AU649785B2

AU649785B2 - Bacillus thuringiensis cryIIIC(b) toxin gene and protein toxic to coleopteran insects

Info

Publication number: AU649785B2
Application number: AU11926/92A
Authority: AU
Inventors: William P. Donovan; Mark J Rupar; Annette C. Slaney
Original assignee: Ecogen Inc
Current assignee: Monsanto Technology LLC
Priority date: 1991-01-31
Filing date: 1992-01-03
Publication date: 1994-06-02
Anticipated expiration: 2012-01-03
Also published as: JPH06502077A; WO1992013954A1; EP0569438A1; AU1192692A; JP2531913B2; CA2101338A1

Description

OPI DATE n7/09/2 AOJP DATE 15/10/92 APPLN. ID 11926 92 PCT NUMBER PCT/IISq2/nnI4 INTERNAMiin V TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 92/13954 C12N 15/32, A01N 63/02 C12Q 1/68, C12N 1/21 Al C07K 13/00 (C12N 1/21 (43) International Publication Date: 20 August 1992 (20.08.92) C12R 1:07, 1:19) (21) International Application Number: PCT/US92/00040 (74) Agents: NADEL, Alan, Panitch Schwarze Jacobs Nadel, 1601 Market Street 36th Floor, Philadelphia, (22) International Filing Date: 3 January 1992 (03.01.92) PA 19103 (US) et al.

Priority data: (81) Designated States: AT (European patent), AU, BE (Euro- 649,562 31 January 1991 (31.01.91) US pean patent), BR, CA, CH (European patent), CS, DE 813,592 23 December 1991 (23.12.91) US (European patent), DK (European patent), ES (European patent), FI, FR (European patent), GB (European patent), GR (European patent), HU, IT (European pa- (71) Applicant: ECOGEN INC. [US/US]; 2005 Cabot Boule- tent), JP, KR, LU (European patent), MC (European pavard West, Langhorne, PA 19047-1810 tent), NL (European patent), NO, PL, RU, SE (European patent).

(72) Inventors: DONOVAN, William, P. 36 Calicobush Road, Levittown, PA 19057 RUPAR, Mark, J. 42 Sturbridge Drive, Wilmington, DE 19810 SLANEY, Published Annette, C. 4 Dunmoor Court South, Hamilton Square, With international search report.

NJ 08690 (US).

(54)Title: BACILLUS THURINGIENSIS CRYIIIC(b) TOXIN GENE AND PROTEIN TOXIC TO COLEOPTERAN IN-

SECTS

(57) Abstract A Bacillus thuringiensis strain isolate, designated EG5144, exhibits insecticidal activity against coleopteran insects, including Colorado potato beetle and insects of the genus Diabrotica. A novel toxin gene in B.t. strain EG5144 produces an irregularly shaped insecticidal crystal protein of approximately 70 kDa that is toxic to coleopteran insects. The cryIII-type gene (SEQ ID NO:1), designated as the cryIIC(b) gene, has a nucleotide base sequence illustrated in Figure 1.

WO 92/13954 PCT/US92/00040 BACILLUS THURINGIENSIS cryIIIC(b) TOXIN GENE AND PROTEIN TOXIC TO COLEOPTERAN INSECTS Field of the Invention The present invention relates to an isolated Bacillus thuringiensis strain, to its novel toxin encoding gene and to the insecticidal crystal protein toxin made by the gene, as well as to insecticidal compositions containing the protein that are toxic to coleopteran insects.

Background of the Invention Bacillus thuringiensis (hereinafter is a gram-positive soil bacterium that produces crystal proteins during sporulation which are specifically toxic to certain orders and species of insects. Many different strains of B.t. have been shown to produce insecticidal crystal proteins. Compositions including B.t. strains which produce insecticidal proteins have been commercially available and used as environmentally acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other nontargeted organisms.

WO 92/13954 PCr/US92/00040 2 A number of genes encoding crystal proteins have been cloned from several strains of B.t. A review of such genes is set forth in H. Hofte et al., Microbiol. Rev., 53, pp.242-255 (1989). This reference provides a good overview of the genes and proteins obtained from B.t. and their uses, adopts a nomenclature and classification scheme for B.t. genes and proteins, and has an extensive bibliography.

The B.t. crystal protein is toxic in the insect only after ingestion. After ingestion, the alkaline pH and proteolytic enzymes in the insect mid-gut solubilize the crystal allowing the release of the toxic components.

These toxic components disrupt the mid-gut cells causing the insect to cease feeding and, eventually, to die. In fact, B.t. has proven to be an effective and environmentally safe insecticide in dealing with various insect pests.

As noted by Hofte et al., the majority of insecticidal B.t. strains are active against insects of the order Lepidoptera, caterpillar insects. Other B.t. strains are insecticidally active against insects of the order Diptera, flies and mosquitoes, or against both lepidopteran and dipteran insects. In recent years, a few B.t. strains have been reported as producing crystal protein that is toxic to insects of the order Coleoptera, beetles.

WO 92/13954 PCT/US92/00040 3 The first isolation of a coleopteran-toxic B.t.

strain is reported by A. Krieg et al., in Z.angew.Ent., 96, pp.500-508 (1983); see also A. Krieg et al., Anz.Schaedlingskde., Pflanzenschutz, Umweltschutz, 57, pp.145-150 (1984) and U.S. Patent 4,766,203, issued August 23, 1988 of A. Krieg et al. The strain, designated B.t.

var. tenebrionis, is reported to be toxic to larvae of the coleopteran insects Agelastica alni (blue alder leaf beetle) and Leptinotarsa decemlineata (Colorado potato beetle). B.t. tenebrionis makes an insecticidal crystal protein reported to be about 65-70 kilodaltons (kDa) (U.S.

Patent 4,766,203; see also K. Bernhard, FEMS Microbiol.Lett., 33, pp.261-265 (1986)).

V. Sekar et al., Proc.Natl.Acad.Sci.USA, 84, pp.7036- 7040 (1987), report the cloning and characterization of the gene for the coleopteran-toxic crystal protein of B.t.

tenebrionis. The size of the protein, as deduced from the sequence of the gene, was 73 kDa, but the isolated protein contained primarily a 65 kDa component. Hbfte et al., Nucleic Acids Res., 15, p.7183 (1987), also report the DNA sequence for the cloned gene from B.t. tenebrionis, and the sequence of the gene is identical to that reported by Sekar et al. (1987).

McPherson et al., Bio/Technology, 6, pp.61-66 (1988), disclose the DNA sequence for the cloned insect control gene from B.t. tenebrionis, and the sequence is identical to that reported by Sekar et al. (1987). E.coli cells and WO 92/13954 PCT/US92/00040 4 Pseudomonas fluorescens cells harboring the cloned gene were found to be toxic to Colorado potato beetle larvae.

PCT International Publication No. WO 91/07481 dated May 30, 1991, of Novo Nordisk A/S, describes B.t. mutants that produce high yields of the same insecticidal proteins originally made by the parent strains at lesser yields.

Mutants of the coleopteran-toxic B.t. tenebrionis strain are disclosed.

A coleopteran-toxic strain, designated B.t. var. san diego, is reported by C. Herrnstadt et al., Bio/Technology, 4, pp.305-308 (1986), to produce a 64 kDa crystal protein that was toxic to various coleopteran insects: strong toxicity to Pyrrhalta luteola (elm leaf beetle),; moderate toxicity to Anthonomus grandis (boll weevil), Leptinotarsa decemlineata (Colorado potato beetle), Otiorhynchus sulcatus (black vine weevil), Tenebrio molitor (yellow mealworm) and Haltica tombacina; and weak toxicity to Diabrotica undecimpunctata undecimpunctata (western spotted cucumber beetle).

The DNA sequence of the cloned coleopteran toxin gene of B.t. san diego is reported in C. Herrnstadt et al., Gene, 57, pp.37-46 (1987); see also U.S. Patent 4,771,131, issued September 13, 1988, of Herrnstadt et al. The sequence of the toxin gene of B.t. san diego is identical to that reported by Sekar et al. (1987) for the cloned coleopteran toxin gene of B.t. tenebrionis.

WO 92/13954 PCT/US92/00040 5 A. Krieg et al., J.Appl.Ent., 104, pp.417-424 (1987), report that the strain B.t. san diego is identical to the B.t. tenebrionis strain, based on various diagnostic tests.

Another new B.t. strain, designated EG2158, is reported by W.P. Donovan et al., in Mol.Gen.Genet., 214, pp.365-372 (1988) and in U.S. Patent No. 5,024,837 issued June 18, 1991, to produce a 73 kDa crystal protein that is insecticidal to coleopteran insects. The toxin-encoding gene from B.t. strain EG2158 was cloned and sequenced, and its sequence is identical to that reported by Sekar et al.

(1987) for the cloned B.t. tenebrionis coleopteran toxin gene. This coleopteran toxin gene is referred to-as the cryIITA gene by H6fte et al., Microbiol.Rev., 53, pp.242- 255 (1989).

The Donovan et al. '837 U.S. patent noted above also describes hybrid B.t. var. kurstaki strains designated EG2424 and EG2421, which are active against both lepidopteran insects and coleopteran insects. The beetle activity of these hybrid strains results from the coleopteran toxin plasmid transferred from B.t. strain EG2158 by conjugal plasmid transfer.

U.S. Patent 4,797,279, issued January 10, 1989, of D.

Karamata et al. (corresponding to EP-A-0 221 024), discloses a hybrid B.t. microorganism containing a plasmid from B.t. var. kurstaki with a lepidopteran toxin gene and a plasmid from B.t. tenebrionis with a coleopteran toxin WO 92/13954 PCT/US92/00040 6 gene. The hybrid B.t. produces crystal proteins characteristic of those made by B.t. kurstaki, as well as those of B.t. tenebrionis.

U.S. Patent No. 4,910,016, issued March 20, 1990, of Gaertner et al. (corresponding to EP-A-0 303 379), discloses a novel B.t. isolate identified as B.t. MT 104 which has insecticidal activity against two orders of insects, Colorado potato beetle (Coleoptera) and cabbage looper (Lepidoptera).

European Patent Application Publication No. 0 318 143, published May 31, 1989, of Lubrizol Genetics, Inc., discloses the cloning, characterization and selective expression of the intact partially modified gene from B.t.

tenebrionis, and the transfer of the cloned gene into a host microorganism rendering the microorganism able to produce a protein having toxicity to coleopteran insects.

Insect bioassay data for B.t. san diego reproduced from Herrnstadt et al., Bio/Technology, 4, pp.305-308 (1986) discussed above, is summarized. The summary also includes data for B.t. tenebrionis from another source; B.t.

tenebrionis is reported to exhibit strong toxicity to Colorado potato beetle, moderate toxicity to western corn rootworm (Diabrotica virgifera) and weak toxicity to southern corn rootworm (Diabrotica undecimpunctata).

European Patent Application Publication No. 0 324 254, published July 19, 1989, of Imperial Chemical Industries PLC, discloses a novel B.t. strain identified WO 92/13954 PCT/US92/00040 7 as A30 which has insecticidal activity against coleopteran insects, including Colorado potato beetle larvae, corn rootworm larvae and boll weevils.

U.S. Patent No. 4,999,192, issued March 12, 1991, of Payne et al. (corresponding to EP A-0 328 383), discloses a novel B.t. microorganism identified as B.t. PS40D1 which has insecticidal activity against Colorado potato beetle larvae. B.t. strain PS40D1 is identified via serotyping as being serovar 8a8b, morrisoni.

U.S. Patent No. 5,006,336, issued April 9, 1991, of Payne et al. (corresponding to EP-A-0 346 114), discloses a novel B.t. isolate designated as PS122D3, which is serotyped as serovar 8a8b, morrisoni and which exhibits insecticidal activity against Colorado potato beetle larvae.

U.S. Patent No. 4,966,765, issued October 30, 1990, of Payne et al. (corresponding to EP-A-0 330 342), discloses a novel B.t. microorganism identified as B.t.

PS86B1 which has insecticidal activity against the Colorado potato beetle. B.t. strain PS86B1 is identified via serotyping as being serovar tolworthi.

The nucleotide sequence of a cryIIIB gene and its encoded coleopteran-toxic protein is reported by Sick et al., in Nucleic Acids Res., 18, p.1305 (1990) but the B.t.

source strain is identified only via serotyping as being subspecies tolworthi. U.S. Patent No. 4,966,155, issued February 26, 1991, of Sick et al. (corresponding to EP-A-0 WO 92/13954 PCT/US92/00040 8 337 604), discloses a B.t. toxin gene obtained from the coleopteran-active B.t. strain 43F, and the gene sequence appears identical to the cryTIIB gene. B.t. strain 43F is reported as being active against Colorado potato beetle and Leptinotarsa texana.

European Patent Application No. 0 382 990, published August 22, 1990, of Plant Genetic Systems discloses two novel B.t. strains (btGSI208 and btGSI245) producing respective crystal proteins of 74 and 129 kDa that exhibit insecticidal activity against Colorado potato beetle larvae. The DNA sequence reported for toxin gene producing the 74 kDa protein appears to be identical to that of the cryIIIB gene of Sick et al.

PCT International Publication No. WO 90/13651, published November 15, 1990, of Imperial Chemical Industries PLC, discloses novel B.t. strains which contain a toxin gene encoding an 81 kDa protein that is stated to be toxic not only to lepidopteran insects but also to coleopteran insects, including Diabrotica.

U.S. Patent No. 5,055,293, issued October 8, 1991, of Aronson et al., discloses the use of B. laterosporous for corn rootworm (Diabrotica) insect control.

The various B.t. strains described in aforementioned literature are reported to have crystal proteins insecticidally active against coleopteran insects, but none has been demonstrated to have significant, quantifiable toxicity to the larvae and adults of the WO 92/13954 P~/US92/000b40 9 insect genus Diabrotica (corn rootworm), which includes the western corn rootworm (Diabrotica virgifera virgifera), the southern corn rootworm (Diabrotica undecimpunctata howardi) and the northern corn rootworm (Diabrotica barberi).

The B.t. strain of the present invention contains a novel toxin gene that expresses protein toxin having quantifiable insecticidal activity against the Diabrotica insects, among other coleopteran insects.

Summary of the Invention One aspect of the present invention relates to a purified and isolated coleopteran toxin gene having a nucleotide base sequence coding for the amino acid sequence illustrated in Figure 1 and hereinafter designated as the cryIIIC(b) gene (SEQ ID NO:1). The cryIIIC(b) gene (SEQ ID NO:1) has a coding region extending from nucleotide bases 144 to 2099 shown in Figure 1.

Another aspect of the present invention relates to the insecticidal protein produced by the cryIIIC(b) gene.

The CryIIIC(b) protein (SEQ ID NO:2) has the amino acid sequence, as deduced from the nucleotide sequence of the cryIIIC(b) gene (SEQ ID NO:1) from nucleotide bases 144 to 2099 that is shown in Figure 1. The protein exhibits insecticidal activity against insects of the order WO 92/13954 PC-r/US92/00040 10 Coleoptera, in particular, Colorado potato beetle and insects of the genus Diabrotica.

Still another aspect of the present ention relates to a biologically pure culture of a B.t. bacterium deposited with the Agricultural Research Culture Collection, Northern Regional Research Laboratory (NRRL) having Accession No. NRRL B-18655 and being designated as B.t. strain EG5144 and a biologically pure culture of a second bacterium deposited with the NRRL having Accession No. NRRL B-18920 and being designated as B.t. strain EG5145. B.t. strain EG5144 is a wild-type B.t. strain that carries the cryIIIC(b) gene (SEQ ID NO:1) and produces the insecticidal CryIIIC(b) protein (SEQ ID NO:2). B.t. strain EG5145 is also a wild-type B.t.

strain, whose characteristics are similar to those of B.t.

strain EG5144 described in more detail below.

Biologically pure cultures of other B.t. bacteria carrying the cryIIIC(b) gene (SEQ ID NO:1) are also within the scope of this invention.

Yet another aspect of this invention relates to insecticidal compositions containing, in combination with an agriculturally acceptable carrier, either the CryIIIC(b) protein (SEQ ID NO:2) or fermentation cultures of a B.t. strain which has produced the CryIIIC(b) protein.

The invention also includes a method of controlling coleopteran insects by applying to a host plant for such WO 92/13954 PCT/US92/00040 11 insects an insecticidally effective amount of the CryIIIC(b) protein (SEQ ID NO:2) or of a fermentation culture of a B.t. strain that has made the CryIIIC(b) protein. The method is applicable to a variety of coleopteran insects, such as the Colorado potato beetle, Japanese beetle larvae (white grubs), Mexican bepn beetle and corn r ootworm.

Still another aspect of the present invention relates to a recombinant plasmid containing the cryIIIC(b) gene (SEQ ID NO:1), a'biologically pure culture of a bacterium transformed with such recombinant plasmid, the bacterium preferably being such as B.t. strain EG7237 described in Example 6, as well as a plant transformed with the cryIIIC(b) gene.

Brief Description of the Drawings Figure 1 comprises Figures 1-1 through 1-3 and shows the nucleotide base sequence of the cryIIIC(b) gene (SEQ ID NO:1) and the deduced amino acid sequence of the CryIIIC(b) protein (SEQ ID NO:2). The putative ribosome binding site (RBS) is indicated. Restriction sites for SspI and HindIII are also indicated.

Figure 2 is a photograph of an ethidium bromide stained agarose gel containing size fractionated native plasmids of B.t. strains EG5144 (lane EG4961 (lane 2), EG2838 (lane 3) and EG2158 (lane The numbers to the WO 92/13954 PCT/US92/00040 12 left of Figure 2 indicate the approximate sizes, in megadaltons (MDa), of the plasmids of B.t. strain EG5144.

Figure 3 is a photograph of an autoradiogram made by transferring size fractionated DNA :fragments from an agarose gel to a nitrocellulose filter, hybridizing the filter with a radioactively labeled 2.4 kilobases (kb) cryIIIB probe, and exposing the filter to X-ray film. The agarose gel contained size fractionated total DNA fragments from B.t. strains EG2158, EG5144, EG2838 and EG4961, that had been obtained in separate digestions with the restriction enzymes SspI, HindIII and EcoRI. The numbers to the left of Figure 3 indicate the sizes, in kb, of B.t. strain EG5144 restriction fragments that hybridized to the cryllIB probe. The lane labeled "stnd" is a size standard.

Figure 4 is a photograph of a Coomassie stained sodium dodecyl sulfate polyacrylamide gel showing crystal proteins solubilized from B.t. strains EG5144 (lane EG4961 (lane EG2158 (lane 3) and EG2838 (lane The numbers to the left of Figure 4 indicate the approximate sizes in kDa of the crystal proteins produced by B.t. strain EG5144. Lane 5 contains protein molecular size standards.

Figure 5 shows a restriction map of plasmid pEG271.

The location and orientation of the cryIIIC(b) gene (SEQ ID NO:1) is indicated by the arrow. Plasmid pEG271 is functional in Escherichia coli (E.coli), since it contains WO 92/13954 PCT/US92/00040 13 E.coli plasmid pUC18 (Apr), indicated by the segment marked pUC18. The abbreviations for the restriction endonuclease cleavage sites are as follows: Ba=BamHI; Bg=BglII; H=HindIII; R=EcoRI; S=SphI; and X=XbaI. A one kilobase scale marker is also illustrated.

Figure 6, aligned with and based on the same scale as Figure 5, shows a restriction map of plasmid pEG272. The location and orientation of the cryIIIC(b) gene (SEQ ID NO:1) is indicated by the arrow shown in Figure Plasmid pEG272 is derived from plasmid pEG271 (Figure and contains the Bacillus plasmid pNN101 (Cmr Tcr), indicated by the segment marked pNNl01 and is incorporated into the SphI site of pEG271; this plasmid is functional in B.t. Abbreviations are the same as those for Figure Figure 7 is a photograph of a Coomassie stained SDSpolyacrylamide gel. The gel shows protein bands synthesized by B.t. strain EG5144 (lane 1) and by recombinant B.t. strain EG7237 containing pEG272 (lane 3).

Lane 2 contains a protein size standard and the numbers on either side of lanes 1 and 3 indicate approximate sizes, in kDa, of the crystal proteins produced by these strains.

Detailed Description of the Preferred Embodiments The isolation and purification of the cryIIIC(b) gene (SEQ ID NO:l) and the coleopteran-toxic CryIIIC(b) crystal prote:H. (SEQ ID NO:2) and the characterization of the new B.t. strain EG5144 which produces the CryIIIC(b) protein WO 92/13954 PCT/US92/00040 14 are described at length in Examples 1-7. The utility of B.t. strain EG5144 and of the CryIIIC(b) crystal protein (SEQ ID NO:2) in insecticidal compositions and methods is also illustrated in Examples 8-11.

The cryIII-type gene of this invention, the cryIIIC(b) gene (SEQ ID NO:1), has the nucleotide base sequence shown in Figure 1. The coding region of the cryIIIC(b) gene (SEQ ID NO:1) extends from nucleotide base position 144 to position 2099 shown in Figure 1.

A comparison of the nucleotide base sequence of the cryIIIC(b) gene coding region with the corresponding coding region of the prior art cryIIIA gene indicates significant differences between the two genes. The cryIIIC(b) gene (SEQ ID NO:1) is only 76% homologous (positionally identical) with the cryIIIA gene.

A comparison of the nucleotide base sequence of the crylIIC(b) gene coding region with the corresponding coding region of the crylIB gene obtained from recently discovered B.t. strain EG2838 (NRRL Accession No. B-18603) indicates that the cryIIIC(b) gene (SEQ ID NO:1) is 96% homologous (positionally identical) with the cryIIIB gene.

The CryIII-type protein of this invention, the CryIIIC(b) protein, that is encoded by the cryIIIC(b) gene (SEQ ID NO:1), has the amino acid sequence (SEQ ID NO:2) shown in Figure 1. In this disclosure, references to the CryIIIC(b) "protein" are synonymous with its description as a "crystal protein", "protein toxin", "insecticidal WO 92/13954 PCT/US92/00040 15 protein" or the like, unless the context indicates otherwise. The size of the CryIIIC(b) protein (SEQ ID NO:2), as deduced from the DNA sequence of the cryIIIC(b) gene (SEQ ID NO:1), is 74,265 Daltons (Da).

The size of the CryIIIB protein, as deduced from the sequence of the cryIIIB gene, is 74,237 Da. The prior art CryIIIA protein, encoded by the cryIIIA gene, has a deduced size of 73,116 Da.

Despite the apparent size similarity, comparison of the amino acid sequence of the CryIIIC(b) protein (SEQ ID NO:2) with that of the prior art CryIIIA protein shows significant differences between the two. The CryIIIC(b) protein (SEQ ID NO:2) is only 68% homologous (positionally identical amino acids) with the CryIIIA protein. The CryIIIC(b) protein (SEQ ID NO:2) is 95% homolgous with the CryIIIB protein. Nevertheless, despite the apparent homology of the CryIIIC(b) and CryIIIB proteins, the CryIIIC(b) protein (SEQ ID NO:2) has been shown to be a different protein than the CryIIIB protein, based on its significantly improved insecticidal activity compared to the CrylIIB protein with respect to insects of the order Coleoptera and in particular, insects of the genus Diabrotica. The CryIIIC(b) protein (SEQ ID NO:2), unlike the CryIIIB protein, exhibits quantifiable insecticidal activity against corn rootworm larvae.

The present invention is intended to cover mutants and recombinant or genetically engineered derivatives, WO 92/13954 PC/US92/00041 16 truncated versions, of the cryIIIC(b) gene (SEQ ID NO:1) that yield a protein with insecticidal properties essentially the same as those of the CryIIIC(b) protein (SEQ ID NO:2).

The cryIIIC(b) gene (SEQ ID NO:1) is also useful as a DNA hybridization probe, for discovering similar or closely related cryIII-type genes in other B.t. strains.

The cryIIIC(b) gene (SEQ ID NO:l), or portions or derivatives thereof, can be labeled for use as a hybridization probe, with a radioactive label, using conventional procedures. The labeled DNA hybridization probe may then be used in the manner described in the Examples.

The cryIIIC(b) gene (SEQ ID NO:1) and the corresponding insecticidal CryIIIC(b) protein (SEQ ID NO:2) were first identified in B.t. strain EG5144, a novel B.t. isolate. The characteristics of B.t. strain EG5144 are more fully described in the Examples. Comparison of the plasmid arrays and other strain characteristics of B.t. strain EG5144 with those of the recently discovered B.t. strains EG2838 and EG4961 and those of the prior art B.t. strain EG2158 and B.t. var. tenebrionis (or the equivalent, B.t. var. san diego) demonstrates that eac of these coleopteran-toxic B.t. strains is distinctly different. The plasmid array of B.t. strain EG5145, another wild-type strain isolated along with B.t. strain EG5144, is similar to that of B.t. strain EG5144, and B.t.

WO 92/13954 PCrIUS92/00040 17 strain EG5145 exhibits the same insecticidal activity against coleopteran insects, Japanese beetle larvae, as that of B.t. strain EG5144 (see Example 11).

The cryIIIC(b) gene (SEQ ID NO:1) may be introduced into a variety of microorganism hosts, using procedures well known to those skilled in the art for transforming suitable hosts under conditions which allow for stable maintenance and expression of the cloned cryIIIC(b) gene.

Suitable hosts that allow the cryIIIC(b) gene (SEQ ID NO:1) to be expressed and the CryIIIC(b) protein (SEQ ID NO:2) to be produced include Bacillus thuringiensis and other Bacillus species such as B. subtilis or B.

megaterium. It should be evident that genetically altered or engineered microorganisms containing the cryIIIC(b) gene (SEQ ID NO:1) can also contain other toxin genes present in the same microorganism and that these genes could concurrently produce insecticidal crystal proteins different from the CryIIIC(b) protein.

The Bacillus strains described in this disclosure may be cultured using conventional growth media and standard fermentation techniques. The B.t. strains harboring the cryIIIC(b) gene (SEQ ID NO:1) may be fermented, as described in the Examples, until the cultured B.t. cells reach the stage of thir growth cycle when CryIIIC(b) crystal protein (SEQ ID NO:2) is formed. For sporogenous B.t. strains, fe.mentation is typically continued through the sporulation stage when the CryIIIC(b) crystal protein WO 92/13954 PCT/US92/00040 18 is formed along with spores. The B.t. fermentation culture is then typically harvested by centrifugation, filtration or the like to separate fermentation culture solids, containing the CryIIIC(b) crystal protein, from the aqueous broth portion of the culture.

The B.t. strains exemplified in this disclosure are sporulating varieties (spore forming or sporogenous strains) but the cryIIIC(b) gene (SEQ ID NO:1) also has utility in asporogenous Bacillus strains, strains that produced the crystal protein without production of spores. It should be understood that references to "fermentation cultures" of B.t. strains (containing the cryIIIC(b) gene (SEQ ID NO:1)) in this disclosure are intended to cover sporulated B.t. cultures, B.t.

cultures containing the CryIIIC(b) crystal protein and spores, and sporogenous Bacillus strains that have produced crystal protein during the vegetative stage, as well as asporogenous Bacillus strains containing the cryIIIC(b) gene (SEQ ID NO:1) in which the culture has reached the growth stage where crystal protein is actually produced.

The separated fermentation solids are primarily CryIIIC(b) crystal protein (SEQ ID NO:2) and B.t. spores, along with some cell debris, some intact cells, and residual fermentation medium solids. If desired, the crystal protein may be separated from the other recovered solids via conventional methods, sucrose density WO 92/13954 PCT/US92/00040 19 gradient fractionation. Highly purified CryIIIC(b) protein (SEQ ID NO:2) may be obtained by solubilizing the recovered crystal protein and then precipitating the protein from solution.

The CryIIIC(b) protein (SEQ ID NO:2), as noted earlier, is a potent insecticidal compound against coleopteran insects, such as the Colorado potato beetle, Japanese beetle larvae (white grubs), Mexican bean beetle and the like. The CryIIIC(b) protein (SEQ ID NO:2), in contrast to the CryIIIA and CryIIIB proteins, exhibits measurable insecticidal activity against Diabrotica insects, corn rootworms, which have been relatively unaffected by other coleopteran-toxic B.t. crystal proteins. The CryIIIC(b) protein (SEQ ID NO:2) may be utilized as the active ingredient in insecticidal formulations useful for the control of coleopteran insects such as those mentioned above. Such insecticidal formulations or compositions typically contain agriculturally acceptable carriers or adjuvants in addition to the active ingredient and are prepared and used in a manner well known to those skilled in the art.

The CryIIIC(b) protein (SEQ ID NO:2) may be employed in insecticidal formulations in isolated or purified form, as the crystal protein itself.' Alternatively, the CryIIIC(b) protein (SEQ ID NO:2) may be present in the recovered fermentation solids, obtained from culturing of a Bacillus strain, Bacillus thuringiensis, or other WO92/13954 PCT/US92/00040 20 microorganism host carrying the cryIIIC(b) gene (SEQ ID NO:l) and capable of producing the CryIIIC(b) protein.

Preferred Bacillus hosts include B.t. strain EG5144 and genetically improved B.t. strains derived from B.t. strain EG5144. The latter B.t. strains may be obtained via plasmid curing and/or conjugation techniques and contain the native cryIIIC(b) gene-containing plasmid from B.t.

strain EG5144. Genetically engineered or transformed B.t.

strains or other host microorganisms containing a recombinant plasmid that expresses the cloned cryIIIC(b) gene (SEQ ID NO:l), obtained by recombinant DNA procedures, may also be used.

An example of such transformants is B.t. strain EG7237, which contains the cloned cryIIIC(b) gene (SEQ ID NO:1) on a recombinant plasmid.

The recovered fermentation solids contain primarily the crystal protein and (if a sporulating B.t. host is employed) spores; cell debris and residual fermentation medium solids may also be present. The recovered fermentation solids containing the CryIIIC(b) protein may be dried, if desired, prior to incorporation in the insecticidal formulation.

The formulations or compositions of this invention containing the insecticidal CryIIIC(b) protein (SEQ ID NO:2) as the active component are applied at an insecticidally effective amount which will vary depending on such factors as, for example, the specific coleopteran W6 92/1.3954 PCr/US92/00040 21 insects to be controlled, the specific plant or crop to be treated and the method of applying the insecticidally active compositions, An insecticidally effective amount of the insecticide formulation is employed in the insect control method of this invention.

The insecticide compositions are made by formulating the insecticidally active component with the desired agriculturally acceptable carrier. The formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral) or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term "agriculturally acceptable carrier" covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in insecticide formulation technology; these are well known to those skilled in insecticide formulation.

The formulations containing the CryIIIC(b) protein (SEQ ID NO:2) and one or more solid or liquid adjuvants are prepared in known manners, by homogeneously mixing, blending and/or grinding the insecticidally active CryIIIC(b) protein component with suitable adjuvants using conventional formulation techniques.

The insecticidal compositions of this invention are applied to the environment of the target coleopteran WO 92/13954 PCr/US92/0040 22 insect, typically onto the foliage of the plant or crop to be protected, by conventional methods, preferably by spraying. Other application techniques, dusting, sprinkling, soaking, soil injection, seed coating, seedling coating or spraying, or the like, are also feasible and may be required for insects that cause root or stalk infestation. These application procedures are well known in the art.

The cryIIIC(b) gene (SEQ ID 90:l) or its functional equivalent, hereinafter sometimes referred to as the "toxin gene," can be introduced into a wide variety of microorganism hosts. Expression of .the cryIIIC(b) gene (SEQ ID NO:1) results in the production of insecticidal CryIIIC(b) crystal protein toxin (SEQ ID NO:2). Suitable hosts include B.t. and other species of Bacillus, such as B. subtilis or B. megaterium, fr,r example. Plantcolonizing or root-colonizing microorganisms may also be employed as the host for the cryIIIC(b) gene (SEQ ID NO:1). Various procedures well known to those skilled in the art are available for introducing the cryIIIC(b) gene (SEQ ID NO:l) into the microorganism host under conditions which allow for stable maintenance and expression of the gene in the resulting transformants.

The transformants, host microorganisms that harbor a cloned gene in a recombinant plasmid, can be isolated in accordance with conventional methods, usually employing a selection technique, which allows growth of WO 92/113954 PCr!US92/00040 23 only those host microorganisms that contain a recombinant plasmid. The transformants then can be tested for insecticidal activity. Again, these techniques are standard procedures.

characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the gene into the host, availability of expression systems, efficiency of expression, stability of the CryIIIC(b) insecticidal protein in the host, and the presence of auxiliary genetic capabilities. The cellular host containing the insecticidal crylIIC(b) gene (SEQ ID NO:1) may be grown in any convenient nutrient medium, where expression of the cryIIIC(b) gene is obtained and CryIIIC(b) protein (SEQ ID NO:2) produced, typically to sporulation. The sporulated cells containing the crystal protein may then be harvested in accordance with conventional methods, centrifugation or filtration.

The cryIIIC(b) gene (SEQ ID NO:l) may also be incorporated into a plant which is capable of expressing the gene and producing CryIIIC(b) protein (SEQ ID NO:2), rendering the plant more resistant to insect attack.

Genetic engineering of plants with the crylIIC(b) gene (SEQ ID NO:1) may be accomplished by introducing the desired DNA containing the gene into plant tissues or cells, using DNA molecules of a variety of forms and origins that are well known to those skilled in plant genetic engineering. An example of a technique for WO 92/13954 PC/US92/00040 24 introducing DNA into plant tissue is disclosed in European Patent Application Publication No. 0 289 479, published November 2, 1988, of Monsanto Company.

DNA containing the cryIIIC(b) gene (SEQ ID NO:I) or a modified' cryIIIC(b) gene capable of producing the CryIIIC(b) protein (SEQ ID NO:2) may be delivered into the plant cells or tissues directly by infectious plasmids, such as Ti, the plasmid from Agrobacterium tumefaciens, viruses or microorganisms like A. tumefaciens, by the use of lysosomes or liposomes, by microinjection by mechanical methods and by other techniques familiar to those skilled in plant genetic engineering.

Variations may be made in the cryIIIC(b) gene nucleotide base sequence (SEQ ID NO:1), since the various amino acids forming the protein encoded by the gene usually may be determined by more than one codon, as is well known to those skilled in the art. Moreover, there may be some variations or truncation in the coding regions of the cryIIIC(b) nucleotide base sequence which allow expression of the gene and production of functionally equivalent forms of the CryIIIC(b) insecticidal protein.

These variations which can be determined without undue experimentation by those of ordinary skill in the art with reference to the present specification are to be considered within the scope of the appended claims, since they are fully equivalent to the specifically claimed subject matter.

W6 92/13954 PCT/US92/00040 25 The present invention will now be described in more detail with reference to the following specific, nonlimiting examples. The examples relate to work which was actually done based on techniques generally known in the art and using commercially available equipment.

The novel B.t. strain EG5144 was isolated following the procedure described in Example 1. The procedures described in Example 1 were also used to isolate the novel B.t. strain EG5145.

Example 1 Isolation of B.t. Strains EG5144 and EG5145 Crop dust samples were obtained from various sources throughout the U.S. and abroad, typically grain storage facilities. The crop dust samples were treated by suspending the crop dust in an aqueous buffer and heating the suspension at 60 0 C for 30 min. to enrich for heat resistant spore forming Bacillus-type bacteria such as B.t. The treated dust suspensions were diluted in aqueous buffer, and the dilutions were spread on agar plates to allow each individual bacterium from the crop dust to grow into a colony on the surface of the agar plate. After growth, a portion of each colony was transferred from the agar plate to a nitrocellulose filter. The filter was treated with NaOH to lyse the colonies and to fix the DNA from each colony onto the filter.

WO 92/13954 PCT/US92/00040 26 A modified treatment procedure was developed for use with B.t. colonies utilized in the colony hybridization procedure, since standard techniques applicable to E.coli were found to be unworkable with B.t. In the treatment described above, special conditions were required to assure that the B.t. colonies were in a vegetative state of growth, making them susceptible to lysis with NaOH.

Accordingly, after a portion of each colony was transferred to the nitrocellulose filter, the filter was placed colony side up on an agar medium containing glucose. The transferred colonies were then allowed to grow on the agar-glucose medium for 5 hours at 30 0

C.

Use of 0.5% glucose in the agar medium and the 0 C growth cycle were critical for assuring that the B.t.

colonies were in a vegetative state and thus susceptible to lysis.

A cloned coleopteran toxin gene was used as a specific probe to find other novel and rare coleopterantoxic strains of B.t. from crop dust samples. A 2.9 kb HindIII DNA restriction fragment containing the cryIIIA gene, formerly known as the cryC gene of B.t. strain EG2158, described in Donovan et al., Mol.Gen.Genet., 214, pp.365-372 (1988), was used as a probe in colony hybridization procedures.

The 2.9 kb HindIII crylllA DNA fragment, containing the entire cryIIIA gene, was radioactively labeled with [alpha-P 32 ]-dATP and Klenow enzyme, by standard methods.

WO 92/13954 PCT/US92/00040 27 The nitrocellulose filters containing the DNA from each lysed colony were incubated at 65 0 C for 16 hours in a buffered solution that contained the radioactively labeled 2.9 kb HindIII crylIIA DNA probe to hybridize the DNA from the colonies with the DNA from the radioactively labeled cryIIA probe. The 65 0 °C hybridization temperature was used to assure that the cryIIIA DNA probe would hybridize only to DNA from colonies that contained a gene that was similar to the cryIIIA DNA probe.

The 2.9 kb cryIIA probe hybridized to many B.t.

colonies from various samples of crop dust. Examination of these colonies revealed, unexpectedly, that they did not contain any cryIXI-type genes. These colonies did contain cryl-type genes. The cryl-type genes encode lepidopteran-toxic, coleopteran-nontoxic crystal proteins with molecular masses of approximately 130 kDa. Computerassisted comparisons of the sequence of the cryiIIA gene with the sequence of several cryl-type genes revealed that the 3'-end of the cryIIIA gene was partially homologous with portion of the cryl-type genes. This finding supported the belief that the 3'-end of the cryIIA gene was causing the 2.9 kb cryIIIA probe to hybridize to B.t.

colonies containing cryl-type genes.

To correct this problem, the 2.9 kb HindIII cryIIIA probe was digested with the enzyme XbaI and a 2.0 kb HindIII-XbaI fragment was purified that contained the cryIIIA gene minus its 3'-end. The 2.0 kb HindIII-XbaI WO 92/13954 PCT/US92/00040 28 fragment contains the 3'-truncated cryllA gene. When the kb fragment was used in repeated colony hybridization experiments, it did not hybridize to cryl gene-containing B.t. colonies.

Approximately 48,000 Bacillus-type colonies from crop dust samples from various locations were probed with the radioactively labeled 2.0 kb HindIII-XbaI cryIIIA probe.

Only one novel B.t. strain from an Illinois crop dust sample was discovered that specifically hybridized to the cryIIIA probe. That novel strain was designated B.t.

strain EG2838, which has been deposited with the NRRL under Accession No. NRRL B-18603.

Subsequently, approximately 50,000 additional Bacillus-type colonies from crop dust samples were also screened with the radioactively labeled 2.0 kb HindIII- XbaI cryIIIA probe, but without success in identifying any other strains containing novel cryllI-type genes.

B.t. strain EG2838 was found to be insecticidally active against coleopteran insects, notably, the Colorado potato beetle. B.t. strain EG2838 did not have substantial insecticidal activity with respect to the southern corn rootworm. A gene, designated the cryllIB gene, was isolated from B.t. strain EG2838, and its nucleotide base sequence determined. The crylXB gene encoded a crystal protein, designated the CryIIIB protein, containing 651 amino acids having a deduced size of 74,237 Daltons. The size of the prior art CryIIIA protein had WO' 92/13954 PCT/US92/00040 29 previously been deduced to be 73,116 Daltons (644 amino acids). The cryIIIB gene is 75% homologous with the cryIIIA gene, and the CryIIIB protein is 68% homologous with the CryIIIA protein.

Thousands of Bacillus-type col-nies from numerous crop dust samples from various locations from around the world were screened with a cryIIIB probe obtained from B.t. strain EG2838. The cryXIIB probe was radioactively labeled using the procedure set forth above with respect to the radioactively labeled cryIIIA probe. The radioactively labeled cryIIIB probe consisted of a 2.4 kb SspI restriction fragment of DNA from B.t. strain EG2838.

The fragment contains the complete protein coding region for the coleopteran toxin cryIIIB gene of B.t. strain EG2838. Ultimately, the B.t. strains of the present invention, designated B.t. strains EG5144 and EG5145, were isolated from a crop dust sample via B.t. colonies that specifically hybridized to the cryIIIB probe.

To characterize B.t. strain EG5144, several studies were conducted. One series of studies was performed to characterize its flagellar serotype. Additional studies were conducted to determine the sizes of the native plasmids in B.t. strain EG5144 and to ascertain which plasmids contained genes that encoded coleopteran-active insecticidal crystal proteins. DNA blot analysis was thereafter performed using size fractionated total DNA restriction fragments from B.t. strain EG5144, compared WO 92/13954 PCT/US92/00040 30 with similarly-processed total DNA from other B.t. strains containing cryIII-type toxin genes, to demonstrate that B.t. strain EG5144 contains a unique coleopteran-active toxin gene. In addition, B.t. strain EG5144 was evaluated further by characterizing the crystal proteins it produces and by measuring the insecticidal activity associated with B.t. strain EG5144 and its crystal proteins. Examples 2 through 7 are directed to the procedures for characterizing B.t. strain EG5144 and its unique cryIIItype gene, and Examples 8 through 11 are directed to the insecticidal activity of B.t. strain EG5144 and of B.t.

strain EG7237, containing the cryIIIC(b) gene (SEQ ID NO1l) of this invention.

Example 2 Evaluation of the Flagellar Serotype of B.t. Strain EG5144 Flagellar serotyping studies were carried out with B.t. strain EG5144, using an antibody mediated cell agglutinization assay (Craigie et al., J.Immunol., 21, pp.417-511 (1936)). Flagellar antibody reagents were prepared using purified flagella from B.t. var. kurstaki, morrisoni and tolworthi type-strains and from the novel coleopteran-active B.t. strain EG4961.

The study included formalin-fixed vegetative cells of B.t. strain EG5144 and of cells of other coleopteranactive B.t. strains and of several common B.t. type- WO 92/13954 WO 9213954PCr/US92/00040 31 strains, each of which were scored for flagellar antibody mediated cell agglutinization.

The other coleopteran-active B.t. strains included B.t. var. tenebr-ionis, B.t. var. san diego, B.t. strain 5EG2158 (all containing the c-ryIIXA gene); B.t. strain EG2838 (containing the cryXXTB gene); and B.t. strain EG4961 (containing a novel coleopteran toxin-encoding gene designated as the cryIIXC gene).

The B.t. flagelJlar type-strains were B.t. var.

kurstaki (HD-l, serotype 3ab), B.t. var. morrisoni (HD-12, serotype 8ab) and B.t. var. tolworthi (HD-13, serotype 9).

Results of this study are shown in Table 1; "1+1 indicates that a cross-reaction occurred and indicates that no cross-reaction occurred.

Table 1 Flagellar Antibody Reagent Cells kurstaki morrisoni toiworthi EG4961 B.t. strain EG5144---- B.t. var. tenebrionis B.t. var. san diego B.t. strain EG2158 B.t. strain EG2838 B.t. strain EG4961---+ WO 92/13954 PCT/US92/00040 32 Other B.t. flagellar type-strains: B.t. var. kurstaki (HD-1) B.t. var. morrisoni (HD-12) B.t. var. tolworthi (HD-13) The results in Table 1 show that cells of B.t. strain EG5144 gave a negative reaction with B.t. type-strain kurstaki, morrisoni and tolworthi flagella antibody reagents. B.t. strain EG5144 cells also gave a negative reaction with flagellar reagent from B.t. strain EG4961, a novel coleopteran-active strain that has been discovered to exhibit Diabrotica toxicity.

These results indicate that B.t. strain EG5144 is not a kurstaki, morrisoni or tolworthi-type B.t. strain.

Furthermore, the flagellar serotype of B.t. strain EG5144, which is yet not known, is apparently different from that of B.t. strain EG4961, which has been serotyped as serovar kumamotoensis (serotype 18). Both B.t. strain EG5144 and B.t. strain EG4961 appear to have flagellar serotypes that are different from those of other coleopteran-toxic B.t.

strains reported in the literature.

Example 3 Size Fractionation and cryvIIB Probing of Native Plasmids of EG5144 B.t. strains may be characterized by fractionating their plasmids according to size by the well-known procedure of agarose gel electrophoresis. This procedure WO 92/13954 PCT/US92/00040 33 involves lysing B.t. cells with lysozyme and SDS, electrophoresing plasmids from the lysate through an agarose gel and staining the gel with ethidium bromide to visualize the plasmids. Larger plasmids, which move more slowly through the gel, appear at the top of the gel and smaller plasmids appear toward the bottom of the gel.

The agarose gel in Figure 2 shows that B.t. strain EG5144 contains native plasmids of approximately 145, 92, 12, 10 and 5.5 MDa, as indicated by the white horizontal bands. Plasmid sizes were estimated by comparison to plasmids of known sizes (not shown). Although not shown on Figure 2, B.t. strain EG5145 contains native plasmids of approximately 145, 92, 12 and 5.5 MDa. The cryptic MDa plasmid found in B.t. strain EG5144 is not present in B.t. strain EG5145.

Figure 2 further shows that the coleopteran-toxic B.t. strain EG4961 contains native plasmids of about 150, 70, 50, 5 and 1.5 MDa and that the coleopteran-toxic B.t. strain EG2838 contains native plasmids of about 100, 90 and 37 MDa. Figure 2 also shows that the coleopterantoxic B.t. strain EG2158 contains native plasmids of about 150, 105, 88, 72, and 35 MDa. Some of the plasmids, such as the 150 and 1.5 MDa plasmids of B.t. strain EG4961 and the 150 MDa plasmid of B.t. strain EG2158, may not be visible in the photograph, although they are visible in the actual gel. Figure 2 demonstrates that the sizes of the native plasmids of B.t. strain EG5144 are different WO 92/13954 PCr/US92/00f40 34 from the sizes of the native plasmids of B.t. strains EG2158, EG2838 and EG4961. B.t. strain EG5144 is therefore distinct from the other coleopteran-toxic B.t.

strains EG2158, EG2838 and EG4961, based on these plasmid array studies and on the serotyping studies described in Example 2. Likewise, B.t. strain EG5145 appears distinct from the coleopteran-toxic B.t. strains noted above based on plasmid array studies.

The plasmids shown in Figure 2 were transferred by blotting from the agarose gel to a nitrocellulose filter using the blot techniques of Southern, J.Molec.Biol., 98, pp.503-517 (1975), and the filter was hybridized as described above with the radioactively labeled 2.4 kb cryIIIB DNA probe. After hybridization, the filter was exposed to X-ray film. Examination of the X-ray film confirmed that the cryIIIB probe specifically hybridized to the 92 MDa plasmid of B.t. strain EG5144. This result demonstrates that the 92 MDa plasmid of B.t. strain EG5144 contains a DNA sequence that is at least partly homologous to the cryIIIB gene and confirms that the 92 MDa plasmid contains a cryIl-type gene. The X-ray film also showed that the cryIIIB probe hybridized, as expected, to the MDa plasmid of B.t. strain EG4961 and to the 100 MDa plasmid of B.t. strain EG2838, and to the 88 MDa plasmid of B.t. strain EG2158. The 88 MDa plasmid of B.t. strain EG2158 has been previously shown to contain the coleopteran-toxin cryIIIA gene (see Donovan et al., w6 92/13954 PC/US92/0004O 35 Mol.Gen.Genet., 214, pp.365-372 (1988)). The inventors have previously determined that the 100 MDa plasmid of B.t. strain EG2838 contains the coleopteran toxin cryIIIB gene and that the 95 MDa plasmid of B.t. strain EG4961 contains the novel coleopteran toxin cryIIIC(a) gene.

Example 4 Blot Analysis of DNA from B.t. Strains EG5144 and EG5145 Both chromosomal and plasmid DNA (total DNA) from B.t. strain EG5144 were extracted and digested with separate restriction enzymes, SspI, HindIII and EcoRI.

The digested DNA was size fractionated by electrophoresis through an agarose gel, and the fragments were then visualized by staining with ethidium bromide. For comparison, total DNA from the coleopteran-toxic B.t.

strains EG2158, EG2838 and EG4961 was processed in an identical manner. Examination of the resultant stained agarose gel showed that restriction digestions of total DNA from these B.t. strains with each of Sspl, HindIII and EcoRI yield hundreds of DNA fragments of various sizes.

The size fractionated DNA restriction fragments were transferred by blotting from the agarose gel to a nitrocellulose filter and were then probed with a cryIIItype DNA hybridization probe. The filter was hybridized at 65°C in a buffered aqueous solution containing a radioactively labeled 2.4 kb cryIIIB DNA probe. After hybridization, the filter was exposed to X-ray film to WO 92/13954 PCT/US92/00040 36 make an autoradiogram. Figure 3 is a photograph of the autoradiogram where the numbers to the left indicate the size, in kb, of the DNA fragments of B.t. strain EG5144 that hybridized to the cryIIB probe. These sizes were determined by comparison with the lane labeled "stnd" which contained phage lambda DNA digested with HindIII and radioactively labelled as size markers. Lanes in Figure 3 marked EG2158, EG5144, EG2838 and EG4961 contain size fractionated DNA fragments from these respective B.t.

strains, obtained by digestion with the restriction enzyme designated above the individual lanes.

In the lanes for each B.t strain in Figure 3, the dark bands represent DNA restriction fragments that hybridized with the cryIIIB probe. Visual inspection of Figure 3 shows that the sizes of the cryIIIB-hybridizing restriction fragments of B.t. strain EG5144 are distinctly different from the sizes of the cryIIB-hybridizing fragments of B.t. strains EG2158, EG2838 and EG4961.

In particular, the size of the cryIIIB-hybridizing SspI restriction fragment for B.t. strain EG5144 is 3.4 kb, and this is unlike the corresponding SspI restriction fragments for the other three B.t. strains: 2.8 kb for B.t. strain EG2158; 2.4 kb for B.t. strain EG2838; and and 6.0 kb for B.t. strain EG4961. Similar differences are apparent for the DNA restriction fragments obtained using HindIII and EcoRI.

WO 92/13954 PCT/US92/00040 37 These restriction pattern results suggest that B.t.

strain EG5144 contains a cryIII-type gene that is different from the crylJXA, cryIIIB and cryIIC(a) genes of B.t. strains EG2158, EG2838 and EG4961, respectively.

The crylI-type gene of B.t. strain EG5144 has been designated cryIIIC(b) (SEQ ID NO:1) by the inventors.

Total DNA from B.t. strain EG5144 and B.t. strain EG5145 was extracted and digested with six separate restriction enzymes (HindIII, EcoRI, AccI, Dral, SspI, XbaI), and size fractionated by electrophoresis on an agarose gel. The size fractionated DNA restriction fragments were then transferred by blotting to a nitrocellulose filter and were then probed with a cryIIItype DNA hybridization probe, specifically a probe containing cryIIA. After hybridization, the filter was exposed to X-ray film to make an autoradiogram. The restriction pattern results were identical for the two B.t. strain evaluated, EG5144 and EG5145, which suggests that the 'wo strains contain the same cryIII-type gene.

Example Characterization of Crystal Proteins of B.t. Strain EG5144 B.t. strain EG5144 was grown in DSMG sporulation medium at room temperature (about 21-250C) until sporulation and cell lysis had occurred (4 to 5 days growth). The DSMG medium is 0.4% Difco nutrient broth, 25 mM K 2 HP0 4 25 mM KH 2 P0 4 0.5 mM Ca(N0 3 2 0.5 mM WO 92/13954 PCr/US92/0040 38 MgS0 4 10 pM FeSO 4 10 pM MnC12 and 0.5% glucose.

The sporulated culture of B.t. strain EG5144 was observed microscopically to contain free floating, irregularly shaped crystals in addition to B.t. spores. Experience has shown that 3.t. crystals are usually composed of proteins that may be toxic to specific insects. The appearance of the crystals of B.t. strain EG5144 differed from the flat, rectangular (or rhomboidal) crystals of B.t. strain EG2158, but partially resembled some of the irregularly shaped crystals of B.t. strains EG2838 and EG4961.

Spores, crystals and residual lysed cell debris from the sporulated culture of B.t. strain EG5144 were harvested by centrifugation. The recovered solids were washed once with aqueous 1N NaCl and twice with TETX (containing 10 mM Tris HC1 pH 7.5, imM EDTA and 0.005% Triton® X-100) and suspended in TETX at a concentration of 50 mg/ml. The washed crystals were specifically solubilized from 250 pg centrifuged fermentation culture solids (containing crystals, spores and some cell debris) by heating the solids mixture in a solubilization buffer (0.14 M Tris pH 6.8, 2% SDS, 2-mercaptoethanol, 10% glycerol and 0.1% bromophenol blue) at 100 0 C for 5 minutes. The solubilized crystal proteins were size fractionated by SDS-PAGE. After size fractionation, the proteins were visualized by staining with Coomassie dye. Cultures of Wd 92/13954 PCT/US92/00040 39 B.t. strains EG4961, EG2158 and EG2838 were processed in an identical manner for purposes of comparison.

Figure 4 shows the results of this protein size fractionation analysis where the numbers to the left indicate the size, in kDa, of the crystal proteins synthesized by B.t. strain EG5144. As shown in lane 1, a major protein of approximately 70 kDa and a minor protein of approximately 30 kDa were solubilized from centrifuged fermentation solids containing B.t. strain EG5144 spores and crystals. The approximately 70 kDa protein of B.t.

strain EG5144 appears similar in size to the approximately kDa coleopteran-toxic crystal proteins of B.t. strains EG4961 (lane EG2158 (lane 3) and to the approximately 74 kDa coleopteran-toxic crystal protein of B.t. strain EG2838 (lane 4).

Previous work by the inventors has shown that the coleopteran-toxic crystal proteins of B.t. strains EG4961, EG2158 and EG2838 are each different. The CryIIIC(a) protein of P.t. strain EG4961 is coded by the cryIIIC(a) gene and has a deduced size of 74,393 Da. The CryIIlA protein of B.t. strain EG2158 is coded by the cryIIIA gene and has a deduced size of 73,116 Da. The CryIIIB protein of B.t. strain EG2838 is coded by the cryIIIB gene and has a deduced size of 74,237 Da. As described in Example 6, the coleopteran-toxic crystal protein of B.t. strain EG5144 produced by the novel cryIIIC(b) gene (SEQ ID NO:1) WVO 92/13954 PCT/US92/00040 40 is clearly different from the CrylIIA, CryIIIB and CryIIIC(a) proteins.

The minor crystal protein of approximately 30 kDi that is produced by B.t. strain EG5144 is roughly similar in size to sr.all c.:ystal proteins produced by B.t. strains EG4961, EG2158 and EG2838. The approximately 30 kDa minor proteins of B.t. strains EG2158, EG2838 and EG4961 appear to be related to each other and none has been found to exhibit measurable insecticidal activity towards coleopteran insects. There is no reason to believe that the approximately 30 kDa protein of B.t. strain EG5144 possesses insecticidal activity against coleopteran insects.

Following the procedure of Example 4, further DNA blot analysis revealed that the 2.4 kb cryIIIB DNA probe specifically hybridized to a single 7.0 kb EcoRI-XbaI restriction fragment of B.t. strain EG5144 DNA. This result suggested that the 7.0 kb fragment contained the complete cryIIIC(b) gene.

The 7.0 kb EcoRI-XbaI fragment of B.t. strain EG5144 was isolated and studies were conducted on the 7.0 kb EcoRI-XbaI restriction fragment to confirm that the fragment contained a cryIII-type gene, in particular, the cryIIIC(b) gene. The procedures set forth in Example 6 describe the determination of the nucleotide base sequence of the cryIIIC(b) gene (SEQ ID NO:1).

WO 92/13954 PCT/US92/00040 41 Example 6 Cloning and sequencing of the crylIIC(b) Gene of B.t. Strain EG5144 In order to isolate the 7.0 kb EcoRI-Xbal fragment described in the previous Example, a plasmid library of B.t. strain EG5144 was constructed by ligating sizeselected DNA EcoRI-XbaI restriction fragments from B.t.

strain EG5144 into the well-known E.coli vector pUC18.

This procedure involved first obtaining total DNA from B.t. strain EG5144 by cell lysis followed by DNA spooling, then double digesting the total DNA with both EcoRI and XbaI restriction enzymes, electrophoresing the digested DNA through an agarose gel, excising a gel slice containing 4-10 kb size selected fragments of DNA, and electroeluting the size selected EcoRI-XbaI restriction fragments from the agarose gel slice. These fragments were mixed with the E.coli plasmid vector pUC18, which had also been digested with EcoRI and XbaI. The pUC18 vector carries the gene for ampicillin resistance (Ampr) and the vector replicates in E.coli. T4 DNA ligase and ATP were added to the mixture of size-selected restriction fragments of DNA from B.t strain EG5144 and of digested pUCl8 vector to allow the pUC18 vector to ligate with the B.t. strain EG5144 restriction fragments.

The plasmid library was then transformed into E. coli cells, a host organism lacking the gene of interest, as follows. After ligation, the DNA mixture was incubated with an ampicillin sensitive E. coli host strain, E. coli WO 92/13954 PCT/US92/00040 42 strain DH5a, that had been treated with CaCl 2 to allow the cells to take up the DNA. E. coli, specifically strain was used as the host strain because these cells are easily transformed with recombinant plasmids and because E. coli strain DH5a does not naturally contain genes for B.t. crystal proteins. Since pUC18 confers resistance to ampicillin, all host cells acquiring a recombinant plasmid would become ampicillin resistant. After exposure to the recombinant plasmids, the E. coli host calls were spread on agar medium that contained ampicillin. After incubation overnight at a temperature of 37 0 C, several thousand E. coli colonies grew on the ampicillincontaining agar from those cells which harbored a* recombinant plasmid. These E. coli colonies were then blotted onto nitrocellulose filters for subsequent probing.

The radioactively labeled 2.4 kb cryIIB gene was then used as a DNA probe under conditions that permitted the probe to bind specifically to those transformed host colonies that contained the 7.0 kb EcoRI-XbaI fragment of DNA from B.t. strain EG5144. Several E. coli colonies specifically hybridized to the 2.4 kb cryIIIB probe. One cryIIIB-hybridizing colony, designated E. coli strain EG7236, was studied further. E. coli strain EG7236 contained a recombinant plasmid, designated pEG271, which consisted of pUCl8 plus the inserted EcoRI-XbaI restriction fragment of DNA from B.t. strain EG5144 of WO 92/13954 PC/US92/00040 43 approximately 7.0 kb. The cryIIIB probe specifically hybridized to the 7.0 kb DNA fragment insert in pEG271. A restriction map of pEG271 is shown in Figure The 7.0 kb fragment of pEG271 contained HindIII fragments of 2.4 kb and 3.8 kb, and a BamHI-XbaI fragment of 4.0 kb that specifically hybridized with the cryIIIB probe. The 2.4 kb HindIII fragment was subcloned into the DNA sequencing vector M13mpl8. The 4.0 kb BamHI-XbaI fragment was subcloned into the DNA sequencing vectors M13mpl8 and M13mpl9.

The nucleotide base sequence of a substantial part of each subcloned DNA fragment was determined using the standard Sanger dideoxy method. For each subcloned fragment, both DNA strands were sequenced by using sequence-specific 17-mer olignucleotide primers to initiate the DNA sequencing reactions. Sequencing revealed that the 7.0 kb fragment contained an open reading frame and, in particular, a new cryIII-type gene.

This new gene, designated cryIIIC(b) (SEQ ID NO:1), is significantly different from the cryIIIA gene. As indicated below, the cryIIIC(b) gene is also clearly distinct from the cryIIIB gene.

The DNA sequence of the cryIIIC(b) gene (SEQ ID NO:1) and the deduced amino acid sequence of the CryIIIC(b) protein (SEQ ID NO:2) encoded by the cryIIIC(b) gene are shown in Figure 1. The protein coding portion of the cryIIIC(b) gene (SEQ ID NO:1) is defined by the WO 92/13954 PCr/US92/00040 44 nucleotides starting at position 144 and ending at position 2099. The probable ribosome binding site is indicated as "RBS" in Figure 1-1. The size of the CryIIIC(b) protein (SEQ ID NO:2) encoded by the cryIIIC(b) gene, as deduced from the open reading frame of the cryIIIC(b) gene (SEQ ID NO:1), is 74,265 Da (652 amino acids). It should be noted that the apparent size of the CryIIIC(b) protein, as determined from SDS-PAGE, is approximately 70 kDa. Therefore, the CryIIIC(b) protein (SEQ ID NO:2) will be referred to in this specification as being approximately 70 kDa in size.

The size of the prior art CryIIIA protein has previously been deduced to be 73,116 Da (644 amino acids).

The size of the CryIIIB protein has previously been determined to be 74,237 Da (651 amino acids).

DNA sequencing revealed the presence of a HindIII restriction site within the cryIIIC(b) gene and a Sspl restriction site downstream of the cryIIIC(b) gene (See Figures 1-2 and 1-3 respectively). Knowledge of the locations of these restriction sites permitted the precise determination of the location and orientation of the cryIIIC(b) gene within the 7.0 kb fragment as indicated by the arrow in Figure The computer program of Korn and Queen Korn and C. Queen, "Analysis of Biological Sequences on Small Computers," DNA, 3, pp. 421-436 (1984)) was used to compare the sequences of the cryIIIC(b) gene (SEQ ID NO:l) WO'92/13954 PCr/US92/00040 45 to the cryIIIB and cryIIIA genes and to compare the deduced amino acid sequences of their respective CryIIIC(b), CryIIIB and CryIIIA proteins.

The nucleotide base sequence of the cryXIIC(b) gene (SEQ ID NO:1) was 96% positionally identical with the nucleotide base sequence of the cryIIIB gene and only 76% positionally identical with the nucleotide base sequence of the cryIIIA gene. Thus, although the cryIIIC(b) gene (SEQ ID NO:1) is related to the cryIIIB and cryIIIA genes, it is clear that the cryIIIC(b) gene is distinct from the cryIIIB gene and substantially different from the cryIIIA gene.

The deduced amino acid sequence of the CryIIIC(b) protein (SEQ ID NO:2) was found to be 95% positionally identical to the deduced amino acid sequence of the CryIIIB protein, but only 68% positionally identical to the deduced amino acid sequence of the CryIIIA protein.

These differences, together with the differences in insecticidal activity as set forth below, clearly show that the CryIIIC(b) protein encoded by the cryIIIC(b) gene (SEQ ID NO:1) is a different protein from the CryIIIB protein or the CryIIIA protein.

Moreover, while not wishing to be bound by any theory, based on a comparison of the amino acid sequences of the CryIIIC(b) protein (SEQ 'ID NO:2) with other CryIIItype proteins known to the inventors, it is believed that the following amino acid residues may be of significance WO 92/13954 PCT/US92/00040 46 for the enhanced corn rootworm toxicity of the CryIIIC(b) protein, where the numbers following the accepted abbreviations for the amino acids indicate the position of the amino acid in the sequence illustrated in Figure 1 and identified in SEQ ID NO:2: His9, His231, Gln339, Ser352, Asn446, His449, Val450, Gly451, Ile600 and Thr624. These amino acid residues were selected as being of probable significance for the corn rootworm toxicity of the CryIIIC(b) protein (SEQ ID NO:2) because, after studying the amino acid sequences of several other CryIII proteins, the amino acids at the indicated positions fairly consistently showed different amino acids than those indicated for the CryIIIC(b) protein.

Based on the same studies, it is also believed that site directed mutagenesis of the cryIIIC(b) gene (SEQ ID NO:1) may result in improved or enhanced corn rootworm toxicity for the resultant protein where one or more of the following amino acid modifications are effected: Pro21 to Gly; Asp97 to Asn; Val289 to Ile; Ser352 to Phe; 417Ile to Val; Phe419 to Leu; Gly451 to Ser; Ile590 to Leu; Ile600 to Lys; Thr624 to Lys.

As is well understood in the art, other changes in the cryIIIC(b) gene (SEQ ID NO:1) may be made, via site directed mutagenesis or gene truncation or the like, that could yield a toxic protein which possesses essentially similar insecticidal activity (to corn rootworm and other coleopteran insects) as that exhibited by the CryIIIC(b) WO 92/13954 PCT/US92/00040 47 protein (SEQ ID NO:2). Modifications to the cryIIIC(b) gene (SEQ ID NO:1) and CryIIIC(b) protein (SEQ ID NO:2) such as described above are intended to be within the scope of the claimed invention.

Example 7 Expression of the Cloned cryIIIC(b) Gene Studies were conducted to determine the production of the CryIIIC(b) protein (SEQ ID NO:2) by the cryIIIC(b) gene (SEQ ID NO:1).

Table 2 summarizes the relevant characteristics of the B.t. and E. coli strains and plasmids used during these procedures. A plus indicates the presence of the designated element, activity or function and a minus indicates the absence of the same. The designations s and r indicate sensitivity and resistance, respectively, to the antibiotic with which each is used. The abbreviations used in the Table have the following meanings: Amp (ampicillin); Cm (chloramphenicol); Cry (crystalliferous); Tc (tetracycline).

Table 2 Strains and Plasmids Strains Relevant characteristics B. thurinqiensis HD73-26 Cry", Cms EG7237 HD73-26 harboring pEG272 (cryIlC(b) WO 92/13954 WO 9213954PC/US92/00 144 E. coli EG7236 Plasmids pUC 18 pNN1Ol pEG271 pEG272 48 cr-ylliC Cry-, Am~ps .Cry-, Amps DH5a harboring PEG271 (cryIIXC(b) Amtpr, Cry-, E. coi vector CMr, Tcr, Cry-, Bacllus vector Ampr, cr-yIIIC(b)+ E. coi recombinant plasmid consisting of the 7.0 kb EcoRI-XbaI cryII.TC(b) fragment of B.t. strain EG5144 ligated into the EcoRI-XbaI sites of pUCiB Tcr CrcryXXTC(b)+ Baclius-E.

coi recombinant plasmid consisting of the Bacillus vector pNNl0l ligated into the SphI site of pEG27l.

coi cells harboring plasmid pEG271 described in Example 16 were analyzed and found n6t to produce detectable amounts of the 70 kDa CryIIIC(b) crystal protein.

Experience has shown that cloned B.t. crystal genes are poorly expressed in E. coli and highly expressed in B.t. from their respective native promoter sequences.

WO 92/13954 PCT/US92/00040 49 Recombinant plasmid pEG271, constructed as set forth in Example 6 and shown in Figure 5, will replicate in E.

coli, but will not replicate in B.t. To achieve a high level of expression of the cloned cryIIIC(b) gene, the Bacillus vector pNN101 (Tcr Cmr Cry") that is capable of replicating in B.t. was ligated into the Sphl site of pEG271. The resultant plasmid was designated pEG272.

Details of the construction of plasmid pEG272 and its subsequent use to transform B.t. are described below.

The isolated plasmid pEG271 DNA was digested with SphI and was then mixed with the Bacillus vector pNN101 that had also been digested with SphI. T4 DNA ligase and ATP were added to the mixture to allow pEG271 to ligate into the SphI site of the pNNl01 vector.

After ligation, the DNA mixture was added to a suspension of E. coli strain DH5a cells that had been treated with calcium chloride to permit the cells to take up plasmid DNA. After exposure to the recombinant plasmids, the E. coli host cells were spread on an agar medium containing tetracycline. Only cells that had taken up a plasmid consisting of pEG271 ligated into the SphI site of pNN101 would grow on the tetracycline agar medium whereas cells that had not absorbed the plasmid would not grow.

Plasmid was isolated from one tetracycline resistant colony, digested with SphI, and electrophoresed through an agarose gel. The plasmid consisted of two SphI DNA WO 92/1:3954 PC/US92/00040 50 fragments of 5.8 kb and 9 kb corresponding to plasmids pNN101 and pEG271, respectively. This plasmid was designated pEG272. A restriction map of pEG272 is shown in Figure 6. Plasmid pEG272 was then used to transform cells of E. coli strain GM2163 made competent by the calcium chloride procedure described earlier in Example 6.

E. coli strain GM2163 is a crystal negative (Cry-) and ampicillin sensitive (Amps) strain, constructed by the procedures of M.G. Marinus et al. in Mol.Gen.Genet., 192, pp.288-289 (1983).

Plasmid pEG272 was then isolated from the transformed E. coli strain GM2163, using the procedures described above. The isolated plasmid pEG272 was next transformed by electroporation into B.t. strain HD73-26. Cells of B.t. strain HD73-26 are crystal-negative (Cry") and chloramphenicol sensitive (Cms). Using a BioRad Gene Pulser" apparatus to carry out the electroporation, cells of B.t. strain HD73-26 in suspension were induced to take up pEG272 which was also added to the mixture.

After electroporation, the transformed B.t. cells were spread onto an agar medium containing 5 Ag chloramphenicol and were incubated about 16-18 hours at Cells that had taken up plasmid pEG272 would grow into colonies on the chloramphenicol agar medium whereas cells that had not absorbed the plasmid would not grow.

One Cmr colony, designated B.t. strain EG7237, contained a WO 92/13954 PCT/US92/00040 51 plasmid whose restriction pattern appeared identical to that of pEG272.

Cells of B.t. strain EG7237 were grown in a sporulation medium containing chloramphenicol (3 pg/ml) at 22-25°C until sporulation and cell lysis had occurred days). Microscopic examination revealed that the sporulated culture of B.t. strain EG7237 contained spores and small free floating irregularly shaped crystals.

These crystals resembled the small, irregularly-shaped crystals observed with a sporulated culture of B.t. strain EG5144 that had been prepared in a similar manner.

Spores, crystals and cell debris from the sporulated fermentation culture of B.t. strain EG7237 were harvested by centrifugation. The centrifuge pellet was washed once with IN aqueous NaCl and twice with TETX (10 mM Tris'HCl pH 7.5, 1 mM EDTA, 0.005% Triton® X-100), and the pellet suspended in TETX at a concentration of 50 mg pellet/ml TETX.

The crystals in the centrifuge pellet suspension were solubilized by heating a portion of the centrifuge suspension (containing 250 pg pellet solids) in solubilization buffer (0.14 M Tris pH 6.8, 2% SDS, 2-mercaptoethanol, 10% glycerol and 0.1% bromophenol blue) at 100 0 C for 5 minutes. After crystal solubilization had occurred, the mixture was applied to an SDS-polyacryamide gel and the solubilized proteins in the mixture were size fractionated by WO 92/13954 PCr/US92/00040 52 electrophoresis. After size fractionization, the proteins were visualized by staining with Coomassie dye. A photograph of the Coomassie stained gel is shown in Figure 7.

Lane 3 of the gel in Figure 7 shows that B.t. strain EG7237 produced a major protein of approximately 70 kDa and a minor protein of approximately 30 kDa. These proteins appeared to be identical in size with the major approximately 70 kDa protein and the minor approximately 30 kDa protein produced by B.t. strain EG5144, which are shown in the lane 1 of Figure 7 and which were prepared in a manner identical to B.t. strain EG7237. This result indicates that the 7.0 kb fragment of pEG272 contains two crystal protein genes: one for the approximately 70 kDa protein and one for the approximately 30 kDa protein.

The gene encoding the approximately 70 kDa protein is the cryIIIC(b) gene, and its encoded protein is the insecticidal CryIIIC(b) protein. The DNA sequence for the cryIIIC(b) gene (SEQ ID NO:1) and the amino acid sequence for its corresponding deduced protein (SEQ ID NO:2) are shown in Figure 1.

B.t. strain EG7237 produced approximately three times more 70 kDa protein, on a weight basis, than did B.t.

strain EG5144, as is evident from the protein bands in Figure 7. Production of the minor 30 kDa protein in recombinant B.t. strain EG7237 was also increased, as compared with B.t. strain EG5144.

WO 92/13954 "'CT/US92/00040 53 The following Examples 8-11 describe the manner in which the insecticidal activities of B.t. strain EG5144, B.t. strain EG7237, and the CryIIIC(b) protein made by these strains were determined.

Example 8 Insecticidal Activity of B.t. Strain EG7237 and its CryIIIC(b) Protein Against Southern Corn Rootworm and Colorado Potato Beetle The insecticidal activity of recombinant B.t. strain EG7237, which contains the cryIIIC(b) gene (SEQ ID NO:1) that produces the CryIIIC(b) toxin protein (SEQ ID NO:2), was determined against southern corn rootworm (Diabrotica undecimpunctata howardi) and Colorado potato beetle (Leptinotarsa decemlineata).

For comparison, two other recombinant B.t. strains containing cryIII-type toxin genes in a B.t. strain HD73- 26 background were also included in the bioassay study.

These were recombinant B.t. strain EG7235, which contains the cryIIIA gene that produces the CryIIIA toxin protein, and recombinant B.t. strain EG7225, which contains the cryIIIB gene that produces the CryIIIB toxin protein.

The three B.t. strains were grown in liquid sporulation media at 30 0 C until sporulation and cell lysis had occurred. The fermentation broth was concentrated by microfiltration. The concentrated fermentation broth was then freeze dried to prepare a B.t. powder suitable for WO 92/13954 PCT/US92/00040 54 insect bioassay. The amount of CryIII-type toxin protein in each of the B.t. powders was quantified using standard SDS-PAGE techniques.

First instar southern corn rootwom larvae were bioassayed via surface contamination of an artificial diet similar to Marrone et al., J.Econ.Entomol., 78, pp.290-293 (1985), but without formalin. Each bioassay consisted of eight serial aqueous dilutions with aliquots applied to the surface of the diet in a bioassay tray. Each 2 ml well of the bioassay tray contained 1 ml diet having a surface area of 175 mm 2 After the diluent (an aqueous 0.005% Triton® X-100 solution) had evaporated, the insect larvae were placed on the diet and incubated at 28°C.

Thirty-two larvae were tested per dose. Mortality was scored after 7 days. A control, consisting of diluent only, was also included in the bioassay study.

First instar Colorado potato beetle larvae were tested using similar techniques, except for the substitution in the artificial diet of BioServe's No. 9830 insect diet with potato flakes added. Thirty-two larvae were tested per dose, and mortality was scored at three days instead of seven days.

The results of the bioassay study are shown below in Table 3, where insecticidal activity is reported as PLC 50 values, the concentration of CryIII-type protein required to kill 50% of the insects tested. Four replications per dose were used in the bioassay studies for both insects WO 92/13954 PCT/US92/00040 55 tested. Data from each of the replicated bioassays were pooled for probit analysis Daum, Bull.Entomol.Soc.Am., 16, pp.10-15 (1970)) with mortality corrected for control death, the control being the diluent only Abbott, J.Econ.Entomol., 18, pp.265-267 (1925)). Results are shown as the dose amount of CryIIItype protein (in ng CryIII protein per mm2 of diet surface) resulting in PLC 50 Confidence intervals, at are given within parentheses below the PLC 50 values.

Table 3 Insecticidal Activity of Recombinant B.t. Strains EG7237. EG7235 and EG7225 Southern Corn Rootworn Colorado Potato Rpptlt B. t. Strain B.t. EG7237 B.t. EG7235 B.t. EG7225 CryIII Protein concentration CryIII Protein CryIIIC(b) 7.2 CryIIIA 28.4 CrylIIB 9.4 P1C50 (nci CrvIII protein/,m-z 1548 (1243-1992) 6% control at 4570 20% control at 4570

PLCS

0 2 n (nci CrvIII protein/mm-I 6.92 (5.15 9.10) 0.34 (0.30 0.39) 1.26 (1.07 1.46) WO 92/13954 PCT/US92/00040 57 The results of this bioassay study demonstrate that B.t. strain EG7237 which produces the CryIIIC(b) toxin protein (SEQ ID NO:2) is insecticidal to southern corn rootworm. In contrast, the CryIIIA and CryIIB toxin proteins of B.t. strains EG7235 and EG7225, respectively, appear to have no measurable activity against this insect at the highest dose level tested.

All three of the B.t. strains exhibit insecticidal activity against Colorado potato beetle larvae, with the CryIIIA toxin protein of B.t. strain EG7235 being significantly more potent than the CryIIIC(b) toxin protein (SEQ ID NO:2) of B.t. strain EG7237 and with the CryIIIB toxin protein of B.t. strain EG7225 having insecticidal activity falling between that shown for CryIIIA and CryIIIC(b).

These results suggest that the insecticidal activity of specific CryIII-type toxin proteins varies for different insect genera within the order Coleoptera.

Example 9 Insecticidal Activity of B.t. Strain EG7237 and its CryIIIC(b) Protein Against Mexican Bean Beetle The insecticidal activity of recombinant B.t. strain EG7237, evaluated in Example 8, was also determined against Mexican bean beetle (Epilachna varivestis). As in Example 8, recombinant B.t. strains EG7235 and EG7225 were WO 92/13954 PCr/US92/00040 58 included for comparison, and all B.t. powders were prepared as in Example 8.

First instar Mexican bean beetle larvae were bioassayed by a leaf dip procedure, since a suitable artificial diet is not available for this insect. Soybean leaves were dipped into known treatment concentrations of the B.t. powder suspended in an aqueous 0.1% Triton® X-100 solution. After excess material had dripped off, the leaves were allowed to dry. Leaves dipped in 0.1% Triton® X-100 served as untreated controls. Twenty insect larvae were confined to a petri dish with treated leaves, incubated at 25 0 C, and allowed to feed for three days, at which time mortality was scored.

The results of the bioassay study are shown below in Table 4, where insecticidal activity is reported as PLC 50 values, the concentration of CryIII-type protein required to kill 50% of the insects tested. The data were handled as described in Example 8, for Table 3. Results are shown as the dose amount of CryIII-type protein (in mg CryIII protein/ml solution used in the leaf dip) resulting in

PLC

50 Confidence intervals, at 95%, are given within parentheses following the PLC 50 values.

WC; 92/13954 PC/US92/000O4 59 Table 4 Insecticidal Activity of B.t. Strains EG7237, EG7235 and EG7225 Against Mexican Bean Beetle CryIII No. of PLC 5 0 B.t. Strain Protein Replications (mc CryIIprotein/ml) B.t. EG7237 CryIIIC(b) 4 4.2 (2.5-6.5) B.t. EG7235 CryIIIA 4 16% control at B.t. EG7225 CryIIIB 4 51.8 (31-209) The results of this bioassay study demonstrate that B.t. strain EG7237 which produces the CryIIIC(b) toxin protein (SEQ ID NO:2) is significantly more insecticidal to Mexican bean beetle than the CryIIIB-producing B.t, strain EG7225. B.t. strain EG7235 which produces CryIIIA toxin protein exhibited no measurable insecticidal activity at the highest dose tested.

These results are further evidence that the insecticidal activity of specific CryIII-type toxin proteins varies widely for insect genera within the order Coleoptera.

Example Insecticidal Activity of B.t. Strain EG5144 Against Southern Corn Rootworm The insecticidal activity of B.t. strain EG5144 was evaluated against Southern corn rootworm (Diabrotica undecimpunctata howardi). For comparison, B.t. strain WO~C 92/139,54 PCT/US92/00040 60 EG4961 which produces the CryIIIC(a) toxin protein was included in the bioassay study.

The bioassay procedure for southern corn rootworm in this Example determined PLC 50 values, the concentration of CryIII-type protein required to kill 50% of the insects tested. The procedure was similar to the artificial diet bioassay carried out in the previous Example, using thirty-two first instar southern corn rootworm larvae per dose. Data from each of the replicated bioassays were pooled for probit analysis Daum, Bull.Entomol.Soc.Am., 16, pp.10-15 (1970)) with mortality corrected for control death, the control being the diluent only Abbott, J.Econ.Entomol., 18, pp.265-267 (1925)). Results are reported for two separate tests as the dose amount of CryIII-type protein (ng CryIII protein per mm 2 of diet surface) resulting in PLC 5 0 Confidence intervals, at 95%, are given within parentheses following the PLC 50 values. In Test 1 four replications per dose were used, and in Test 2, carried out at a later date, two replications were used.

The B.t. strains used in this Example were prepared as described for the B.t. strains in Example 8, except that the fermentation broth was concentrated by centrifugation.

The results of this bioassay study with southern corn rootworm are shown below in Table WO 92/13954 PCT/US92/00040 61 Table Ilsecticidal Activity of B.t. Strains EG5144 and EG4961 Against Southern Corn Rootworm CryIII Protein PLC 50 Concentration (ng CryIII B.t. Strain CrvIII Protein protein/mm 2 B.t. EG5144 CryIIIC(b) Test 1: 4.0 944 (690-1412) Test 2: 6.4 1145 (773-2185) B.t. EG4961 CryIIIC(a) Test 1: 11.6 102 (86-119) Test 2: 11.6 165 (121-220) This bioassay study demonstrates that both B.t. strain EG5144 and B.t. strain EG4961, which produce CryIIIC-type proteins, provide quantifiable insecticidal activity against southern corn rootworm.

Example 11 Insecticidal Activity of B.t. Strain EG5144 Against Japanese Beetle Larvae The insecticidal activity of B.t. strain EG5144 was evaluated against Japanese beetle larvae, also known as white grubs (Popillia japonica). For comparison, B.t.

strain EG4961 which produces the CryIIIC(a) toxin protein was included in the bioassay study, as were B.t. strain EG2158 which produces the CryIIIA toxin protein and B.t.

strain EG2838 which produces the CryIIIB toxin protein.

The bioassay procedure in this Example was a screening assay, at a single dose of CryIII-type protein WO 92/13954 CT/US92/00040 62 in a diet incorporation assay (1 mg CryIII-type protein per ml diet). B.t. powder to be tested, suspended in a diluent (an aqueous 0.005% Triton® X-100 solution) was incorporated into 100 ml of hot (50 0 -60 0 liquid artificial diet (based on the insect diet described by Ladd, Jr. in J.Econ.Entomol., 79, pp.668-671 (1986)). The mixture was allowed to solidify in petri dishes, and one 19 mm diameter plug of this material then placed in each well of a plastic ice cube tray. One grub was introduced per well of the trays, the wells were covered with moist germination paper ,-,rlaid with aluminum foil, and the trays were held at 25 0 C for seven days before mortality was scored. The insects tested were third instar Japanese beetle grubs. Two replications of sixteen insects each were carried out in this study.

The results of this screening bioassay study are shown below in Table 6, where insecticidal activity is reported as percentage insect mortality, with the mortality being corrected for control death, the control being diluent only incorporated into the diet plug.

Results were obtained at a single dose rate of CryIII-type protein: 1 mg CryIII-type protein per ml of diet; percentage CryIII-type protein present in the respective B.t. powders is also shown in Table 6.

Table 6 Insecticidal Activity of B.t. Strain. BG5144. EG4961, EG2158 and EG2838 Against Japanese Beetle Grubs CrylIl-type Protein in B Powder (wt. 11 CryIII-type Protein Dose (mg CryIII-type protein/mi d-iet) B.t6. strain B. t. EG5144 B. t. EG4961 B. t. EG2158 B.t. EG2838 crylIl Protein CryIIIC(b) CrylIIC (a) CrylIIA CrylIIB 5.4 18.0 14 .0 7.2 Insect Mortality 62. 9 44 48 WO 92/13954 PCT/US92/00040 64 The insecticidal performance against Japanese beetle grubs of B.t. strain EG5144 with its CryIIIC(b) toxin protein (SEQ ID NO:2) is clearly superior to that of B.t.

strain EG4961 with its CryIIIC(a) protein.

With respect to B.t. strains EG2158 and B.t. strain EG2838, B.t. strain EG5144 exhibited superior insecticidal performance against Japanese beetle grubs.

B.t. strain EG5145, whose characteristics are similar to those of B.t. strain EG5144, has been found to exhibit insecticidal activity against Japanese beetle grubs equivalent to that of B.t. strain EG5144, although the bioassay data are not presented in this Example 11.

Microorganism Deposits To assure the availability of materials to those interested members of the public upon issuance of a patent on the present application, deposits of the following microorganisms were made prior to the filing of present application with the ARS Patent Collection, Agricultural Research Culture Collection, Northern Regional Research Laboratory (NRRL), 1815 North University Street, Peoria, Illinois 61604, as indicated in the following Table 7: WO 92/13954 PCrIUS92/0OO40 65 Table 7 Bacterial Strain B.t. EG2158 B.t. HD73-26 B.t. EG2838 B.t. EG5144 B.t. EG7237 E.coli EG7236 B.t. EG5145 NRRL Accession No.

B-18213 B-18508 B-18603 B-18655 B-18736 B-18662 B-18920 Date of Deposit April 29, 1987 June 12, 1989 February 8, 1990 May 22, 1990 October 17, 1990 June 6, 1990 November 21, 1991 These microorganism deposits were made under the provisions of the "Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure". All restrictions on the availability to the public of these deposited microorganisms will be irrevocably removed upon issuance of a patent based on this application.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification as indicating the scope of the invention.

WO 92/13954 PCT/US92/00040 66 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Donovan, William P.

Rupar, Mark J.

Slaney, Annette C.

(ii) TITLE OF INVENTION: BACILLUS THURINGIENSIS cryIIC(b) TOXIN GENE AND PROTEIN TOXIC TO COLEOPTERAN INSECTS (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Panitch Schwarze Jacobs Nadel c/o A.S.

Nadel STREET: 1601 Market Street, 36th Floor CITY: Philadelphia STATE: Pennsylvania COUNTRY: U.S.A.

ZIP: 19103 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 07/649,562 FILING DATE: 31-JAN-1991 (viii) ATTORNEY/AGENT INFORMATION: NAME: Egolf, Christopher REGISTRATION NUMBER: 27633 REFERENCE/DOCKET NUMBER: 7205-29 P1 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 215-757-1590 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 2430 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS WO 92/13954 PCY/1JS92/00040 67 LOCATION: 144. .2099 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i: CCATATAC1AA CTTATCAGGA AGGGGGGGAT GCACAAAGAA GAAAAGAATA AGAAGTGAAT GTTTATAATG TTCAATAGTT TTATGGGAAG GCATTTTATC AGGTAGAAAG TTATGTATTA TGATAAGAAT GGGAGGAAGA AAA ATG AAT CCA AAC AAT CGA AGT GAA CAT Met Asn Pro Asn Asn Arg Ser Glu His 1 120 170

GAT

Asp ACG ATA AAG GTT Thr Ile Lys Val

ACA

Thr 15 CCT AAC AGT GAA TTG CCA ACT AAC CAT Pro Asn Ser Glu Leu Pro Thr Asn His

AAT

Asn CAA TAT CCT TTA Gin Tyr Pro Leu

GCT

Ala GAC AAT CCA AAT TCG ACA CTA GAA GAA Asp Asn Pro Asn Ser Thr Leu Giu Glu 35 TTA AAT La~u Asn 266 TAT AAA GAA Tyr Lys Glu GAC AAC TCT Asp Asn Ser TTA AGA ATG ACT Leu Arg Met Thr

GAA

Giu GAC AGT TCT ACG Asp Ser Ser Thr GAA GTG CTA Giu Val Leu TCT GTT 6TA Ser Val Val ACA GTA AAA GAT Thr Val Lys Asp

GCA

Ala GTT GGG ACA GGA Val Gly Thr Gly GGG CAG Gly Gin ATT TTA GGT GTT Ile Leu Gly Val

GTA

Val GGA GTT CCA TTT Gly Val Pro Phe GGG GCA CTC P-T Gly Ala Leu ;':ar 410 458 TCA Ser TTT TAT CAA TCA Phe Tyr Gin Ser

TTT

Phe 95 CTT GAC ACT ATA Leu Asp Thr Ile

TGG

Trp 100 CCA AGT GAT GCT Pro Ser A, Ala

GAC

Asp 105 CCA TGG AAG GCT Pro Trp Lys Ala

TTT

Phe 110 ATG GCA CAA GTT Met Aia Gin Vai

GAA

Glu 115 GTA CTG ATA GAT Val Leu le Asp AAG AAA Lys Lys 120 ATA GAG GAG le Glu Giu CAA AAT AAT Gin Asn Asn 140

TAT

Tyr 125 GCT AAA AGT AAA Ala Lys Ser Lys

GCT

Ala 130 CTT GCA GAG TTA Leu Ala Giu Leu CAG GGT CTT Gin Gly Leu 135 TGG AAG AAA Trp Lys Lys TTC GAA GAT TAT Phe Giu Asp Tyr AAT GCG TTA AAT Asn Ala Leu Asn

TCC

S er 150 ACA CCT Thr Pro 155 TTA AGT TTG CGA Leu Ser Leu Arg

AGT

Ser 160 AAA AGA AGC CAA Lys Arg Ser Gin GAT CGA ATA AGG Asp Arg Ile Arg 165 TCZ ATG CCG TCA Ser Met Pro Ser

GAA

Glu

TTT

Phe 185

CTT

Leu 170 TTT TCT CAA GCA Phe Ser Gin Ala

GAA

Giu 175 AGT CAT TTT CGT Ser His Phe Arg GCA GTT TCC AAA Ala Val Ser Lys TTC GAA GTG CTG TTT CTA Phe Glu Val Leu Phe Leu 190 195 CCA ACA TAT GCA Pro Thr Tyr Ala CAA GCT Gin Ala 200 WO 92/13954d PC/US92/000040 68 GCA AAT ACA Ala Asn Thr GAA TGG GGA Glu Trp Gly 220

CAT

.His 205 TTA TTG CTA TTA Leu Leu Leu Leu

AAA

Lys 210 GAT GCT CAA GTT Asp Ala Gin Val TTT GGA GAA Phe Giy Glu 215 CAT AGA CAA His Arg Gin 794 842 TAT TCT TCA GAA Tyr Ser Ser Glu

GAT

Asp 225 GTT GCT GAA TTT Val Ala Giu Phe

TAT

Tyr 230 TTA AAA Leu Lys 235 CTT ACG CAA CAA Leu Thr Gin Gin

TAC

Tyr 240 ACT GAC CAT TGT Thr Asp His Cys GTC AAT TGG TAT AAT Val Asn Trp Tyr Asn 245 GA2 CCA TGG GTC AAA Asp Ala Trp Val Lys

GTT

Val 250 GGA TTA AAT GGT Gly Leu Asri Gly

TTA

Leu 255 AGA GGT TCA ACT Arg Gly Ser Thr

TAT

Tyr 260 TTT AAC (jT TTT Phe Asn Arg Phe AGA GAA ATG ACT Arg Giu Met Thr

TTA

Leu 275 ACT GTA TTA GAT Thr Val Leu Asp CTA ATT Leu Ile 280 986 GTA CTT TTC Val Leu Phe &CA GAA CTA ITr Glu Leu 300

CCA

Pro 285 TTT TAT GAT GTT Phe Tyr Asp Vai

CGG

Arg 290 TTA TAC TCA AAA Leu Tyr Ser Lys GGT GTT AAA Gly Val Lys 295 TCA CTC AAT Ser Leu Asn ACA AGA GAC ATT Thr Arg Asp Ile

TTT

Phe 305 ACG GAT CCA ATT Thr Asp Pro Ile

TTT

Phe 310 ACT CTT Thr Leu 315 CAG GAG TAT GGA Gin Giu Tyr Gly

CCA

Pro 320 ACT TTT TTG AGT Thr Phe Leu Ser ATA GAA AAC TCT ATT Ile Giu Asn Ser Ile 325 ATT GAA TTT CAT ACG Ile Glu Phe His Thr

CGA

Arg 330 AAA CCT CAT TTA Lys Pro His Leu

TTT

Phe 335 GAT TAT TA CAG Asp Tyr Leu Gin

GGT

Gly 340 CGT CTT CAA CCT Arg Leu Gin Pro

GGT

Giy 350 TAC TCT GGG AAA Tyr Ser Gly Lys

GAT

Asp 355 TCT TTC AAT TAT Ser Phe Asn Tyr TGG TCT Trp Ser 360 1034 1082 1130 1178 1226 1274 1322 1370 1418 1466 GGT AAT TAT Gly Asn Tyr ACT TCC CCA Thr Ser Pro 380

GTA

Val 365 GAA A' AGA CCT Glu Thr Arg Pro

AGT

Ser 370 ATA GGA TCT AGT Ile Giy Ser Ser TTT TAT GGA GAT Phe Tyr Gly Asp

AAA

Lys 385.

TCT ACT GAA CCT Ser Thr Glu Pro

GTA

Val 390 AAG ACA ATT Lys Thr lie 375 CAA AAG TTA Gin Lys Leu ACA GAC GTA Thr Asp Val AGC TTT Ser Phe 395 GAT GGA CAA AAA Asp Gly Gin Lys GTT TAT Val Tyr 400 CGA ACT ATA Arg Thr le GCT AAT Ala-Asn 405

GCG

Ala 410 GCT TGG CCG AAT Ala Trp Pro Agn AAG ATA TAT TTT Lys Ile Tyr Phe GTT ACG AAA GTT Val Thr Lys Val TTT AGT CAA TAT Phe Ser Gln Tyr

GAT

Asp 430 GAT CAA AAA AAT Asp Gln Lys Asn GAA ACT AGT ACA CAA ACA TAT Glu Thr Ser Thr Gin Thr Tvr 43t 440 WO 92/13954 WO 9213954PC/US92/00040 69 GAT TCA AAA Asp Ser Lys CAA TTA CCA Gin Leu Pro 460

AGA

Arg 445 AAC AAT GGC CAT Asn Asn Gly His

GTA

Val 450 GGT GCA CAG GAT Gly Ala Gin Asp TCT ATT GAC Ser Ile Asp 455 GCA TAT AGT Ala Tyr Ser CCA GAA ACA ACA Pro Glu Thr Thr

GAT

Asp 465 GAA CCA CTT G2?JA Giu Pro Leu 07u~

AAA

Lys 470 CAT CAG His Gin 475 CTT AAT TAC GCG Leu Asn Tyr Ala

GAA

Giu 480 TGT TTC TTA ATG Cys Phe Leu Met CAG GAC CGT CGT Gin Asp Arg Arg 485 GTA GAC TTT TTT Vai Asp Phe Phe

GGA

-Gly

AAT

Asn 505

ACA

Thr 490 ATT CCA TTT TTT Ile Pro Phe Phe

ACT

Thr 495 TGG ACA CAT AGA Trp Thr His Arg

AGT

Ser 500 ACA ATT GAT GCT Thr Ile Asp Ala GAA AAG ATT ACT CAA CTT CCA GTA GTG AAA GCA TAT Giu Lys Ile Thr Gin Leu Pro Val Vai Lys Aia Tyr 510 515 520 GCC TTG TCT Ala Leu Ser GGA AAT TTA Giy Asn Leu 540

TCA

Ser 525 GGT GCT TCC ATT Gly Ala Ser Ile

ATT

Ile 530 GAA GGT CCA GGA Giu Gly Pro Gly TITC ACA GGA Phe Thr Gly 535 GCT AAA TTT Ala Lys Phe 1514 1562 1610 1658 1706 1754 1802 1850 1898 1946 1994 2042 CTA TTC CTA AAA Leu Phe Leu Lys

GAA

Giu 545 TCT AGT AAT TCA Ser Ser Asn Ser

ATT

Ile 550 AAA GTT Lys Val 555 ACA TTA AAT TCA Thr Leu Asn Ser

GCA

Ala 560 GCC TTG TTA CAA Ala Leu Leu Gin

CGA

Arg 565 TAT CGT GTA AGA Tyr Arg Val Arg

ATA

Ile 570 CGC TAT GCT TCT Arg Tyr Ala Ser

ACC

Thr 575 ACT AAC TTA CGA Thr Asn Leu Arg

CTT

Leu 580 TTT GTG CAA AAT Phe Val Gin Asn

TCA

Ser 585 AAC AAT GAT TTT Asn Asn Asp Phe

ATT

Ile 590 GTC ATC TAC ATT Val Ile Tyr Ile

AAT

Asn 5195 AAA ACT ATG AAT Lys Thr Met Asn ATA GAT Ile Asp 600 GAT GAT TTA Asp Asp Leu ATG GGG TTC Met Gly Phe 620 TAT CAA ACA TTT Tyr Gin Thr Phe CTC GCA ACT ACT Leu Ala Thr Thr AAT TCT AAT Asn Ser Asn 615 GCA GAA TCT Ala Giu Ser TCG GGT GAT ACG Ser Gly Asp Thr GAA CTT ATA ATA Giu Leu Ile Ile

GGA

Gly 630 TTC GTT TCT AAT GAA AAA ATC TAT ATA GAT AAG ATA GAA TTT A'rC CCA Phe Val Ser Asn Giu Lys Ile Tyr Ile Asp Lys Ile Giu Phe Ile Pro 635 *640 645 GTA CAA TTG TAAGGAGATT TTGAAATGTA GGGCGATGGT CAAAATGAAA Val Gin Leu 650 GAATAGGAAG GTGAATTTTG ATGGTTAGGA AAGATTCTTT TAAGAAAAGC AACATGGAAA AGTATACAGT ACAAAT,' 2LTA GAAATAAAAT TTATTAACAC AGGGGAAGAT GGTAAACCAG 2090 2139 2199 2259 WO 92/13954 PCT/US9/00o4 AACCGTATGG TTATATTGAC TTTTATTATC AACCTGCTCC TAACCTGAGA GAAGAAAAAG 2319 TAAGAATTTG GGAAGAGAAA, AATrAGTAGCT CTCCACCTTC AATAGAAGTT ATTACGGGGC 2379 TAACTTTTAA TATCATGGCT ACTTCACTTA GCCGATTATG TTTTGAAGGT T 2430 INFOR~MATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 652. amino~ acids TYPE: amnino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Asn Pro Asn Asn Arg Ser Giu His Asp Thr Ile Lys Val Thr Pro 1 5 10 Asn Ser Giu Leu Pro Thr Asn His Asn Gin Tyr Pro Leu Ala Asp Asn 25 Pro Asn Ser Thr Leu Giu Giu Leu Asn Tyr Lys Giu Phe Leu Arg Met 40 Thr Giu Asp Ser Ser Thr Giu Val Leu Asp Asn Ser Thr Val Lys Asp 55 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gin Ile Leu Gly Val Val 70 75 Gly Val Pro The Ala Gly Ala Leu Thr Ser Phe Tyr Gin Ser Phe Leu 90 Asp Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gin Val Giu. Vai Leu Ile Asp Lys Lys Ile Giu Giu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala Giu Leu Gin Gly Leu Gin Asn Asn Phe Giu Asp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 Lys Arg Ser Gin Asp Arg Ile Arg Giu Leu Phe Ser Gin Ala Giu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gin Ala Ala Asn Thr His Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gin Val Phe Gly Glu Giu Trp Gly Tyr Ser Ser Giu 210 215 220 WO 92/13954 WO 9213954PC1T/US92/00040 71 Asp 225 Val Ala Giu Phe Tyr His Arg Gin Leu Lys Leu Thr Gin Gin Tyr 230 235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Vai Giy Leu Asn Gly Leu Arg 255 Gly Met Val Plie 305 Thr Tyr Gly S er Thr Arg 290 Thr Phe Leu Lys 245 Thr Tyr Asp 260 Leu Thr Val 275 Leu Tyr Ser Asp Pro Ile Leu Ser Ile 325 Gin Gly Ile 340 Asp Ser Phe 355 250 Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Giu 270 Pro Ser Ile Gly Lys 385 Tyr Ile Lys His Asp 465 Cys Thr 370 Ser Arg Tyr Asn Vai 450 Giu Phe His Thr Thr Phe Giu 435 Giy Pro Leu Arg Glu Ile Gly 420 Thr Ala Lei: Mel Se~ Ser Pro Ala 405 Val Ser Gir IGil.

Gii 48~ Va 0 Leu Asp Leu 280 Lys Gly Val 295 Phe Ser Leu 310 Glu Asn Ser Giu Phe His Asn Tyr Try 360 Ser Lys Thi 375 Val Gin Ly~ 390 Asn Thr Asj Thr Lys Va Thr Gin Th: 44 Asp Ser 11 455 1Lys Aia Ty 470 i Asp Arg Ar 5 L Asp Phe Ph Lys 'J Asn Ile Thr 345 Ser Ile Leu SVai 1 Asp 425 r Tyr 0 e Asp r Ser g Giy .e Asn 505 Lhr Lhr krg 330 Arg Gly Thr S er Ala 410 Phe Asp Gir HiE ThJ 49( Th: Giu I Leu C 315 Lys Leu Asn Ser Phe 395 Ala Ser Ser 1Leu Gin 475 Ile 0*li ~00 ;1n ?ro 31n Tyr Pro 380 Asp Trp Gin Lys Pro 460 Leu 265 Ile Val Leu Phe Pro Phe Tyr Asp rhr .31u His Pro Val 365 Phe Gly Pro Tyr Arg 445 Prc Asr Arg 7 TyrC Leu Gly 350 Giu Tyr Gin Asn Asp 430 Asn Giu Tyr a Phe a Giu ~sp ;ly he 335 Tyr I'hr Gly Lys Giy 415 Asp Asn Thy Ai2 Th2 49~ Ly~ Ile Pro 320 Asp S er Arg Asp Val 400 Lys Gin Gly Thr Giu 480 Trp Ile Pro Asp PhE Al~ Thr Gin Leu Pro Val Vai Lys Ala Tyr Ala Leu Ser Ser 510 Gly Ala Ser 515 520 525 Ile Ile Giu Gly Pro Gly 530 Phe Thr Gly Gly Asn 535 Leu Leu Phe Leu Lys 540 WO 92113954 WO 9213954PCT/US92/00040 72 Giu 545 Ala Asn Tyr Phe Asn 625 Tyr Ser Leu Leu Ile Asp 610 Giu Ile Ser Leu Arg Asn 595 Leu Leu Asp Asn Gin Leu 580 Lys Ala Ile Lys Ser Ile 550 Arg Tyr 565 Phe Val Thr Met Thr Thr Ile Gly 630 Ile Giu 645 Ala Arg Gin Asn Asn 615 Ala Phe Lys Val Asn Ile 600 Ser Giu Ie Phe Arg Ser 585 Asp Asn Ser Pro Lys Val 555 Ile Arg 570 Asn Asn Asp Asp Met Giy Phe Val 635 Val Gin 650 Thr Tyr Asp Leu Phe 620 Ser Leu Leu Ala Phe Thr 605 Ser Asn Asn Ser Ile 590 Tyr Gly Glu Ser Thr 575 Val Gin Asp Lys Ala 560 Thr Ile Thr Thr Ile 640 WO 92/13954 73 WO 9213954PCr/US92/00040 PCT Applicant's Guide Volum I -'Annex mi ANNEX M3 lntsmstionml Application No: PCT/ I MICROORGANISMS See Attachment Oei Sbee 1i caseecfn with the .nkrorgz 1i m e d to en 104,1111 110,0 of the descrptienI A. 111HURIPIWATION OF DIPOSIT I Futhar doeeolt we Wenoloald on an additional ehee M.

Nor" 4 dpeWrl islte American Research Culture Collection (NRRL) Addrose W1 depnftq loiotdon (nclding Peatea eft ontrg 4 1815 N. University Street Peoria, Illinois 61604 United States of America DOW of Aecesade Numbers See Attachment ISee Attachment SL ADDITIONAL INDICAIONS'I (leave blank III hot applicable). This Informatilon io continued enas eparate, atched sheet 0 In respect of those designations in which a European patent is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample (Rule 28(4) EPC).

C. D3SSMATI STATSIS FOR WHICH INDICATIONS ARIE MAUREI (it the Indicats are hot lot all 11800M8at1 Stats) 0. SEIPARATII FURNISHING OF INDICATIONS'I (leave blank it ,ot applicable) !he W dcatiens listed below wllt be subitted to the International Bureau later'a (Specify the veneral nature of V4" Indicationa Acceeete Number *I Depesit") 9. Q This ahee wes received with the International application when filod (to be checked by the receivingp Office) (Authorized Officer) 0 The date of recsipt (irom, the applicant) by the intornatonal Bureau to waes (Authorized Officer) WO 92/13954 74 PCT/US92/00040 ATTACHMENT TO FORM PCT/RO/134 CONTINUATION OF 1 MICROORGANISM" BOX: page 10, lines 3-11 page 26, lines 21-22 page 28, lines 10-12 page 42, lines 22-24 page 47, lines 26-27 CONTINUATION4 OF IDE1NTIFICATION OF DEPOSIT BOX A: The following microorganisms were deposited in the depository institution listed in Box A on the dates listed below: Bacterial Strain NURL Acession No. Date of Deposit thuringiensis thuringiensis thuringiernsis thuringierisis ,thuringiensis thuringiensis coli EG7236 EG2 158 HD73-26 EG2838 EG5 144 EG7237 EG5145 B-18213 B-18508 B-18603 B-18655 B-18736 B-18920 B-18 662 29 April 1987 12 June 1989 8 February 1990 22 May 1990 17 October 1990 21 November 1991 6 June 1990

Claims

1. A purified and isolated cryIIIC(b) gene characterized in that its nucleotide base sequence encodes the amino acid sequence illustrated in Figure 1 (SEQ ID NO:2).

2. A purified and isolated cryIIIC(b) gene according to claim 1 further characterized in that the gene has a coding region extending from nucleotide bases 144 to 2099 in the nucleotide base sequence illustrated in Figure 1 (SEQ ID NO:l).

3. A recombinant plasmid containing the gene of claim 1 or claim 2.

4. A coleopteran-toxic protein produced by the gene of claim 1 or claim 2.

5. A biologically pure culture of a bacterium transformed with the recombinant plasmid of claim 3.

6. The bacterium of claim 5 further characterized in that the bacterium is Bacillus thuringiensis.

7. The Bacillus thuringiensis bacterium of claim 6 deposited with the NRRL with accession number NRRL B-

18736.

8. An insecticide composition characterized in that the composition comprises the protein of claim 4 and an agriculturally acceptable carrier.

9. An insecticide composition characterized in that the composition comprises the bacterium of claim 5, a coleopteran-toxic protein produced by such bacterium, and an agriculturally acceptable carrier.

10. A plant characterized in that the plant is transformed with the gene of claim 1 or claim 2.

11. The cryIIIC(b) gene of claim 2 further characterized in that the gene or a portion thereof is labelled for use as a hybridization probe.

12. A biologically pure culture of a Bacillus thuringiensis bacterium deposited with the NRRL with accession number NRRL B-18655.

13. A coleopteran-toxic protein characteristic of that made by the Bacillus thuringiensis bacterium of claim 12 and having the amino acid sequence illustrated in Figure 1 (SEQ ID NO:2).

14. An insecticide composition characterized in that the composition comprises the coleopteran-toxic protein of claim 13, in combination with an agriculturally acceptable carrier. The insecticide composition of claim 14 further characterized in that the coleopteran-toxic protein is associated with a Bacillus thuringiensis bacterium which has produced such protein. 16. A method of controlling coleopteran insects characterized by applying to a host plant for such insects an insecticidally effective amount of the coleopteran- toxic protein of claim 4. 17. The method of claim 16 further characterized in that the coleopteran-toxic protein is associated with a Bacillus thuringiensis bacterium which has produced such protein. 18. The method according to claim 16 further characterized in that the insects are selected from the group consisting of corn rootworms, Mexican bean beetles and Japanese beetle larvae. 19. A method of controlling coleopteran insects characterized by applying to a host plant for such insects an insecticidally effective amount of the coleopteran- toxic protein of claim 13. The method of claim 19 further characterized in that the coleopteran-toxic protein is associated with a Bacillus thuringiensis bacterium which has produced such protein. 21. The method of claim 19 further characterized in that the insects are selected from the group consisting of corn rootworms, Mexican bean beetles and Japanese beetle larvae. 22. A biologically pure culture of a Bacillus thuringiensis bacterium deposited with the NRRL with accession number NRRL B-18920. 23. An insecticide composition characterised in that the composition comprises the coleopteran-toxic protein obtainable from the Bacillus thuringiensis bacterium of claim 12 or claim 22, in combination with an agriculturally acceptable carrier. 24. A purified and isolated crylIIC(b) gene substantially as hereinbefore described with reference to any one of the Examples. A coleopteran-toxic protein produced by the gene of claim 24. 26. A recombinant plasmid containing the gene of claim 24. 27. A biologically pure culture of a bacterium transformed with the recombinant plasmid of claim 26. 1o 28. An insecticide composition substantially as hereinbefore described with reference to any one of the Examples. 29. A plant characterised in that the plant is transformed with the gene of claim 24. Dated 3 August, 1993 is Ecogen Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON :jf nj .Iwb\d075:GsA L 1 of 1