AU685516B2 - Integrative DNA segment comprising gene encoding insecticidal protein - Google Patents

Description

WO 94/25611 PCTIEP94/01249 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 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 8-endotoxins, of 27 to 140Kd. Most crystal proteins are protoxins 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 the 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, Ivicrbiol. 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 Lepidopteraspecific Lepidoptera- and Diptera-specific Coleoptera-specific and Dipteraspecific (IV) genes.

The Lepidoptera-specific genes (cryI) encode 130 to 140Kd molecular weight proteins which accumulate in bipyramidal crystalline inclusions during the sporulation of B.t. The cryl I _LL WO 94/25611 PCTIEP94/01249 genes can be distinguished from other cry genes by sequence homology amino acid identity). Three of these genes, crylA(a), crylA(b), and crylA(c), show more than amino acid identity and have therefore been considered as a separate subgroup. The more recently identified crylB, cryIC, and cryID genes differ from each other and from the crylA genes. The CrylA, CryIB, and CryIC 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 wa cloned from B.t. subsp. kurstaki HD-263 and expressed in Bacillus megaterium. Cells producing the CryIIA protein were toxic for the lepidopteran species Heliothis virescens and Lymantria dispar as well as for larvae of the dipteran Aedes aegypti.

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 cryIV 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 cryIVA, cryIVB, cryIVC, and cryIVD 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 crylV 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 I WO 94/25611 PCT/EP94/01249 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 togene 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 a WO 94/25611 PCT/EP94/01249 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 cryIIIA 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 proteinencoding 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 CryIA(c) coding region of B.t. kurstaki HD73 (Calogero, et al., Appl. Env. Microbial.

55:446-453 (1989)). The plasmid vector was found to express the HD73 cryIA(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 WO 94/25611 PCT/EP94/01249 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 insecticideencoding 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 havng 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 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.

s~-~ll lP I~ WO 94/25611 PCT/EP94/01249 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 insecticideencoding 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 WO 94/25611 PCT/EP94/01249 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 insecticideencoding 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), cryIA(b), cryIA(c), crylB, crylC, crylD, crylE, crylF, cryIIA, cryllB, cryIIA, cryIlB, cryImC, crylVA, crylVB, cryIVC, crylVD and cytA genes of nmy 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 a-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 uch a tho lslisted 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 WO 94/25611 PCT/EP94/01249 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 standed 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 c' replication for a gramnegative bacterium. Any origin of replication capable of functioning in one or more gramnegative 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 WO 94/25611 PCT/EP94/01249 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 gramnegative 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 gramnegative 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 sel-ction 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 gramnegative 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 Tn1545 and Tn916, all of which are described by Camilli et al. (Camilli et al., J. Bacterial. 172:3738-3744 (1990)) or other tram-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 WO 94/25611 PCT/EP94/01249 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 pLTV1, as exemplified below among others. In a preferred embodiment, temperature-sensitive plasmids are used to select transposed host cells. Plasmids such as pTV51Ts and pLTV1 are unable to replicate above a certain temperature. Thus, a DNA segment carried on a temperaturesensitive 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 necesary for host cell expression of the transposed insecticide-encoding DNA sequence. In other embodiments, the transposable element may be designed to create an operor 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, "Plasmid Vectors for Recovering and Exploiting Tn917 Transpositions in Bacillus and Other Gram Positive Bacteria", In Plasmids: A Practical Approach, Handy, ed. IRL Press 79-103 (1973)).

The transposable element of the invention may be utilized in the preparation of a transformed i WO 94/25611 PCT/EP94/01249 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, 'gation, 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 grampositive bacteria. Appropriate vectors include any vector capable of self-replication in gram- WO 94/25611 PCTEP94/01249 negative bacteria, yeast or any monocellular host in addition to gram-positive bacteria. Such shuttle ve:ors 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 pHT1330 B.t. plasmid and pUC18 to move a crylA(a) gene isolated from B.t. 407 into the cryB strain (Lereclus, et al., FEMS Microbiol. Lett., 49:117 (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, HD1 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, 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 thereofset- forth in Table 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 segr- A:, 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 insecticideencoding 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.

12 h. WO 94/2561.1 PCT/EP94/01249 It will be appreciated that the DNA segment of the invention may be used to stably incorporate any exogenous DNA sequence into the chromost 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. irn 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 insectic'de-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 syntheoized 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.

WO 94/25611 PCT/EP94/01249 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 insecticideencoding 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-encodirig DNA sequence. The techniques for constructing operon and gene fusions are described by Sambrook et al. (Sambrook, Fritsch, E.F. Maniatis, 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 gramnegative hosts. The selectable marker for a monocellular organism is utilized to clone arnd 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 WO 94/25611 PCTIEP94/01249 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 thi 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 10 6 to about 10 13 hybrid microorganisms/g carrier, and more preferably about 10 1 0 to about 10" 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 WO 94/25611 PCT/EP94/01249 present purpose. Typically, the carriers are inert compounds or compositions that neither 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, and Pflanzenkrankh, 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 10 8 to about 1016 hybrid microorganisms/acre and more preferably about 1013 to about 1014 hybrid microorganisms/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 WO 94/25611 PCTJEP94/01249 intended to limit the invention or any embodiment thereof, unless so specified.

WO 94/25611 PCT/EP94/01249

EXAMPLES

Example 1: Construction of Plasmids Competent E.coli DH5a (Gibco BRL) and GM2163 (New England Biolabs) were prepared by the method of Alexander (Alexander, "A Method for Cloning Full-Length cDNA in Plasmid Vectors", In Wu. R. and Grossman, Eds., Recombinant DNA part E. Meth.

Enzymol. 154:41-63 (1987)). Plasmids were extracted from E. coli by the method of Bimboim and Doly (Birnboim and Doly, "A Rapid Alkaline Extraction Procedure for 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 for erythromycin resistance and the phospholipase C region as described by Lechner et al. as a target for integration (Lechner et al., "Molecular Characterization and Sequence of 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 in Figure 1 was constructed from the pSB140 plasmid shown in Figure 2 and described below in Example 2 by adding a multiple cloning site (MCS) at the EcoRI and HindlI sites. The MCS was created by annealing oligonucleotides KK14 and KK14B, the sequences of which are described in Table 2 below, which had been purified using oligonucleotide purification cartridges from Applied Biosystems, following the manufacturer's directions.

WO 94/25611 WO 94/5611 CT/'EP94/01249 Table 2: sequences of Oligonucleotides Name: Sequence Phos I GGAACGCTACATACTAGTGATAGAGTAG Phos4 GCTGTACACCGCAACTGTITCGCATG KK1I4B AGCIGCGGCCGCGTCGACCCCGGGCCATGGGGGCCG KK 14 AA7TCGGGCCCCCATGGCCCGGGGTCGACGCGGCCGCA GaIP I CCACAGTTACAGTCTGTAGCTCAATfACC GaIP2 CCGCI'ACTAATAGAACCTGCACCA CAATACATITATCCATGGAAAATrCCTCCIAAATATCATG NHS39 GAGCAATGAAAGAGTTAGGGCCrTGTrTAAGGTGTCATG NH1S42 GAGTGAATTATGCGG NHS43 A'1IGTAU7AAACGG crylA 1 ACTATITGTGATGCGTATAATGTA cryIIA2 AAITCCCCATITCATCTGC PG2 GAAATCGGCTCAGQAAAAGG PG4 CCIAAAACATGCAGGAAT7GACG CTArFGGrrGGAATGGCGTG TYIAA GAGCCAAGCAGCTGGAGQAGTITACACC TYLAC TCAC1CCCATCGACATCTACC TYIUN 12 ATCACTGAGTCGCTTCGCATGTTGACT=CTC TY6 GGTCGTGGCTATATCC1CGTGTCACAGC TY1 3 ACAGAAGAA'ITGCTICATAGGCTC TY 14 GAATTGcTICATAGGCTCCGTC Tet3 CAACAAACGOQCCATAAGCTTGTATAAG Tet4 GCCGTCTGTAACGGTACCTAAGG CPO I.Rev CACCCAGMIGTACTCGCAGG Hybridizing Gene phospholipase C phospholipase C

(MCS)

(MCS)

cryiC cryIC cryfLA cryllA cryILA cryfIB cryLA cryfLA errnC ermnC ermnC crylA(a) crylA(c) cryI-type5 cryl-type cryl-type cry'-type tet tet tet

SEQ

11D No.

2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 In the second step, the phos C gene was added to pSB2IO. The phos C region had been amplified from HD1173 total DNA by PCR using primers Phos 1 and Phos4 described above in Table 2. The PCR product was cloned into the Sinai site of pUC18 to construct pSB 139. The phos C target region was isolated on a 2.2kb blunted-Kpnl, BaMInil fragment from pSB 139, gel-purified and ligated into pSB210, which had been digested with MscI and BamMH and purified using the Geneclean Kit (Biol~l), following the manufacturer's directions. The resulting plasmid designated pSB2 10.1 and is shown in Figure 3.

The final step was to add a crystal gene. The pSB21O.2 plasmid contains the cry1C gene on a 4.2kb ApaI-NotI fragment from pSB6l9, described below in Example 3. pSB619 was digested with Apal and NotI, and the 4.2kb Apal-NotI fragment was isolated. The 4.2kb Apal-NotI fragment was ligated to pSB2 10.1 cut with Apal and Noti to form pSB2 10.2 which is shown in Figure 4. The pSB2lO.3 plasmid contains the crylC gene isolated on a 6kb fragment from pSBO13 (described below in Example 4) cut with Apal and NotI. The 6kb __j WO 94/25611 PCT/EP94/01249 ApaI-NotI fragment was purified by electroelution and ligated to pSB210.1 cut with Apal and NotI to form pSB210.3 which is shown in Figure 5. pSB210.3 differs from pSB210.2 in that the cryIC gene is placed behind the cryIIA promoter rather than the native cryIC promoter found in pSB210.2. In both plasmids, the cryIC 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 crylIA 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, 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 Hindi/ClaI fragment from the plM13 Baciilus subtilis plasmid described by Monod et al. (Monod et al., J. Bacteriol. 167: 138- 147(19861). The ermC Hindlll/ClaI fragment was ligated to pUC18 cut with Hindm and AccI.

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 HindIII and the large fragment was purified away from the tet' gene fragment. Plasmid pSB901 was digested with Smal followed by HindlI and the fragment carrying the ermC SmaI-HindII fragment was purified. The ermC gene was ligated into the pBR322 HindIn large fragment to generate pSB140. The derivation of the pSB140 plasmid is shown in Figure 2.

Example 3: Construction of pSB619 PlasmiJ The crylC gene isolated as an 8kb EcoRI DNA fragment was cloned into the EcoRI site of Lambda ZAP II 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 frm the gel. Alternatively, the crylC gene can be obtained by following the cloning protocol described by Honee et al. (Honee, van der Salm, and Visser, Nucl. Acids Res.

16:6240 (1988)). The cryIC clone was digested in two separate reactions, one with HindII and

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WO 94/25611 PCT/EP94/01249 KpnI and the other with KpnI and EcoRI. The digestion created a 2.6kb HindfI-KpnI fragment containing the promoter and N-terminal crylC sequence and a 2.3kb Kpnl-EcoRI fragment containing the C-terminal sequence and terminator. The cryIC gene was reconstructed by ligating the 2.6kb Hindml-KpnI fragment to the 2.3kb KpnI-EcoRI fragment in pTZ19R obtained from Pharmacia. The unique NcoI site was engineered at the translation start site of the cryIC 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 Hindll-EcoRI fragment containing the crylC promoter and entire protein coding region was then cloned into a pBluescript vector with the PCR generated 350bp cryIA(c) terminator.

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

Primerl: GTCTCATGCAAACTCAGG, SEQ ID NO.: Primer2: CTCTGGCGCTCCATCTAC, SEQ ID NO.: 26 A cryIA(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 Hindll, EcoRI fragment containing the crylC promoter and coding region was then cloned into the HindIm-EcoRI sites of pBluescript KS The cloning and sequencing of cryIA(c) are described by Adang et al. (Adang, Staver, Rocheleau, Leighton, Barker, R. F. and Thompson, "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 USDA. 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 2 were used as primers in a PCR reaction to generate from B.t. kurstaki HD-1 total DNA the 1800bp fragment containing the entire cryllA operon. Vent polymerase was used according to the manufacturer's i WO 94/25611 PCT/EP94/01249 recommendations (New England Biolabs, Beverly, MA). The PCR reaction products were isolated from a gel and ligated into pTZ19R which had been digested with HindII, The ligation product was used to transform competent E. coli DH5a. The transformants were selected on LB plates containing 75tg/ml ampicillin (amp) and 40mM Xgal. Plasmid DNA was screened by Apal and NcoI digestion and the orientation of the 1800bp PCR fragment within the pTZ19R multicloning site was determined by AfI digestion. A clone with the desired 1800bp fragment orientation was designated pSB009.

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

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

Both pSB009 containing the cryllA operon fragment in pTZ19R and pSB070 containing cryIIIA coding region with the crylC promoter and the cryIA(c) terminator were digested with Apal and Ncol. A 5667 bp fragment of pSB070 and the 1800 bp fragment from pSBOO9 containing the cryIlA operon were isolated. The fragments were ligated together. Competent E. coli DH5a cells were transformed and colonies were selected on LB plates containing ampicillin at 37 0 C overnight. The DNA from twelve colonies was digested with AfIIl+NotI to identify the isolate containing the desired operon cassette. The plasmid was designated pSBOIO.

The cryIC gene was cloned into the pSBOO10 cryIIA ope,- cassette, pSB619 was obtained as described above in Example 3. The full length cryIC coding region was obtained by digesting pSB619 with Ncol, EcoRI and Bgffl and isolating the 3900 bp NcoI EcoRI fragment. The WO 94/25611 PCTIEP94/01249 operon cassette vector, pSBOO1, was digested with NcoI+EcoRI and purified. The 3900 bp cryIC fragment vas ligated to the appropriate pSB010 NcoI-EcoRI fragment. The ligation reaction product was used to transform DH5a and colonies were selected on LB plates containing 75pg/ml ampicillin. The plasmid DNA from twelve colonies was analyzed by restriction digests (AfIII EcoRI and AfII BgII). The plasmid cassette containing the full length cryIC gene downstream of the cryIIA operon was designated pSB013.

Example 5: Construction of pSB304 Plasmid pSB304 was obtained by cloning the cryIIA 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 HindmI, and fragments of about 5 kb were purified by gel electrophoresis. The gel-purified fragments were ligated with HindIII-cut pTZ18R (Pharmacia) and transfected into E.coli DH5at. The clone containing cryllA was probed with a cryIHA-specific oligonucleotide (CCCATGGATAATGTATTGAATAGTGGAAG), SEQ. ID.:27. The clone containing the cryIIA 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 cryIIA operon was removed to produce pSB304. Further information on the sequence of the cryIIA operon and its 5' region can be found in Widner et al. (Widner, and Whiteley, "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 cryIIA operon was added to pSB140. The source of the cryIIA operon was plasmid pSB304 described above in Example pSB304 contains the cryllA operon from B.t. galleriae HD232 cloned as a BamHI/HindIII fragment in pTZ18R. Plasmid pSB30 and plasmid pSB140 were digested with EcoRI and HindIII. The large pSB140 fragment was purified and the cryIIA operon EcoRI-HindI 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 (plc) from the HD73 strain of B.t. kurstaki obtained from the USDA. This DNA fragment was isolated from WO 94/25611 PCT/EP94/01249 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 plc region from B.t. strain ATCC 10792 was obtained from Genbank (Accession number X14178) and is described by Lechner et al., (Lechner, et al., Mol.

Microbiol. 3: 6Z1-626 (1989)). In addition to the plc gene, this 2254bp sequence contained 454bp upstream of the plc gene and 810bp downstream. Two primers, Phosl and Phos4, described above in Table 2, were designed to hybridize to the sequences that flank the plc gene. These primers were used in a polymerase chain reaction with HD73 total DNA template to generate a 2.2kb fragment carrying the HD73 plc 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 KpnI and BamHI and the plc region was isolated from the vector sequences. Plasmid pSB141 was also cut with KpnI and BamHI and the resulting fragments were ligated with the isolated plc region. The resulting construct, pSB141.5, carried the pBR322 portion of plasmid pSB141 and the plc region from pSB139.

Next, plasmids pSB141 and pSB141.5 were used to generate a pla:mid that contained ermC, the plc region, and the cryllA operon. 'Piasmid pSB141.5 was digested with BamHI. Plasmid pSB141 was digested with BamHI and the fragment carrying the cryIlA operon and the ermC gene was isolated. The cryIIA/ermC BamHI fragment was ligated to the linearized pSB141.5 to form pSB147. A map of pSB14', 3 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 (electrotranzformation). 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 selectae 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.

WO 94/25611 PCTIEP94/01249 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 sucrose (BHIS) with a white disposable loop of cells from a fresh overnight LB plate.

The cells were gro'-n in a 1 baffled flask at 37 0 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 transfe-red 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 2 00pl of competent cells. Pulse parameters for HD73 cells were kV 1.25, pF= 3 and 0 After the pulse was delivered, the cells were transferred to 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 10pg/ml erythromycin.

pSB098 was used as control plasmid DNA to determine the transformation efficiency of the electroporation procedure. pSB098 is a shuttle vector containing pTZl9R (Pharmacia) and pBC16.1. pBC16.1 is a B. cereus vector constructed by Kreft (Kreft, Mol. Gen. Genet.

162:59 (1978)). pTZ19R and pBC16.1 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).

WO 94125611 PCT/EP94/01249 Table 3: Transformation Efficiency of B. thuringiensis kurstaki HD73 Efficiency Plasmid Amount Amount Number of (transformants/ (pg) (pl) transfectants pg pSB098 DNA) HD73 STRAIN pSB210.2 9 10 3 3.5x10 pSB147 15 10 8 2 x The efficiency given in the final column of the table was determined for each experiment by electroporating 0.51 pg pSB098 and calculating the number of colony-forming units obtained per pg 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 cryIIA gene using primers cryIIAl and cryIIA2 and the ermC gene with PG2 and PG4. The primer sequences are provided in -4--tbe e. 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 Thore (1978), supra. The strain was revived by inoculating 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 0 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 0.45pM filter and stored at 16 0

C.

WO 94/25611 PCT/EP94/01249 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 106 cells of 569 were mixed with 100.l 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 NaCI, 0.2g MgSO 4 7Hz0, 0.05g MnSO 4

H

2 O, 0.15g CaCL,2H,;0/, pH 5.9-6.0) which had been dried overnight at room temperature. The plates were incubated at overnight and plaques were counted.

To test for susceptibility to infection by the phage, approximately 106 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 the 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 tube. The culture was grown at 37 0 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 4X10 6 PFU's and either 1X10 6 or 3X10 6 cells. The plates were incubated overnight at 30 0 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 0.45pm filter and stored at 15 0

C.

To determine the titer, 5X10 6 CFU of strain HD73 were mixed with 100lO of 10.2 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 0 C, the 10 4 plate had 1200 plaques and the titer was estimated to be 1.2x10' 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.

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WO 94/25611 PCTIEP94/01249 The titers of CP51 phage stocks were determined as follows. 0.1 ml of the phage diluted in 1 Peptone and approximately 2X10 7 spores of B. cereus 569 were added to 2ml of PA soft agar and the 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 B. cereus strain 569 5 x 107 B. thuringiensis strains HD73 8.6 x 10 8 SAll and SA12 2.6 x 10 s S287 1.1 x 10 7 PFU PFU/ml CP51: 8x 10 6 CP210.2: 1.2 x 108 Example 11: Construction of pSB136 Plasmid pSB 136 (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.

.awai HD229 gene and the pBC16-1 B. cereus plasmid tetracycline resistance gene (tet) were cloned. The integration target site was a fragment of DNA of unknown function from the B.t.

kurstaki HD1 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 104 transformants per microgram transforming DNA).

Competent E.coli DH5ac were prepared by the method of Alexander (Alexander (1987), supra).

The transformation was conducted by methods well known in the art using Library Efficiency

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WO 94/25611 PCT(EP94/01249 Competent Cells (Gibco, BRL, Life Technologies, Inc., Gaithersburg, MD).

The selection for transformants was conducted on LB containing 75pg/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 the Klenow fragment of E.coli DNA polymerase I were conducted by the 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 HD1 cry-B is a plasmid cured strain of HD1 described by Stahly et al. (Stahly, et al., Biochem. Biophys. Res. Comm. 84:581 (1978)). Total DNA was isolated from B.t.

kurstaki HD1 cry-B by the following method. 200ml of 2XTY were inoculated with 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 0 C with baffling at 300rpm. Cells were collected by centrifugation at 10,000 rpm for 5 min. at 4 0 C when the optical density of the culture reached an OD6 0.8 to 1. The cells were washed with TES (TE+100 mM NaCI) and suspended in 18ml 25% sucrose +25mM TrisHCI (pH8)+25mM EDTA. 2ml of lysozyme were added to the sucrose solution and mixed gently. 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 0 C for 15 min. 5.5ml of 5M NaCI were then added, mixed gently and incubated at 50 0 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 gently, 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 0 C overnight. The two tubes were then combined into one tube. 200pl of 1 mg/ml RNase and 1 200 ]l of proteinase K were added and the mixture was incubated at 37 0 C for 30 min. 20ml of phenol/chloroform were added and the 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.

WO 94/25611 PCT/EP94/01249 The plasmid DNA for cloning was isolated from E.coli cells by alkaline lysis (Birnboim, H.C, and Doly, "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 tetf gene functional in Bacilli. Next, an integration target site was added, a piece of DNA of unknown function isolated from the HD1 cryB genome. In step three, a NotI linker was added to facilitate cloning the cr'J.C gene. In the final cloning step, the cryIC gene was added to the integration vector. Each of these steps is described in more detail below.

Plasmid pSB206 was constructed by cloning the tef gene from pBC16 (Bernhard, et al., J. Bacteriol., 133:897-903 (1978)) into pUC18. Plasmid pBC16-1 was generated from plasmid pBC16 by removal of an EcoRI fragment by the method of Kreft et al. (Kreft, et al., Mol.

Gen. Genet., 162:59-67 (1978)). The tet' gene was isolated from pBC16-1 using the polymerase chain reaction with primers Tet3 and Tet4 described in Tb4-~b Primer Tet3 introduced a HindIl site upstream of the tet gene and primer Tet4 introduced a KpnI site downstream of the tetr gene. The PCR product was inserted into pUC18 at the polylinker cartridge HindI site.

To remove the tet' gene, plasmid pSB206 was digested with SmaI and HindII. To remove the tet' gene from pBR322, this plasmid was cut with Aval, treated with the Klenow fragment of E. coli DNA polymerase I to generate blunt ends, and then digested with HindIm. 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 pSB 131. The source of the target site was a 1.lkb DNA fragment isolated from 10 the HD1 cryB genome. This fragment was cloned in pUC18 and the construct was named pSB132. The 1.1kb DNA fragment from HD1 cryB was isolated as follows. Total HD1 cryB DNA was restricted with HaeIm or EcoRV and the two digests were mixed and subjected to electrophoresis on an 0.8% agarose gel. Three size 15 fractions 2 f? 3 0 -o i u' 7-r 0 WO 94/25611 PCT/EP94/01249 were cut from the gel: 0.5kb -0.9kb; 0.9kb -1.8kb; 1.8kb-2.7kb.

The DNA fractions and were purified. The DNA from fraction was ligated into pUC18 cut with Smal and the resulting clones were characterized by EcoRI/HindII digests.

The plasmid called pSB132 had a 1.1kb 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 with HindII. Plasmid pSB131 was cut with SspI and HindIII and ligated with the purified 1.1 kb fragment from plasmid pSB132 to yield plasmid pSB134.

A NotI linker was added to pSB134 to facilitate addition of the cryIC gene. The sequence of the NotI linkers was pAGCGGCCGCT (New England Biolabs #1125, SEQ ID No. 28).

Plasmid pSB134 was digested with BamHI and blunt ends were generated by treatment with the Klenow fragment of E.coli DNA polymerase I. The NotI 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 cryIC gene to pSB134.5. 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 crylA(c) terminator. The crylC gene with the promoter and terminator were cloned as an Apal/NotI cassette in bluescript To isolate crylC, pSB619 was cut with Apal, filled with T4 DNA polymerase, and digested with NotI. Plasmid pS 134.5 was cut with EcoRI, filled by treatment with the Klenow fragment of 15 E.coli DNA polymerase I, and then cut with NotI.

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 HD1 cryB and HD73 Plasmid pSB136 was constructed zs described in Example 11 above. It contains the crylC gene, the gene encoding tetracycline resistance from pBC16.1, and a portion of DNA from the HD1 CryB chromosome of unknown function which acts as an integration target site. These fragments were ligated into the plasmid pBR322.

WO 94/25611 PCT/EP94/01249 Competent B.t. kurstaki HD1 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 0 C. The pulse parameters for the HDI Cry B cells were 1.05kV, 25pF, R The pulse parameters for HD73 cells were 1.25kV, 3pF, R o.

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 pg of pSB098 plasmid.

In the HD1 CryB cells, one tetracycline-resistant colony was obtained when 7.5pg of the plasmid pSB136 was electroporated into the cells. The overall efficiency of transformation of the cells was 6 xl0 5 CFU/pg (using pSB098). This new recombinant strain, CryB::pSB136, 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 5X10 8 spores/ml for HD1 CryIB in the same experiment. The tetracyclineresistance gene was 98% stable through sporulation and germination.

In HD73 cells, two colonies were obtained when 15pg of plasmid pSB136 was electroporated into the cells in an experiment where the overall efficiency was 2x10 6 CFUpg DNA. Both colonies, designated HD73::pSB136, were positive for the 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 cryIC gene and tetracycline resistance marker in the CryB::pSB136 and HD73::pSB136 5 recombined strains was confirmed by PCR. Cells from a fresh overnight plate were boiled for 10 min. in 8pl of a solution containing the necessary primers (0.5l of 20pM stock solution) and the dNTP mix (1.6pl of 1.25mM stock solution) in 1 x Taq polymerase buffer. Cell debris was pelletted and 2pl 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

'L

WO 94/25611 PCT/EP94/01249 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 galP1 and galP2 were used to produce a 0.8kb size fragment. Primer sequences are given in Table 2 above.

Example 14: Introduction of cryIC and cryHlA Genes into B.t kurstaki Strain HD73 Plasmid pSB304 was constructed as described in Example 5 above. The cryllA gene was isolated from pSB304 as a NotI-EcoRI fragment. pSB134.5 was constructed as described in Example 10 above The cryllA NotI-EcoRI fragment was ligated to pSB134.5 cut with NotI and EcoRI to form plasmid pSB134.5.2.

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 0 C. Pulse parameters for HD73 cells were 1.25kV, 3pF, R o. 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/g of pSB098 DNA.

Ten colonies were obtained from the electroporation of the HD73 cells with 20pg of the pSB134.5.2 plasmid. The transformation efficiency was lx10 6 CFU/pg DNA. Two of the transfectants proved positive for the tetracycline resistance and cryIIA 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 cryIIA gene and tetracycline resistance marker in the HD73::pSBI34.5.2 recombinants was confirmed by PCR as described on Example 13. To screen for the cryIIA gene, the primers crylA1 and cryIIA2 were used to produce a 0.57kb fragment. Primer sequences are given in the Tabfe-=4=abve.

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 Sz 33

C,

WO 94/25611 PCT/EP94/01249 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 Thorne (1978) (Margolin, "Escherichia coli and Salmonella typhimurium", Cell. and Mol. Biol., (1987); Thorne, "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 Thorne, (Thorne, 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 method described in Example 8 above and diluted to approximately 10 7 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 10 s plaque forming units (PFU) per ml. A sterile HA filter (Millipore) was placed on the surface of an LB plate then 100l each of phage lysate CP210.2 and cells were pipetted onto the filter and gently mixed using a sterile wire spreader. The plates were incubated at 37 0 C for 3 hrs. The filters were transferred to LB plates with erythromycin (10pg/ml), returned to 37 0 C, and allowed to grow for 36 hrs. The results of the plate transductions are given in Table 5 below.

WO 94/25611 PCT/EP94/01249 Table 5: Results of Transduction Experiments Strain CFU's plated Multiplicity Ery r of Infection Colonies Efficiency HD73 1 x 10 6 10 2 1.6 x 10 7 SAl1 4 x 10 6 3 4 3 x 10- 7 SA12 4 x 10 6 3 2 1.6 x 10 7 S287 1 x 10 6 40 3 2 x 10 The efficiency is expressed as the number of erythromycin resistant colonies obtained per plaque-forming unit. For HD73, SAll and SA12, 1.2x107 plaque-forming units were plated.

For S287, 4x107 PFU were plated.

Example 17: Polymerase-Chain Reaction Screeling 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 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(b) IA(c) IIA IC ermC SAllWT SA11CP1 SA11CP2 SA11CP3 SAllCP4 SA12WT SA12CP 1 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 cryIC and ermC genes. When screening for the cryIA(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

I

WO 94/25611 PCT/EP94/01249 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 0 C and restreaked on fresh SA (lX Spizizen salts, 1% casaminoacids, 5% glucose, 0.0005mM MnSO 4 HO) plates, and grown for 3 to 4 hours at 37°C. For each strain, 2 to 3 loopfuls of cells were suspended in (100l TESL 100mM Tris pH8, 10 mMEDTA, 20% sucrose, 2mg/ml lysozyme) on ice and incubated for 15 min. at 37 0

C.

200pl 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. 150gl 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 0 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 0 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 2 0 pl 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 general phospholipase C (phosC probe extending 1800 bp from BamHI to Clal of the known phosC sequence.

Eco left (EL) extending 850 bp mHI to EcoRI.

WO 94/25611 PCT/EP94/01249 Eco right a 1427 bp EcoRI fragment.

Chromosomal DNA from wild type HD73 as described in Example 11 was isolated from 100ml samples of cultures in 2XTY medium (5g yeast extract, 5g tryptone, 2.5g NaCl/L) Chromosomal DNA from wild type SAll, 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 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 EL 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 ER 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 WO 94/25611 PCT/EP94/01249 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 EL and ER probes also revealed 2.4 kb and 4.3 kb internal EcoPJ 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 SAll, SA12, HD73 and HD1-51 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 SA11CP1 and SA12CP2 were analyzed for stability. 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 0 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 A6 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 -r i I WO 94/25611 PCTIEP94/01249 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 10 9 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 0 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 107 and 1.5 x 10 S spores per ml were estimated for SA1l (WT) and the recombinant SA11CPI, respectively. 3.5 x 107 and 3.4 x 10 7 spores per ml were estimated for SA12(WT) and the recombinant SA12CP2, respectively. The introauced 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 (10g casitone, 5g glucose, 2g yeast extract, Ig

KHPO

4 Iml 50mM MgCl1, nml 50mM MnCl1, 1 ml 50 mM ZnSO 4 1 ml 50 mM FeCI,, 1 ml 200mM CaCl 2 L) cultures of the recombinants and parental strains were grown for 36 hrs.

at 30°C. 50l were mixed with an equal volume of 2x sample oading buffer (0.125MI Tris-HCl pH8, 4% SDS, 0.005% Bromophenol Blue, 20% glycerol, B iiercapteothanol) and immediately boiled for 5 min. 2.5, 5, and 10pl 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 CryllA protein. An additional band running at 135Kd, WO 94/25611 PCT/EP94/01249 the size expected for the CrylC protein, was seen for HD73::pSB210.2, SA11CP1, 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 f~in Lethality Bioassay For most bioassays the samples were grown in 100ml Fishmeal Fishmeal, 4% Starch, 0.1 NH 4 CI, 0.125% KH2PO 4 0.05% MgSO 4 0.001% FeSO 4 0.001% MnClJL) 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 0 C. The SA11 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 SAll, 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, llg/L vitamin mix (Moorehead Co. Van Nuys, CA), 8.8g/L salt mix (BioServ, Frenchtown, NJ), 2.3g/L sorbic acid, 1.lg/L methyl paraben, 13g/L agar and formaldehyde. The mixture was then fed to late third instar larvae incubated at 25 0 C. The mortality was recorded after 4 days, and the LC 5 o 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 cryIC gene has been introduced into the chromosomes of several different strains of WO 94/25611 PCT/EP94/01249 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 CryIC protein contributed to the bioactivity against S. exigua. Introducing the cryIC 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 pLTV1 Replication in B.t.

B. subtilis PY1177 (pLTV1) was obtained from Dr. Phil Youngman is described by Camilli et al. (Camilli et al., J. Bacteriol. 172: 3738-3744 (1990)). pLTV1 DNA was isolated from PY1177 according to the method of Birnboim and Doly (1979), supra. B.t. kurstaki HD73 was transformed with pLTV1 DNA by electroporation as described in Example 7 above and the transformant was designated HD73 pLTV1.

The temperature required to abolish the replication of pLVT1 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 f.ask containing 10 ml of LB teto was inoculated with a single colony of HD73 +pLTV1. This primary culture was grown to OD6 0.4 at 0 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 10ml LB cultures prewarmed to 30, 37, 40 and 42 0 C. After inoculation, the cultures were maintained at their respective temperatures until the cultures reached an ODo value between 0.6 to 0.8. The cultures were then diluted by a factor of 104 to 107 and 100l aliquots of the dilutions were spread in onto LB eryo.0 plates. Each plate was incubated overnight at the incubation temperature of the corresponding primary culture. After overnight incubation, the colonies from the LB eryo.

0 plates were replica-patched onto four different LB plates containing no antibiotics as well as LB er.lo, LB cm,2 and LB teto plates. The plates were incubated at 30 0 C overnight and scored the following day for antibiotic sensitivity.

Experiments in which the primary cultures were grown in LB liquid which contained i WO 94/25611 PCTIEP94/01249 tetracycline produced the following results. At the pLTVI replication-permissive temperature of 30 0 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 0 C, 98% of the colonies were sensitive to all three antibiotics. At 40 0 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 0 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 0 C and 42 0

C

exhibited plasmid losses of 94%, 90% and 99%, respectively. Therefore, it was concluded that plasmid replication of pLTV1 was inhibited at 37 0 C or above under the experimental conditions used in this study. Since it can be lost even at its replication-permissive temperature, the pLTV1 plasmid appears to be somewhat unstable.

Example 24: Determination of Conditions for Transposition of pLTV1-Borne Tn917 in B.t Retrieving transposed colonies is a three-day, three-step procedure. This method was first tested with HD73 pLTV1. First, 10ml of liquid LB containing ery,, cm, and tet, was inoculated with an single colony of HD73 containing pLTVI and gro'"n to an ODo 0.7 at 30 0 C with baffling at 300 rpm. This primary culture was then centrifuged and the pelleted cells were washed in 10m] LB to remove the antibiotics. Two secondary flasks containing 100ml LB containing both ery, and cm, (but no tet) were inoculated with 100pl 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, cm 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 0 C LB containing ery, cm 5 plates were replica-patched onto LB alone. LB containing ery, cmr, LB containing eryo 0 LB containing cm 7 and LB containing teto plates to determine what percentage of the colonies were resistant to erythromycin, chloramphenicol but sensitive to tetracycline, indicating that a transposition event had occurred.

I 4,, WO 94/25611 PCT/EP94/01249 The ery'cm'tets colonies obtained were designated HD73::pLTV1.

Almost 100% of the HD73 colonies derived from these experiments with pLTV1 showed the transposition of the lacZ gene from pLTV1, as expected. PCR analysis of the ery'cmfrtets HD73 colonies using primers LACNHS1 and LACNHS2 indicated that the lacZ gene from pLVT1 was present in all cases. Further PCR analysis using primers TY6 and TY7 indicated that 38 out of 40 transposed colonies retained the native crylA(c) gene. The primer sequences are providec in Table 7 below.

Table 7: Sequences of Oligonucleotides SEQ.ID No.

LACNHS1 GGCTTTCGCTACCTGGAGAGACGCGCCCGC 29 LACNHS2 CCAGACCAACTGGTAATGGTAGAGACCGGC TY6 GGTCGTGGCTATATCATTCGTGTCACAGC 31 TY7 CCACGCTATCCACGATGAATGTTCCTTC 32 NHS21 GATATTAGCTCATGATCTTTTCCTCCTATTAAC 33 NHS37 CATTACGCATTTGGAATACC 34 The pSBO50 plasmid was assembled in order to introduce the cryIIA operon from B.t. galleriae HD232 onto the HD73 genome. The B.t. galleriae strain HD232 was obtained from the USDA.

The entire cryllA operon from HD232 was isolated as a 4kb BamHI HincII fragment from pSB304 described in Example 5. The plasmid pLTV1 was cut with restriction enzymes Smal and BamHI to produce a 20.6kb fragment and the 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 and then replica-patched onto a series of agar plates containing either no antibiotics, cm 7 eryo or tetio. 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 pLTV1. The resistant colonies were further screened by PCR amplification of the 1400 bp product between the LACNHS 1 and LACNHS2 primers. The sequences of these primers are shown above in Table 7, and correspond to sequences which are within the lacZ gene contained in pLTV1. Amplification with PCR and the NHS37 and NHS21 primers, whose sequences are shown in Table 7 above produced a WO 94/25611 PCT/EP94/01249 region from the erythromycin gene to the end of the first open reading frame of the cryllA operon. The DNA from the resistant colonies was analyzed by restriction analysis with BgmII 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, 3pF, and a resistance at oo ohms with 5pg of pSBO50 DNA isolated from GM2163. The cells were allowed to recover at the permissive temperature of 30 0 C for three hours in BHIS medium with baffling at 300 rpm. The cells were then concentrated, plated onto LB plates containing 10pg/ml tetracycline'and incubated at 30 0 C overnight. The presence of 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 CryIIA 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 cryllA operon from pSBO50 onto a large resident plasmid of HD73. The HD73 pSBO50 isolates were incubated at a non-permissive temperature and selected for chloramphenicol and erythromycin resistance and tetracycline sensitivity. The erytcm'tet 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 cryIIA) and the 1400bp product between the LACNHS 1 and LACNHS2 primers. The presence of an intact cryIA(C) coding region was also confirmed by PCR amplification with the TY6 and TY7 primers, whose sequences are provided in Table 7 above.

Example 27: Expression of CryIA(c) and CryIIA Genes in Transposed B.L Strain HD73::050 The microscopic observation of HD73 pSB050 and HD73::050 at 1000x magnification showed that both strains produced two crystal types. Both the bipyramidal CryIA(c) crystals

-I

WO 94/25611 PCT/EP94/01249 of HD73 and the cuboidal CrylIA 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 CryIA(c) and CryllA in HD73::050 was analyzed by SDS-PAGE.

100l samples of sporulated cultures (40 to 50 hours old) were pelleted, resuspended in EDTA and mixed 1 to 1 on ice with 2x SDS loading and boiled for three minutes. A pre-cast Novex gel was run and stained with Coomassie blue.

The SDS-PAGE analysis revealed the presence of the 135Kd CryIA(c) and the 65Kd CryIIA proteins. These were confirmed by Western blot analysis using specific antisera made against CryIA(c) or CryllA 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 pLTV1, 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 10pg/ml tetracycline, lug/ml erythromycin and 7pg/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 2X10' spores/ml. Most 97 to 100%) of HD73::050 maintained the correct ery' cm' tet' 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 WO 94/25611, PCT/EP94/01249 transposon had been inserted into t"ro 50Mdalton plasmids present in the natural HD73 strain.

The gels showed an increase in the 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 crylA(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.L Strains HD73::050 and HD73::pLTV1 Total DNA samples from wild type HD73, HD73::pLTVI 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 the 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 plasmids. No preferred transposition site was observed. The restriction map of pLTVl indicated that the DNA from HD73::pLTVl contained BamHI fragments of 9kb or larger which hybridize to the probe. Bands of this size represent the transposed portion of pLTV1 in one of the 50Mdalton plasmids. Similarly, the HD73::pLTVI DNA 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 pLTV1 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::pLTVI and HD73::050 is due to the presence of the cryIIA I I WO 94/25611 PCT/EP94/01249 gene within the transposed region of HD73::050. This confirms that the transposed cryI.A 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 0 C with baffling at 300 rpm (Yamamoto, T. 1990. ACS Symposium Series 432: 46-60). The cells were resuspended in 1/20volume of Buffer A Tris pH8.0,0.25% Triton), and the cell lysate was electrophoresed on 8% Novex SDS-PAGE gels. The concentration of the CryIA(c) protein was determined by densitometer scanning, Nineteen two-fold dilutions were made in Buffer A and the amount of CryIA(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 0 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 LCs 0 while the HD73::050 exhibited an LC 5 0 2ppm. The results with S. exigua showed an LCs 0 475ppm for wild type HD73 and an LC 5 s= 225ppm for HD73::050.

The present invention may be summarised by the following clauses numbered 1 and 2 and is defined by the clims 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.

I Il A. J .4 NO I A, 11 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 replicated in the host.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word S"comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

S

S

ee Se 13- i 1 C, 1 WO 94/25611 PCTIEP94/01249 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Sandoz Ltd STREET: Lichtstrasse CITY: Basel STATE: BS COUNTRY: Switzerland POSTAL CODE (ZIP): CH-4002 TELEPHONE: 061-324-1111 TELEFAX: 061-322-7532 TELEX: 96 50 50 NAME: Sandoz Patent GmbH STREET: Humboldstrasse 3 CITY: Loerrach COUNTRY: Germany POSTAL CODE (ZIP): D-79540 NAME: Sandoz Erfindungen Verwaltungs GmbH STREET: Brunnerstrasse 59 CITY: Vienna COUNTRY: Austria 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: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (EPO) INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GGAACGCTAC ATACTAGTGA TAGAGTAG 28 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear I- WO 94/25611 1'CT/EP94/C 1249 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GCTTGTACAC CGCAACTGTT TTCGCATG 28 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: AGCTTGCGGC CGCGTCGACC CCGGGCCATG GGGGCCG 37 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AATTCGGGCC CCCATGGCCC GGGGTCGACG CGGCCGCA 38 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID CCACAGTTAC AGTCTGTAGC TCAATTACC 29 WO 94/25611 PCT/EP94/01249 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CCGCTACTAA TAGAACCTGC ACCA 24 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CAATACATTA TCCATGGAAA ATTCCTCCTT AAATATCATG INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GAGCAATGAA AGAGTTAGGG CCCTGTTTAA GGTGTCATG 39 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 16 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

-II

WO 94/25611 PCT/EP94/01249 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GAGTGAATTA TGGGGG 16 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 16 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID ATTTTGTATT AAACGG 16 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: ACTATTTGTG ATGCGTATAA TGTA 24 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: AATTCCCCAT TCATCTGC 18 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid WO 94/25611 PCT/EP94/01?19 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GAAATCGGCT CAGGAAAAGG INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CCTTAAAACA TGCAGGAATT GACG 24 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID CTATTGGTTG GAATGGCGTG INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO WO 94/25611 PCT/EP94/01249 (xi) SEQUENCE DESCRIPTION: SEQ ID NO;16: GAGCCAAGCA GCTGGAGGAG TTTACACC 28 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: :ENGTH: 22 base pairs mYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ-ID NO:17: TCACTTCCCA TCGACATCTA CC 22 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ATCACTGAGT CGCTTCGCAT GTTTGACTTT CTC 33 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (x4) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGTCGTGGCT ATATCCTTCG TGTCACAGC 29 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 94/25611 PCT/EP94/01249 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID ACAGAAGAAT TGCTTTCATA GGCTC INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GAATTGCTTT CATAGGCTCC GTC 23 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CAACAAACGG GCCATAAGCT TGTATAAG 28 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: WO 94/25611 PCT/EP94/01249 GCCGTCTGTA ACGGTACCTA AGG 23 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CACCCAGTTT GTACTCGCAG G 21 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQU24CE DESCRIPTION: SEQ ID GTCTCATGCA AACTCAGG 18 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CTCTGGCGCT CCATCTAC 18 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 94/25611 PCT/EP94/01249 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: CCCATGGATA ATGTATTGAA TAGTGGAAG 29 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 10 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: AGCGGCCGCT INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GGCTTTCGCT ACCTGGAGAG ACGCGCCCGC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID CCAGACCAAC TGGTAATGGT AGAGACCGGC -p -s WO 94/25611 PCT/EP94/01249 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GGTCGTGGCT ATATCATTCG TGTCACAGC 29 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: CCACGCTATC CACGATGAAT GTTCCTTC 28 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: GATATTTTAG CTCATGATCT TTTCCTCCTA TTAAC INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) -L I I WO 94/25611 PCT/EP94/01249 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: CATTACGCAT TTGGAATACC

Claims (20)

1. A genetically transformed Bacillus thuringiensis bacterium and progeny thereof comprising at least one exogenous DNA segment containing at least one insecticide encoding DNA sequence and further having at least one native crystal protein gene contained on a host resident plasmid, wherein the insecticide encoding sequence is stably integrated into the bacterial chromosome and is capable of replication and expression in said bacterium,

2. The transformed Bacillus thuringiensis bacterium and progeny thereof according to claim 1, wherein the said DNA segment further comprises an origin of replication of a gram negative bacterium.

3. The transformed Bacillus thuringiensis bacterium and progeny thereof according to any one of claims 1 or 2 having high spodoptera activity and retained potency agaiust Lepidopteren insects.

4. The transformed Bacillus thuringiensis bacterium and progeny thereof according to any one of claims 1 to 3, wherein said bacterium is a strain of Bacillus thuringienqis kurstaki. o

5. The transformed Bacillus thuringiensis bacterium and progeny thereof according to any one of claims 1 to 3, wherein said bacterium is a strain of Bacillus thuringiensis HD73.

6. The transformed Bacillus thuringiensis bacterium and progeny thereof according to any one of claims 1 to 3, wherein said bacterium is a strain of Bacillus thuringiensis aizawai.

7. The transformed Bacillus thuringiensis bacterium and progeny thereof according to any one of claims 1 to 6, wherein the insecticide encoding DNA sequences are crystal toxin encoding DNA sequences. 0 rAR A 8. The transformed Bacillus thuringiensis bacterium and progeny thereof according to Tr C, I 'P:\OPIR\ I.R\7879694.150 IS301/97 -61 claim 7, wherein said crystal protein encoding DNA sequence is selected from the group consisting of cryIA(a), cryIA(b), cryIA(c), cryIB, crylC, cryID, cryIE, cryIF, cryllA, crylIB, cryIIIA, cryIIIB, cryIIIC, cryIVA, cryIVB, cryIVC, cryIVD, and cytA gene of any subspecies and/or strain of B.t. and hybrid toxine thereof.

9. The transformed Bacillus thuringiensis bacterium and progeny thereof according to claim 8, wherein said crystal protein encoding DNA sequence is cryIC, cryIE or a hybrid thereof or sequence homologous thereto. The transformed Bacillus thuringiensis bacterium and progeny thereof according to claim 8, wherein said crystal protein encoding DNA sequence is cryIC or sequence homologous thereto. *1 l

11. The transformed Bacillus thuringiensis bacterium and progeny thereof according to claim 7 further comprising additional crystal encoding DNA sequences in its genome.

12. The transformed Bacillus thuringiensis bacterium and progeny thereof according to any one of claims 1 to 11 further comprising native crystal toxin encoding genes which are stable and expressed in the transformed bacterium in the presence of the chromosomally integrated crystal toxin encoding DNA sequences.

13. The transformed Bacillus thuringiensis bacterium and progeny thereof according to any one of claims 1 to 12, wherein the transformed bacterium is a crystal minus strain.

14. The transformed Bacillus thuringiensis bacterium and progeny thereof according to any one of claims 1 to 3, further comprising a native crystal tcxin encoding gene wherein the native crystal toxin encoding gene encodes a toxin active against lepidopteran insects and the insecticidal encoding DNA sequence of the exogenous DNA segment encodes a toxin active ,,;nagainst spodoptera insects. s ~s PA:OP1'R)ULR \787M94,150 *30/5/97 -62- The transformed Bacillus thuringiensis bacterium and progeny thereof according to any one of claims 1 to 3, wherein the insecticide encoding DNA sequence is from the insecticidal protein-encoding genes of any strain of B. larvae and B papillae or a sequence homologous thereto and all other insecticidal protein-encoding genes known to be expressed in bacilli or other gran-positive bacteria.

16. The transformed Bacillus thuringiensis bacterium and progeny thereof according to any one of claims 1 to 3, wherein the insecticide encoding DNA sequence is from a gene encoding tor an a-amylase inhibitor, or a proteinase inhibitor.

17. An insecticidal composition comprising an insecticidally effective amount of a transformed bacterium according to any one of claims 1 to 16 and a pesticidally acceptable carrier thereof.

18. A method of preparing a transformed Bacillus thuringiensis bacterium comprising the i. steps of i) introducing 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 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 the regions both 5' and 3' of the said sequence comprise nucleotide sequences that are homologous to sequences present in Bacillus thuringiensis chromosomal DNA to an extent sufficient to permit said insecticide encoding DNA sequence to be inserted into the bacterial chromosomal DNA; and ii) further transforming said linear or circular DNA segment according to 1) into a S Bacillus thuringiensis host bacterium and preparing a recipient Bacillus thuringiensis -'C I'P:\OERULR\7796 91. ISO 30I/5/ -63- bacterium.

19. A method according to claim 18, wherein said DNA segment further comprises an origin of replication from a gram-negative bacterium. A method according to claim 18, wherein said DNA segment further comprises a transposon sequence.

21. A method according to claim 18, wherein transformation is effected by a method selected from the group consisting of transfection, electroporation, transduction or conjugation.

22. A method according to any one of claims 18 to 21, wherein the recipient is selected from strains of Bacillus thuringiensis kurstaki.

23. A method of controlling insect pests comprising applying an effective amount of the insecticidal composition of claim 17 to the insects pests or their habitats.

24. A transformed Bacillus thuringiensis bacterium obtainable by a method according to claim 18. An insecticidal composition according to claim 17 further comprisih ther insecticidal compounds. DATED this 30th day of May 1997. SANDOZ LTD. by their Patet Attorneys DAVIES COLLISON CAVE -Ils