Isolates and Genes Encoding

Europäisches Patentamt *EP000612218B1* (19) European Patent Office Office européen des brevets (11) EP 0 612 218 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.7: A01N 63/00, A01N 63/02, of the grant of the patent: C12N 15/32, C12N 1/20 04.10.2001 Bulletin 2001/40 (86) International application number: (21) Application number: 92925087.6 PCT/US92/09510 (22) Date of filing: 06.11.1992 (87) International publication number: WO 93/08693 (13.05.1993 Gazette 1993/12) (54) NOVEL COLEOPTERAN-ACTIVE $i(BACILLUS THURINGIENSIS) ISOLATES AND GENES ENCODING COLEOPTERAN-ACTIVE TOXINS NEUES KOLEOPTERENAKTIVES ISOLAT VON BACILLUS THURINGIENSIS UND GENE DIE FÜR KOLEOPTERENAKTIVE TOXINE KODIEREN NOUVEAUX ISOLANTS DE $i(BACILLUS THURINGIENSIS) ACTIFS CONTRE LES COLEOPTERES, ET GENES CODANT LES TOXINES ACTIVES CONTRE LES COLEOPTERES (84) Designated Contracting States: • FU, Jenny, M. AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL San Diego, CA 92130 (US) SE (74) Representative: Perry, Robert Edward et al (30) Priority: 06.11.1991 US 788638 GILL JENNINGS & EVERY Broadgate House (43) Date of publication of application: 7 Eldon Street 31.08.1994 Bulletin 1994/35 London EC2M 7LH (GB) (73) Proprietor: MYCOGEN CORPORATION (56) References cited: San Diego, California 92121 (US) EP-A- 0 382 990 EP-A- 0 501 650 WO-A-90/13651 WO-A-91/16433 (72) Inventors: US-A- 4 996 155 • PAYNE, Jewel, M. San Diego, CA 92126 (US) Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 612 218 B1 Printed by Jouve, 75001 PARIS (FR) EP 0 612 218 B1 Description Background of the Invention 5 [0001] The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These often appear microscopically as distinctively shaped crystals. The proteins are highly toxic to pests and specific in their activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products produced and approved. In addition, with the use of genetic engineering techniques, new approaches for delivering B.t. endotoxins to agricultural environments are under development, includ- 10 ing the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner, F.H., L. Kim [1988] TIBTECH 6:S4-S7). Thus, isolated B. t. endotoxin genes are becoming commercially valuable. [0002] Bacillus thuringiensis produces a proteinaceous paraspore or crystal which is toxic upon ingestion by a sus- ceptible insect host. Over most of the past 30 years, commercial use of B.t. pesticides has been largely restricted to 15 a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of B. thuringiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces a crystal called a delta endotoxin which is toxic to the larvae of a number of lepidopteran insects. [0003] In recent years, however, investigators have discovered B.t. pesticides with specificities for a much broader 20 range of pests. For example, other species of B.t., namely israelensis and san diego (a.k.a. B.t. tenebrionis), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F.H. [1989] "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms," in Controlled Delivery of Crop Protection Agents, R.M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255). See also Couch, T.L. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis," Developments in Industrial Micro- 25 biology 22:61-76; Beegle, C.C., (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in Indus- trial Microbiology 20:97-104. Krieg, A., A.M. Huger, G.A. Langenbruch, W. Schnetter (1983) Z. ang. Ent. 96:500-508, describe a B.t. isolate named Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and Agelastica alni. [0004] Recently, many new subspecies of B.t. have been identified, and many genes responsible for active δ-endo- 30 toxin proteins have been isolated (Höfte, H., H.R. Whiteley [1989] Microbiological Reviews 52(2):242-255). Höfte and Whiteley classified 42 B.t. crystal protein genes into 14 distinct genes, grouped into 4 major classes based on amino- acid sequence and host range. The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to protozoan path- ogens, animal-parasitic liver flukes (Trematoda), or mites (Acari) has broadened the potential B.t. product spectrum 35 even further (see Feitelson, J.S., J. Payne, L. Kim [1992] Bio/Technology 10:271-275). With activities against unique targets, these novel strains retain their very high biological specificity; nontarget organisms remain unaffected. The availability of a large number of diverse B.t. toxins may also enable farmers to adopt product-use strategies that min- imize the risk that B.t.-resistant pests will arise. [0005] The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in the pub- 40 lished literature (see, for example, Schnepf, H.E., H.R. Whitely [1981] Proc. Natl. Acad. Sci. USA 78:2893-2897). U. S. Patent 4,448,885 and U.S. Patent 4,467,036 both disclose the expression of B.t. crystal protein in E. coli. U.S. Patent 4,853,331 discloses B. thuringiensis strain san diego (a.k.a. B.t. tenebrionis) which can be used to control coleopteran pests in various environments. [0006] EP-A-0 382 990 discloses a coleopteran-active crystal toxin. Further B.t. toxins are disclosed in WO-A- 45 90/13651, WO-A-91/16433 and US-A-4 996 155. Brief Summary of the Invention [0007] The subject invention concerns the discovery that certain known and publicly available strains of Bacillus 50 thuringiensis (B.t.) are active against coleopteran pests. This is a surprising discovery since these B.t. microbes were not known to have any insecticidal properties. [0008] The microbes of the subject invention were obtained from the Howard Dalmage collection held by the NRRL culture repository in Peoria, Illinois and are designated B.t. HD511 and B.t. HD867. These microbes, and variants of these microbes, as well as genes and toxins obtainable therefrom, can be used to control coleopteran pests. The 55 procedures for using these microbes are similar to known procedures for using B.t. microbes to control coleopteran pests. [0009] Further, the invention also includes the treatment of substantially intact B.t. cells, and recombinant cells con- taining a gene of the invention, to prolong the pesticidal activity when the substantially intact cells are applied to the 2 EP 0 612 218 B1 environment of a target pest. Such treatment can be by chemical or physical means, or a combination of chemical or physical means, so long as the technique does not deleteriously affect the properties of the pesticide, nor diminish the cellular capability in protecting the pesticide. The treated cell acts as a protective coating for the pesticidal toxin. The toxin becomes available to act as such upon ingestion by a target insect. 5 [0010] Disclosed herein are specific toxins, and nucleotide sequences encoding these toxins, obtainable from the exemplified isolates. Advantageously, these nucleotide sequences can be used to transform other microbes or plants to create insecticidal compositions and plants. Brief Description of the Sequences 10 [0011] SEQ ID NO. 1 is the nucleotide sequence encoding toxin HD511. [0012] SEQ ID NO. 2 is the amino acid sequence of toxin HD511. [0013] SEQ ID NO. 3 is the nucleotide sequence encoding toxin HD867. [0014] SEQ ID NO. 4 is the amino acid sequence of toxin HD867. 15 Detailed Disclosure of the Invention [0015] A summary of the characteristics of the B. thuringiensis microbes of the subject invention is shown in Table 1. 20 Table 1. A comparison of the novel coleopteran-active strains with B.t.s.d. Strain Crystal Type Approx. Molecular Weight of Protein* Serotype HD511 Bipyramid 130, 143 15, dakota 25 HD867 Bipyramid 130 18, kumamotoensis * as shown on a standard polyacrylamide gel. [0016] The cultures disclosed in this application are on deposit in the Agricultural Research Service Patent Culture 30 Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA. [0017] In a preferred embodiment, the nucleotide sequence information provided herein can be used to make primers which, when using standard PCR procedures, can be used to obtain the desirable genes from the disclosed isolates. These procedures are well known and commonly used in this art. Alternatively, synthetic genes, or portions thereof, can be made using a "gene machine" and the sequence information provided herein. 35 [0018] The subject invention pertains not only to the specific genes and toxins exemplified herein, but also to genes and toxins obtainable from variants of the disclosed isolates. These variants would have essentially the same coleopteran activity as the exemplified isolates. Furthermore, using the DNA and amino acid sequence provided herein, a person skilled in the art could readily construct fragments or mutations of the genes and toxins disclosed herein.

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