WO1989007605A1 - Bacillus thuringiensis var. israelensis cryd toxin gene, protein and related insecticide compositions - Google Patents

Abstract

The present invention discloses a nucleic acid and amino acid sequence for the cryD protein of Bacillus thuringiensis var. israelensis. The protein encoded by the gene is shown to have mosquitocidal activity. Also provided are insecticidal compositions employing the protein.

Description

BACILLUS THURINGIENSIS VAR. ISRAELENSIS CRYD TOXIN GENE, PROTEIN AND RELATED INSECTICIDE COMPOSITIONS

1. INTRODUCTION

This invention relates to a crystalline protein which is useful as a biological insecticide and is known as the 67-kDa Bacillus thuringiensis var. israelensis toxin, or 67-kDa toxin. The 67-kDa toxin is naturally produced by B. thuringiensis var. israelensis . More specifically, this invention relates to the cloning and expression in various microorganisms of the cryD gene coding for the 67-kDa toxin, and related novel insecticide compositions incorporating the

67-kDa toxin itself and microorganisms transformed with the cryD gene.

2. BACKGROUND OF THE INVENTION 2.1. COMMERCIAL PESTICIDES: GENERAL CONSIDERATIONS Each year, significant portions of the world's commercially important agricultural crops are lost to insects and other pest infestation. The damage wrought by these pests extends to all areas of commercially important plants including foods, textiles, and various domesticplants, and the economic damage runs well into the millions of dollars. Thus, protection of crops from such infestations is of paramount concern.

Broad spectrum pesticides are most commonly used for crop protection, but indiscriminate use of these agents can lead to disruption of the plant's natural defense agents. Furthermore, because of their broad spectrum of activity, the chemical pesticides may destroy non-target organisms such as beneficial insects and parasites of destructive pests. These are also frequently toxic to animals and humans and, thus, pose environmental hazards when applied.

Additionally, insects and other organisms have frequently developed resistance to these pesticides when repeatedly exposed to them. In addition to reducing the utility of the pesticide, resistant strains of minor pests may become major infestation problems due to the reduction of beneficial parasitic organisms.

This is a major problem encountered in using broad spectrum pesticides. What is needed is a biodegradable pesticide that combines a narrower spectrum of activity with the ability to maintain its activity over an extended period of time, i.e., to which resistance develops much more slowly, or not at all. Biopesticides appear to be useful in this regard.

2.2. BIOLOGICAL PESTICIDES

Biopesticides, also called biorationals, make use of naturally occurring pathogens to control insects, fungal, and weed infestations of agricultural crops. Such substances may comprise a bacterium which produce a substance toxic to the infesting agent (such as a toxin), with or without a bacterial growth medium. Such bacteria can be applied directly to the plants by standard methods of application and will typically persist on the crops for an extended period of time, decreasing the need for repeat applications.

The use of biological methods of pest control was first suggested in 1895 when a fungal disease was discovered in silkworms. It was not until 1940, however, when spores of the milky disease bacterium Bacillus popilliae were used to control the Japanese beetle, that successful biological pest control was first achieved. In the late 1960^,s, the discovery of a new strain of bacterium that secreted a toxin fatal to caterpillars set the stage for commercial biopesticides. The bacterium, named Bacillus thuringiensis (hereinafter referred to alternatively as "B.t.") is currently the most widely used biopesticide. 2.3. BACILLUS THURINGIENSIS AND CRYSTAL TOXINS

Bacillus thuringiensis is a widely distributed, rod shaped, aerobic and spore forming microorganism. During its sporulation cycle B. thuringiensis synthesizes proteins that aggregate to form parasporal crystals. The pathogenicity of B. thuringiensis to a variety of sensitive insects, such as those in the order Lepidoptera (caterpillars) and Diptera (mosquitos), is essentially due to these parasporal crystals, which may represent over 20% of the dry weight of the B. thuringiensis cell at the time of sporulation.

The parasporal crystal is active in the insect only after ingestion. For instance, after ingestion by a lepidopteran insect, the alkaline pH and proteolytic enzymes in the mid-gut activate the crystal allowing the release of the toxic components. These toxic components poison the mid-gut cells causing the insect to cease feeding and, eventually, to die. In fact, B. thuringiensis has proven to be an effective and environmentally safe insecticide in dealing with lepidopteran and dipteran pests.

It has been reported that different varieties of B. thuringiensis produce serologiσally different parasporal cystals. Many varieties of B. thuringiensis produce a bipyramidal crystal composed of one or more closely related 130-kDa proteins (P1 proteins) that are lepidopteran toxic. Several varieties also produce a flat, cuboidal crystal composed of a 66-kDa protein (P2 protein) that is both lepidopteran and dipteran toxic. B. thuringiensis var. israelensis produces an irregularly shaped parasporal crystal that is composed of three major proteins of approximately 130, 67 and 28 kDa. These sizes are only estimations based on the rates of migration of the proteins during electrophoresis through polyacrylamide gels. The var. israelensis crystal is toxic to dipteran larvae although there are conflicting data as to the toxic activity of the individual proteins that comprise the crystal.

2.4. B. THURINGIENSIS TOXIN GENE CLONING Since B. thuringiensis toxin gene products have proven to be effective insecticides which are readily isolated when in crystalline form or when associated with spore formation, they have been the subject of a great deal of scientific study, particularly with regard to gene isolation and cloning procedures.

Several genes encoding P1 crystal proteins have been isolated from different varieties of B. thuringiensis (Schnepf et al. U.S. Patent 4,467,036; Schnepf et al. J. Biol. Chem. (1985) 260:6264-6272; Adang et al. Gene (1985) 36:289-300; Klier et al. EMBO J. (1982) 1:791-799; Shibano et al. Gene (1985) 34:243-251. The cryBl gene encoding the P2 crystal protein has recently been isolated from B. thuringiensis strain HD263 (Donovan et al. J. Biol. Chem. (1988) 263:561-567;) for Bacillus thuringiensis P-2 Toxin Gene, Protein and Related Insecticide Compositions.

As mentioned above, the parasporal crystal of B. thuringiensis var. israelensis that is toxic to mosquito larvae is composed of three proteins of approximately 130, 67 and 28 kDa. Visser et al. (FEMS Microbiology Lett.

(1986) 30:211-214) reported that the mosquito toxicity of the var. israelensis crystal could be attributed to the 130-kDa protein. Other researchers have attributed mosquito toxicity to the 28-kDa protein (Yamamoto et al. Current Micro. (1983) 9:279-284; Armstrong et al. J. Bacteriol.

(1985) 161:39-46; Ward et al. J. Mol. Biol. (1986) 191:13- 22). Lee et al. (Biochem. Biophys. Res. Comm. (1985) 126:953-960) and Hurley et al. (Applied Env. Microbiol.

(1987) 53:1316-1321) presented evidence that the 28-kDa protein was haemolytic and that the 67-kDa protein was mosquitocidal. Wu and Chang (FEBS LETT. (1985) 190:232-236) found that mixtures of either the 130-kDa and 28-kDa proteins or of the 67-kDa and 28-kDa proteins were significantly more toxic to mosquito larvae that any of the proteins alone. They suggested that the mosquito toxicity was due to a synergistic interaction among the proteins.

The gene encoding the 130-kDa crystal protein of var. israelensis has been isolated (Angsuthanasombat et al. Mol. Gen. Genet. (1987) 208:384-389). Escherichia coli cells harboring the cloned gene were toxic to Aedes aegypti larvae. The gene encoding the 28-kDa crystal protein of var. israelensis has also been isolated (Waalwijk et al. Nucleic Acids Research (1985) 13:8207-8217; Bourgouin et al. Mol. Gen. Genet. (1986) 205:390-397) and recombinant Bacillus subtilis cells containing the 28-kDa protein gene were haemolytic for sheep red blood cells.

A number of recent papers refer to nucleic acid and amino acid sequences, nominally coding for various dipteran-active proteins from B^ thuringiensis var. israelensis (Bti). For example, U.S. Patent No. 4,652,628, to Walfield, discloses a sequence coding for a 50-100 Kda protein which is said to have antidipteran activity (see also, Thome, et al., J. Bacteriol, 166:801-811, 1986). Although the identity of this peptide is not specifically established relative to the known var. israelensis endotoxin proteins, it appears that the protein described is a portion of the 130 Kda endotoxin. Ward et al. (Nucl. Acid Res. 15:7195, 1987) have also described a gene sequence which nominally codes for the 130 Kda toxin. European Patent Application No. 0 216 481, to Repligen Corporation,, describes a 66 Kda protein which is described as being the precursor to a second, 35 Kda protein which is said to have antidipteran toxicity. This patent application does not specifically identify the larger protein with the 67 Kda protein recognized in the art, and notes that the entire domain of dipteran toxicity is found within the 35 Kda fragment. There is no disclosure of either nucleotide or amino acid sequence.

From the above discussion, it is apparent that there has been no conclusive characterization of the 67 Kda toxin protein of var. israelensis. The present inventor has, however, been able to isolate and define the gene sequence and amino acid sequence of the 67 Kda protein, and thus has provided a means by which this valuable protein can be produced readily in large quantities by cloning of the gene and transformation of host microorganisms other than B. thuringiensis. This invention also conclusively demonstrates that the 67 Kda cryD protein is highly toxic to mosquito larvae. In this regard, the present inventor also has discovered nucleotide probes which are useful in identifying and isolating the gene, and identifying plasmids containing same. Thus, there is now established a practical, easily obtained, insecticidal formulation which can be usefully employed against members of the order Diptera, and particularly against mosquitos.

To date, the gene (herein referred to as cryD) encoding the 67-kDa crystal protein of var. israelensis has not been isolated. This fact has rendered it impossible to provide a means for expressing this crystal protein in an organism other than var. israelensis. The availability of the cloned cryD gene would enable the enhanced production of the 67 kDa protein in B._ thuringiensis and also enable 67 kDa protein synthesis in a heterologous organism free of other crystal proteins.

3. SUMMARY OF THE INVENTION This invention relates to the 67-kDa protein produced by Bacillus thuringiensis var. israelensis, the DNA sequence for the gene (designated cryD) which codes for this protein and novel insecticides incorporating this protein and/or organisms transformed with the cryD gene. More specifically, this invention relates to the cloning and transformation of microorganisms with the gene coding for the 67-kDa protein toxin of var. israelensis. This invention is particularly useful in enabling the expression in organisms other than B. thuringiensis of the cryD protein crystal toxin in quantities greater than that produced by a native 67-kDa protein-producing B. thuringiensis organism during sporulation. In addition, this invention is useful in permitting the transformation of a non-sporulating microorganism with the gene coding for the 67-kDa protein toxin so that this toxin may be produced during virtually all stages of microorganism growth and, thereby, not be limited to production only during a sporulation stage. It is an additional object of this invention to provide a homogenous 67-kDa protein produced by the isolated gene. This protein may be produced by the process of transforming a microorganism, sporulating or non-sporulating, such as Bacillus megaterium or Escherichia coli or a different strain of B. thuringiensis with the cloned cryD gene. This process, by virtue of selection of the appropriate host and vector, would permit high yield production of the 67-kDa protein such that it is possible to derive a substantially homogenous preparation of the 67-kDa protein, i.e. minus any contamination by other varieties of crystal toxins typically produced in conjunction with or concurrently with the 67-kDa protein in its native B. thuringiensis var. israelensis host. The 67-kDa protein and/or the transformed host may be utilized in a variety of insecticidal compositions.

It is further an object of this invention to provide an organism, other than the native B. thuringiensis host, transformed with the DNA coding for the 67-kDa protein. This foreign transformed host enables the production of the 67-kDa protein under more desirable and/or selective culturing conditions.

The present invention also provides oligonucleotide probes which may be used to identify both DNA restriction fragments as well as plasmids containing the cryD gene.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1 is a restriction map of the recombinant plasmids pEG214, pEG215, pEG216, pEG217, pEG218 and pEG219 containing either all or part of the cryD gene encoding the 67-kDa protein of var. israelensis and all or part of the gene encoding the 28-kDa var. israelensis crystal protein. The location and direction of transcription of the cryD gene and the 28-kDa protein gene are indicted by arrows.

FIGURE 2 shows the DNA sequence of the cryD gene and the deduced amino acid sequence of the 67-kDa protein.

FIGURE 3 is a photograph of an SDS/polyacrylamide gel which shows that a recombinant host strain of Bacillus megaterium harboring the cloned cryD gene synthesizes large quantities of a protein having a similar size as that of authentic var. israelensis 67-kDa protein. Proteins were extracted from strains; lane 2, B. thuringiensis var. israelensis HD567; lane 3, B. megaterium EG1325; lane 4, B. megaterium EG1315; lane 5, B. megaterium EG1320. Lane 1 contains molecular weight standards. Numbers to the left indicate the sizes, in kilodaltons, and locations of the three crystal proteins of var. israelensis.

FIGURE 4 is comprised of 4A and 4B. Fig. 4A is a photograph of an ethidium bromide stained agarose gel that contains Hindlll digested DNA from various B. thuringiensis var. israelensis strains. Fig. 4B is a photograph of an autoradiogram that was made by hybridizing the radioactively labeled cloned cryD gene with the Hindlll fragments shown in 4A. Fig. 4B demonstrates that the cloned cryD gene hybridized exclusively to an 11.0 kb Hindlll fragment from a strain of var. israelensis known to produce the 67 kDa protein.

FIGURE 5 is comprised of 5A and 5B. Fig. 5A is a photograph of an ethidium bromide stained Eckhardt gel containing native plasmids from various B. thuringiensis var. israelensis strains. Fig. 5B is a photograph of an autoradiogram that was made by hybridizing the radioactively labeled cloned cryD gene with the plasmids shown in 5A. Fig. 5B illustrates that the cloned cryD gene hybridized specifically to a plasmid of 75 MDa in two strains of B. thuringiensis that were known to produce the 67 kDa protein (HD567 and HD567-61-9).

5. DESCRIPTION OF THE INVENTION Generally stated, the present invention provides for a cloned gene coding for Bacillus thuringiensis var. israelensis 67-kDa protein toxin and comprising the DNA nucleotide sequence shown in Fig. 2. This gene (which comprises double stranded DNA wherein the nucleotide strands have a complementary base sequence to each other) codes for a protein (or as also used herein equivalently, polypeptide) having the amino acid sequence of the 67-kDa protein which amino acid sequence is shown in Fig. 2. The 67-kDa protein encoded by the cloned gene has insecticidal activity against dipteran larvae.

Methods of producing the 67-kDa protein are also provided by this invention. In this method of production the cryD gene is inserted into a cloning vector or plasmid which plasmid is then utilized to transform a selected microorganism. The gene may be used with its native promoter, or with a foreign promoter. The cloning vectors, as described herein, are generally known in the art and are commercially available. The choice of a particular plasmid is within the skill of the art and would be a matter of personal choice. Plasmids suitable for use in this invention are, for instance, pBR322, plasmids derived from B. thuringiensis, and plasmids derived from Bacillus microorganisms. Microorganisms suitable for use with this invention are both sporulating and non-sporulating such as E. coli, B. thuringiensis, and B. megaterium. The microorganisms utilized are also known in the art and are generally available. The choice of any particular microorganism for use in the practice of this invention is also a matter of individual preference. In a preferred embodiment of this invention the microorganism would comprise Bacillus megaterium.

5.1. RECOMBINANT DNA TECHNOLOGY AND GENE EXPRESSION Generally stated, recombinant DNA technology involves insertion of specific DNA sequences into a DNA vehicle (plasmid or. vector) to form a chimeric DNA molecule which is capable of replication in a host cell. The inserted DNA sequence is typically foreign to the recipient host. In recent years several general methods have been developed which enable construction of recombinant DNA molecules. For example, U.S. Pat. No. 4,237,224 to Cohen and Boyer describes production of such recombinant plasmids using restriction enzymes and methods known as ligation. These recombinant plasmids are then introduced and replicated in unicellular organisms by means of transformation. Because of the general applicability of the techniques described therein, U.S. Pat. No. 4,237,224 is hereby incorporated by reference into the present specification. Regardless of the method used for construction, the recombinant DNA molecule must be compatible with the host cell, i.e., capable of autonomous replication in the host cell. The recombinant DNA molecule should also have a marker function which allows the selection of host cells so transformed by the recombinant DNA molecule. In addition, if all of the proper replication, transcription and translation signals are correctly arranged on the chimeric DNA molecule, the foreign gene will be expressed in the transformed cells and their progeny.

These different genetic signals and processing events control many levels of gene expression, i.e., DNA transcription and messenger RNA translation. Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes transcription.

Translation of messenger RNA (mRNA) in procaryotes depends upon the presence of the proper procaryotic signals. Efficient translation of mRNA in procaryotes, such as B.t., requires a ribosome binding site called the Shine Dalgarno (SD) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon (AUG) which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3 '-end of the 16S RNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the mRNA to allow correct positioning of the ribosome (Roberts and Lauer, 1979, Methods in Enzymology, 68:473).

One method widely employed for the cloning of a particular gene is to prepare a "library" of recombinant plasmids. Each recombinant plasmid is comprised of a plasmid vector, which usually confers antibiotic resistance to cells that harbor it, plus a fragment of DNA from the donor organism, an organism that contains the gene. The plasmid library is commonly prepared by digestion of both the plasmid vector and total DNA from the donor organism with a restriction enzyme, inactivation of the enzyme and ligation of the DNA mixture. The ligated DNA is a plasmid library. The key feature of this plasmid library is that it contains many different recombinant plasmids. It is highly likely that at least one of the recombinant plasmids in the library will contain a fragment of DNA from the donor organism on which the gene of interest resides. The plasmid library is transformed into the cells of a host organism that does not contain the gene. The host cells are spread on a selective solid medium, usually one containing an antibiotic, that allows only transformed cells, those containing recombinant plasmids, to grow into colonies. Individual transformed host colonies are tested for the acquisition of the gene from the donor organism. In host colonies the acquired gene is carried on the recombinant plasmid.

One of the most direct methods of testing for acquisition of a gene is to use a gene-specific hybridization probe, a fragment of DNA that is homologous to the gene. A characteristic of homologous DNA fragments is that they will bind tightly to each other during hybridization. Typically a radioactively labeled DNA probe is used during hybridization so that binding of the probe to the gene can be easily monitored.

A recent advance in molecular biology is the use of synthetic oligonucleotides as gene-specific probes. The basis for the use of the oligonucleotides is that in all biological systems a particular sequence of nucleotides encodes a precise sequence of amino acids. Conversely if the sequence of amino acids is known for a particular protein then the nucleotide sequence encoding the protein can be inferred, although not precisely. In practice, the partial amino acid sequence of a protein, the product of the gene of interest, is determined by chemical methods. Based on the protein amino acid sequence a gene-specific oligonucleotide probe is synthesized that may be, to varying degrees, homologous to the gene. Exact homology cannot be guaranteed because knowledge of' the amino acid sequence of a protein does give exact knowledge of the nucleotide sequence of the gene encoding the protein. Nevertheless, even though the homology between the oligonucleotide probe and the gene may not be precise, hybridization conditions can usually be found that will permit the oligonucleotide probe to bind specifically to the gene.

The procedure used to isolate the gene encoding the 67-kDa protein was to purify the protein from var. israelensis strain HD567, determine its NH2-terminal amino acid sequence, synthesize a gene-specific oligonucleotide probe based on the amino acid sequence and use the probe in colony hybridization experiments as described below.

5.2. CLONING THE GENE (CRYD) ENCODING THE 67-KDA CRYSTAL PROTEIN FROM B. THURINGIENSIS VAR. ISRAELENSIS STRAIN HD567

More specifically, in order to clone the cryD toxin gene of this invention, cells of B. thuringiensis var. israelensis strain HD567 (U.S.D.A. Cotton Insect Research

Unit, Brownsville, Texas 78520), were grown in C2 medium (1% glucose, 0.2% peptone, 0.5% N Z amine A (Sheffield

Products), 0.2% yeast extract, 15 mM (NH₄)₂SO₄, 23 mM KH₂PO₄, 27 mM K₂HPO₄, 1 mM MgSO₄.7H₂O), 600 μΑ CaCl₂, 17 μϋt ZnSO₄.7H₂O, 17 μM. CuSO₄.5H₂O, 2 μM FeSO₄.7H₂O) at 30°C for 72 hours and spores plus crystals were harvested by centrifugation. The spore/crystal pellet was washed with several changes of 1 M NaCl and then several changes of deionized water. Toxin proteins were solubilized by incubating the spore/crystal preparation in 5% 2-mercaptoethanol, 2% NaDodeSO₄, 60 mM Tris pH 6.8, 10% glycerol at 70°C for 7 min., and spores were removed by centrifugation. The supernatant was electrophoresed through polyacrylamide gels containing NaDodeSO₄ to separate proteins. The gel was stained with Coomassie dye and gel slices containing the 67-kDa protein were cut out with a razor blade. The homogeneous 67-kDa protein preparation was electroeluted from gel slices and, after acetone precipitation, the NH2-terminal amino acid sequence of the 67-kDa protein was determined by automated Edman degradation carried out on an Applied Biosystems Gas Phase Sequenator (model 470 A) and analyzed on a DuPont Zorbax C 18 column in a Hewlett-Packard HPLC (model 1090) with a 1040 diode array detector. The amino acid sequence of the NH2-terminal portion of the homogeneous 67-kDa protein was determined to be:

1 2 3 4 5 6 7 8 9 10 MET GLU ASP SER SER LEU ASP THR LEU SER

11 12 13 14 15 16 17 18 19 20 ILE VAL ASN GLU THR ASP PHE PRO LEU TYR

21 22 23 24 25 26

ASN ASN TYR THR GLU PRO

5.3. CRYD-SPECIFIC OLIGONUCLEOTIDE PROBE A 47-mer oligonucleotide probe encoding amino acids 11 through 26 of the NH2 terminus of the 67 kDa protein was synthesized on an Applied Biosystems DNA synthesizer (model 380A) . It was recognized that because of the codon degeneracy (certain amino acids are each encoded by several slightly different codons) the sequence of the synthetic oligonucleotide would probably be different from the actual NH2-terminal sequence of the cryD gene. However, the fact that the B. thuringiensis genome is 64% A:T (Laskin and Lechevalier, Handbook of Microbiology, 2nd Ed., Vol. Ill, (1981) p. 579, CRC Press, Inc., Boca Raton, FL) was used in designing an oligonucleotide probe that would have the highest probability of matching the actual sequence of the cryD gene. The oligonucleotide probe was designed to bind only to the NH2-terminal coding region of the cryD gene. The sequence of the cryD gene-specific oligonucleotide probe was:

5'-ATT GTA AAT GAA ACA GAT TTT CCA TTA TAT AAT AAT TAT ACA GAA CC-3' .

In addition to enabling the original isolation of the cryD gene herein, this DNA probe also comprises another preferred embodiment of this invention. This DNA probe permits the screening of any B. thuringiensis strain to determine whether the cryD gene (or possibly a related gene) is naturally present or whether a particular transformed organism includes the cryD gene. In this fashion it is also possible to estimate the insecticidal activity of that strain of B. thuringiensis. It is also within the scope of this invention that this probe may comprise a smaller or larger oligonucleotide. The probe may be labeled by any number of techniques known in the art (such as radioactively or enzymatically labeled) and as described below.

5.4. CONSTRUCTION OF A PLASMID LIBRARY ENRICHED FOR THE CRYD GENE

Cells of the B. thuringiensis var. israelensis strain HD567 (U.S.D.A., Cotton Insect Research Unit,

Brownsville, Texas 78520) were grown to mid-log phase at

30ºC in LB medium (1% Difco tryptone, 0.5% Difco yeast extract, 0.5% NaCl, pH 7.0). Cells were harvested by centrifugation, resuspended in 50 mM Tris HCl, pH 7.8, 10 mM

EDTA, 1 mg/ml lysozyme and incubated at 37°C for 60min. Cells were lysed by adding NaDodeSO₄. to a final concentration of 0.2%. Cell lysates were extracted twice with an equal volume of phenol and once with an equal volume of chloroform/isoamyl alcohol (24/1).

One tenth volume of 3 M NaAcetate and 2 volumes of EtOH were added to the lysates and DNA was extracted by spooling on a glass rod. The spooled DNA was soaked in 66% EtOH for 5 min and in diethyl-ether for 1 min. The spooled DNA was air dried and resuspended in deionized water.

The DNA was digested with Hindlll and EcoRI, electrophoresed through a 0.8% agarose gel and blotted (Southern, J. Molec. Bio. (1975) 98:503-517) to a nitrocellulose filter. The filter was hybridized with approximately 1 μg of the radioactively labeled oligonucleotide probe in 15 ml of a solution of 3 X SSC (1 X SSC = 0.15M NaCl, 0.015 M Sodium Citrate), 0.1% NaDodeSO₄, 200 μg/ml heparin, 10 X Denhardt solution (1 X Denhardt = 0.02% polyvinyl-pyrrolidone 0.02% bovine serum albumin, 0.02% Ficoll) for 16 h at 30°C. The filter was washed with 300 ml of a solution containing 3 X SSC, 0.1% NaDodeSO₄ at 30ºC for 20min. The washing was repeated twice. After 16 h exposure to the filter. X-ray film revealed numerous hybridizing DNA fragments. The filter was rewashed in 3 X SSC, 0.1% NaDodeSO₄ at 35°C for 20 min and exposed to X-ray film. The washings were repeated at temperatures higher by 5ºC, each wash being followed by exposure of the filter to X-ray film, until a temperature (47ºC) was reached at which the oligonucleotide hybridized to a unique EcoRI fragment of 5.7 kb and to a unique Hindlll fragment of 11.0 kb. Therefore, it was determined that at least the NH₂-terminal coding region of the cryD gene resided on a 5.7 kb EcoRI and a 11.0 kb Hindlll fragment from HD567. We estimated that it would be easier to clone a fragment of 5.7 kb than one of 11.0 kb. Therefore, the 5.7 kb EcoRI fragment was cloned as described below. A cryD gene-enriched plasmid library was constructed by digesting HD567 total DNA with EcoRI, electrophoresing the digested DNA on an agarose gel and excising gel slices containing EcoRI fragments ranging in size from approximately 4.8 to 7.4 kb. HD567 EcoRI fragments were electroeluted from agarose gel slices, phenol plus chloroform extracted, ethanol precipitated and ligated into the EcoRI site of plasmid pBR322 that had been digested with EcoRI and treated with alkaline phosphatase. Alkaline phosphatase greatly increased the probability that recombinant plasmids were formed consisting of pBR322 plus a EcoRI fragment of HD567 DNA. The resulting ligation mix consisted of a library of recombinant plasmids enriched for the cryD toxin gene from strain HD567.

5.5. COLONY HYBRIDIZATION AND ISOLATION OF A 5.7 kb ECORI FRAGMENT CONTAINING PART OF THE CRYD GENE

The cryD gene-enriched plasmid library was transformed into an ampicillin sensitive host strain of

Escherichia coli, HB101, by the CaCl₂ procedure. E. coli strain HB101 is not toxic to mosquito larvae and, therefore, it would not be expected to contain the cryD gene. E. coli was used as the host strain because these cells are easily transformed with recombinant plasmids. All host cells acquiring a recombinant plasmid would become ampicillin resistant. After exposure to the recombinant plasmids the

E. coli host cells were spread onto solid medium containing ampicillin and those cells that harbored a recombinant plasmid were able to grow into colonies. It was expected that each individual ampicillin resistant host colony would harbor many identical copies of a recombinant plasmid comprised of pBR322 plus a unique EcoRI fragment from the donor strain HD567 DNA. However, the donor strain EcoRI fragment in the recombinant plasmid would differ from one colony to the next. Approximately 2,000 individual ampicillin resistant colonies were blotted onto nitrocellulose filters. Replicas of the colonies were saved for later use as described below. The recombinant plasmids contained in the colonies were bound to the nitrocellulose filters by treating the colonies with NaOH and NH₄Acetate. The resulting nitrocellulose filters contained an array of recombinant plasmids each of which was physically separated from other recombinant plasmids. The nitrocellulose filters were hybridized at 47°C for 16 h in a solution of 3 X SSC, 200 μg/ml heparin, 0.1% NaDodeSO₄, 10 X Denhardt solution and approximately 1 μg of the cryD gene-specific oligonucleotide probe that had been radioactively labeled. The filters were washed three times for 30 min at 47ºC in 3 X SSC, 0.1% NaDodeSO₄ and were exposed to X-ray film. The resulting autoradiogram showed that the oligonucleotide probe had hybridized to colonies containing recombinant plasmids at four different locations on the nitrocellulose filters.

By aligning the autoradiogram with the colony replicas it was possible to identify four colonies whose recombinant plasmids had apparently hybridized with the oligonucleotide probe.

Recombinant plasmids were extracted from three of the four colonies (one colony was no longer viable), plasmids were digested with EcoRI and electrophoresed on an agarose gel. Each of the three plasmids consisted of a 4.3 kb EcoRI fragment corresponding to pBR322 plus, in each case, a 5.7 kb fragment of HD567 DNA. The plasmids were transferred from the agarose gel to a nitrocellulose filter. The nitrocellulose filter was hybridized with the radioactively labeled oligonucleotide probe and exposed to X-ray film. The resulting autoradiogram showed that the oligonucleotide probe hybridized exclusively to the 5.7 kb EcoRI fragment in each of the three recombinant plasmids. One of these recombinant plasmids, designated pEG214, was selected for further experimentation and evaluation. The original E. coli colony harboring pEG214 was designated EG1318.

5.6. LOCATION OF THE CRYD GENE ON THE CLONED 5.7 KB ECORI FRAGMENT

It was likely that the cloned 5.7 kb EcoRI fragment contained at least the NH₂-terminal coding region of the cryD gene. Presence of the cryD gene on the 5.7 kb fragment was verified using DNA sequencing to search for a region in the cloned 5.7 kb fragment that encoded the NH2 terminus of the cryD protein. Since it is time consuming to sequence a fragment of DNA longer than 2 kb it was necessary to identify a small fragment of DNA within the 5.7 kb fragment that would be expected to contain the cryD gene. Accordingly, plasmid pEG214 was digested with various restriction enzymes, electrophoresed through an agarose gel and plasmid restriction fragments were blotted from the gel to a nitrocellulose filter. Hybridization of the filter with the radioactively labeled oligonucleotide probe revealed that the probe specifically hybridized to a 1.1 kb Dral restriction fragment of DNA. Therefore, it was expected that the 1.1 kb fragment would contain at least the NH2-terminal coding region of the cryD gene.

The 1.1 kb Dral fragment was subcloned from pEG214 into the vectors mpl8 and mpl9 (Bethesda Research Laboratories) and sequenced. A DNA sequence encoding the NH2 terminus of the cryD protein was located thirty nucleotides from one Dral site. This conclusively demonstrated that the cloned 5.7 kb EcoRI fragment from the donor strain HD567 contained at least the NH₂-terminal coding region of the cryD gene. Additional DNA sequencing revealed the presence of a PvuII restriction site within the 1.1 kb fragment. The position of this PvuII site served as a marker. It allowed the location of the cryD gene in the 5.7 kb fragment to be precisely determined as described below.

The location and direction of transcription of the cryD gene on the cloned 5.7 kb fragment was determined by digesting the 5.7 kb fragment with PvuII in combination with various other restriction enzymes. The restriction fragments were electrophoresed through an agarose gel and blotted onto a nitrocellulose filter. By hybridizing the filter with the radioactively labeled cryD gene-specific oligonucleotide probe it was possible to determine the location and orientation of various restriction fragments on the larger 5.7 kb fragment. From this knowledge the precise position and direction of transcription of cryD on the 5.7 kb fragment was determined as indicted by the arrow in Figure 1. Figure 1 shows a restriction map of plasmid pEG214. The length of the cryD gene was assumed to be approximately 2.0 kb based on the estimated size (67 kDa) of the cryD protein.

Subcloning of a 0.8-kb PvuII-EcoRI fragment from pEG214 into mpl8 and mp19 permitted the determination of the complete nucleotide sequence between the Dral site and the EcoRI in the 5.7 kb fragment (Fig. 1). A long open reading frame began 30 nucleotides from the Dral site and continued to the EcoRI site at the end of the 5.7 kb fragment. The open reading frame encoded a protein of approximately 64 kDa. The size of the open reading frame (64 kDa versus and expected size of approximately 67 kDa) and the absence of a translation stop codon indicated that the 5.7 kb fragment did not contain the complete cryD gene. It was estimated that the 3' terminus of the cryD gene was located beyond the EcoRI site of the 5.7 kb fragment. The fact that recombinant Bacillus megaterium cells harboring the 5.7 kb fragment were not toxic to mosquito larvae (described below) further indicated that the 5.7 kb fragment did not contain the complete cryD gene.

In order to clone the complete cryD gene it was necessary to isolate a DNA fragment that contained DNA sequences beyond the EcoRI site of the 5.7 kb fragment. The 5.7 kb EcoRI fragment did not contain a Hindlll site (see pEG214, Fig. 1). Therefore, it was expected that the 5.7 kb fragment would be contained within a large Hindlll fragment. The 5.7 kb EcoRI fragment was radioactively labeled and hybridized with a DNA blot. of total Hindlll digested DNA from HD567. The 5.7 kb fragment specifically hybridized to a Hindlll fragment of 11 kb. As discussed above, the cryDspecific oligonucleotide probe hybridized to an 11 kb Hindlll fragment of HD567 DNA. The same 11 kb fragment was probably involved in both cases. This 11 kb fragment was expected to contain the 5.7 kb EcoRI fragment as well as the complete cryD gene.

In order to isolate the 11 kb fragment a sizeselected Hindlll library of HD567 DNA was constructed. HD567 total DNA was digested with Hindlll and the resulting Hindlll fragments were electrophoresed through an agarose gel. Gel slices containing fragments of 9 to 14 kb in size were excized, fragments were electroeluted from the gel slices and ligated into the plasmid pBR322 which had been digested with Hindlll and treated with alkaline phosphatase. The resulting recombinant plasmids were transformed into E. coli HB101 cells with selection for ampicillin resistance. Approximately 3000 amp^r colonies were blotted to nitrocellulose filters and hybridized with the 5.7 kb EcoRI fragment that had been radioactively labeled. The 5.7 kb fragment hybridized to 13 colonies. Plasmids were extracted from the hybridizing colonies, digested with Hindlll and electrophoresed through an agarose gel. The Hindlll fragments were blotted to a nitrocellulose filter and the filter was hybridized with radioactively labeled 5.7 kb fragment. The 5.7 kb fragment specifically hybridized with an 11 kb fragment that was present in two plasmids. One plasmid, consisting of pBR322 plus and 11 kb Hindlll fragment, was designated pEG216. As expected, pEG216 contained a 5.7 kb EcoRI fragment corresponding to the 5.7 kb fragment in pEG214 (Fig. 1). pEG216 also contained a 0.8 kb EcoRI fragment adjacent to the 5.7 kb fragment (Fig. 1). It was estimated that the 0.8 kb fragment would contain the 3'-end of the cryD gene.

5.7. DNA SEQUENCE OF THE CLONED CRYD GENE The 0.8 kb fragment was subcloned, in both orientations, into mp18 and sequenced. An open reading frame encoding 76 amino acids was located at one end of the 0.8 kb fragment. The open reading frame was terminated by a UAG translation stop codon. We estimated that this open reading frame was the 3'-end of the cryD gene, missing from the 5.7 kb fragment. In order to confirm that the open reading frame in the 0.8 kb fragment contained the 3'-end of the cryD gene, it was necessary to determine the continuous DNA sequence extending from the 0.8 kb fragment, across the EcoRI site, and into the 5.7 kb fragment. A 1.9 kb ClalPvuII fragment (Fig. 1), which was estimated to contain the EcoRI junction between the 0.8 kb fragment and the 5.7 kb fragment, was subcloned from pEG216 into mpl8. Sequencing of the 1.9 kb fragment confirmed that the open reading frame (3 '-end of cryD) in the 0.8 kb fragment was continuous with the open reading frame (5'-portion of cryD) in the 5.7 kb fragment. The complete sequence of the cryD gene is presented in Figure 2. The cryD gene encodes a protein of 72,357 Da (644 amino acids). This size (approximately 73 kDa) is 6 kDa larger than the protein size (67 kDa) estimated from SDS/polyacrylamide gels. The size discrepancy may be explained in two ways. Protein size estimations, based on SDS/polyacrylamide gels, frequently differ by 10 to 20% from the protein size deduced from the gene sequence. Alternatively, the 73-kDa protein may be proteolytically processed, either prior to incorporation into the crystal or during crystal solubilization, to yield a protein of 67 kDa.

THE 11 KB HINDIII FRAGMENT CONTAINS THE CRYD GENE AS WELL AS THE GENE FOR THE 28-KDA CRYSTAL PROTEIN

McLean and Whiteley, J. Bacteriol. (1987) 169:1017-1023) have cloned a var. israelensis Hindlll fragment of approximately 10 kb that contained the gene for the 28-kDa crystal protein. We observed that several of the restriction sites reported by McLean and Whiteley in the 10 kb.fragment appeared to match restriction sites in the 11 kb Hindlll fragment of this application. Therefore, we estimated that the 11 kb fragment might contain the gene for the 28-kDa var. israelensis crystal protein. A 2.6-kb PvulI-BamHI fragment from the 11-kb fragment (contained in pEG216, Fig. 1) was subcloned into mp18 and partially sequenced. An open reading frame of 33 codons, terminated by the BamHI site, was found in the 2.6-kb fragment that exactly matched the NH2-terminal amino sequence of the 28-kDa crystal protein as reported by Waalwijk et al. (Nucleic Acids Res. (1985) 13:8207-8217). Therefore, as shown in Figure 1, the 11-kb fragment reported in this application contains the cryD gene as well as the var. israelensis gene for the 28-kDa protein.

5.8. USE OF THE CLONED CRYD GENE AS A SPECIFIC HYBRIDIZATION PROBE 5.8.1. IDENTIFICATION OF DNA RESTRICTION FRAGMENTS CONTAINING THE CRYD GENE

Cloned genes are valuable as specific probes for identifying homologous genes in uncharacterized DNA samples. In the following example we demonstrate that the cloned cryD gene can be used as a probe to identify DNA restriction fragments carrying the gene.

The cryD gene is contained on an 11.0 kb Hindlll restriction fragment (Fig. 1, pEG216). The fragment contains approximately 9 kb of DNA in addition to cryD (Fig. 1, pEG216). Within the 11.0 kb fragment a 2.5 kb EcoRI-EcoRV fragment contains the coding region of the cryD gene, minus 76 COOH-terminal codons, plus approximately 1200 upstream nucleotides (Fig. 1, pEG216) . Therefore the 2.5 kb fragment is suitable as a cryD-specific probe.

Total DNA from B. thuringiensis strains HDl (67-kDa negative), EG2158 (67-kDa negative) and HD567 (67-kDa positive) was digested with Hindlll restriction enzyme and the digested DNA was electrophoresed through an agarose gel. Figure 4A is a photograph of an ethidium bromide stained agarose gel that contains Hindlll digested DNA from strains HDl (lanes 1 and 2), EG2158 (lanes 3 and 5) and HD567 (lanes 6 and 7). Lane 4 contains lambda phage DNA digested with Hindlll to serve as molecular weight size markers. Figure 4A illustrates that total Hindlll digested B_^ thuringiensis DNA can be resolved into hundreds of different size fragments. The digested DNA was transferred from the gel to a nitrocellulose filter. The filter was hybridized at low stringency (50ºC) with the radioactively labeled 2.5 kb EcoRI-EcoRV cryD fragment and, after washing at 50°C, the filter was exposed to x-ray film.

Figure 4B is a photograph of the exposed x-ray film (autoradiogram). Figure 4B shows that the 2.5 kb cryD fragment hybridized specifically to a Hindlll fragment of 11.0 kb from strain HD567 (67 kDa-positive) (lanes 6 and 7) but failed to hybridize to any DNA from the 67 kDa-negative strains HD1 (lanes 1 and 2) and EG2158 (lanes 3 and 5).

The above demonstrates that the cloned cryD gene can be used as a very specific probe for detecting DNA fragments containing the gene. The complete specificity of the probe is shown by the fact that the probe did not hybridize to any DNA from the 67 kDa-negative B. thuringiensis strains HDl and EG2158. The probe specifically hybridized to an 11.0 kb fragment, that was known to contain the cryD gene, from hundreds of DNA fragments present on the nitrocellulose filter.

5.8.2. IDENTIFICATION OF NATIVE B. THURINGIENSIS PLASMIDS CONTAINING CRYD GENES

In this application we demonstrate that the cloned cryD gene can be used as a probe to identify B. thuringiensis cryD-containing plasmids. The procedure of

Eckhardt (Eckhardt, T. Plasmid 1:584-588, 1978) was used to separate native E. huringiensis plasmids according to size.

Figure 5A is a photograph of an Eckhardt gel displaying plasmids for var. israelensis strains HD567, (lane 1);

HD567-42, (lane 2); HD567-16, (lane 3); and HD567-61-9,

(lane 4). HD567 contains large plasmids of approximately

135, 105, 75 and 68 MDa (indicated by numbers to the left of

Fig. 5A) , and also several small plasmids. Strains HD567- 42, HD567-16 and HD567-61-9 were derived from strain HD567 by the loss of the 75 MDa plasmid (HD567-42, Fig. 5A, lane

2), by the loss of both the 75 and the 68 MDa plasmids

(HD567-16, Fig. 5A, lane 3) and by the loss of all except the 75 MDa plasmid (HD567-61-9, Fig. 5A, lane 4), respectively. The plasmids were transferred from the gel to a nitrocellulose filter. The filter was hybridized at moderate stringency (65 °C) with the radioactively labeled

2.5 kb EcoRV-EcoRI fragment containing most of the cryD coding region from pEG216 (Fig. 1). Autoradiography of the nitrocellulose filter revealed that the cryD gene fragment hybridized exclusively to one plasmid of approximately 75 MDa in the 67 kDa-producing strains HD567 and HD567-61-9 (Fig. 5B, lanes 1 and 4). The cryD gene also hybridized to a broad band of DNA in strains HD567 and HD567-61-9 that is seen below the 75-MDa plasmid (Fig. 5B, lanes 1 and 4). The cloned cryD gene did not hybridize to any plasmids or DNA in the 67-kDa-negative strians HD567-42 and HD567-16 (Fig. 5B, lanes 2 and 3). Therefore, the broad band of hybridizing DNA seen below the 75-MDa plasmid is most likely due to the presence of linearized or degraded 75-MDa plasmid. Linearization or degradation of the 75-MDa plasmid could occur during cell lysis or electrophoresis. This experiment demonstrated that the cloned cryD gene can be used in a direct manner to identify native plasmids containing cryD genes in B. thuringiensis strains. DNA hybridization with the cloned cryD gene allowed direct identification of a single plasmid of 75 MDa carrying the cryD gene out of several such plasmids existing in var. israelensis.

5.9. ADDITIONAL PURIFICATION OF 67 KDa TOXIN

The cryD gene can be inserted in any appropriate plasmid which may then be utilized to transform an appropriate microorganism. It is clearly within the scope of this invention that microorganisms other than B. thuringiensis var. israelensis may be transformed by incorporation of the cryD gene i.e., generally stated, organisms from the genera Bacillus, Escherichia, and Cyanobacteria. Preferred for use with this invention are spore-forming organisms such as Bacillus megaterium. It is also within the scope of this invention that different strains of B. huringiensis var. israelensis may also be transformed by the incorporation of the cryD gene. The microorganisms so transformed will preferably produce the cryD toxin in quantities that are far in excess of the quantity of the toxin produced in a B. thurincfiensis natural host strain. The cryD toxin produced by a transformed organism is preferably the only delta-endotoxin produced by that organism. In this manner, the organism itself may be utilized alone or as part of an insecticidal composition. Since the cryD toxin would preferably be the only delta-endotoxin produced by the organism, it is a straightforward process to purify the cryD toxin from other cellular material by methods known in the art such as renografin density gradients.

It will also be recognized that the present protein toxin may be synthesized chemically in accordance with the sequence depicted in Figure 2. The synthesis of the protein may be achieved by any of the peptide synthetic techniques known in the art. The synthesized protein may contain deletions, additions and substitutions of amino acid residues within the sequence depicted in Figure 2, which result in a silent change, thus producing a bioreactive product.

5.10. TRANSFORMATION OF CRYD INTO CYANOBACTERIA

Cyanobacteria (blue green algae) thrive in most aquatic environments and are a natural food of mosquito larvae. It is within the scope of this invention that the cryD gene (Fig. 2) be inserted directly into cyanobacteria so that the bacteria itself produces the cryD toxin. Recent advances in genetic engineering have allowed the construction of so-called "shuttle" plasmids that are able to replicate in both Escherichia coli and cyanobacteria (Buzby, J.S., Porter, R.D., and Stevens, S.E., Jr. Expression of the Escherichia coli lac Z gene on a plasmid vector in a cyanobacteria. Science 230:805-807, 1985). Genetic engineering of these shuttle plasmids is initially accomplished in E. coli which is easy to work with. After construction, the plasmid is isolated from E. coli and transformed into cyanobacteria. This example is similar to the construction of the Bacillus - E. coli shuttle plasmids pEG215, pEG217 and pEG219 described in this application. pEG215, pEG217 and pEG219 were initially constructed in E. coli and, after isolation from E. coli, transformed into B. megaterium.

It was within the state of the art to subclone the cryD gene into a E. coli - cyanobacteria shuttle plasmid. Typically such plasmids are engineered such that inserted genes are expressed under the control of a cyanobacteria promoter. If the cloned cryD gene were expressed on a recombinant plasmid in cyanobacteria it would be expected that the cyanobacteria would be highly toxic to mosquito larvae.

5.11. PRODUCTS AND FORMULATIONS INCORPORATING THE CRYD PROTEIN

The cryD delta-endotoxin is a potent insecticidal compound with activity against dipteran insects. It is, therefore, within the scope of the invention that the cryD protein toxin be utilized as an insecticide (the active ingredient) alone, preferably in homogenous or pure form and having the amino acid sequence of Figure 2, or as included within or in association with a transformed microorganism which expresses a cloned cryD gene or in a mixture of B. thuringiensis or other transformed sporulating microorganisms containing cryD in spores or otherwise. The compositions of the invention containing cryD toxin are applied at an insecticidally effective amount which will vary depending on such factors as, for example, the specific dipteran insects to be controlled, and the method of applying the insecticidally active compositions. The preferred insecticide formulations are made by mixing the toxin alone or incorporated in or associated with a transformed organism, with the desired carrier. The formulations may be administered as a dust or as a suspension in oil (vegetable or mineral) or water, a wettable powder or in any other material suitable for application, using the appropriate carrier adjuvants. Suitable carriers can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g., natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders, fertilizers or any other appropriate medium.

The formulations containing a solid or liquid adjuvant, are prepared in known manner, e.g., by homogenously mixing and/or grinding the active ingredients with extenders, e.g., solvents, solid carriers, and in some cases surface active compounds (surfactants).

Suitable liquid carriers are vegetable oils, such as coconut oil or soybean oil, mineral oils or water. The solid carriers used, e.g., for dusts and dispersible powders, are normally natural mineral fibers such as calcite, talcum, kaolin, or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated absorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite. Suitable nonsorbent carriers are materials such as silicate or sand. In addition, a great number of pregranulated materials or inorganic or organic mixtures can be used, e.g., especially dolomite or pulverized plant residues.

Depending on the nature of the active ingredients to be formulated, suitable surface-active compounds are non-ionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "surfactants" will also be understood as comprising mixtures or surfactants.

Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface active compounds.

Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted ammonium salts of higher fatty acids (C₁₀-C₁₁), e.g., the sodium or potassium salts of oleic or stearic acid, or natural fatty acid mixtures which can be obtained, e.g., from coconut oil or tallow oil. Further stable surfactants are also the fatty acid methyltaurin salts as well as modified and unmodified phospholipids.

More frequently, however, so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates.

The fatty sulfonates or sulfates are usually in the forms of alkali metal salts, alkaline earth metal salts or unsubstituted ammonium salts and generally contain a C₈-C₂₂ alkyl, e.g., the sodium or calcium salt of dodecylsulfate, or of a mixture of fatty alcohol sulfates, obtained from fatty acids. These compounds also comprise the salts of sulfonic acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing about 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde condensation product. Also suitable are corresponding phosphates, e.g., salts of the phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of ethylene oxide.

Nonionic surfactants are preferably a polyglycol ether derivative or aliphatic or cycloaliphatic alcohol or saturated or unsaturated fatty, acids and alkylphenols, said derivative containing 3 to 10 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.

Other suitable non-ionic surfactants are the water soluble adducts of polyethylene oxide with alkylpropylene glycol, ethylenediaminopolypropylene glycol and alkylpolypropylene glycol contain 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups.

Representative examples of non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil, glycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, ethylene glycol and octylphenoxypolyethoxynethanol. Fatty acid esters of polyoxyethylene'sorbitan, such as polyoxyethylene sorbitan trioleate, are also suitable non-ionic surfactants.

Cationic surfactants are preferably quaternary ammonium salts which contain, as substituents on the nitrogen, at least one C₈-C₂₂ alkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl benzyl, or hydroxylated lower aikyl radicals. The salts are preferably in the form of halides, methyl sulfates or ethylsulfates, e.g., stearyltrimethylammonium chloride. The present compositions have been shown to be highly toxic to dipteran larvae, in particular, to mosquito larvae. 6. EXAMPLES

6.1. TRANSFORMATION OF THE CRYD GENE INTO BACILLUS MEGATERIUM

As discussed above, the cloned 11-kb Hindlll fragment contained the gene for the 28-kDa crystal protein of var. israelensis as well as the cryD gene. In order to assess the toxicity of the cryD gene it was necessary to construct a recombinant plasmid containing only the cryD gene. Accordingly, a 6.8-kb Hindll-BamHI fragment, containing the complete cryD gene and only 33 NH2-terminal codons of the 28-kDa gene, was subcloned from pEG216 into the Hindlll-BamHI sites of pBR322 yielding plasmid pEG218 (Fig. 1). Plasmid pEG218 encoded only a small part of the 28-kDa protein (33 out of 280 amino acids) and therefore would not synthesize the 28-kDa protein. Plasmids pEG214, pEG216 and pEG218 (Fig. 1) will replicate only in gramnegative strains such as E. coli. Experience has shown that cloned B. thuringiensis toxin genes are expressed at high levels in Bacillus megaterium and at low levels in E. coli. The purpose of this example was to determine whether the cloned cryD gene would be expressed at a high level in Bacillus megaterium. In order to test for the expression of the cloned cryD gene in B. megaterium it was first necessary to construct a recombinant plasmid that contained the cryD gene and that was capable of replicating in Bacillus. Bacillus-E. coli "shuttle vectors" that contained the cryD gene were constructed. The term "shuttle vector" indicates that the plasmid is capable of replication both in Bacillus and in E. coli. The Bacillus-E. coli shuttle vectors were constructed by digestion of the Bacillus plasmid pNN101 (tetracycline and chloramphenicol resistance) with Sphl, ligation of the digested plasmid into the Sphl sites of the ampicillin resistance plasmids pEG214, pEG216 and pEG218 and transformation of E. coli to ampicillin (amp), tetracycline (tet) and chloramphenicol (cam) resistance. Three amp, tet and cam resistant E. coli transformants, designated EG1324, EG1316 and EG1323, harboring plasmids pEG215, pEG217 and pEG219, respectively, were isolated. Figure 1 shows the restriction maps of plasmids pEG215, pEG217 and pEG219. The arrows in Fig. 1 denote the coding regions of the cryD gene and the 28-kDa crystal protein gene. Plasmids were isolated from EG1324(pEG215), EG1316 (pEG217) and EG1323 (pEG219) and were transformed into the Bacillus megaterium strain VT1660 (ATCC No. 35985). The resulting cam , tet^r B. megaterium transformants were designated EG1321(pEG215), EG1322 (pEG217) and EG1319 (pEG219). A negative control plasmid (pEG220:cryD , 28-kDa ) was constructed by ligating Sphldigested pNNlOl into the Sphl site of pBR322. pEG220 was transformed into B. megaterium strain VT1660 yielding the negative control strain EG1325(cryD-, 28-kDa-).

This example determined if the clone cryD and 28-kDa crystal protein genes were expressed in the recombinant B. megaterium strains EG1322 (pEG217:cryD , 28-kDa ) and EG1319 (pEG219:cryD , 28-kDa^"). Gene expression was measured by the technique of NadodeSO₄/polyacrylamide gel electrophoresis. The gel technique involved preparation of cell lysates, electrophoresis of cell lysates through a NadodeSO /polyacrylamide gel and staining of the gel to permit visualization of proteins.

Specifically, the gel technique was carried out as follows: recombinant B. megaterium strains were grown on DS plates containing 10 ug/ml tetracycline for 48 h at 30°C. B. thuringiensis var. israelensis strain HD567 was grown similarly to B. megaterium except the DS plates contained no tetracycline. Cells were harvested with a spatula and suspended in deionized water. The cell suspension was incubated with an equal volume of cell lysis solution (2 mg/ml lysozyme, 50 mM Tris:HCl pH 7.4, 20 mM EDTA) for 20 min at 37°C. This mixture was incubated with 4 volumes of preheated gel loading buffer (5% 2-mercaptoethanol, 2% NaDodeSO₄, 60 mM Tris pH 6.8, 10% glycerol) for 7 min at 85ºC. The mixture was loaded onto an NadodeSO₄/polyacrylamide gel and the proteins were resolved by gel electrophoresis according to the method of Laemmli (J. Mol. Biol. (1973), 80:575-599). The proteins in the gel were visualized by staining the gel with Coomassie dye.

Figure 3 shows the results of this analysis. Figure 3 is a photograph of a NadodeS04/polyacrylamide gel that had been prepared as described above. The gel contained: lane 1, protein molecular weight standards; lane 2, B. thuringiensis var. israelensis HD567; lane 3, B. megaterium EG1325 (pEG220:cryD-, 28-kDa-); lane 4, B. megaterium EG1322 (pEG217:cryD⁺, 28-kDa⁺); lane 5, B. megaterium EG1319 (pEG219:cryD⁺, 28-kDa-). Numbers to the left indicate the 130-kDa, 67-kDa and 28-kDa var. israelensis proteins. Strains EG1322 (cryD ⁺, 28kDa⁺) (lane 4) and

EG1319(cryD⁺, 28-kDa-) (lane 5) synthesized a major protein of approximately 67 kDa corresponding to the 67-kDa crystal protein of var. israelensis (lane 2). The 67-kDa protein was not detected in the negative control strain EG1325(cryD-, 28-kDa-) (lane 3). Strains EG1325(cryD-, 28-kDa-) (lane 3), and EG1319 (cryD⁺, 28-kDa-) (lane 5) synthesized a minor protein whose size (28-kDa) was, coincidentally, approximately that of the var. israelensis 28-kDa crystal protein. Strain EG1322 (cryD⁺, 28-kDa⁺) (lane 4) synthesized an additional 28-kDa protein corresponding to the 28-kDa var. israelensis crystal protein. This demonstrated that B. megaterium harboring the cloned cryD gene synthesized high levels of the 67-kDa var. israelensis protein. For an unknown reason, the level of expression of the 28-kDa var. israelensis gene was low in B. megaterium. 6.2. BIOASSAY OF THE EXPRESSION PRODUCT OF THE CLONED CRYD GENE IN B. MEGATERIUM

This example determined if B. megaterium cells harboring the cloned cryD gene were toxic to mosquito larvae. The insecticidal activity of transformed or nontransformed Bacillus megaterium was determined by including various amounts of these microorganisms in a test diet which was fed to mosquito larvae. After feeding, insect mortality was measured. Specifically, this involved growing the Bacillus strains to stationary phase on solid DS agar base medium (0.8% Difco nutrient broth, 13 mM KCl, 1 mM Ca(NO₃)₂, 0.5 mM MgSO₄, 10 μM MnCl₂, 10 μM FeSO₄, 1.5% agar, pH 7.0) for two days at 30°C. For B. megaterium strains the DS medium contained 10 ug/ml tetracycline. The microorganisms were harvested from the solid medium by scraping with a spatula. The wet weight of the harvested bacteria was determined and bacterial cells were resuspended to a known concentration in deionized water. Serial dilutions of the suspended bacterial cells were made and 100 μl of each dilution was added to 100 ml of deionized water in a plastic cup. Twenty third or fourth instar Aedes aegypti larvae were added to each cup. Mortality was scored after 24 h. The results (Table 1) showed that strain EG1319 (cryD , 28-kDa ) was highly toxic to A. aegypti larvae in contrast to the control strain EG1325(cryD-, 28-kDa-) which was not toxic. This demonstrates that the 67-kDa protein encoded by the cloned cryD gene is highly toxic to mosquito larvae. Strain EG1322 (cryD⁺, 28-kDa⁺) was no more toxic than EG1319(cryD , 28-kDa-). This demonstrates that the 28-kDa protein is not necessary for the mosquitocidal activity of the 67-kDa protein. Strain EG1321 contains a truncated form of the cryD gene as well as the complete gene for the 28 kDa var. israelensis protein on a multicopy plasmid (pEG215, Fig. 1). The truncated cryD gene lacks 76 codons at the 3' terminus (described above). Strain EG1321 was not toxic to mosquito larvae. This demonstrates that the 3'-end of the cryD gene is necessary for toxicity.

TABLE 1. Mosquitocidal activity of Bacillus megaterium cells harboring the cloned cryD gene.

Strain Dose A. aegypti

(μg cells/ml) # dead/total

EG1325(cryD-, 28-kDa ) 50 0/40 EG1316(cryD⁺, 28-kDa⁺) 50.0 19/20

25.0 20/20

12.5 19/20

6.0 15/20

3.0 13/20

1.5 9/20

0.8 3/20

0.4 0/20

0.2 0/20

EG1319(cryD⁺, 28-kDa ) 50.0 19/20

25.0 19/20

12.5 17/20

6.0 12/20

3.0 10/20

1.5 3/20

0.8 1/20

0.4 0/20

0.2 0/20

EG1321(cryD^a, 28-kDa⁺) 20.0 1/20

The cryD gene in EG1321 is missing 76 codons at the COOH-terminus.

TABLE 2. Description of plasmids.

Plasmid Toxin Genes Replication Cloned Fragment

pEG220 none E. coli/Bacillus none pEG214 cryD^a, 28-kDa E. coli 5.7-kb EcoRI pEG215 cryD^a, 28-kDa E. coli/Bacillus 5.7-kb EcoRI pEG216 cryD, 28-kDa E. coli 11.0-kb Hindlll PEG217 cryD, 28-kDa E. coli/Bacillus 11.0-kb Hindlll pEG218 cryD E. coli 6.7-kb Bam-Hindlll PEG219 cryD E. coli/Bacillus 6.7-kb Bam-Hindlll

The cloned cryD gene in pEG214 and pEG 215 is missing 76 COOH-terminal codons.

TABLE 3. Description of strains.

Strain Genotype Plasmids

B. thuringiensis HD567 130-kDa⁺ cryD⁺ 28-kDa⁺ Several native

E. coli

EG1318 cryD^a, 28-kDa⁺ PEG214

EG1324 cryD^a, 28-kDa⁺ PEG215

EG1315 cryD , 28-kDa⁺ pEG216

EG1316 cryD , 28-kDa⁺ pEG217

EG1320 cryD , 28-kDa- pEG218

EG1323 cryD , 28-kDa- pEG219

B. megaterium

EG1325 cryD , 28-kDa- pEG220

EG1321 cryD^a, 28-kDa⁺ pEG215

EG1322 cryD , 28-kDa⁺ pEG217

EG1319 cryD , 28-kDa- pEG219

The clone cryD gene in EG1318 and EG1321 is missing 76 COOH-terminal codons.

DEPOSIT OF MICROORGANISMS Biologically pure cultures of the following microorganisms have been deposited with the Agricultural Research Culture Collection, Northern Regional Research Center, under the corresponding accession numbers: Bacillus thuringiensis var. israelensis HD567 NRRL B-18304 Escherichia coli EG1315, with plasmid pEG216 NRRL B-18305

Bacillus megaterium EG1322 with plasmid pEG217 NRRL B-18306

Bacillus megaterium EG1319 with plasmid pEG219 NRRL B-18327

The present invention is not to be limited in scope by the deposited strains, since these are intended as an illustration of one aspect of the invention and any cell lines which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Form 34 Continued

Name of Depository: Agricultural Research Culture Collection

Address of Depository: 1815 North University Street Peoria, Illinois U.S. A 61604

Date of Deposit Accession Number February 5, 1988 B-18304 February 5, 1988 B-18305 February 5, 1988 B-18306

Claims

WHAT IS CLAIMED IS:

1. A gene for Bacillus thuringiensis cryD toxin having the DNA sequence of FIGURE 2 or any portions or derivatives thereof.

2. The gene of claim 1 wherein said gene codes for a protein having the amino acid sequence of FIGURE 2.

3. The gene of claim 2 wherein said protein has insecticidal activity.

4. The gene of claim 3 wherein said insecticidal activity is effective against insects of the order Diptera.

5. The gene of claim 1 wherein said DNA sequence is inserted into a recombinant plasmid.

6. The gene of claim 5 wherein said plasmid is comprised of DNA from at least two different species of microorganisms after insertion of said DNA sequence.

7. The gene of claim 5 wherein said plasmid is comprised of DNA from at least two different subspecies of the same species of microorganism after insertion of said DNA sequence.

8. The gene of claim 1 wherein said DNA sequence is attached to its native promoter DNA sequence.

9. The gene of claim 1 wherein said DNA sequence is attached to a foreign promoter DNA sequence.

10. A protein having the amino acid sequence of FIGURE 2 or any portions or derivatives thereof.

11. The protein of claim 10 wherein said protein has insecticidal activity.

12. The protein of claim 11 wherein said insecticidal activity is effective against insects of the order Diptera.

13. The protein of claim 10.wherein said protein is produced by the process comprising: a) transforming a microorganism with the gene of FIGURE 2; b) growing said transformed microorganism whereby the protein encoded by said gene of step a) is expressed in said microorganism; and c) extracting and separating said protein expressed in step b) from said organism.

14. The protein of claim 13 wherein said gene of step a) is located on a plasmid.

15. The protein of claim 14 wherein said plasmid iscomprised of DNA from at least two different species of microorganisms when including said gene.

16. The protein of claim 14 wherein said plasmid is comprised of DNA from at least two different subspecies of microorganism when including said gene.

17. The protein of claim 13 wherein said protein is expressed in a non-sporulating microorganism.

18. The protein of claim 15 and 16 wherein the gene is controlled by its native promoter.

19. The protein of claim 15 and 16 wherein the gene is controlled by a foreign promoter.

20. The protein of claim 19 wherein said protein is expressed in a sporulating microorganism.

21. The protein of claim 18 wherein the protein is expressed during non-sporulating growth phases of said microorganism.

22. The protein of claim 13 wherein said protein is extracted in step c) by lysis of said microorganism.

23. The protein of claim 10 wherein said protein is in substantially pure form.

24. A method for producing of Bacillus thuringiensis cryD toxin comprising: a) inserting into a plasmid a gene for said cryD toxin having the DNA sequence of Figure 2; b) transforming a microorganism with the plasmid of step a); and c) growing the transformed microorganisms of step b) whereby said cryD toxin is expressed in said microorganisms.

25. The method of claim 24 wherein said gene codes for a protein having the amino acid sequence of FIGURE 2.

26. The method of claim 24 wherein said plasmid is comprised of DNA of at least two different species of microorganism after insertion of said cryD gene.

27. The method of claim 24 wherein said plasmid is comprised of DNA from at least two different subspecies of the same species of microorganism after insertion of said cryD gene.

28. The method of claim 24 wherein said DNA sequence is attached to its native promoter DNA sequence.

29. The method of claim 24 wherein said DNA sequence is attached to a foreign promoter DNA sequence.

30. The method of claim 24 wherein said microorganism is a non-sporulating microorganism.

31. The method of claim 24 wherein said microorganism is a sporulating microorganism.

32. The method of claim 30 wherein the cryD toxin is expressed during non-sporulating growth phases of said microorganism.

33. The method of claim 24 wherein said cryD toxin is extracted from the microorganism by lysis of said microorganism.

34. An insecticide suitable for use against Diptera comprising a mixture of a Bacillus thuringiensis cryD toxin and a suitable carrier.

35. The insecticide of claim 34 wherein the cryD toxin is associated with Bacillus thuringiensis spores.

36. The insecticide of claim 34 wherein the cryD toxin is a homogeneous protein preparation.

37. The insecticide of claim 34 wherein the cryD toxin is contained in a mixture of Bacillus thuringiensis spores and cultured Bacillus thuringiensis organisms.

38. The insecticide of claim 34 wherein the cryD toxin is associated with a non-sporulating microorganism.

39. The insecticide of claim 34 wherein the cryD toxin is associated with a sporulating microorgamism.

40. The insecticide of claim 34 wherein the carrier is a liquid carrier.

41. The insecticide of claim 40 wherein the liquid carrier contains one or more surfactants.

42. The insecticide of claim 34 wherein the carrier is a solid carrier.

43. The insecticide of claim 43 wherein the solid carrier is selected from the group consisting of calcite, talcum, kaolin, attapulgite, silicate, sand, dolomite, and pulverized plant residue.

44. The insecticide of claim 43 wherein the solid carrier is a granulated adsorptive carrier.

45. The insecticide of claim 45 wherein the granulated adsorptive carrier is selected from the group consisting of pumice, broken brick, sepiolite, and bentonite.

46. A recombinant vector containing the DNA sequence of claim 1.

47. A non-sporulating microorganism containing the DNA sequence of claim 1.

48. The non-sporulating microorganism of claim 47 wherein said microorganism is E. coli.

49. An Escherichia coli bacterium assigned Accession No. NRRL B-18305, or a mutant, recombinant or genetically engineered derivative thereof.

50. A sporulating microorganism containing the DNA sequence of claim 1.

51. The sporulating microorganism of claim 50 wherein said microorganism is Bacillus megaterium.

52. A microorganism containing the DNA sequence of claim 1 selected from the group consisting of Bacillus, Escherichia and Cyanobacteria.

53. A Bacillus megaterium bacterium assigned Accession No. NRRL B-18306, or a mutant, recombinant, or genetically engineered derivative thereof.

54. A Bacillus megaterium bacterium assigned Accession No. NRRL B-18327, or a mutant, recombinant, or genetically engineered derivative thereof.

55. An oligonucleotide probe for the gene coding for cryD delta-endotoxin comprising the sequence:

5'ATT GTA AAT GAA ACA GAT TTT CCA TTA TAT AAT AAT TAT ACA GAA CC-3' or derivative thereof.

56. The oligonucleotide probe of claim 55 wherein said probe is labeled.

57. The oligonucleotide probe of claim 56 wherein said probe is labeled with a radioactive label.

58. The DNA sequence of FIGURE 2 wherein said DNA or a portion or derivative thereof is labeled.

59. The DNA sequence of Claim 58 wherein said DNA or portion or derivative thereof is labeled with a radioactive label.

60. A protein having the amino acid sequence of a fragment of cryD as in FIGURE 2.