US 5,356,623 AGrant
Bacillus Thuringiensis cryET1 Toxin Gene and Protein Toxic to Lepidopteran Insects
Issue Date:1994-10-18
•12 Claims
•13 Drawing Sheets
Abstract
A Bacillus thuringiensis strain isolate, designated EG5092, exhibits insecticidal activity against lepidopteran insects. A purified and isolated novel cryET1 toxin gene product from B.t. strain EG5092 exhibits specific insecticidal activity against Plutella xylostella (Diamondback moth). The cryET1 gene (SEQ ID NO:1) has a nucleotide base sequence illustrated in FIG. 1 and produces a CryET1 gene product (SEQ ID NO:2) having a deduced amino acid sequence illustrated in FIG. 1.
Metadata
Assignee
- Ecogen Inc.
Inventors
- Michael A. von Tersch
- Jose M. Gonzalez
Application Information
Application Number:US 0323640
Filing Date:1993-03-17
Priority Date:1993-03-17
Art Unit:184
Classifications
IPC:
C12N 121C12N 122C12N 1532A01N 6300
Field of Search:
53643580042423.71320.1;252.1;252.3;252.31;172.3;69.120593 L;93 A
Patent Drawings (13 sheets)
Description
FIELD OF THE INVENTION
The present invention relates to a lepidopteran-toxic protein and the gene coding therefor. In particular, the present invention is directed to a gene designated as cryET1 (SEQ ID NO:1) and its protein designated as CryET1 (SEQ ID NO:2).
BACKGROUND OF THE INVENTION
B. thuringiensis (commonly known as B.t.) is a Gram-positive soil bacterium that often produces cellular inclusions during sporulation which are specifically toxic to certain orders and species of insects. Many different strains of B.t. have been shown to produce these inclusions of insecticidal crystal protein (ICP). 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 non-targeted organisms.
B. thuringiensis ICP toxins are active in the insect only after ingestion. After ingestion by an insect, the alkaline pH and proteolytic enzymes in the mid-gut solubilize the crystal allowing the release of the toxic components. These toxic components disrupt the mid-gut cells resulting in cessation of feeding and, eventually, death of the insect. B.t. has proven to be an effective and environmentally safe insecticide in dealing with various insect pests.
A number of genes encoding crystal proteins have been cloned from many strains of B.t. A good overview is set forth in H. Hofte and H. R. Whiteley, Microbiol. Rev., 53, pp. 242-255 (1989), hereinafter "Hofte and Whiteley (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 nucleotide sequences of ICP genes responsible for a given crystal phenotype and active against the same insect order are generally more related than are the nucleotide sequences of B.t. genes encoding delta-endotoxin proteins active against different orders of insects. Hofte and Whiteley (1989) defines an ordered classification of genes encoding B.t. delta-endotoxin proteins based on homology of delta-endotoxin amino acid sequences, as well as similarities in insecticidal activity; a subranking has also been established based upon further refinement of sequence relationship. As noted by Hofte and Whiteley (1989), the majority of insecticidal B.t. strains are active against insects of the order Lepidoptera, i.e., caterpillar insects. Insecticidal crystal proteins specifically active against Lepidoptera have been designated CryI proteins. These ICPs are encoded by cryI genes. Other B.t. strains produce different classes of crystal proteins, e.g., CryII proteins are active against lepidopteran and (for CryIIA) dipteran insects; CryIII proteins are insecticidal to insects of the order Coleoptera, i.e., beetles; and CryIV proteins are active against insects of the order Diptera, i.e., flies and mosquitoes. A compilation of the amino acid identities for several CryI proteins as well as CryII, CryIII and CryIV proteins has been determined in Hodgman and Ellar, J. DNA Sequencing and Mapping, 1, pp. 97-106 (1990).
The CryI family of ICPs contains the largest number of known toxin genes derived from B.t., as evidenced by the survey in Hofte and Whiteley (1989) and by subsequent reports of CryI-type ICPs.
Schnepf et al., J. Biol. Chem., 260, pp. 6264-6272 (1985), reported the complete nucleotide sequence for a toxin gene from B.t. kurstaki HD-1. This gene was subsequently classified as cryIA(a)by Hofte and Whiteley (1989). The published open reading frame extends 1176 amino acids and encodes a protein with a calculated molecular mass of 133,500 Daltons (Da). Another gene, also classified as cryIA(a), was isolated from B.t. subsp. kurstaki HD-1 Dipel.RTM. by Shibano et al., Gene 34, pp. 243-251 (1985). As detailed in Table 2 of Hofte and Whiteley (1989), this gene is highly related, especially in the N terminal moiety, to cryIA(a) reported by Schnepf et al. (1985). CryIA(a) protein is broadly active against Lepidoptera; Hofte and Whiteley (1989) reports that four of five tested lepidopteran insects were sensitive to this toxin.
Other ICP genes subsequently identified as cryIA(a) that are greater than 99% identical to the holotype cryIA(a) gene have been identified in B. thuringiensis subspecies aizawai, (Shimizu et al., Agric. Biol. Chem. 52, pp. 1565-1573 (1988)), subspecies kurstaki, (Kondo et al., Agric. Biol. Chem. 51, pp. 455-463 (1987)), and subspecies entomocidus (Masson et al, Nucleic Acids Res. 17, p. 446 (1989)). The CryI-type nucleotide sequence disclosed in European Patent Application Publication No. 0 367 474, published May 9, 1990, of Mycogen Corporation, reveals a DNA sequence related to the cryIA(a) gene and its encoded protein that is 92% positionally identical to the holotype CryIA(a) ICP.
Wabiko et al., DNA, 5, pp. 305-314 (1986), describe the DNA sequence of an insecticidal toxin gene from B.t. subsp. berliner 1715, subsequently classified as cryIA(b) by Hofte and Whiteley (1989). The molecular mass of the protein encoded is 130,615 Da and sequential deletions indicate that the NH.sub.2 -terminal 612 amino acid polypeptide is toxic to lepidopteran insects. Hofte et al., Eur. J. Biochem., 161, pp. 273-280 (1986), describe the cloning and nucleotide sequencing of a variant crystal protein gene from B.t. subsp. berliner 1715, subsequently also classified as cryIA(b). The cloned gene produces an approximately 130,000 Da protein which coincides with the mass of the major protein observed in the strain. The gene has an open reading frame of 3465 bases which would encode a protein 1155 amino acids in length having a mass of 130,533 Da. Similarities of this sequence to the previously reported sequences for the cloned crystal genes from B.t. kurstaki HD-1, B.t. kurstaki HD- 73 and B.t. sotto are discussed in the Hofte et al. (1986) paper. Data identifying a minimal toxic fragment required for insecticidal activity are also presented. The cryIA(b) gene discussed in Hofte et al. (1986) differs in its deduced amino acid sequence by only two amino acids from the CryIA(b) protein reported by Wabiko et al.
Other cryIA(b) genes have been disclosed in Geiser et al., Gene, 48, pp. 109-118 (1986), Hefford et al., J. Biotechnol., 6, pp. 307-322 (1987), Oeda et al., Gene, 53, pp. 113-119 (1987), Kondo et al., supra, Fischhoff et al., Bio/Technology 5, pp. 807-813, (1987) and Haider and Ellar, Nucl. Acids Res., 16, p. 10927 (1988). Each of these six CryIA(b) ICPs is greater than 99% positionally identical to the holotype CryIA(b) toxin.
Adang et al., Gene, 36, pp. 289-300 (1985), report the cloning and complete nucleotide sequence of a crystal protein gene harbored on the 75 kilobase (kb) plasmid of strain B.t. subsp. kurstaki HD-73. The restriction map in the article identified this gene as holotype cryIA(c) under the current classification system of Hofte and Whiteley (1989). The complete sequence of the gene, spanning 3537 nucleotide base pairs (bp), coding for 1178 amino acids and potentially encoding a protein of 133,330 Da, is shown in the article. Toxicity data against Manduca sexta for the protein made by the CryIA(c) gene are also presented. CryIA(c) toxins have been isolated from several strains of B.t. subsp. kenyae that are highly related to the above-noted CryIA(c) toxin from B.t. subsp. kurstaki (greater than 99% positionally identical in deduced amino acid sequence) but whose protein products, although broadly active against lepidopteran insects, nonetheless show quantitatively different toxicities for individual insect species (Yon Tersch et al., Appl. Environ. Microbiol., 57, pp. 349-358 (1991 )).
Brizzard et al., Nucleic Acids Res., 16, pp. 2723-2724 (1988), describe the nucleotide sequence of crystal protein gene cryA4 (subsequently classified as cryIB by Hofte and Whiteley (1989)) isolated from B.t. subsp. thuringiensis HD-2. Hofte and Whiteley (1989) report an insecticidal specificity of CryIB toxin for Pieris brassicae.
Honee et al., Nucleic Acids Res., 16, p. 6240 (1988), describe the complete DNA sequence for the BTVI crystal protein gene isolated from B.t. subsp. entomocidus 60.5 (holotype CryIC by Hofte and Whiteley (1989)). This protein is reported to exhibit enhanced insecticidal activities against Spodoptera species.
Sanchis et al., Mol. Microbiol., 3, pp. 229-238 (1989) report the nucleotide sequence for the N-terminal coding region (2470 nucleotides) and 5' flanking region of a gene from B.t. subsp. aizawai 7.29 now classified as the cryIC gene under the classification system of Hofte and Whiteley (1989). Sanchis et al. disclose similar information about the cryIC gene in European Patent Application Publication No. 0 295 156, published Dec. 14, 1988. The open reading frame encodes a truncated polypeptide 824 amino acids long with a calculated mass of 92,906 Da.
A gene isolated from B.t. subspecies aizawai and now classified as holotype cryID under the Hofte and Whiteley (1989) system is disclosed in European Patent Application Publication No. 0 358 557, published Mar. 14, 1990 of Plant Genetic Systems, N.V. Hofte and Whiteley (1989) report selective lepidopteran toxicity against Manduca sexta for the CryID protein, the CryID toxin being largely inactive against other lepidopteran insects tested.
The holotype cryIE gene, found in a B.t. subspecies darmstadiensis strain, is disclosed in European Patent Application Publication No. 0 358 557, supra. A highly related cryIE gene from B.t. subsp. kenyae is disclosed by Visser et al., J. Bacteriol. 172, pp. 6783-6788 (1990).
Visser et al., Mol. Gen. Genet., 212, pp. 219-224 (1988) report the isolation and analysis of five toxin genes belonging to four different gene families from B.t. entomocidus 60.5, one of which is reported by Honee et al. (1988), Supra. Two of these genes, BTIV and BTVIII, are cryIA(a)-type genes according to the Hofte and Whiteley (1989) classification scheme. The BTVI gene, also reported by Honee et al. (1988) supra, is a cryIC gene according to the Hofte and Whiteley (1989) classification scheme. The authors state that the restriction map for another gene, designated BTV, closely resembles that identified for the cryID gene isolated from B.t. strain HD68 subsp. aizawai, and disclosed in European Patent Application Publication No. 0 358 557, supra. A fifth gene, designated BTVII, is also identified and its restriction map differs significantly from the other four genes described. Toxicity data against several lepidopteran insects, S. exigua, S. littoralis, H. virescens and P. brassicae, are presented for each of the isolates. The BTV gene product was inactive against all insects tested. The BTVI protein is highly active against Spodoptera larvae, and the BTVII protein is toxic to P. brassicae.
Additional genes within the cryI family have been reported in the literature. A gene found in B.t. subsp. aizawai and described as cryIF is disclosed by Chambers et al. in J. Bacteriol., 173, pp. 3966-3976 (1991) and in PCT International Publication No. WO91/16434, published Oct. 31, 1991. A gene described as cryIG from B.t. subsp. galleria is disclosed by Smulevitch et al., FEBS Lett., 293, pp. 25-28 (1991). A gene that is highly related to the cryIG gene has been isolated from B.t. DSIR 517 by Gleave et al., J. Gen. Microbiol., 138, pp. 55-62 (1992).
A novel gene related to cryI-type genes is disclosed in PCT International Publication No. WO 90/13651, published Nov. 15, 1990, of Imperial Chemical Industries PLC. This gene encodes an 81 kDa polypeptide (Cry pJH11) that is broadly insecticidal and more distantly related to the family of cryI sequences than are most other reported cryI-type sequences. Four CryI-type sequences are disclosed in European Patent Application Publication No. 0 405 810, published Jan. 2, 1991, of Mycogen Corporation. Inspection of the cryI-type sequences revealed that one of the disclosed genes (cry 81IB2) belongs to the cryIC class, one (cry 81IB) belongs to the cryID class, and one (cry 81IA) belongs to the CryIF class. The fourth disclosed cryI sequence (cry 81IA2) appears to belong to a new class. Two cryI sequences are disclosed in European Patent Application Publication No. 0 401 979, published Dec. 12, 1990, of Mycogen Corporation. One of the disclosed sequences (PS82A2) appears to encode a novel gene, the other sequence (PS82RR) is highly related to the novel sequence cry 81IA2 disclosed in European Patent Application Publication No. 0 405 810.
Five novel cry sequences are disclosed in European Patent Application Publication No. 0 462 721, published Dec. 27, 1991, of Mycogen Corporation. These Cry proteins are reported to be nematocidal.
SUMMARY OF THE INVENTION
Briefly stated, the present invention relates to a purified and isolated cryET1 gene having a nucleotide base sequence coding for the amino acid sequence (SEQ ID NO:2) illustrated in FIG. 1.
The purified and isolated cryET1 gene may have a coding region extending from nucleotide bases 177 to 3689 (including the stop codon) in the nucleotide base sequence illustrated in FIG. 1 (SEQ ID NO:1).
The present invention also relates to the CryET1 protein which is obtainable from the CryET1 gene and which is insecticidal to lepidopteran insects and, in particular, to Plutella xylostella (diamondback moth).
Additionally, the present invention relates to a biologically pure culture of a Bacillus thuringiensis bacterium designated as strain EG7094 transformed with a cryET1 gene having a coding region (SEQ ID NO:1) illustrated in FIG. 1, or mutants thereof having insecticidal activity against lepidopteran insects.
The invention also relates to a biologically pure culture of a Bacillus thuringiensis bacterium designated as strain EG5092, or mutants thereof having insecticidal activity against lepidopteran insects. B.t. strain EG5092 is a wild type isolate and is the B.t. strain from which the cryET1 gene was isolated.
Additional aspects of the present invention relate to recombinant plasmids containing the cryET1 gene; bacteria transformed with the recombinant plasmid; insecticide compositions comprising the protein and/or the transformed bacterium and/or other bacteria containing the CryET1 protein, with an agriculturally acceptable carrier; a method of controlling lepidopteran insects using the insecticide; plants transformed with the cryET1 gene; and hybridization probes containing the cryET1 gene wherein the gene or a portion of it is labeled for such use.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 comprises FIGS. 1A through 1J and shows the nucleotide sequence of the cryET1 gene (SEQ ID NO:1) and the deduced amino acid sequence of the CryET1 protein (SEQ ID NO:2).
FIG. 2 is a photograph of an ethidium bromide stained agarose gel containing size-fractionated plasmids of B.t. strain EG5092, with plasmid sizes in megadaltons (MDa) being shown.
FIG. 3 is a photograph of a Coomassie blue stained gel containing size-fractionated proteins from B.t. strains EG5092 and EG7094, obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
FIG. 4 is a restriction map of the 6.3 kb HindIII fragment of plasmid pEG744 and of four EcoRI fragments utilized in the cloning of plasmids pEG750, pEG751, pEG752 and pEG753. The location and orientation of the cryET1 gene are indicated by the arrow.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A novel Bacillus thuringiensis (B.t.) toxin gene cryET1 was obtained from a novel B.t. isolate designated EG5092. Isolation of B.t. strain EG5092, isolation of the novel toxin gene cryET1, construction of a Bacillus/E. coli shuttle vector containing cryET1 (pEG744) and transformation of pEG744 into a B.t. host (B.t. strain EG7566) to produce a recombinant B.t. EG7094 expressing the cryET1 gene product, a toxin protein designated as CryET1, are described generally in the Examples.
Subcultures of B.t. EG5092 and B.t. EG7094 were deposited in the permanent collection of the Agricultural Research Service Culture Collection, Northern Regional Research Laboratory (NRRL), U.S. Department of Agriculture, 1815 North University Street, Peoria, Ill. 61604, U.S.A. The accession numbers and deposit dates are as follows:
These microorganism deposits were made under the provisions of the "Budapest Treaty on the International Recognition of the Deposit of Microorganism 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 United States patent based on this application.
The present invention is intended to cover mutants and recombinant or genetically engineered derivatives, e.g., truncated versions, of the CryET1 gene (SEQ ID NO:1) shown in FIG. 1 that yield a protein with insecticidal properties essentially the same as those of the CryET1 protein (SEQ ID NO:2), also shown in FIG. 1. Likewise, the present invention covers those gene nucleotide base sequences that encode the amino acid sequence (SEQ ID NO:2) of the CryET1 protein. Variations may be made in the cryET1 gene nucleotide base sequence (SEQ ID NO:1) which do not affect the amino acid sequence of the gene product, since the degeneracy of the genetic code is well known to those skilled in the art. Moreover, there may be some variations or truncations in the coding regions of the cryET1 nucleotide base sequence which allow expression of the gene and production of functionally equivalent forms of the CryET1 insecticidal protein. These variations or truncations, 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.
It has been shown that proteins of identical structure and function may be constructed by changing the amino acid sequence, if such changes do not alter the protein secondary structure (Kaiser and Kezdy, Science, 223, pp. 249-255 (1984)). Single amino acid substitutions have been introduced by site-directed mutagenesis at various positions of CryIA(a) toxin protein without altering the insecticidal properties of the parent toxin (Ge et al., Proc. Natl. Acad. Sci. USA, 86, pp. 4037-4041 (1989)). The present invention includes mutants of the amino acid sequences disclosed herein which have an unaltered protein secondary structure or, if the structure is altered, where the mutant has the substantially equivalent biological activity retained.
The cryET1 gene (SEQ ID NO:1) is also useful as a DNA hybridization probe, for discovering similar or closely related cryET1-type genes in other B.t. strains. The cryET1 gene (SEQ ID NO:1), or portions or derivatives thereof, can be labeled for use as a hybridization probe, e.g., with a radioactive label, using conventional procedures.
The cryET1 gene (SEQ ID NO:1) and the corresponding insecticidal CryET1 protein (SEQ ID NO:2) were first identified in B.t. strain EG5092, a novel B.t. isolate. The characteristics of B.t. strain EG5092 are more fully described in the Examples.
The Bacillus strains described herein may be cultured using conventional growth media and standard fermentation techniques. The B.t. strains harboring the cryET1 gene (SEQ ID NO:1) may be fermented, as described in Example 1, until the cultured B.t. cells reach the stage of their growth cycle when CryET1 crystal protein (SEQ ID NO:2) is formed. For sporogenous B.t. strains, fermentation is typically continued through the sporulation stage when the CryET1 crystal protein 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 CryET1 crystal protein from the culture medium.
The separated fermentation solids are primarily CryET1 crystal protein (SEQ ID NO:2) and B.t. spores (if a sporulating host is employed), 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, e.g., density gradient fractionation.
The B.t. strains exemplified in this disclosure are sporulating varieties (spore forming or sporogenous strains) but the cryET1 gene (SEQ ID NO:1) also has utility in asporogenous Bacillus strains, i.e., strains that produce the crystal protein without production of spores. It should be understood that references to "fermentation cultures" of B.t. strains containing the cryET1 gene (SEQ ID NO:1) in this disclosure are intended to cover sporulated B.t. cultures, i.e., B.t. cultures containing the CryET1 crystal protein and spores, and sporogenous Bacillus strains that have produced crystal proteins during the vegetative stage, as well as asporogenous Bacillus strains containing the cryET1 gene (SEQ ID NO:1) in which the culture has reached the growth stage where the crystal protein is actually produced.
Mutants of B.t. strains harboring the cryET1 gene (SEQ ID NO:1) can be made by procedures well known in the art. For example, an asporogenous mutant can be obtained through ethylmethane sulfonate mutagenesis. Mutants can also be made using ultraviolet light and nitrosoguanidine by procedures that are well known to those skilled in the art. References in this specification to "mutants" of wild-type or recombinant B.t. strains harboring the cryET1 gene refer to those derivatives which are capable of producing a toxin protein exhibiting insecticidal activity against lepidopteran insects.
The CryET1 protein (SEQ ID NO:2) is an insecticidal compound active against lepidopteran insects, particularly Plutella xylostella (diamondback moth). The CryET1 protein (SEQ ID NO:2) may be used as the active ingredient in insecticidal formulations useful for controlling lepidopteran insects such as the diamondback moth. 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 CryET1 protein (SEQ ID NO:2) may be employed in insecticidal formulations in isolated or purified form, e.g., as the crystal protein itself. Alternatively, the CryET1 protein (SEQ ID NO:2) may be present in the recovered fermentation solids, obtained from culturing of a Bacillus strain, e.g., Bacillus thuringiensis, or other microorganism host carrying the cryET1 gene (SEQ ID NO:1) and capable of producing the CryET1 protein. The recovered fermentation solids containing the CryET1 protein may be dried, if desired, prior to incorporation in the insecticidal formulation. Preferred Bacillus hosts include B.t. strain EG7566, a plasmid-free strain lacking crystal toxin proteins (Cry.sup.-), described in Example 2. Genetically engineered or transformed B.t. strains or other host microorganisms containing a recombinant plasmid that expresses the cloned cryET1 gene (SEQ ID NO:1), obtained by recombinant DNA procedures, may also be used.
The formulations or compositions of this invention containing the insecticidal CryET1 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 lepidopteran insects to be controlled, the specific plant or crop to be treated and the method of applying the insecticidally active compositions.
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, e.g., 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 CryET1 protein (SEQ ID NO:2) and one or more solid or liquid adjuvants are prepared in known manners, e.g., by homogeneously mixing, blending and/or grinding the insecticidally active CryET1 protein component with suitable adjuvants using conventional formulation techniques.
The insecticidal compositions of this invention are applied to the environment of the target lepidopteran insect, typically onto the foliage of the plant or crop to be protected, by conventional methods, preferably by spraying. Other application techniques, e.g., 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 cryET1 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 cryET1 gene. Suitable hosts that allow the CryET1 gene (SEQ ID NO:1) to be expressed and the CryET1 protein (SEQ ID NO:2) to be produced include B.t. and other Bacillus species such as B. subtilis or B. megaterium. Genetically altered or engineered microorganisms containing the cryET1 gene (SEQ ID NO:1) can also contain other toxin genes present in the same microorganism; these genes could concurrently produce ICPs different from the CryET1 protein.
Plant-colonizing or root-colonizing microorganisms may also be employed as the host for the cryET1 gene (SEQ ID NO:1). Various procedures well known to those skilled in the art are available for introducing the cryET1 gene (SEQ ID NO:1) into the microorganism host under conditions which allow for stable maintenance and expression of the gene in the resulting transformants.
The transformants, i.e., 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 only those host microorganisms that contain a recombinant plasmid. The transformants then can be tested for insecticidal activity. These techniques are standard procedures well known to those skilled in the art.
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 CryET1 insecticidal protein in the host, and the presence of auxiliary genetic capabilities. The cellular host containing the insecticidal cryET1 gene (SEQ ID NO:1) may be grown in any convenient nutrient medium, where expression of the cryET1 gene is obtained and CryET1 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, e.g., centrifugation or filtration.
The cryET1 gene (SEQ ID NO:1), particularly the toxin portion (N-terminal moiety) thereof, may also be incorporated into a plant which is capable of expressing the gene and producing CryET1 protein (SEQ ID NO:2), rendering the plant more resistant to insect attack. Genetic engineering of plants with the cryET1 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. Examples of techniques for introducing DNA into plant tissue are disclosed in European Patent Application Publication No. 0 289 479, published Nov. 1, 1988, of Monsanto Company and by Perlak et al. in "Modification of the Coding Sequence Enhances Plant Expression of Insect Control Protein Genes," Proc. Natl. Acad. Sci. USA, 88, pp 3324-3328 (1991).
DNA containing the cryET1 gene (SEQ ID NO:1) or a modified CryET1 gene capable of producing the CryET1 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. Additionally, the use of lysosomes or liposomes, microinjection by mechanical methods and by other techniques familiar to those skilled in plant genetic engineering may be used.
The characteristics of the CryET1 protein (SEQ ID NO:2), sequencing of the cryET1 gene (SEQ ID NO:1), comparison of sequence data to known B.t. toxin genes and insecticidal activity of B.t. EG7094 and the CryET1 protein are described in the following specific, non-limiting examples.
EXAMPLE 1
Characterization of B.t. EG5092 and B.t. EG7094
B.t. strain EG5092 is a wild-type isolate, identified by visual examination of the colony as exhibiting a unique crystal morphology, and was isolated as a colony from maize dust. The colony contained endospores and bipyramidal, apparently "jagged" crystalline inclusions. Subsequent insect bioassay of this wild-type B.t. strain confirmed its insecticidal activity towards lepidopteran insects.
The complement of native plasmids contained within isolated B.t. EG5092 was determined by modified Eckhardt agarose gel electrophoresis as described by Yon Tersch et al., in Appl. Environ. Microbiol., 57, pp. 349-358 (1991). The results, as shown in FIG. 2, revealed the presence of 4.9, 6.4, 8.7, 49, 59, 98 and 115 MDa plasmids. This pattern of native plasmids did not correspond to patterns typical of known serovars (Carlton and Gonzalez, pp. 246-252, in Molecular Biology of Microbial Differentiation, J. A. Hoch and P. Setlow, ed., American society for Microbiology, Washington, D.C. (1985).
Wild-type B.t. strain EG5092 was grown 5 days at 25.degree. C. on Luria plates (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl, 15 g/l agar). Recombinant B.t. strain EG7094, containing the cryET1 gene, was likewise grown on Luria plates, but containing 10 .mu.g of tetracycline per ml. Cells were recovered from the surfaces of the plates. B.t. strain EG5092 was observed to have fully lysed naturally. The partially lysed culture of B.t. strain EG7094 was fully lysed by repeated intermittent agitation with 0.1 mm zirconium beads. Both cultures were washed two times with high salt TNT buffer (50 mM Tris HCl pH 7.5, 1.0M NaCl, 0.05% Triton.RTM. X-100) followed by two washes with TNT buffer (50 mM Tris HCl pH 7.5, 100 mM NaCl, 0.05% Triton.RTM. X-100). Aliquots of the washed crystals were solubilized by heating in Laemmli buffer (10% (w/w) glycerol, 5% (w/w) 2-mercaptoethanol, 1% (w/v) SDS, 0.188M Tris HCl pH 6.8, 0.01% (v/v) bromphenol blue) at 100.degree. 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 Blue R-250 dye.
FIG. 3 shows the results of these protein size fractionation analyses where lane 1 is a molecular mass size standard, lanes 2 and 3 are duplicate runs of B.t. strain EG5092 and lane 4 is B.t. strain EG7094. The numbers on the right side indicate the apparent molecular masses, in kilodaltons (kDa), of the crystal proteins synthesized by B.t. strains EG5092 and EG7094. As shown in lane 4 for EG7094, a major protein having an apparent molecular mass estimated to be approximately 135,000 Da was solubilized from centrifuged fermentation solids containing B.t. strain EG7094 spores and crystals. B.t. EG5092, as shown in lanes 2 and 3, exhibited two major proteins whose apparent molecular masses were estimated to be about 145,000 and about 135,000 Da.
EXAMPLE 2
Cloning of the cryET1 Gene
Genomic DNA was isolated from B.t. strain EG5092 and then digested with HindIII. Resulting fragments of approximately 4-8 kilobase pairs (kb) in size were purified by gel electrophoresis and electroelution. These fragments were ligated to E. coli and Bacillus vector pEG147 (described by Yon Tersch et al., Appl. Environ. Microbiol., 57, pp. 349-358 (1991)), and the ligation mixture was then used to transform E. coli. Colony hybridization to a cryI-type gene probe (the cryIA(b) gene was used) identified positive colonies. One colony containing a recombinant plasmid designated pEG744 was selected for further analysis. HindIII digestion of pEG744 revealed an insert fragment 6.3 kb in size, whose restriction map is shown in FIG. 4. Plasmid pEG744 was transformed into B.t. strain EG7566; B.t. strain EG7566 is a plasmid-free strain obtained by curing B.t. strain HD73-26, to eliminate this latter strain's single, 4.9 MDa plasmid. The resulting strain was designated B.t. strain EG7094, and this strain contained the cryET1 gene as its sole B.t. toxin gene.
EXAMPLE 3
Sequencing of the cryET1 Gene
Plasmid pEG744 (Example 2) was digested with EcoRI, and four of the five generated fragments were cloned separately into pUC18. The four plasmids were designated pEG750, pEG751, pEG752 and pEG753. Mapping of the EcoRI fragment inserts in each of these plasmids is also shown in FIG. 4. DNA sequences were determined for the entire EcoRI fragments cloned in pEG751 and pEG752 and for parts of the EcoRI fragments cloned in pEG750 and pEG753. The DNA sequences were joined at the EcoRI junctions to give a continuous sequence of 3766 nucleotides which is shown in FIG. 1. Inspection of the sequence revealed an open reading frame beginning at position 177 and extending to position 3689 (including the stop codon). The deduced 1170 amino acid sequence of the gene product is also shown in FIG. 1. The gene has been designated cryET1. The mass of the CryET1 protein (SEQ ID NO:2) encoded by the cryET1 gene (SEQ ID NO: 1), as deduced from the open reading frame, is 33,566 Da.
EXAMPLE 4
Homology of CryET1 to Other Insecticidal Crystal Proteins
The deduced amino acid sequence of the protein product of the cryET1 gene was compared to a database of holotype and other known B.t. toxin protein amino acid sequences. The results shown in Table 1 indicate that CryET1 is a novel ICP different from all previously described ICPs. The amino acid sequence homologics show that the protein product of the cryET1 gene is most closely related to the CryI class of ICP gene products. Most CryI toxins showed between 60 and 66% amino acid identity to CryET1 in pairwise alignments of the complete protoxin amino acid sequence. Alignments limited to the N-terminal toxic fragments showed lower percent identities, which is consistent with the generally higher conservation of C-terminal sequence among the family of CryI toxins. Alignments limited to the N-terminal toxic fragment of CryET1 showed lesser homologics to CryII, CryIII and CryIV ICPs.
CryET1 amino acid sequence divergences from the family of ICP sequences imply corresponding divergences in the nucleotide sequences of the corresponding genes.
EXAMPLE 5
Insecticidal Activity of CryET1 Protein
Insecticidal activities of solubilized CryET1 toxin from a culture of recombinant B.t. strain EG7094 containing the cryET1 gene were determined at a single toxin dose level against a diluent control after solubilization of crystalline inclusions in base and elimination of non-soluble spores and cell debris by centrifugation. A Luria broth culture of B.t. strain EG7094 containing crystal toxin proteins was solubilized by addition of NaOH to 0.1N. After 5 minutes at room temperature, spores and cell debris were removed by centrifugation. Solubilized toxin was diluted 1:5 with 0.005% Triton.RTM. X-100, then 50 .mu.l aliquots were applied to the surfaces of 32 wells (1.8 cm.sup.2 surface area) containing insect diet and dried for 1 hour at 30.degree. C. A general purpose Noctuidae artificial diet as described by E. G. King et al. in Handbook of Insect Rearing, Vol. 2, P. Singh and R. F. Moore (eds.), pp. 323-328, Elsevier Science Publishers B.V., Amsterdam (1985) was used for Trichoplusia ni, Ostrinia nubilalis and Heliothis virescens. Other standard diets were used for the other lepidopteran insects tested. One neonate larva (third-instar larvae in the case of P. xylostella) was added to each cup, and the cups were incubated at 30.degree. C. Mortality was recorded after seven days.
The results (Table 2) indicate that diamondback moth, soybean looper, and to a lesser extent, cabbage looper were sensitive to the solubilized CryET1 protein (SEQ ID NO:2), i.e., these lepidopteran insects were readily killed at the dose levels of CryET1 protein tested. Other insects such as tobacco budworm and European corn borer that are usually sensitive to other known CryI toxins were either not sensitive or only slightly sensitive to the CryET1 toxin protein at the dose levels tested.
In a separate procedure, to illustrate the dose response of CryET1 toxin against various lepidopteran insect larvae, crystal inclusions from B.t. strain EG7094 cultures were first separated from spores and cell debris by equilibrium centrifugation on Renografin-76 gradients by the method of P.C. Fitz-James et al in J. Invertebr. Pathol. 43, pp. 47-58 (1984). The amount of CryET1 protoxin recovered from the gradients was quantified by laser densitometry after solubilization of the recovered crystal protein with base and a reducing agent.
Dilutions of purified CryET1 crystals were prepared in 0.005% Triton.RTM. X-100. Aliquots of appropriate dilutions (150 .mu.l) were applied to the surfaces of 32 wells and assayed as indicated above. Control assays of CryIA(b) and CryIA(c) crystals were included. The results are shown in the following Table 3.
The results indicate the same spectrum of activity as was observed in the assays of solubilized CryET1 (Table 2). CryET1 protein (SEQ ID NO:2) shows a specificity of activity unlike that of any other CryI toxin. The CryET1 toxin protein was shown to be insecticidal to diamondback moth, soybean looper and cabbage looper. Diamondback moth was observed to be the most sensitive. The activity observed for CryET1 protein appeared to be selective for diamondback moth, and Example 6 describes additional work performed to determine the LC.sub.50 value for CryET1 against this insect. Insecticidal activity observed against the other lepidopteran insects tested was decreased relative to activity exhibited by CryIA(b) and CryIA(c).
EXAMPLE 6
Bioassay of CryET1 Crystals Against Diamondback Moth
The insecticidal activity of CryET1 protein was determined against larvae of Plutella xylostella (diamondback moth), an insect pest that causes significant damage to vegetables, particularly cold crops. In this procedure, the LC.sub.50 value of CryET1 was measured against P. xylostella larvae, the LC.sub.50 dose being that which killed half of the insects tested, i.e., the median lethal concentration.
Crystal inclusions from a B.t. strain EG7094 culture were first separated from spores and cell debris by equilibrium centrifugation on Renografin-76 gradients, as described in Example 5. The amount of recovered CryET1 protoxin was then quantified by a commercial dye binding assay after solubilization of the crystals with base and a reducing agent.
To quantify the insecticidal activity, dilutions of purified CryET1 crystals were prepared in 0.005% Triton.RTM. X-100. Aliquots of eight two-fold serial dilutions (50 .mu.l) were applied to the surfaces of 32 wells, one P. xylostella larva was placed in each well, and the bioassay was performed as indicated in Example 5.
Composite results for two such bioassays showed an LC.sub.50 value of 354 ng/well for CryET1 crystals against diamondback moth (95% confidence intervals were 311-403 ng/well ).
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.
Claims
We claim:
1. A purified and isolated cryET1 gene having a nucleotide base sequence coding for the amino acid sequence (SEQ ID NO:2) illustrated in FIG. 1.
2. A purified and isolated cryET1 gene according to claim 1 wherein the gene has a coding region extending from nucleotide bases 177 to 3689 in the nucleotide base sequence illustrated in FIG. 1 (SEQ ID NO:1).
3. A recombinant plasmid containing the gene of claim 1 or 2.
4. A biologically pure culture of a bacterium transformed with the recombinant plasmid of claim 3 which expresses the cryET1 gene.
5. The bacterium of claim 4 wherein the bacterium is Bacillus thuringiensis.
6. The Bacillus thuringiensis bacterium of claim 5 deposited with the NRRL having accession number NRRL B-18972 and being designated as strain EG7094, or mutants thereof having activity against Plutella xylostella insects.
7. A biologically pure culture of a Bacillus thuringiensis bacterium deposited with the NRRL having accession number NRRL B-18973 and being designated as strain EG5092, or mutants thereof having insecticidal activity against Plutella xylostella.
8. An insecticide composition comprising the bacterium of claim 4, an insecticidal protein having insecticidal activity against Plutella xylostella insects and produced by such bacterium, and an agriculturally acceptable carrier.
9. An insecticide composition comprising the bacterium of claim 7, an insecticidal protein produced by such bacterium, and an agriculturally acceptable carrier.
10. A method of controlling susceptible lepidopteran insects comprising applying to a host plant for such insects an insect-controlling effective amount of an insecticidal protein identical to a protein produced by a Bacillus thuringiensis bacterium deposited with the NRRL having accession number NRRL B-18972 and being designated as strain EG7094, having activity against Plutella xylostella insects.
11. The method of claim 10 wherein the insecticidal protein is associated with a Bacillus thuringiensis bacterium which has produced such protein.
12. The method of claim 10 wherein the lepidopteran insect is Plutella xylostella.
Patent Citations (10)
| Patent | Date | Inventor | Cited By |
|---|---|---|---|
| US5064648 | 1991-11-01 | Hickle et al. | |
| EPX289479 | 1988-11-01 | ||
| EPX295156 | 1988-12-01 | ||
| EPX358557 | 1990-03-01 | ||
| EPX367474 | 1990-05-01 | ||
| EPX401979 | 1990-12-01 | ||
| EPX405810 | 1991-01-01 | ||
| EPX462721 | 1991-12-01 | ||
| WOX90-13651 | 1990-11-01 | ||
| WOX91-16434 | 1991-10-01 |
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