis host with the DNA segment wherein the DNA segment randomly integrates into the B.t. host chromosome; and e) isolating the transformed host and wherein the insecticide encoding DNA sequences is expressed as transformed host. This invention also encompasses the preparation of a hybrid B.t. host expressing at least one exogenous insecticide by transforming a B.t. host with the DNA segment or hybrid vector described above and allowing homologous recombination to occur and the insecticide- encoding DNA sequence to become stably incorporated into the B.t. chromosome and isolating the transformed host.

Disclosed herein is a broad spectrum, insecticidal composition comprising the hybrid host of the invention, and a carrier, optionally including other insecticidal products. In one embodiment the insecticidal range of the B.t. may be increased by transforming B.t. as described above insecticide-encoding DNA sequence remains expressible.

Also encompassed is a method of protecting a plant from insect damage, comprising applying to the plant or the soil around the plant an effective amount of an insecticidal composition of the invention.

The invention will be further apparent from the following discussion, including the associated Examples, drawings and Sequence Identifications.

Of the drawings:

Figure 1 describes plasmid pSB210 including a gram-negative origin of replication.

Figure 2 shows the derivation of the pSB147 plasmid and its family tree.

Figure 3 describes plasmid pSB210.1 including the pSB210 sequences.

Figure 4 describes plasmid pSB210.2 including the pSB210.1 sequences.

Figure 5 shows a map of the pSB210.3 plasmid.

Figure 6 shows a map of the pSB147 plasmid.

Figure 7 depicts the construction of the pSB136 plasmid.

The Sequence Identifications are given in Tables 2 and 5, and in Examples 3, 5 and 11.

The present invention improves upon the narrow insecticidal range of the prior art B.t. Hybrid B.t. strains possessing more than one insecticide-encoding gene with broader host ranges may be constructed by introducing foreign insecticide-encoding genes into the chromosomes of known bacterial strains. Accordingly, the present invention provides a method of increasing the host range of a B.t. strain by increasing the number of different insecticidal crystal proteins produced by the strain.

Introduced plasmids in recombinant B.t. strains can cause instability to resident plasmids of the cell, and the introduced plasmids themselves may also be unstable. This often results in loss of expression of the crystal genes contained in the cell. The present invention circumvents this problem by introducing a crystal gene into the host chromosome for expression without disturbing existing crystal genes contained on the host resident plasmids.

Accordingly, the invention provides a DNA segment comprising one or more insecticide- encoding DNA sequences; and a DNA sequence homologous to a chromosomal DNA sequence of a B.t. wherein said insecticide encoding DNA sequence inserted into the chromosome is replicated and expressed in said B.t.

The insecticide-encoding DNA sequence may be any DNA sequence encoding an insecticidal protein capable of being expressed in a B.t. bacterium. Insecticide-encoding DNA sequences suitable for use in the invention include but are not limited to cryLA(a), crylA(b), cryΙA(c), crylB, crylC, crylD, crylE, crylF, cryllA, cryllB, crylllA, crylllB, crymC, crylVA, crylVB, crylVC, crylVD and cytA genes of any subspecies and/or strain of B.t. and the insecticidal protein-encoding genes of any strain of B. larvae and B. papillae and all other insecticidal protein-encoding genes known to be expressed in bacilli or other gram-positive bacteria among others. Also suitable are DNA sequences encoding insecticides such as α-amylase inhibitor, proteinase inhibitor and any other toxins from bacteria, among others.

Homologous DNA sequences suitable for use in the DNA segment of the invention include any DNA sequence substantially homologous to any chromosomal DNA fragment of any B.t. species or strain such as those listed in Table 1 above. The homologous DNA sequence permits and directs the integration of the DNA segment of the invention into the host's DNA by homologous recombination thereof with bacterial DNA. When the DNA segment of the invention is provided as a circular, covalently closed DNA segment, homologous recombination may occur by means of a single cross-over event between the host DNA and the homologous DNA sequence. When the DNA segment is provided as a linear DNA segment, homologous recombination may occur by means of a double cross-over event between the host's DNA and the homologous DNA sequences flanking the desired insecticide-encoding DNA. Thus, the homologous DNA sequences may be provided as one or two flanking DNA sequences. Moreover, the DNA segment of the invention is provided in double stranded and single stranded form. The single stranded form may be obtained by heat or chemical denaturation of the double stranded form as is known in the art. The double stranded form may be obtained by enzyme restriction of the desired sequence, and ligation as is known in the art.

The homologous DNA sequence is homologous to a fragment of the bacterial chromosome in the range of about 5 bases to about 20 kbases. More preferably the sequence is homologous to about 500 bases to about 10 kbases. Also preferred is a DNA sequence homologous to about 2250 bases of the phospholipase C-encoding region of B.t. Particularly preferred are DNA sequences homologous to B.t. host chromosome portions outside the endogenous insecticide-producing gene(s). Where the DNA segment contains a DNA sequence homologous to the bacterial DNA both 5' and 3' of the sequence encoding the insecticidal protein, in the light of the above, the skilled man will recognize the size of the homologous region either side of the insecticide encoding region. In this manner, even the addition of a single new gene will increase the insecticidal range of the bacterium.

The DNA segment of the invention may further comprise an origin of replication for a gram- negative bacterium. Any origin of replication capable of functioning in one or more gram- negative bacterial species or strains of the Neisseria, Veillonella, Brucella, Pasteurella, Hemophilus, Bordetella, Escherichia, Erwinia, Shigella, Salmonella, Proteus, Enterobacter, Serratia, Azotobacter, Rhizobuim, Nitrosomonas, Nitrobacter, Thiobacillus, Pseudomonas, Acetobacter, Photobacterium, Zymomonas, Aeromonas, Vibrio, Desulfovibrio, or Spirillum genera, among others may be used. After cloning the DNA segment in a gram-negative bacterium such as E. coli and transforming a Bacillus thuringiensis, the only surviving insecticidal DNA sequences will be those integrated into the host's chromosome. Since the gram-negative origin of replication will not function in a B.t. bacterial host, a B.t. host transformed with the DNA segment will neither replicate, nor express the insecticide unless the DNA segment becomes integrated into the host chromosome.

The DNA segment of the invention may also further comprise a selectable marker expressible in a monocellular organism other than a gram-positive bacterium, a selectable marker expressible in a gram-positive bacterium, and/or a selectable marker expressible in a gram- negative and a gram-positive bacterium. A selectable marker expressible in a monocellular host other than a gram-positive bacterium is defined as any DNA sequence capable of expressing a phenotype in a monocellular host other than a gram-positive host, that is useful in the detection or selection of the host carrying the DNA sequence. A selectable marker capable of being expressed in a gram-positive bacterium is defined as any DNA sequence capable of expressing a phenotype in a gram-positive bacterial host useful in the detection or selection of a gram-positive host carrying the DNA sequence. A selectable marker capable of being expressed in a gram-negative and a gram-positive bacterium is defined as any DNA sequence capable of expressing a phenotype in a gram-positive and a gram- negative bacterial host useful in the detection or selection of the host carrying the DNA sequence. In general, examples are markers for drug resistance, chemical resistance, amino acid auxotrophy or prototrophy, or other phenotypic variations useful in the selection or detection of mutant or recombinant organisms. The presence of the selectable markers facilitates the cloning and/or maintenance of the DNA segment of the invention in gram- negative bacteria and improves the selection and/or detection of recombinant B.t. bacteria carrying the DNA segment of the invention.

Further provided herein is a DNA segment comprising at least one insecticide-encoding DNA sequence capable of being replicated and expressed in a B.t. bacterial host, and a DNA sequence capable of randomly integrating into the B.t. host's genomic DNA. Randomly integrating DNA sequences suitable for use herein are insertion sequences or transposon sequences capable of inserting or copying themselves and DNA sequences operatively linked thereto at random locations in the chromosomal or plasmid DNA of a B.t. host. Examples include transposons, such as Tn917 exemplified below, and Tnl545 and Tn916, all of which are described by Camilli et al. (Camilli et al., J. Bacterial. 172:3738-3744 (1990)) or other gram-positive transposons known in art. Although the existence of randomly integrating DNA sequences or cassettes has been known, it is being applied for the first time to the insertion of an insecticidal gene in a gram-positive bacteria.

Suitably the DNA segment of the invention comprising a transposon sequence may be carried on a plasmid vector. Plasmids appropriate for use herein include pTV51Ts and pLTVl, as exemplified below among others. In a preferred embodiment, temperature-sensitive plasmids are used to select transposed host cells. Plasmids such as pTV51Ts and pLTVl are unable to replicate above a certain temperature. Thus, a DNA segment carried on a temperature- sensitive plasmid will be maintained in the host at a non-permissive temperature only if the DNA segment transposes into the host genome. The transposition efficiency may be increased by selecting for a marker contained within the transposable element, such as drug resistance, amino acid auxotrophy or protrophy, and the like.

In a particularly preferred embodiment, the DNA segment of the invention is used to generate multiple transposition events within a single B.t. host. Multiple insertions of the DNA segment into host genomic DNA may be obtained by a rapid temperature upshift in the case of temperature-sensitive plasmid vectors. If transposable elements carrying drug resistance markers are used, increased drug levels will encourage multiple transposition events. The addition of mitomycin C will also increase trasnposition frequency. Alternatively, the gram-positive host may be transformed with an array of different transposable elements in which each element carries a unique selectable marker.

In one embodiment, the transposable element of the invention carries all control elements necessary for host cell expression of the transposed insecticide-encoding DNA sequence. In other embodiments, the transposable element may be designed to create an operon or gene fusion in which the insecticide-encoding sequence is placed under the transcriptional and/or translational control of the host DNA. Operon and gene fusions may be constructed according to the methods for Tn917-mediated operon and gene fusions described by Youngman or other methods known in the art (Youngman, P., "Plasmid Vectors for Recovering and Exploiting Tn917 Transpositions in Bacillus and Other Gram Positive Bacteria", In Plasmids: A Practical Approach, Handy, K.G., ed. IRL Press 79-103 (1973)).

The transposable element of the invention may be utilized in the preparation of a transformed B.t. host expressing at least one exogenous insecticide by operatively linking thereto at least one insecticide-encoding DNA sequence capable of being replicated and expressed in B.t. to obtain a DNA segment, so that when B.t. is transformed with the DNA segment the insecticide-encoding DNA sequence becomes integrated in the B.t. chromosome and may be expressed, transforming a Bacillus thuringiensis host with the DNA segment and allowing the DNA segment to randomly integrate into the B.t. chromosome, and isolating the transformed host.

The transposable element or DNA sequence capable of randomly integrating into the B.t. genome may be obtained by methods known in the art including enzyme restriction, ligation, cloning, and/or chemical synthesis. The insecticidal gene or DNA sequence may be obtained and be operatively linked to the randomly integrating DNA sequence similarly by methods known in the art.

Transformation may be conducted by for example transfection, electroporation, transduction or conjugation. Host isolation may be conducted by selecting from the selectable marker on the transformed host. In an embodiment of this invention, the randomly integrated DNA comprises the Tn917 transposon.

The insecticidal range of B.t. may be increased by maintaining the expression of an endogenous insecticide-encoding DNA sequence and operatively linking one or more exogenous insecticidal genes to the transposable element which may be incorporated into the B.t. chromosome.

The DNA segment of the invention may be provided as a hybrid plasmid. It will be appreciated that any plasmid sequences suitable for carrying the DNA segment of the invention may be used in the construction of the hybrid plasmid. Particularly suitable for use in the present invention are plasmids pSB210.1, pSB210.2, pSB210.3, pSB136 and pSB147 described below in Examples 1, 6 and 11, among others.

The DNA segment of the invention may also be provided as a hybrid shuttle vector for gram- positive bacteria. Appropriate vectors include any vector capable of self-replication in gram- negative bacteria, yeast or any monocellular host in addition to gram-positive bacteria. Such shuttle vectors are well known in the art. The utility of this shuttle vector was first demonstrated by moving a B.t. crystal toxin gene into a crystal minus B.t. strain called cryB. The resulting transformant expressed the 130Kd crystal protein. In addition, Lereclus et al. constructed another shuttle vector incorporating pHT1030 B.t. plasmid and pUC18 to move a cryΙA(a) gene isolated from B.t. 407 into the cryB strain (Lereclus, D., et al., FEMS Microbiol. Lett., 49:417 (1988)). When transferred into a Cry-derivative of the 407 strain, the level of expression of this toxin increased significantly over that seen in the wild-type strain, possibly the result of an increased gene copy number. Miteva et al. constructed a shuttle vector by incorporating the 3.65Mdalton plasmid of B.t. israelensis and pBR322 (Miteva, V.I. et al., Arch. Microbiol. 150:496 (1988)). Shuttle vectors constructed using B.t. plasmids from B.t. kurstaki HD263, HD73, HDl and B.t. israelensis and E. coli vectors have also been described. One such shuttle vector was used to move a coleopteran-active toxin gene into a B.t. israelensis strain, widening the spectrum of insecticidal activity to include both Diptera and Coleoptera (Crickmore, N., et al., Biochem. J. 270:133 (1990)).

Also provided herein is a B.t. host comprising at least one insecticidal DNA sequence of the invention stably incorporated in its chromosome. Suitable B.t. hosts include B.t. subspecies and strains thereof set forth in Table 1 and those exemplified below, as well as any other B.t. subspecies or strain known in the art. As used herein, the term "host" includes both vegetative and spore forms of B.t. bacteria. The stable incorporation of the DNA segment of the invention into a host chromosome is defined as the maintenance of the DNA segment within the host chromosome through many generations of progeny and through the sporulation and germination phases of B.t. hosts.

In a preferred embodiment, the transformed B.t. host comprises multiple exogenous expressible insecticide encoding DNA sequences stably integrated into its chromosome. The multiple sequences may comprise any combination of the above described insecticide- encoding sequences, including multiple copies of the same insecticide-encoding sequence. In a particularly preferred embodiment the host is capable of expressing two or more different insecticidal proteins. It will be appreciated that the DNA segment of the invention may be used to stably incoφorate any exogenous DNA sequence into the chromosome of a B.t. host. Exogenous DNA is defined as any DNA which alters the chromosomal DNA of a B.t. host upon integration into the host's chromosome. The desired DNA sequence may be introduced into B.t. in a similar manner as the insecticide-encoding DNA sequences described herein. Accordingly, the invention encompasses a B.t. host having incorporated into its chromosome an exogenous DNA sequence capable of being replicated and expressed by the host.

The DNA segment of the invention may be prepared by obtaining a DNA sequence homologous to a chromosomal DNA sequence of a B.t. bacterium, operatively linking thereto at least one insecticide-encoding DNA sequence so that when a Bacillus thuringiensis is transfected with the DNA segment, the insecticide-encoding DNA sequence is expressed.

The homologous DNA sequence may be obtained by screening known genomic libraries of B.t. organisms. If a genomic library does not exist for the B.t. bacterium of interest, one may be constructed by methods known in the art. Screening methods are also known in the art. Once the sequences of interest are determined, they may be excised with restriction enzymes. If the DNA or peptide sequence is known, the DNA fragments may be synthesized by methods known in the art. Alternatively, if the DNA or peptide sequence is not known, genomic restriction fragments can be used to randomly clone homologous fragments.

The insecticide-encoding DNA sequence may be operatively linked to the homologous DNA sequence by joining of the DNA sequences so that upon homologous recombination and integration of the insecticidal DNA into the host's chromosome, the insecticide-encoding DNA is capable of being expressed in Bacillus thuringiensis. In one embodiment the DNA segment comprises regulatory sequences capable of directing the transcription and translation of the insecticide-encoding DNA within the B.t. host. Such regulatory sequences may include promoter, operator, repressor and/or enhancer sequences, transcription initiation and termination sites, ribosome binding sites, translation start and stop codons, and/or other regulatory sequences known in the art. For example, the insecticide-encoding DNA sequence may have operatively linked thereto regulatory sequences which control expression of the insecticide-encoding DNA within its native bacterial host. Also within the scope of the invention are DNA segments designed to create operon or gene fusions within the host's DNA. In the case of an operon fusion the DNA segment may comprise control elements capable of directing the translation of the insecticide-encoding mRNA. The homologous DNA sequence may be designed to integrate the insecticide- encoding DNA sequence into an operon within the host's DNA so that upon insertion in the host's chromosome, the insecticide-encoding DNA sequence is placed under the transcriptional control of the host's operon. In the case of a gene fusion, the homologous DNA may be designed to integrate the insecticide-encoding DNA into a structural gene in the host's chromosome so that the host's control elements direct both the transcription and the translation of the insecticide-encoding DNA sequence. The techniques for constructing operon and gene fusions are described by Sambrook et al. (Sambrook, J., Fritsch, E.F. & Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989)).

Once constructed the DNA segment of the invention may be isolated by methods known in the art such as centrifugation or agarose-gel electrophoresis, among others.

The method for preparing the DNA segment of the invention may further comprise a selectable marker selected from the group consisting of those capable of being expressed in a monocellular host other than a gram-positive host, those capable of being expressed in a gram-positive host, and those capable of being expressed in both gram-positive and gram- negative hosts. The selectable marker for a monocellular organism is utilized to clone and purify the DNA segment in such organism.

The selectable markers of the invention may be operatively linked to the DNA segment by concatenating the selectable marker to, or inserting the gram-positive selectable marker in, the DNA segment so that, upon insertion into the B.t. host's chromosome, the selectable marker does not interfere with the expression of the insecticide-encoding DNA sequence in the B.t. host. In addition, linkage of a gram-positive selectable marker should not interfere with the functioning of the selectable marker for the cloning organism (the non-gram-positive host), and should be expressible in the B.t. host. In one embodiment, a gram-positive selectable marker carries all control elements necessary for its expression. The gram-positive selectable marker may also be designed to function in an operon or in a gene fusion within the host DNA in a manner similar to that described.

The method of the invention may further comprise operatively linking to the DNA segment described above an origin of replication for the monocellular organism in which it is cloned. This organism may be an insect cell, CHO cells, gram-negative bacteria, yeast and the like. Origins of replication such as those described above may be operatively linked to the DNA segment of the invention by placing the origin in a location within the DNA segment in which it will not disrupt the functioning of any other elements in the DNA segment, e.g. outside the insecticidal DNA and the homologous DNA sequences.

A B.t. host having stably incorporated into its chromosome a DNA segment encoding at least one insecticide may be prepared by a) obtaining a DNA segment of the invention; b) transforming a B.t. host with the DNA segment; c) allowing for homologous recombination to occur and the insecticide-encoding DNA sequence to become stably incorporated into the host's chromosome; and d) isolating the transformed host.

B.t. hosts may be transformed with the DNA segment by methods well known by one skilled in the art, including electroporation, transfection, transduction and conjugation or any combination of these methods.

Further provided herein is a broad range insecticidal composition comprising the hybrid host of the invention in an insecticidally effective amount and a carrier thereof.

The composition may contain about IO⁶ to about IO¹³ hybrid microorganisms/g carrier, and more preferably about IO¹⁰ to about IO¹¹ microorganisms/g carrier. However, other amounts are also suitable.

B.t. hosts may be present in the composition in either vegetative or spore form. Suitable carriers are known in the art and an artisan would be able to select those suitable for the present purpose. Typically, the carriers are inert compounds or compositions that neithe interact with the host in the insecticidal compositions nor with the plants to be treated. However, certain carriers may be metabolized by the plants or the soil organisms and are therefore biodegradable. The effectiveness and persistence of the insecticidal composition is enhanced by the addition of carriers such as spreaders, stickers, wetting agents, and including corn meal baits, Loco® (amine stearate) spray additives, Plyac®, Triton B-1956, polybutenes L-100 and H-35, corn oil, Triton B-1946, and Cellosize QP 4400, boric acid, surfactant oils, Pinolene® and other adjuvants known in the art. The ingredients are admixed and compounded as is known in the art, and the composition provided in powder, liquid or aerosol form. The hybrid host may best be prepared at low temperature and thawed prior to use.

The insecticidal compositions of this invention may further comprise other insecticidal compounds. Compositions including B.t. are known to be compatible with a wide range of chemical insecticides, such as those reported by Herfs and Pflaanzenkrankh (Herfs, W., and Pflanzenkrankh, Z., Pflanzenshutz 72(10):584-599 (1965)). Accordingly the insecticidal composition of the invention may comprise one or more of the insecticides identified by Herfs or other insecticidal B.t. hosts known in the art or hybrids thereof prepared in accordance with this invention.

The invention also provides a method of protecting a plant from insect damage, comprising applying to the plant or the soil around the plant an effective amount of the insecticidal composition of the invention. Methods known in the art for the application of commercial insecticidal microorganism preparations are suitable for applying the insecticidal composition of the invention.

Typically the present composition may be applied by spreading about 10⁸ to about IO¹⁶ hybrid microorganisms/acre and more preferably about 10¹³ to about IO¹⁴ hybrid micro¬ organisms/acre. The compositions are best applied by spraying the plants, and subsequent reapplications may also be undertaken.

Specific examples are described hereinbelow for purposes of illustration only and are not intended to limit the invention or any embodiment thereof, unless so specified.

EXAMPLES Example 1: Construction of Plasmids

Competent E.coli DH5a (Gibco BRL) and GM2163 (New England Biolabs) were prepare by the method of Alexander (Alexander, D.C., "A Method for Cloning Full-Length cDN in Plasmid Vectors", In Wu. R. and Grossman, L., Eds., Recombinant DNA part E. Meth Enzymol. 154:41-63 (1987)). Plasmids were extracted from E. coli by the method o Birnboim and Doly (Birnboim and Doly, "A Rapid Alkaline Extraction Procedure fo Screening Recombinant Plasmid DNA", Nucl. Acids Res. 7:1513-1523 (1979)).

The pSB210.2 plasmid which carries the crylC gene, the B. subtilis ermC gene fo erythromycin resistance and the phospholipase C region as described by Lechner et al. as target for integration (Lechner et al., "Molecular Characterization and Sequence o Phoshpatidylinositol-Specific Phospholipase C of Bacillus thuringiensis, Mol. Microbiol 3:621-626 (1989)) was constructed in a 3-step process. First, the pSB210 plasmid shown i Figure 1 was constructed from the pSB140 plasmid shown in Figure 2 and described belo in Example 2 by adding a multiple cloning site (MCS) at the EcoRI and HinaTfl sites. Th MCS was created by annealing oligonucleotides KK14 and KK14B, the sequences of whic are described in Table 2 below, which had been purified using oligonucleotide purificatio cartridges from Applied Biosystems, following the manufacturer's directions.

Table 2: sequences of Oligonucleotides

SEQ

Name: Sequence (5'-3') Hybridizing Gene ID No

Phosl GGAACGCTACATACTAGTGATAGAGTAG phospholipase C 1

Phos4 GCTTGTACACCGCAACTGTTTTCGCATG phospholipase C 2

KK14B AGCTTGCGGCCGCGTCGACCCCGGGCCATGGGGGCCG (MCS) 3

KK14 AATTCGGGCCCCCATGGCCCGGGGTCGACGCGGCCGCA (MCS) 4

GalPl CCACAGTTACAGTCTGTAGCTCAATTACC crylC 5

GalP2 CCGCTACTAATAGAACCTGCACCA crylC 6

NHS20 CAATACATTATCCATGGAAAATTCCTCCTTAAATATCATG cryHA 7

NHS39 GAGCAATGAAAGAGTTAGGGCCCTGTTTAAGGTGTCATG cryHA 8

NHS42 GAGTGAATTATGGGGG cryHA 9

NHS43 ATTTTGTATTAAACGG cryHB 10 cryHAl ACTATTTGTGATGCGTATAATGTA cryHA 11 cryHA2 AATTCCCCATTCATCTGC cryHA 12

PG2 GAAATCGGCTCAGGAAAAGG ermC 13

PG4 CCTTAAAACATGCAGGAATTGACG ermC 14

PG5 CTATTGGTTGGAATGGCGTG ermC 15

TYIAA GAGCCAAGCAGCTGGAGGAGTTTACACC cryLA(a) 16

TYIAC TCACTTCCCATCGACATCTACC cryΙA(c) 17

TYTUN12 ATCACΓGAGTCGCTTCGCATGTTTGACTTTCTC cryl-type5 18

TY6 GGTCGTGGCTATATCCTTCGTGTCACAGC cryl-type 19 TY13 ACAGAAGAATTGCTTTCATAGGCTC cryl-type 20 TY14 GAATTGCTTTCATAGGCTCCGTC cryl-type 21 Tet3 CAACAAACGGGCCATAAGCTTGTATAAG tet 22 Tet4 GCCGTCTGTAACGGTACCTAAGG tet 23 CPOLRev CACCCAGTTTGTACTCGCAGG tet 24

In the second step, the phos C gene was added to pSB210. The phos C region had been amplified from HD73 total DNA by PCR using primers Phosl and Phos4 described above in Table 2. The PCR product was cloned into the Smal site of pUC18 to construct pSB139. The phos C target region was isolated on a 2.2kb blunted-Kpnl, BamHl fragment from pSB139, gel-purified and ligated into pSB210, which had been digested with Mscl and BamHI and purified using the Geneclean Kit (BiolOl), following the manufacturer's directions. The resulting plasmid designated pSB210.1 and is shown in Figure 3.

The final step was to add a crystal gene. The pSB210.2 plasmid contains the crylC gene on a 4.2kb Apal-Notl fragment from pSB619, described below in Example 3. pSB619 was digested with Apal and Notl, and the 4.2kb Apal-Notl fragment was isolated. The 4.2kb Apal-Notl fragment was ligated to pSB210.1 cut with Apal and Notl to form pSB210.2 which is shown in Figure 4. The pSB210.3 plasmid contains the crylC gene isolated on a 6kb fragment from pSB013 (described below in Example 4) cut with Apal and Notl. The 6kb Apal-Notl fragment was purified by electroelution and ligated to pSB210.1 cut with Apal and Nod to form pSB210.3 which is shown in Figure 5. pSB210.3 differs from pSB210.2 in that the crylC gene is placed behind the cryHA promoter rather than the native crylC promoter found in pSB210.2. In both plasmids, the ciylC gene is followed by the crylA(c) terminator. The plasmid pSB147 was constructed as described below in Example 5. It carries the phospholipase C region as an integration target, the cryHA operon, and the ermC gene which conveys resistance to erythromycin.

The plasmid DNA used in the electroporation experiments was purified from GM2163, a dam-, dcm-, E. coli strain (Woodcock, D.M., Nucleic Acids Res. 17:3469 (1989)).

Example 2: Construction of pSB140 Plasmid

The pSB901 plasmid was constructed to provide an erythromycin resistance gene, ermC. To construct pSB901, the ermC gene was isolated as a HindHI/Clal fragment from the plM13 Baciilus subtilis plasmid described by Monod et al. (Monod et al., J. Bacteriol. 167: 138- 147(19861) . The ermC HindHI/Clal fragment was ligated to pUClδ cut with HindHI and Accl. To replace the tetr gene in pBR322 with the ermC gene from pSB901, pBR322 was digested with Aval and the linearized vector was treated with the Klenow fragment of E. coli DNA polymerase I to generate a blunt end. Following Klenow treatment, pBR322 was digested with HindlH and the large fragment was purified away from the tet' gene fragment. Plasmid pSB901 was digested with Smal followed by HindHI and the fragment carrying the ermC Smal-HindlH fragment was purified. The ermC gene was ligated into the pBR322 HindHI large fragment to generate pSB140. The derivation of the pSB140 plasmid is shown in Figure 2.

Example 3: Construction of pSB619 Plasmid

The crylC gene isolated as an 8kb EcoRI DNA fragment was cloned into the EcoRI site of Lambda ZAP H vector obtained from Stratagene. To isolate the gene, a plasmid preparation of Bacillus thuringiensis aizawai HD229 obtained from the USDA was digested with EcoRI, the fragments were separated by gel electrophoresis, and fragments of about 8kb were isolated from the gel. Alternatively, the crylC gene can be obtained by following the cloning protocol described by Honee et al. (Honee, G., van der Salm, T., and Visser, B., Nucl. Acids Res. 16:6240 (1988)). The crylC clone was digested in two separate reactions, one with HindHI and Kpnl and the other with Kpnl and EcoRI. The digestion created a 2.6kb HindHI-Kpnl fragment containing the promoter and N-terminal crylC sequence and a 2.3kb Kpnl-EcoRI fragment containing the C-terminal sequence and terminator. The crylC gene was reconstructed by ligating the 2.6kb HindHI-Kpnl fragment to the 2.3kb Kpnl-EcoRI fragment in ρTZ19R obtained from Pharmacia. The unique Ncol site was engineered at the translation start site of the crylC gene. An additional EcoRI site was also engineered right after the stop codon. These restriction sites enable cleavage of the entire coding region of crylC in one continuous fragment. The 3.8kb HindHI-EcoRI fragment containing the crylC promoter and entire protein coding region was then cloned into a pBluescript vector with the PCR generated 350bp crylA(c) terminator.

The cryΙA(c) terminator was obtained by PCR using two primers synthesized based on the published cryLA(c) sequence as follows.

Primer 1: GTCTCATGCAAACTCAGG, SEQ ID NO.: 25

Primer2: CTCTGGCGCTCCATCTAC, SEQ ID NO.: 26

A crylA(c) gene cloned from B.t. kurstaki HD73 was used as the template. The PCR generated terminator was cloned in an Xbal site, after treatment with Klenow to make it a blunt end, of pBluescript KS+(Stratagene) in the same orientation as the T3 promoter. The HindHI, EcoRI fragment containing the crylC promoter and coding region was then cloned into the HindHI-EcoRI sites of pBluescript KS +. The cloning and sequencing of cryΙA(c) are described by Adang et al. (Adang, M.J., Staver, M.J., Rocheleau, T.A., Leighton, J., Barker, R. F. and Thompson, D.V., "Characterized Full-length and Truncated Plasmid Clones of the Crystal Protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their Toxicity to Manduca sexta.", Gene 36: 289-300 (1985)). This construct was designated as pSB619.

Example 4; Construction of pSB013 Plasmid

The HD-1 strain of B.t. kurstaki was obtained from the USD A. Total DNA was extracted from HD-1 using the ASAP kit according to the protocol from Boehringer Mannheim. The kinased oligonucleotides NHS39 and NHS20 described above in Table 4 were used as primers in a PCR reaction to generate from B.t. kurstaki HD-1 total DNA the 1800bp fragment containing the entire cryHA operon. Vent polymerase was used according to the manufacturer's recommendations (New England Biolabs, Beverly, MA). The PCR reaction products were isolated from a gel and ligated into pTZ19R which had been digested with HindH. The ligation product was used to transform competent E. coli DH5α. The transformants were selected on LB plates containing 75μg/ml ampicillin (amp) and 40mM Xgal. Plasmid DNA was screened by Apal and Ncol digestion and the orientation of the 1800bp PCR fragment within the pTZ19R multicloning site was determined by AflHI digestion. A clone with the desired 1800bp fragment orientation was designated pSBOO9.

pSB070 is a plasmid similar to pSB619 that contains the coding region of cryHIA instead of CrylC. To construct pSB070, the CryHIA gene from B.t. tenebrionis or B.t. san diego was cloned as described by Herrnstadt, C, et al. (Herrnstadt, C, Gilroy, T.E., Sobieski, D.A., Bennett, B.D. and Gaertner, F.H., Gene 57: 37-46 (1987)).

The 3.0 kb HindHI containing cryHIA was cloned in pTZ18R (Pharmacia). A clone was selected that had the crylHA C-terminal coding region ligated to the multiple cloning site sequence containing the EcoRI site. In order to clone cryHIA in pSB070, a unique Ncol site was engineered at the translation start site utilizing the ATG codon. After the Ncol site was engineered, the cryHIA coding region was excised from pTZ18R with Ncol and EcoRI, and cloned into pSB619, from which the CrylC coding region had been removed.

Both pSB009 containing the cryHA operon fragment in pTZ19R and pSB070 containing cryHIA coding region with the crylC promoter and the cryΙA(c) terminator were digested with Apal and Ncol. A 5667 bp fragment of pSB070 and the 1800 bp fragment from pSBOO9 containing the cryHA operon were isolated. The fragments were ligated together. Competent E. coli DH5α cells were transformed and colonies were selected on LB plates containing 75/μg/ml ampicillin at 37°C overnight. The DNA from twelve colonies was digested with AflH+Notl to identify the isolate containing the desired operon cassette. The plasmid was designated pSBOlO.

The crylC gene was cloned into the pSBOlO cryHA operon cassette. pSB619 was obtained as described above in Example 3. The full length crylC coding region was obtained by digesting pSB619 with Ncol, EcoRI and BgHI and isolating the 3900 bp Ncol EcoRI fragment. The operon cassette vector, pSBOlO, was digested with NcoI+EcoRI and purified. The 3900 bp crylC fragment was ligated to the appropriate pSBOlO NcoI-EcoRI fragment. The ligation reaction product was used to transform DH5α and colonies were selected on LB plates containing 75μg/ml ampicillin. The plasmid DNA from twelve colonies was analyzed by restriction digests (AfHI + EcoRI and AfHI + BglH). The plasmid cassette containing the full length crylC gene downstream of the cryHA operon was designated pSB013.

Example 5; Construction of pSB304 Plasmid pSB304 was obtained by cloning the cryHA operon from B.t. galleriae HD232, a B.t. strain available from the USDA. To clone the operon, DNA from B.t. galleriae HD232 was digested with HindHI, and fragments of about 5 kb were purified by gel electrophoresis. The gel-purified fragments were ligated with HindHI-cut pTZ18R (Pharmacia) and transfected into E.coli DH5α. The clone containing cryHA was probed with a cryHA-specific oligonucleotide (CCCATGGATAATGTATTGAATAGTGGAAG), SEQ. ID.:27. The clone containing the cryHA and lacZ genes in the same orientation was chosen. The DNA was purified from the selected clone and a BamHI fragment encompassing the unwanted upstream sequence of the cryHA operon was removed to produce pSB304. Further information on the sequence of the cryHA operon and its 5' region can be found in Widner et al. (Widner, W.R., and Whiteley,^" H.R., "Two Highly Related Insecticidal Crystal Proteins of Bacillus thuringiensis subsp. kurstaki Possess Different Host Range Specificities", J.Bacteriol. 171: 965-974 (1989)).

Example 6: Construction of pSB147 Plasmid pSB140 was obtained as described above in Example 2. Next, the cryHA operon was added to pSB140. The source of the cryHA operon was plasmid pSB304 described above in Example 5. pSB304 contains the cryHA operon from B.t. galleriae HD232 cloned as a BamHI/HindlH fragment in pTZ18R. Plasmid pSB30 and plasmid pSB140 were digested with EcoRI and HindlH. The large pSB140 fragment was purified and the cryHA operon EcoRI-HindHI fragment was ligated to the large pSB140 fragment to yield pSB141.

The next step was to add an integration target site to the vector. The target site was a fragment of DNA that carried the phosphatidylinositol-specific phospholipase-C gene (pic) from the HD73 strain of B.t. kurstaki obtained from the USDA. This DNA fragment was isolated from HD73 total DNA using the polymerase chain reaction. Total DNA was extracted from B.t. kurstaki HD73 using the ASAP kit according to the protocol from Boehringer Mannheim.

The DNA sequence of the pic region from B.t. strain ATCC 10792 was obtained from Genbank (Accession number X14178) and is described by Lechner et al., (Lechner, M., et al., Mol. Microbiol. 3: 6Z1-626 (1989)). In addition to the pic gene, this 2254bp sequence contained 454bp upstream of the pic gene and 810bp downstream. Two primers, Phosl and Phos4, described above in Table 2, were designed to hybridize to the sequences that flank the pic gene. These primers were used in a polymerase chain reaction with HD73 total DNA template to generate a 2.2kb fragment carrying the HD73 pic region. The PCR product was treated with the Klenow fragment of E. coli DNA polymerase I and cloned into pUC18 cut with Smal to generate pSB139.

Plasmid pSB139 was digested with Kpnl and BamHI and die pic region was isolated from the vector sequences. Plasmid pSB141 was also cut with Kpnl and BamHI and the resulting fragments were ligated with the isolated pic region. The resulting construct, pSB141.5, carried the pBR322 portion of plasmid pSB141 and the pic region from pSB139.

Next, plasmids pSB141 and pSB141.5 were used to generate a plasmid that contained ermC, the pic region, and the cryHA operon. Plasmid pSB 141.5 was digested with BamHI. Plasmid pSB141 was digested with BamHI and the fragment carrying the cryHA operon and the ermC gene was isolated. The cr HA ermC BamHI fragment was ligated to die linearized pSB141.5 to form pSB147. A map of pSB147 is shown in Figure 6 and the derivation of pSB147 is shown in Figure 2.

Example 7: Introduction of Hybrid Plasmid into B.t by Electroporation

Crystal genes were integrated into the B.t. cells by electroporating plasmids (electrotransformation). The plasmids did not contain a gram-positive origin necessary for replication in the cell. Instead they carried a region of DNA that acts as a target for integration into the chromosome, the phos C region; and a selectable marker providing resistance to erythromycin. The plasmid, electroporated in high concentrations was forced into the chromosome via a single cross-over event, which causes a duplication of the target site. Chromosomal integrants of strain HD73, using plasmids pSB147 and pSB210.2, were obtained using this technique. Other strains, however, proved recalcitrant to electroporation, and could not be transformed at high efficiencies to allow chromosomal integration to occur. Competent cells were prepared by inoculating 100ml of Brain Heart Infusion media (Difco) containing 0.5M sucrose (BHIS) with a white disposable loop of cells from a fresh overnight LB plate. The cells were grown in a 1 baffled flask at 37°C and 300rpm to an O.D. of 0.2 at 600nm, after which point, the cells were kept on ice. All wash solutions used were cold. Cells were transferred to sterile 250ml bottles and pelleted at 6000rpm for 7 min. The cell pellet was washed once in one volume and washed twice in 1/10 volume of 0.5M sucrose, 5mM HEPES pH7. The pellet was resuspended in a final volume of 10ml of the HEPES-sucrose solution. Freshly-prepared cells were used for electroporation of integrative plasmids.

Plasmid DNA was mixed with 200μl of competent cells. Pulse parameters for HD73 cells were kV = 1.25, μF= 3 and Ω = ∞. After the pulse was delivered, the cells were transferred to 5ml BHIS in a 125ml flask and were allowed to recover at 30°C, with shaking at 250rpm for 3 hrs. For the integrative vector samples, the cultures were pelleted at 7,000rpm for 5 min and plated on LB with lOμg/ml erythromycin.

pSB098 was used as control plasmid DNA to determine the transformation efficiency of the electroporation procedure. pSB098 is a shuttle vector containing pTZ19R (Pharmacia) and pBClό.l. pBClό.l is a B. cereus vector constructed by Kreft (Kreft, J., Mol. Gen. Genet. 162:59 (1978)). pTZ19R and pBClό.l were both digested with EcoRI. The linearized plasmids were ligated into one plasmid, pSB098, in which the ampicillin and tetracycline resistance genes have opposite polarities.

Competent cells of strain HD73 were electrotransformed with plasmid DNA isolated from a dam-, dcm- strain GM2163. The results are given in Table 3 below. Additionally strain HD1- 51 was transformed with plasmid 210.1 (data not shown). Table 3: Transformation Efficiency of B. thuringiensis kurstaki HD73

Efficiency

Plasmid Amount Amount Number of (transformants/

(μg) (μl) transfectants μg pSB098 DNA)

HD73 STRAIN pSB210.2 9 10 3 3.5x10^s pSB147 15 10 8 2 x IO⁶

The efficiency given in the final column of the table was determined for each experiment by electroporating 0.5 lμg pSB098 and calculating the number of colony-forming units obtained per μg DNA. This gave an estimate of how well the cells responded to the conditions of the electroporation. No transformants of pSB210.3 were obtained.

The HD73 strain of B.t. kurstaki was obtained from USDA (Bacillus thuringiensis Cultures Available from the U.S. Department of Agriculture, USDA/ARS Agricultural Reviews and Manuals: ARM-S-30 October 1982).

The transformants, also referred to as recombinants or transfectants, were analyzed by PCR for gene content. Recombinants of HD73 containing the pSB210.2 sequences (HD73::pSB210.2) were screened for the presence of the crylC gene using primers galpl and galp2 and the ermC gene using primers PG2 and PG4. Two of the eight HD73::pSB147 clones were confirmed to have the cryHA gene using primers cryHA 1 and cr HA2 and the ermC gene with PG2 and PG4. The primer sequences are provided in Table 4 above. A profile of the plasmids showed the HD73::pSB210.2 5 recombinant to be identical to its parental strain, HD73.

Example 8: Preparation of Phage for Encapsulation of Hybrid Plasmid

Phage CP51 was obtained in filter discs impregnated with infected spores of B.cereus strain 569 according to the method of Thome (1978), supra. The strain was revived by inoculating 25ml NBY (8g Difco Nutrient broth, 3g Difco yeast extract/L) broth containing 0.4% glycerol (NBYG) with one of the discs and growing at 37°C for 16 hrs. The culture was harvested, the cell debris spun down at 10,000rpm, and the phage lysate sterilized by passing through a 0.45μM filter and stored at 16°C. The titer of the lysate was determined by assaying against a phage-free isolate of strain 569. The lysate was diluted 10 and 100-fold in 1% peptone. Approximately IO⁶ cells of 569 were mixed with lOOμl of the diluted phage and added to 2ml of TBAB (Difco Tryptose blood agar Base) soft agar. This was plated as an overlay onto Phage Assay (PA) plates (8 g Difco nutrient broth, 59 NaCl, 0.2g MgSO₄7H₂0, 0.05g MnSO₄H₂0, 0.15g CaCl₂2H₂0/fi, pH 5.9-6.0) which had been dried overnight at room temperature. The plates were incubated at 30°C overnight and plaques were counted.

To test for susceptibility to infection by the phage, approximately IO⁶ cells of each strain of interest (SA11, SA12, S287 and HD73) were plated as an overlay on PA plates. The surface of the agar was gently touched with an inoculating loop of the phage stock and the plates incubated at 30°C. The next day clearing was observed on die cell lawns of all four strains.

Example 9: Propagation of Phage

Cells from a fresh overnight plate of HD73::pSB210.2 were used to inoculate 6ml LB in a 20mm tube. The culture was grown at 37°C for 4 to 6 hrs., its optical density determined, and the cells diluted with LB to a concentration of 3xl0⁶cells/ml. NBYG plates were overlayed with 4ml NBY soft agar with 0.5ml CP51 phage stock containing 4X10⁶ PFU's and either 1X10⁶ or 3X10⁶ cells. The plates were incubated overnight at 30°C, and the phage were harvested in 5ml PA broth. The top agar was macerated in the PA broth and transferred to 18mm plastic tubes. The cell debris was pelleted. The lysate, labeled CP210.2, was sterilized by passing through a 0.45μm filter and stored at 15°C.

To determine the titer, 5X10⁶ CPU of strain HD73 were mixed with lOOμl of 10² and 10^-4 dilutions of lysate CP210.2 and poured as an overlay on PA plates with 2ml NBY soft agar. After overnight incubation at 30°C, the IO^"4 plate had 1200 plaques and the titer was estimated to be 1.2xl0⁸ PFU/ml.

Example 10: Determination of Conditions for Transduction

The methods used for handling the phage were based on those described by Thorne (1978), supra. The colony-forming units (CFU) per ml and the titer of phage stocks in plaque forming units (PFU) per ml of each B.t. strain were determined by serial dilution. The titers of CP51 phage stocks were determined as follows. 0.1 ml of the phage diluted in 1 % Peptone and approximately 2X10⁷ spores of B. cereus 569 were added to 2ml of PA soft agar and die inoculated soft agar was overlayed onto PA agar plates. The overlayed plates were incubated at 30°C for 16 to 20 hrs. The plaques were counted and the PFU/ml of phage stocks were determined from the dilutions used. The cell concentrations of B.t. cultures were determined by standard methods used in the art. The results are shown in Table 4 below.

Table 4: Determination of Colonies (CFU) of B.t. Cells and Stock of Phage Titer (PFU)

CFU CFU/ml

. cereus strain 569 5 x IO⁷

. thuringiensis strains HD73 8.6 x 10⁸

SA11 and SA12 2.6 x 10⁸ S287 1.1 x IO⁷

PFU PFU/ml

CP51: 8 x IO⁶

CP210.2: 1.2 x 10⁸

Example 11; Construction of pSB136 Plasmid pSB136 (described in Figure 7) is an integration vector which facilitates insertion of the crylC gene into the B.t. chromosome. The pSB136 vector carries the crylC gene, an integration target site, a tetracycline resistance gene, and a portion of the pBR322 vector. The crylC B.t. aizawai HD229 gene and the pBC16-l B. cereus plasmid tetracycline resistance gene (tef) were cloned. The integration target site was a fragment of DNA of unknown function from the B.t. kurstaki HDl cryB chromosome.

The pSB136 plasmid may be used to place the crylC gene in the chromosome of any B.t. strain that is not already tetracycline resistant. For integration to occur, however, the recipient strain must have sequences homologous to the integration target and the strain must be efficiently transformed (> IO⁴ transformants per microgram transforming DNA).

Competent E.coli DH5α were prepared by the method of Alexander (Alexander (1987), supra). The transformation was conducted by methods well known in me art using Library Efficiency DH5α Competent Cells (Gibco, BRL, Life Technologies, Inc., Gaithersburg, MD).

The selection for transformants was conducted on LB containing 75μg/ml ampicillin. Restriction enzyme digestions, ligations, ethanol precipitation, phenol extractions, kinase reactions and the treatment of DNA with T4 DNA polymerase, calf intestinal alkaline phosphatase, and die Klenow fragment of E.coli DNA polymerase I were conducted by d e methods of Maniatis et al. (Maniatis.T., et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)).

B.t. kurstaki HDl cry-B is a plasmid cured strain of HDl described by Stahly et al. (Stahly, D.P., et al., Biochem. Biophys. Res. Comm. 84:581 (1978)). Total DNA was isolated from B.t. kurstaki HDl cry-B by the following method. 200ml of 2XTY were inoculated witii B.t. and incubated at 30°C overnight with baffling at 200 rpm. 200ml of 2XTY were inoculated with 2ml of the above culture and incubated at 30°C witii baffling at 300rpm. Cells were collected by centrifugation at 10,000 rpm for 5 min. at 4°C when the optical density of the culture reached an OD^ = 0.8 to 1. The cells were washed with TES (TE+100 mM NaCl) and suspended in 18ml 25% sucrose +25mM TrisHCl (pH8)+25mM EDTA. 2ml of lOmg/ml lysozyme were added to the sucrose solution and mixed gentiy. The mixture was incubated at 37°C for 30 to 60 min. and checked for protoplasts. 2.2ml of 20% SDS were added, mixed gently and incubated at 50°C for 15 min. 5.5ml of 5M NaCl were then added, mixed gently and incubated at 50°C for 5 min. and at 4°C overnight. The mixture was centrifuged at 10,000 rpm for 10 min. at 4°C and the supernatant placed into 2 tubes. 28ml (56ml total) of cold EtOH were added, mixed gentiy, incubated overnight at -20°C and then centrifuged at 13,000 rpm for 30 min. The precipitate was recovered and washed in 70% EtOH. The precipitate was then dissolved in 10ml (20ml total) of 1M NaCl in TE and incubated at 4°C overnight. The two tubes were then combined into one tube. 200μl of 1 mg/ml RNase and 1200μl of lOmg/ml proteinase K were added and the mixture was incubated at 37°C for 30 min. 20ml of phenol/chloroform were added and die mixture was centrifuged. The aqueous layer was collected and washed twice more with 20ml of phenol chloroform. 20ml of chloroform were added to the aqueous layer and centrifuged.. The aqueous layer was collected and dialyzed against 2000ml of TE overnight. The plasmid DNA for cloning was isolated from E.coli cells by alkaline lysis (Birnboim, H.C. and Doly, J., "A Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA", Nucl. Acids Res. 7:1513-1523 (1979)) or with Qiagen columns obtained from Qiagen Inc.

The construction of pSB136 was divided into the four parts (reference is made to Figure 7). In the first step, the tetracycline resistance gene from pBR322 (which functions in E.coli) was replaced by a tef gene functional in Bacilli. Next, an integration target site was added, a piece of DNA of unknown function isolated from the HDl cryB genome. In step three, a Notl linker was added to facilitate cloning the crylC gene. In the final cloning step, the crylC gene was added to die integration vector. Each of these steps is described in more detail below.

Plasmid pSB206 was constructed by cloning the tef gene from pBC16 (Bernhard, K., et al., J. Bacteriol., 133:897-903 (1978)) into pUC18. Plasmid pBC16-l was generated from plasmid pBC16 by removal of an EcoRI fragment by me method of Kreft et al. (Kreft, J., et al., Mol. Gen. Genet., 162:59-67 (1978)). The tet^r gene was isolated from pBC16-l using the polymerase chain reaction witii primers Tet3 and Tet4 described in Table 4 above. Primer Tet3 introduced a HindHI site upstream of the tef gene and primer Tet4 introduced a Kpnl site downstream of the tet^r gene. The PCR product was inserted into pUC18 at the poly linker cartridge HindH site.

To remove the tet¹ gene, plasmid pSB206 was digested witii Smal and HindHI. To remove the tet^r gene from pBR322, this plasmid was cut witii Aval, treated with the Klenow fragment of E. coli DNA polymerase I to generate blunt ends, and tiien digested witii HindHI. The desired fragments were purified and the pBR322 vector was ligated with the tet^r gene from pSB206 to generate pSB131.

An integration target site was then added to pSB131. The source of the target site was a 1.1 kb DNA fragment isolated from 10 the HDl cryB genome. This fragment was cloned in pUC18 and the construct was named pSB132. The l.lkb DNA fragment from HDl cryB was isolated as follows. Total HDl cryB DNA was restricted with HaeHI or EcoRV and the two digests were mixed and subjected to electrophoresis on an 0.8% agarose gel. Three size 15 fractions were cut from the gel: (1) 0.5kb -0.9kb; (2) 0.9kb -1.8kb; (3) 1.8kb-2.7kb.

The DNA fractions (1) and (2) were purified. The DNA from fraction (2) was ligated into pUC18 cut witii Smal and die resulting clones were characterized by EcoRI/HindHI digests. The plasmid called pSB132 had a l.lkb insert.

The l.lkb cryB fragment was then transferred from pSB132 to pSB131. Plasmid pSB132 was digested with EcoRI, filled by 25 treatment with the Klenow fragment of E. coli DNA Polymerase 1, and digested witii HindHI. Plasmid pSB131 was cut with Sspl and HindHI and ligated with the purified 1.1 kb fragment from plasmid pSB132 to yield plasmid pSB134.

A Notl linker was added to pSB134 to faciUtate addition of the crylC gene. The sequence of the Notl linkers was pAGCGGCCGCT (New England Biolabs #1125, SEQ ID No. 28). Plasmid pSB134 was digested witii BamHI and blunt ends were generated by treatment with the Klenow fragment of E.coli DNA polymerase I. The Notl linkers were then ligated to the linearized pSB134 in a 200:1 molar ratio and the resulting construct was named pSB 134.5.

The final step was to add the crylC gene to pSB 134.5. The source of crylC was plasmid pSB619, described above in Example 3. pSB619 carries the crylC gene from B.t. aizawai HD229 preceded by its native promoter and followed by the B.t. kurstaki 10 HD73 cryΙA(c) terminator. The crylC gene with the promoter and terminator were cloned as an Apal/Notl cassette in bluescript KS(+). To isolate crylC, pSB619 was cut with Apal, filled witii T4 DNA polymerase, and digested witii Notl. Plasmid pSB 134.5 was cut with EcoRI, filled by treatment with the Klenow fragment of 15 E.coli DNA polymerase I, and then cut with Notl. The crylC cassette was purified from the vector portion of pSB619 and ligated into pSB 134.5 to generate pSB136.

Example 12: Introduction of crylC Gene into B.t kurstaki Strains HDl cryB and HD73

Plasmid pSB136 was constructed as described in Example 11 above. It contains the crylC gene, the gene encoding tetracycline resistance from pBClό.l, and a portion of DNA from the HDl CryB chromosome of unknown function which acts as an integration target site. These fragments were ligated into d e plasmid pBR322. Competent B.t. kurstaki HDl cryB and HD73 cells were prepared according to the BHIS protocol described in Example 6 above. After delivering the electrical pulse, the cells recovered in 5ml BHIS for 3 hrs. at 37°C. The pulse parameters for the HDl Cry B cells were 1.05kV, 25μF, R = ~. The pulse parameters for HD73 cells were 1.25kV, 3μF, R = ∞. Following electroporation, the entire culture was pelleted, resuspended in a small volume and plated on selective media. pSB098 DNA was used as the standard for determining transformation efficiency, and for each experiment the efficiency was expressed as colony-forming units (CFU's) per μg of pSB098 plasmid.

In e HDl CryB cells, one tetracycline-resistant colony was obtained when 7.5μg of the plasmid pSB136 was electroporated into the cells. The overall efficiency of transformation of the cells was 6 xlO⁵ CFU/μg (using pSB098). This new recombinant strain, CryB ::pSB 136, was shown to contain the tetracycline-resistance gene and the crylC gene by PCR analysis described below in Example 13.

CryB::pSB136 was grown in CYS medium to sporulation. It had 4X10⁸ spores/ml, compared to 5X10⁸ spores/ml for HDl CrylB in the same experiment. The tetracyclineresistance gene was 98% stable through sporulation and germination.

In HD73 cells, two colonies were obtained when 15μg of plasmid pSB136 was electroporated into the cells in an experiment where the overali efficiency was 2xl0⁶ CFUμg DNA. Both colonies, designated HD73::pSB136, were positive for die crylC gene and the tetracycline-resistance gene by PCR as described in Example 13 below.

Example 13: PCR Screening of CryB::pSB136 and HD73::pSB136 Recombinant Strains

The presence of the introduced crylC gene and tetracycline resistance marker in the CryB ::pSB 136 and HD73::pSB136 5 recombined strains was confirmed by PCR. Cells from a fresh overnight plate were boiled for 10 min. in 8μl of a solution containing the necessary primers (0.5μl of 20μM stock solution) and the dNTP mix (l.όμl of 1.25mM stock solution) in 1 x Taq polymerase buffer. Cell debris was pelletted and 2μl of a solution containing 0.05 unit Taq polymerase in 1 x Taq polymerase buffer was added. Two primer sets were used to screen for the tetracycline resistance gene. The combination of Tet3 with Tet4 produced a fragment approximately 1.4kb, and the combination of Tet3 with CPOl.Rev gave a fragment approximately 0.35kb in size. To screen for the crylC gene, primers galPl and galP2 were used to produce a 0.8kb size fragment. Primer sequences are given in Table 2 above.

Example 14: Introduction of crylC and cryHA Genes into B.t kurstaki Strain HD73

Plasmid pSB304 was constructed as described in Example 5 above. The cryHA gene was isolated from pSB304 as a Notl-EcoRI fragment. pSB 134.5 was constructed as described in Example 10 above. The cryHA Notl-EcoRI fragment was ligated to pSB 134.5 cut witii Notl and EcoRI to form plasmid pSB 134.5.2.

Competent HD73 cells were prepared according to the BHIS protocol described in Example 6 above. After delivering the electrical pulse, the cells recovered in 5ml BHIS for 3 hrs. at 37°C. Pulse parameters for HD73 cells were 1.25kV, 3μF, R = ∞. Following electroporation, the entire culture was pelleted, resuspended in a small volume and plated on selective media. As in Example 12 above, pSB098 DNA was the standard for determining transformation efficiency and the transformation efficiency was expressed as colony-forming units (CFU's) per/μg of ρSB098 DNA.

Ten colonies were obtained from the electroporation of the HD73 cells with 20μg of the pSB 134.5.2 plasmid. The transformation efficiency was lxlO⁶ CFU/μg DNA. Two of the transfectants proved positive for the tetracycline resistance and cryHA genes by PCR analysis described in Example 15 below. The two transformants were designated HD73::pSB134.5.2.

Example 15: PCR Screening of HD73::pSB134.5.2 Recombinant Strain

The presence of the introduced cryHA gene and tetracycline resistance marker in the HD73::pSB 134.5.2 recombinants was confirmed by PCR as described on Example 13. To screen for the cryHA gene, the primers cry HA 1 and cryHA2 were used to produce a 0.57kb fragment. Primer sequences are given in the Table 4 above.

Example 16: Transduction of B.t Strains

Generalized transduction was conducted to transfer a crystal gene which had been integrated into the chromosome of one strain to the chromosome of a strain not easily transformed by electroporation. In these cases one strain containing an integrated crystal gene, HD73::pSB210.2, was used as a donor for DNA to transduce several B.t. strains including strains SA11, SA12 and S287.

Generalized transduction as a means of genetic exchange has been widely used and well characterized for E. coli and S. typhimurium by Margolin and used somewhat for Bacillus thuringiensis as described by Thome (1978) (Margolin, "Escherichia coli and Salmonella typhimurium", Cell, and Mol. Biol., (1987); Thome, "Transduction in Bacillus thuringiensis", Applied and Environmental Microbiology, 35:1109-115 (1978)).

The generalized transducing phage CP51 isolated from soil and used in Bacillus cereus chromosomal mapping experiments was obtained from Thome, C.B., (Thome, C.B., "Transducing Bacteriophage for Bacillus cereus", J. of Virol., 2:657-662 (1968) and "Transduction of Bacillus cereus and Bacillus anthracis", Bacteriological Reviews,32:358-361 (1968)). Thorne et al. further tested the phage against other strains of Bacilli, including B. thuringiensis as described by Thome (1978), supra.

Cells of strains SA11, SA12, S287 and HD73 were grown according to the metiiod described in Example 8 above and diluted to approximately 10⁷ CFU/ml.

The recombinant strain HD73::pSB210.2, isolate number 2, was used to propagate phage CP51. The titer of the resulting lysate, named CP210.2, was estimated to be 1.2 x 10⁸ plaque forming units (PFU) per ml. A sterile HA filter (Millipore) was placed on the surface of an LB plate then lOOμl each of phage lysate CP210.2 and cells were pipetted onto the filter and gentiy mixed using a sterile wire spreader. The plates were incubated at 37°C for 3 hrs. The filters were transferred to LB plates with erythromycin (lOμg/ml), returned to 37°C, and allowed to grow for 36 hrs. The results of the plate transductions are given in Table 5 below. Table 5: Results of Transduction Experiments

Strain CPU's plated Multiplicity Ery^r Efficiency of Infection Colonies

HD73 1 x IO⁶ 10 2 1.6 x IO^"7

SA11 4 x IO⁶ 3 4 3 x IO^"7

SA12 4 IO⁶ 3 2 1.6 x IO^'7

S287 1 x IO⁶ 40 3 2 x IO^"8

The efficiency is expressed as the number of erythromycin resistant colonies obtained per plaque-forming unit. For HD73, SA11 and SA12, 1.2xl0⁷ plaque-forming units were plated. For S287, 4x107 PFU were plated.

Example 17: Polvmerase-Chain Reaction Screening of Recombinant Strains

PCR screening of all recombinant strains was done using whole cells as described in Example 13. The erythromycin-resistant colonies, denoted by the abbreviation "CP", were analyzed by PCR for gene content compared to wild type strains. The results are displayed in Table 6 below.

Table 6: Gene content of transductants by PCR

Strain: IA(a) IA(b) IA(c) ΠA IC ermC

SA11WT + + + + - -

SA11CP1 + + + + + +

SA11CP2 + + + + + +

SA11CP3 + + + + + +

SA11CP4 + + + + + +

SA12WT + + + + - -

SA12CP1 + + + + + +

SA12CP2 + + + + + +

S287WT + + + + - -

S287CP1 + + + + + +

S287CP2 + + + + + +

The recombinants retained the array of crystal genes found in the parental strains. Only the recombinant strains were positive for the primers specific to the introduced crylC and ermC genes. When screening for the cryΙA(b) gene, the combination of TY6 and TY14 probes (described above in Table 2) was used initially but this pair showed some cross-reaction with the control plasmid pSB210.2, which contains only the crylC gene. In subsequent experiments, TY13 described in Table 2 above was substituted for TY14.

Example 18: Comparison of Wild Type and Recombinant B.t Strain Plasmids

The plasmids of the recombinant B.t. strains were isolated by the alkaline lysis procedure of Birnboim and Doly (1979), supra, modified as follows. The strains were streaked on LB+tetracycline and grown overnight at 30°C and restreaked on fresh SA (IX Spizizen salts, 1% casaminoacids, 5% glucose, 0.0005mM MnSO₄H₂O) plates, and grown for 3 to 4 hours at 37°C. For each strain, 2 to 3 loopfuls of cells were suspended in (lOOμl TESL lOOmM Tris pH8, 10 mMEDTA, 20% sucrose, 2mg/ml lysozyme) on ice and incubated for 15 min. at 37°C. 200μl lysis solution (0.2N NaOH, 1% SDS) was added, mixed delicately by tube inversion, and the mixture was incubated for 5 min. at room temperature. 150μl ice cold potassium acetate solution was added and mixed by tube inversion. Next, the solution was centrifuged for 20 min at 15000 rpm and 4°C. The supernatant was recovered with a disposable, wide-bore transfer pipet and then added to 1ml of 100% ethanol and mixed by tube inversion. The mixture was then centrifuged for 20 min. at 15000rpm and 4°C. The supernatant was removed by aspiration, and the pellet was resuspended in 1 ml of 70% ethanol, mixed by tube inversion and then centrifuged for 5 min. at room temp. The supernatant was removed by aspiration and the pellet was vacuum dried for 2 min. in a Speedvac. The dried pellet was suspended in 20μl of TE, incubated on ice for about 15 min., then mixed delicately by tapping the tube on its side.

The DNA was electrophoresed in lxTAE, 0.8% agarose at 70V/32mAmp for 3 hrs. A plasmid profile of these recombinants showed them to be identical to their wild-type parental strains, confirming that they did not carry unwanted plasmids.

Example 19: Comparison of Wild Type and Hybrid B.t. Strain Chromosomal DNA

The chromosomal DNAs of the integrants in strain HD73, and transductants in strains SA11, SA12, and HD73 were analyzed by DNA to DNA hybridization experiments. Three probe fragments were isolated from pSB139.

(a) A general phospholipase C (phosC) probe extending 1800 bp from BamHI to Clal of the known phosC sequence.

(b) Eco left (E_L) extending 850 bp from BamHI to EcoRI. (c) Eco right (ER), a 1427 bp EcoRI fragment. Chromosomal DNA from wild type HD73 as described in Example 11 was isolated from 100ml samples of cultures in 2XTΥ medium (5g yeast extract, 5g tryptone, 2.5g NaCl L)

Chromosomal DNA from wild type SA11, SA12 and HD73 and the corresponding transductants was isolated by using the ASAP kit from Boehringer Manheim, according to the manufacturer's directions. Chromosomal DNAs were digested to completion with EcoRI or Apal, separated on a 0.8% agarose gel in TBE buffer containing EtBr, depurinated, denatured, neutralized and transferred to Hy-Bond nylon membrane by overnight capillary blotting in 20X SSC according to the method of Sambrook et al. (Sambrook et al., "Molecular Cloning: A Laboratory Manualn, Cold Spring Harbor Laboratory Press (1989)). The DNA was fixed to the membrane using 0.4M NaOH for 20 min. Southern hybridizations were performed using the Amersham ECL kit according to protocol.

The analysis by DNA hybridization of EcoRI digested DNA from HD73 and HD73::pSB210.2 using the large phosC probe revealed the expected 2.4kb and 4.3kb internal fragments from the integration vector, pSB210.2, but did not definitively show the chromosomal regions on either side of the integration site or flanking regions. A large discrepancy between the difference in intensity of the internal vector bands and the bands which were later determined to be the flanking regions indicated that multiple integration events may have occurred. Further analysis of the same filter using the E_L probe revealed not only the expected internal EcoRI fragments but also a 1.8kb band in the chromosomal DNA of integrants and wild type samples. This result shows that there is an EcoRI site 1.8kb upstream of the EcoRI present within the phosC gene. Hybridizing with the E_R probe showed the internal fragments in the integrant samples and an approximately 9kb band in both the integrants and wild type DNA, identifying the next EcoRI site downstream from the internal phosC EcoRI site.

The presence of the same EcoRI bands upstream and downstream of the phosC EcoRI site in both the wild type and all integrants proves that the integration occurred at the chromosomal phospholipase C region as expected.

A similar Southern blot analysis of the same DNA samples digested with Apal proved that multiple integrations had occurred. Chromosomal DNA from an isolate in which a single integration event had occurred would show just two bands, each corresponding to Apal fragments extending from the Apal site within the integration vector, pSB210.2 to a wild type Apal site on the chromosomes, either upstream or downstream of the integration site. Multiple integrations would produce the same upstream and downstream bands plus an additional internal band corresponding to DNA extending between Apal sites introduced by two tandem integrated vectors.

Southern analysis did show a 10.4kb fragment corresponding to the full length of the integrated plasmid, indicating that multiple integration events had occurred in the region. However, the flanking Apal chromosomal fragments were not observed nor could it be determined if more than two integration events had occurred. Southern analyses of EcoRI digested DNA from the transductants, SA11 CP1, SA11 CP2, SA12CP1 and SA12CP2 using the E_L and E_R probes also revealed 2.4 kb and 4.3 kb internal EcoRI bands, indicating that the DNA carried in the transducing particles was derived from the desired integrated phosC site. These internal bands were not present in the lanes containing wild type SA11 and SA12 DNA. The probes also hybridized to the same size flanking bands, 1.8kb and approximately 9kb, as they did in wild type HD73, HD1-51, and the corresponding integrants. The chromosomes of SA11, SA12, HD73 and HDl -51 are similar in the phosC area. The actual site of crossover events cannot be determined using these probes.

Example 20: Stability and Viability of Hybrid B.t Strains

The recombinant strains SAUCPl and SA12CP2 were analyzed for stabihty. The stability of the introduced genes was determined by growing the strains without antibiotic selection through sporulation and germination.

The recombinant SA11CP1 and SA12CP2 and wild type SA11 and SA12 strains were streaked on LB plates and incubated overnight at 30°C. A single colony from each plate was used to inoculate a separate 100 ml CYS culture in a 500ml baffled flask. The cultures were grown at 30°C with baffling at 300 rpm. When cultures reached an A^ of 0.8 they were diluted 1:10. The growth of the cultures was monitored each half hour to determine growth curves. Following the transition from log phase to stationary phase, the cultures were grown for an additional 48 hrs. 1 :10 dilutions were made for each sporulated culture and the dilutions were heated at 65°C for 45 minutes.

It was assumed that the samples contained approximately IO⁹ spores/ml. The samples were diluted and plated on LB. The numbers of germinated spores of the recombinant strain were compared to those obtained for the wild type. Fifty to one hundred of the colonies were replica-plated onto LB with erythromycin and onto LB alone and incubated at 30°C overnight. The percentage of colonies retaining the selectable marker was determined against the number of viable colonies.

The newly introduced genes were found to be 100% stable through sporulation and germination, as determined by the continued resistance to erythromycin and by the presence of the crylC and ermC genes detected by PCR. No spontaneous erythromycin- resistant colonies were obtained. The rate of growth of these recombinants in CYS was essentially the same as their parental strains over the first 8 hrs. 9.3 x IO⁷ and 1.5 x 10⁸ spores per ml were estimated for SA11 (WT) and the recombinant SAUCPl, respectively. 3.5 x IO⁷ and 3.4 x 10⁷ spores per ml were estimated for SA12(WT) and the recombinant SA12CP2, respectively. The introduced genes had no deleterious effect on the viability of the recombinant strains.

Example 21: Expression of Gene Product in Hybrid B.t Strains

To test for gene expression, 10 ml CYS (lOg casitone, 5g glucose, 2g yeast extract, lg KH₂PO₄, 1ml 50mM MgCl₂, 1ml 50mM MnCl₂, 1 ml 50 mM ZnSO₄, 1 ml 50 mM FeCl₃, 1 ml 200mM CaCl-^L) cultures of the recombinants and parental strains were grown for 36 hrs. at 30°C. 50μl were mixed with an equal volume of 2x sample loading buffer (0.125MI Tris-HCl pH8, 4% SDS, 0.005% Bromophenol Blue, 20% (v/v) glycerol, 4%(v/v), B mercapteothanol) and immediately boiled for 5 min. 2.5, 5, and lOμl aliquots were loaded on a 10% acrylamide gel (Novex) and electrophoresed for 1.5 hrs. at 125 volts.

Expression of the introduced crystal genes could clearly be detected by SDS-polyacrylamide gel electrophoresis of all the recombinant strains. In the case of HD73::pSB147, a new band at approximately 65Kd was detected in addition to the band at 130Kd seen in the wild-type strain. 65Kd is the size expected for the CryHA protein. An additional band running at 135Kd, the size expected for the CrylC protein, was seen for HD73::pSB210.2, SAUCPl, SA11CP2, SA11CP3, SA11CP4, SA12CP1, SA12CP2, S287CP1 and S287CP2, and was not seen for wild type HD73, SA11, SA12 and S287 strains. The recombinant strains continued to express the other Cryl-type proteins expressed by their parental wild-type strains, detected as a band running at 130Kd.

Example 22: Hybrid B.t Strain Lethality Bioassay

For most bioassays the samples were grown in 100ml Fishmeal (5.5% Fishmeal, 4% Starch, 0.1 % NH₄CI, 0.125% KH₂PO₄, 0.05% MgS0₄, 0.001% FeSO₄, 0.001% MnCl-JL) in a 500ml baffled flask at 30°C for 72 hrs., 300rpm. For bioassay No. 1087, the samples were again grown in 100ml Fish Meal in a 500ml baffled flask at 30°C, but used a 5% inoculum from a 100ml Fish meal starter culture which had been inoculated with a loop of spores from a fresh slant and grown for 6 hrs. at 30°C. The SAl l samples (control) and its recombinants were harvested after 41 hrs., and the SA12 samples (control) and its recombinants were harvested after 47 hrs. of growth. All samples were examined for protein expression by diluting 1:10 with water, and then treated as the CYS cultures described above in Example 18. After harvesting, the cultures were stored at 4°C.

The recombinant SAl l, SA12 and S287 strains grown in Fishmeal medium were assayed against Trichopulsia ni and Spodoptera exigua according to the following protocol. Samples of the recombinant strains were mixed with an artificial insect diet containing 132g/L wheat germ, 28g/L casein, l lg/L vitamin mix (Moorehead & Co. Van Nuys, CA), 8.8g/L salt mix (BioServ, Frenchtown, NJ), 2.3g/L sorbic acid, 1.1 g/L methyl paraben, 13g/L agar and 1.5ml L formaldehyde. The mixture was then fed to late third instar larvae incubated at 25°C. The mortality was recorded after 4 days, and the LC_J0 was determined by probit analysis as is known in the art.

All the recombinant strains had higher activity against S. exigua than the wild type strains from which they were derived. The increase in activity against Spodoptera exigua ranged from 1.6 to 2.2 fold.

The crylC gene has been introduced into the chromosomes of several different strains of Bacillus thuringiensis at a known site using the techniques of electrotransformation and transduction with a phage lysate. For the recombinant strains produced by transduction, the expression of the crylC gene was detected by SDS-PAGE, and the CrylC protein contributed to the bioactivity against S. exigua. Introducing the crylC in the chromosome did not cause instability of the resident plasmids, and is itself stably maintained through sporulation and germination.

Example 23: Determination of Temperature For Inhibition of pLTVl Replication in B.t

B. subtilis PY1177 (pLTVl) was obtained from Dr. Phil Youngman is described by Camilli et al. (Camilli et al., J. Bacteriol. 172: 3738-3744 (1990)). pLTVl DNA was isolated from PY1177 according to die method of Birnboim and Doly (1979), supra. B.t. kurstaki HD73 was transformed with pLTVl DNA by electroporation as described in Example 7 above and the transformant was designated HD73 + pLTVl.

The temperature required to abolish the replication of pLVTl in B.t. kurstaki HD73 was determined by two consecutive heattreatments at different temperatures, the first in liquid medium and the second on a solid medium according to the method of Bohall, (Bohall, N. A., J. Bacteriology 167: 716-718 (1986)). A flask containing 10 ml of LB tet,₀ was inoculated with a single colony of HD73 +pLTVl. This primary culture was grown to OO₆₀₀ = 0.4 at 30°C with baffling at 300rpm. The cells were recovered by centrifugation, washed in LB (containing no antibiotics) to remove tetracycline and resuspended in 10ml of LB with no antibiotics. The resuspended cells were used to inoculate (1%) 10ml LB cultures prewarmed to 30, 37, 40 and 42°C. After inoculation, the cultures were maintained at their respective temperatures until the cultures reached an OD^ value between 0.6 to 0.8. The cultures were then diluted by a factor of IO⁴ to IO⁷ and lOOμl aliquots of the dilutions were spread in onto LB ery₀₀₅ plates. Each plate was incubated overnight at d e incubation temperature of the corresponding primary culture. After overnight incubation, the colonies from the LB ery₀₀₅ plates were replica-patched onto four different LB plates containing no antibiotics as well as LB ery₁₀, LB cm₁₂ and LB tet_I0 plates. The plates were incubated at 30°C overnight and scored the following day for antibiotic sensitivity.

Experiments in which the primary cultures were grown in LB liquid which contained tetracycline produced the following results. At the pLTVl replication-permissive temperature of 30°C, 20% of the colonies examined were sensitive to erythromycin, chloramphenicol and tetracycline indicating the loss of the plasmid due to inhibition of replication. At 37°C, 98% of the colonies were sensitive to all three antibiotics. At 40°C, 94% of the colonies were sensitive all three antibiotics and at 42°C, 100% of the colonies were sensitive. Experiments in which the liquid primary cultures contained no tetracycline exhibited heat-induced plasmid loss at the same temperatures. At the permissive temperature of 30°C, tetracycline-free cultures exhibited a 38% plasmid loss (significantly higher than the 20% loss exhibited by LB + tet cultures incubated at 30°C). Tetracycline-free cultures incubated at 37°C, 40°C and 42°C exhibited plasmid losses of 94%, 90% and 99%, respectively. Therefore, it was concluded that plasmid replication of pLTVl was inhibited at 37°C or above under the experimental conditions used in this study. Since it can be lost even at its replication-permissive temperature, the pLTVl plasmid appears to be somewhat unstable.

Example 24: Determination of Conditions for Transposition of pLTVl-Borne Tn917 in B.t

Retrieving transposed colonies is a tiiree-day, three-step procedure. This method was first tested with HD73 + pLTVl. First, 10ml of liquid LB containing ery„ cvn- and tetj was inoculated with an single colony of HD73 containing pLTVl and grown to an OD^ = 0.7 at 30°C with baffling at 300 rpm. This primary culture was then centrifuged and the pelleted cells were washed in 10ml LB to remove the antibiotics. Two secondary flasks containing lOOml LB containing both ery, and cπ^ (but no tet) were inoculated witii lOOμl of the washed primary culture. One of these flasks was incubated at 30°C (permissive) while the other was grown at 37°C (non-permissive) at 300 rpm overnight.

After overnight growth, both cultures were diluted and plated onto LB alone and LB ery, cm₅ plates. The plates were incubated overnight at the same temperatures at which the corresponding secondary flasks had been incubated. After this second overnight heat treatment, individual colonies from the 37°C LB containing ery, cm₅ plates were replica-patched onto LB alone, LB containing ery, cπ^, LB containing ery₁₀, LB containing cm₇ and LB containing tet,₀ plates to determine what percentage of the colonies were resistant to erythromycin, chloramphenicol but sensitive to tetracycline, indicating that a transposition event had occurred. The ery^rcm^rtet^s colonies obtained were designated HD73::pLTVl.

Almost 100% of the HD73 colonies derived from these experiments with pLTVl showed the transposition of the lacZ gene from pLTNl, as expected. PCR analysis of the ery^rcmr^rtet^s HD73 colonies using primers LACΝHSl and LACΝHS2 indicated tiiat the lacZ gene from pLVTl was present in all cases. Further PCR analysis using primers TY6 and TY7 indicated that 38 out of 40 transposed colonies retained the native cryΙA(c) gene. The primer sequences are provided in Table 7 below.

Table 7: Sequences of Oligonucleotides

SEQ.H No.

LACNHSl GGCTTTCGCTACCTGGAGAGACGCGCCCGC 29

LACNHS2 CCAGACCAACTGGTAATGGTAGAGACCGGC 30

TY6 GGTCGTGGCTATATCATTCGTGTCACAGC 31

TY7 CCACGCTATCCACGATGAATGTTCCTTC 32

NHS21 GATATTTTAGCTCATGATCTTTTCCTCCTATTAAC 33

NHS37 CATTACGCATTTGGAATACC 34

The pSB050 plasmid was assembled in order to introduce the cryHA operon from B.t. galleriae HD232 onto the HD73 genome. The B.t. galleriae strain HD232 was obtained from the USDA. The entire cryHA operon from HD232 was isolated as a 4kb BamHI HincH fragment from pSB304 described in Example 5. The plasmid pLTVl was cut with restriction enzymes Smal and BamHI to produce a 20.6kb fragment and die fragments were ligated together to form pSB050. The ligation products were used to transfect the dam-, dcm- GM2163 E.coli strain. Individual GM2163 colonies containing the desired construct were first selected on LB amp₇₅, and then replica-patched onto a series of agar plates containing either no antibiotics, amp_7s, cm₇, ery₁₀ or tet,₀. Of fifty-five colonies examined, five were resistant to tetracycline, ampicillin and erythromycin indicating that the correct fragments had been ligated. All others were sensitive to tetracycline indicating a lack of pLTNl. The resistant colonies were further screened by PCR amplification of the 1400 bp product between the LACΝHSl and LACΝHS2 primers. The sequences of these primers are shown above in Table 7, and correspond to sequences which are within the lacZ gene contained in pLTVl. Amplification with PCR and the NHS37 and NHS21 primers, whose sequences are shown in Table 7 above produced a region from the erythromycin gene to the end of the first open reading frame of the cryHA operon. The DNA from the resistant colonies was analyzed by restriction analysis with BgHI and those colonies showing the expected PCR results and 2kb, 5.3kb and 16kb Bglll fragments were considered to contain the plasmid designated pSB050.

The transformation of HD73 with the pSB050 plasmid was achieved by electroporation of the host cells at 1.2kV, 3μF, and a resistance at ~ ohms with 5μg of pSB050 DNA isolated from GM2163. The cells were allowed to recover at the permissive temperature of 30°C for tiiree hours in BHIS medium witii baffling at 300 rpm. The cells were then concentrated, plated onto LB plates containing lOμg/ml tetracycline and incubated at 30°C overnight. The presence of pSB050 in the tetracycline resistant HD73 colonies was confirmed by PCR analysis using the LACNHS 1 and LACNHS2 primers (1400 bp product) and the NHS37 and NHS21 primers (600 bp product) under the conditions recommended by Perkin Elmer-Cetus. These isolates were designated as HD73+pSB050.

Example 26: Introduction of CryHA Into 50 Mdalton Plasmids of B.t HD73 by Transposition of pSB050-Borne Tn917

Several HD73 + pSB050 isolates obtained as described in Example 25 above were used to transpose the cryHA operon from pSB050 onto a large resident plasmid of HD73. The HD73 + pSB050 isolates were incubated at a non-permissive temperature and selected for chloramphenicol and erythromycin resistance and tetracycline sensitivity. The ery^rcm^ttet^s colonies obtained were designated HD73::050 indicating that the transposition event had occurred. The transposition was confirmed by PCR amplification of the expected 600bp fragment between the NHS37 and NHS21 primers (ery gene to cryHA) and the 1400bp product between the LACNHS 1 and LACNHS2 primers. The presence of an intact cryΙA(C) coding region was also confirmed by PCR amplification witii the TY6 and TY7 primers, whose sequences are provided in Table 7 above.

Example 27: Expression of CryΙA(c) and CryHA Genes in Transposed B.t Strain HD73::050

The microscopic observation of HD73 + pSB050 and HD73::050 at lOOOx magnification showed that both strains produced two crystal types. Both the bipyramidal CryΙA(c) crystals of HD73 and the cuboidal CryHA crystals, were observed in the HD73+pSB050 and HD73::050 cells. No such cuboidal crystals were seen in the wild-type HD73 cells.

The protein expression of CryΙA(c) and CryHA in HD73::050 was analyzed by SDS-PAGE. lOOμl samples of sporulated cultures (40 to 50 hours old) were pelleted, resuspended in lOmM EDTA and mixed 1 to 1 on ice with 2x SDS loading and boiled for three minutes. A 10% pre-cast Novex gel was run and stained witii Coomassie blue.

The SDS-PAGE analysis revealed the presence of the 135Kd CryΙA(c) and the 65Kd CryHA proteins. These were confirmed by Western blot analysis using specific antisera made against CryΙA(c) or CryHA on two separate blots.

Example 28: Spore Counts and Stability of B.t Strain HD73::050

As a gross indication of the relative health of the cultures, the number of spores per milliliter of culture was compared in samples of the following B.t. strains: the HD73 wild type, and the HD73 + pLTVl, HD73 + pSB050 and HD73::050 hybrids. The sporulated cultures were treated at 65°C for 45 min. to kill any remaining vegetative cells. Serial dilutions were plated onto LB agar plates, and colonies were counted the following day. Stability of the transposed DNA in HD73::050 was determined by plating the spore dilutions used above onto LB agar plates containing lOμg/ml tetracycline, 1 μg/ml erythromycin and 7μg/ml chloramphenicol or LB without antibiotics. The number of growing colonies with selection was compared to the number of growing colonies without selection. The spore counts for HD73::050 were slightly lower than those for the wild type HD73. They ranged from 1 to 2X10⁸ spores/ml. Most (i.e., 97 to 100%) of HD73::050 maintained the correct ery¹ cm^r tef antibiotic resistance, indicating that the transposed region did not become unstable and excised from DNA after transposition.

Example 29: Plasmid Profile of B.t Strain HD73::050

The plasmid content of HD73::050 was determined to asses if the transposition event had occurred onto the chromosome or a large size plasmid of HD73. The plasmid preparation procedure used was a slight adaptation of the Birnboim and Doly alkaline lysis protocol (Birnboim, H. C. and Doly (1979), supra) as previously described in Example 18. The plasmid profile analysis of the DNA preparations from HD73::050 and wild-type HD73 showed that the transposon had been inserted into two 50Mdalton plasmids present in the natural HD73 strain. The gels showed an increase in die molecular weight of the plasmid bands, which make them coincide with the molecular weight of the transposed DNA. In some HD73::050 isolates the high copy number 50Mdalton plasmid carrying the cryΙA(c) gene was the transposition target. In other isolates, the lower copy number 50Mdalton plasmid was the transposition target. All other plasmids within HD73::050, which are also normally present in HD73, showed no change in apparent molecular weight.

Example 30: Southern Analysis of Transposition in B.t Strains HD73::050 and HD73::pLTVl

Total DNA samples from wild type HD73, HD73::pLTVl and HD73::050 were prepared using the ASAP kit according to the protocol from Boehringer Mannheim. The DNA was digested overnight with excess BamHI or EcoRI, electrophoresed for 18 hrs. on a 20cm 0.8% TBE agarose gel and transferred to a Zeta-Probe membrane from Bio-Rad by overnight capillary blot in 20xSSC. The membrane was then treated and probed as described in the Amersham ECL kit. The 1400 bp lacZ gene PCR product described in Example 25 above was used to probe the transferred DNA. The bands corresponding to die HD73::050 DNA were compared to those bands corresponding to DNA from wild type HD73.

Southern blot analysis showed that the transposon had integrated into different locations on the 50Mdalton plasmids. No preferred transposition site was observed. The restriction map of pLTNl indicated that the DΝA from HD73::pLTv^*l contained BamHI fragments of 9kb or larger which hybridize to the probe. Bands of this size represent the transposed portion of pLTVl in one of the 50Mdalton plasmids. Similarly, the HD73::pLTv^"l DΝA contained 14kb or larger EcoRI fragments which hybridize to the probe. In all cases, the expected bands exhibited hybridization in the Southern analysis. No hybridized bands were of the same size, thus confirming that the pLTVl transposition event occurred at random places within the HD73 genome.

Southern blot analysis of the HD73::050 isolates showed the expected bands of 12kb or larger for BamHI digestion and 17kb or larger for EcoRI digestion. The difference in size of the hybridized bands between HD73::pLTVl and HD73::050 is due to die presence of the cryHA gene within the transposed region of HD73::050. This confirms that the transposed cryHA gene was inserted randomly within the HD73 genome.

Example 31; Lethality of B.t Strain HD73::050

Fresh overnight colonies of HD73::050 and wild type HD73 were separately inoculated into 500ml of the CYS medium described by Yamamoto and harvested by centrifugation and allowed to grow for 55 hrs. at 30°C with baffling at 300 rpm (Yamamoto, T. 1990. ACS Symposium Series 432: 46-60). The cells were resuspended in l/20volume of Buffer A (5mM Tris ρH8.0,0.25% Triton), and the cell lysate was electrophoresed on 8% Novex SDS-PAGE gels. The concentration of the CryΙA(c) protein was determined by densitometer scanning. Nineteen two-fold dilutions were made in Buffer A and the amount of CryΙA(c) in ppm was calculated for each dilution. Known amounts of protein were mixed with the insects' diet and fed to late third instar Trichoplusia ni and Spodoptera exigua larvae, the larvae were incubating for 4 days at 25°C and their mortality was then scored.

The bioassay results indicated that the HD73::050 hybrid had higher activity than the wild type HD73. The results with T. ni showed that the wild type HD73 exhibited an LC₅₀= 3.5ppm while the HD73::050 exhibited an LC₅₀= 2ppm. The results with S. exigua showed an LC₅₀= 475ppm for wild type HD73 and an LC₅₀= 225ppm for HD73::050.

The present invention may be summarised by the following clauses numbered 1 and 2 and is defined by the dims appended therafter. 1. A DNA segment comprising a) one or more insecticide-encoding DNA sequences capable of being replicated and expressed in Bacillus thuringiensis and a DNA sequence homologous to chromosomal Bacillus thuringiensis DNA whereby said homologous DNA sequence directs insertion of said DNA segment into the chromosome and whereby said insecticide-encoding DNA sequence is inserted into the Bacillus thuringiensis chromosomal DNA; or b) one or more insecticide encoding DNA sequence capable of being replicated and expressed in Bacillus thuringiensis and a DNA sequence capable of randomly integrating into chromosomal Bacillus thuringiensis DNA whereby said DNA segment is stably integrated into the chromosomal DNA including said insecticide-encoding DNA sequence. 2. A method of preparing a transformed Bacillus thuringiensis host comprising a) obtaining a DNA sequence either homologous to chromosomal Bacillus thuringiensis DNA or capable of randomly integrating into chromosomal Bacillus thuringiensis DNA; b) operatively linking to said DNA sequence one or more insecticide-encoding DNA sequences; c) obtaining a DNA segment; d) transforming a Bacillus thuringiensis strain whereby the DNA segment is incorporated into the chromosomal DNA; and e) isolating a transformed host wherein the insecticide encoding DNA sequence is stably integrated into the host chromosomal DNA and is capable of being expressed and rephcated in d e host.

SEQUENCE LISTING

( 1 ) GENERAL INFORMATION :

(i) APPLICANT:

(A) NAME: Sandoz Ltd

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(G) TELEPHONE: 061-324-1111 (H) TELEFAX: 061-322-7532 (I) TELEX: 96 50 50 55

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(E) COUNTRY: Austria

(F) POSTAL CODE (ZIP) : A-1235

(ii) TITLE OF INVENTION: DNA segment comprising gene encoding insecticidal protein

(iii) NUMBER OF SEQUENCES: 34

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)

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(2) INFORMATION FOR SEQ ID NO:19:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GCCGTCTGTA ACGGTACCTA AGG 23

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CACCCAGTTT GTACTCGCAG G 21

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: GTCTCATGCA AACTCAGG 18

(2) INFORMATION FOR SEQ ID NO:26:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CTCTGGCGCT CCATCTAC 18

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: CCCATGGATA ATGTATTGAA TAGTGGAAG 29

(2) INFORMATION FOR SEQ ID NO:28:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: AGCGGCCGCT 10

(2) INFORMATION FOR SEQ ID NO:29:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: CCAGACCAAC TGGTAATGGT AGAGACCGGC 30 (2) INFORMATION FOR SEQ ID NO:31:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GGTCGTGGCT ATATCATTCG TGTCACAGC 29

(2) INFORMATION FOR SEQ ID NO:32:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: CCACGCTATC CACGATGAAT GTTCCTTC 28

(2) INFORMATION FOR SEQ ID NO:33:

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(2) INFORMATION FOR SEQ ID NO:34:

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(D) TOPOLOGY: linear

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: CATTACGCAT TTGGAATACC 20

Claims

1. A linear DNA segment comprising at least one insecticide encoding DNA sequence capable of being replicated and expressed in Bacillus thuringiensis, wherein the regions both 5' and 3 ' of the said sequence comprise nucleotide sequences homologous to sequences present in Bacillus thuringiensis chromosomal DNA so that the said insecticide encoding DNA sequence is capable of being inserted into the bacterial chromosomal DNA, or a circular DNA segment comprising at least one insecticide encoding DNA sequence capable of being replicated and expressed in Bacillus thuringiensis, wherein a region either 5' or 3' of the said sequence comprises a nucleotide sequence homologous to a sequence present in Bacillus thuringiensis chromosomal DNA so that the said insecticide encoding DNA sequence is capable of being inserted into the bacterial chromosomal DNA.

2. A DNA segment according to Claim 1 further including an origin of replication from a gram negative bacterium and a selectable marker.

3. A hybrid vector comprising the DNA segment according to Claims 1 and 2.

4. A Bacillus thuringiensis host comprising the DNA segment of the preceding claims.

5. An insecticidal composition comprising an insecticidally effective amount of a host according to Claim 4 and a carrier therefor.

6. A method of preparing a transformed Bacillus thuringiensis host comprising the steps of introducing the vector of claim 3 or the DNA segment according to either of claims 1 or 2, into a Bacillus thuringiensis strain, and isolating the resulting transformants wherein the insecticide encoding DNA sequence is stably integrated into the host chromosomal DNA and is capable of being expressed and replicated therein.

7. A method according to Claim 6 further comprising transducing the transformed host and preparing a recipient Bacillus thuringiensis including a) exposing the Bacillus thuringiensis host of Claim 6 to a transducing phage; b) allowing said phage to replicate in said host wherein one or more insecticide encoding DNA sequences integrated in the host chromosomal DNA are incorporated into said phage; and c) introducing said insecticide encoding DNA sequence from the phage into a recipient Bacillus thuringiensis wherein said introduced insecticide encoding DNA sequence is stably incorporated into the chromosomal DNA of said recipient and is expressed.

8. A method according to Claims 6 and 7 wherein the Bacillus thuringiensis host and recipient are selected from strains of Bacillus thuringiensis kurstaki.

9. A method according to Claims 6, 7 or 8 wherein the insecticide encoding DNA sequence is the crylC sequence or a sequence homologous thereto.