Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1998-12-22
2001-04-03
Nelson, Amy J. (Department: 1638)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
Reexamination Certificate
active
06210953
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel
Bacillus thuringiensis
strains, to novel toxin genes, to the proteins encoded by the genes, and to the use of genes and proteins.
BACKGROUND OF THE INVENTION
Bacillus thuringiensis
is a gram-positive soil bacterium characterized by its ability to produce crystalline inclusions during sporulation. The crystalline inclusions can, in some subspecies, account for 20 to 30 percent of the dry weight of the sporulated cell and may be composed of more than one protein. Crystals are composed primarily of a single polypeptide, a protoxin, which may also be a component of the spore coat. The protoxin genes are located mainly on large plasmids, although chromosomally encoded endotoxins have been reported.
The crystal proteins exhibit a highly specific insecticidal activity. Many
B. thuringiensis
strains with different insect host spectra have been identified. They are classified into different serotypes or subspecies based on their flagellar antigens.
The protoxin does not exhibit its insecticidal activity until after oral intake of the crystalline body. The crystal is dissolved in the intestinal juice of the target insects. In most cases, the actual target component (toxin) is released from the protoxin as a result of proteolytic cleavage caused by the action of proteases from the digestive tract of the insects. The activated toxin interacts with the midgut epithelium cells of susceptible insects.
Electrophysiological and biochemical evidence suggests that the toxins generate pores in the cell membrane, thus disrupting the osmotic balance. Consequently, the cells swell and lyse. For several
B. thuringensis
toxins, specific high-affinity binding sites have been demonstrated to exist on the midgut epithelium of susceptible insects. Nucleotide sequences have been recorded for a large number of
B. thuringiensis
(Bt) crystal protein genes. Several sequences are nearly identical, and have been designated as variations of the same gene. The crystal protein (Cry) genes specify a family of related insecticidal proteins. The genes are divided into major classes and subclasses characterized by both the structural similarities and the insecticidal spectra of the encoded proteins. The classification, explained by Höfte and Whiteley (1989) Microbiol. Rev., 53:242-255, placed the known insecticidal crystal proteins into four major classes. The four major classes were Lepidoptera-specific (I), Lepidoptera- and Diptera-specific (II), Coleoptera-specific (III), and Diptera-specific (IV) genes. Additional classes have since been added.
The Cry1 genes are undoubtedly the best-studied crystal proteins. The Cry1 proteins are typically produced as 130 to 140 kDa protoxin proteins which are proteolitically cleaved to produce active toxin proteins about 60 to 70 Kda. The active portion or toxic domain is localized in the N-terminal half of the protoxin. Six groups of Cry1 proteins were known in 1989 when the Höfte and Whiteley article was published. These groups were designated IA(a), IA(b), IA(c), IB, IC, and ID. Since 1989, additional proteins have been discovered and classified as Cry1E, Cry1F, Cry1G, Cry1H, and Cry1X.
The spectrum of insecticidal activity of an individual protoxin from Bt tends to be quite narrow. That is, a given crystal protein is active against only a few insects. None of the crystal proteins active against Coleopteran larvae such as colorado potato beetle (
Leptinotarsa decemlineata
) or yellow mealworm (
Tenebrio molitor
) have demonstrated significant effects on members of the genous Diabrotica particularly
D. virgifera virgifera
, the western corn rootworm (WCRW) or D.
longicornis barberi
, the northern corn rootworm.
Insect pests are a major factor in the loss of the world's commercially important agricultural crops. Broad spectrum chemical pesticides have been used extensively to control or eradicate pests of agricultural importance. However, there is substantial interest in developing effective alternative pesticides.
Microbial pesticides have played an important role as alternatives to chemical pest control. The most extensively used microbial product is based on the bacterium
Bacillus thuringiensis
. However, as noted above, the majority of Bt strains have a narrow range of activity. There is therefore needed microbial strains with a broad range of insecticidal activity for use as broad spectrum insecticides and as a source for additional toxin genes and proteins.
SUMMARY OF THE INVENTION
A broad spectrum Bacillus strain is provided. The strain is active against insects from at least the orders Lepidoptera, Diptera, and Coleoptera. Additionally, the strain is active against nematodes, and rootworms. Disclosed in this invention is the isolation and partial purification of the crystal protein complex from this strain. The crystal protein complex is demonstrated to be active against rootworms and other pests. Genes, proteins, and their use, as well as the use of the strain are provided. The complete nucleotide sequence of a Cry5-like gene herein designated as Cry1I which was isolated from this strain is also provided.
The methods and compositions of the invention may be used in a variety of systems for controlling pests, particularly plant pests.
REFERENCES:
patent: 4948734 (1990-08-01), Edwards et al.
patent: 5080897 (1992-01-01), Gonzalez et al.
patent: 0 537 105 A1 (1993-04-01), None
patent: 0537105 (1993-04-01), None
patent: WO 95/02693 (1995-01-01), None
patent: WO 97/12980 (1997-04-01), None
Chestukhina GG, et al., “Bacillus thuringiensisssp. galleriae simultaneously produces two gamma-endotoxins differing strongly in primary structure and entomocidal activity.” FEBS. Lett. 232: 249-251, May 1988.*
Yan B, et al. “Protein components and toxicity of delta-endotoxin crystals fromBacillus thuringiensisTm 13-14.” Kunchong Xuebao 38: 138-145 (Abstract only), 1995.*
Schnepf HE, et al. (1995) “Bacillus ThuringiensosToxins: Regulation, Activities, and Structural Diversity”Curr. Opinion Biotech.6: 305-312.
Shin, et al. “Distribution ofcryV-Type Insectiial Protein GenesBacillus thuringiensisand Cloning ofcryv-TypeGenes from Bacillus thuringiensis subsp.kurstakiandBacillus thuringiensissubsp.entomocidus”,Applied and Enviromental Microbiology, Jun. 1995, p. 2402-2407, vol. 61, No. 6.
Chambers, et al., “Isolation and Characterization of a Novel Insecticdial Crystal Protein Gene fromBacillus thuringiensissubsp.aizawai” ,Journal of Bacteriology, Jul. 1991, p. 3966-3976, vol. 173, No. 13.
Lee, et al., “Diversity of Protein Inclusion Bodies and Identification of Mosquitocidal Protein inBacillus thuringiensissubsp.Israelensis”,Biochemical and Biophysical Research Communications, Jan. 31, 1985, p. 953-960, vol. 126, No. 2.
Shin et al. “Distribution of CryV-type Insectcdial Protein Genes inBacillus Thuringiensisand cloning of CryV-type Genes fromBacillus Thuringiensissubp. Kurstaki andBacillus Thuringiensissubsp. Entomocidus”,Applied and Environmental Microbiology61:2402-2407 (1995).
Chambers et al. Isolation and Characterization of a Novel Insecticdial Crystal Protein Gene fromBacillus Thuringiensissubsp. Aizawai,Journal of Bacteriology173:3966-3976 (1991).
Lee et al. Diversity of Protein Inclusion Bodies and Idenfication of Mosquitocidal Protein inBacillus Thuringiensissubsp. israelensis ,Biochemical and Biophysical Research Communications12:953-960 (1985).
Bulla, Jr. Lee A.
Madkour Magdy A.
Osman Yehia A.
Alston & Bird LLP
Nelson Amy J.
University of Wyoming
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