CA2080684C - Bacillus thuringiensis cryif and cryix genes and proteins toxic to lepidopteran insects - Google Patents
Bacillus thuringiensis cryif and cryix genes and proteins toxic to lepidopteran insects Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal protein (delta-endotoxin)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
Abstract
Two purified and isolated cryI-type genes were obtained from a novel B.t. strain. One gene, designated cryIF, has a nucleotide base sequence coding for the amino acid sequence illustrated in Figure 1. The 134 kDa crystal protein, designated CryIF, produced by this gene is toxic to European corn borer larvae and other lepidopteran insects.
The second gene, designated cryIX, produces a crystal protein of about 81 kDa, designated CryIX, that is also toxic to lepidopteran insects.
The second gene, designated cryIX, produces a crystal protein of about 81 kDa, designated CryIX, that is also toxic to lepidopteran insects.
Description
BACILLUS THURINGIENSIS cryIF AND cryIX GENES AND
PROTEINS TOXIC TO LEPIDOPTERAN INSECTS
Field of the Invention The present invention relates to two genes isolated from Bacillus thuringiensis (hereinafter "B.t.") encoding insecticidal crystal proteins designated CryIF and CryIX, respectively, as well as insecticidal compositions containing the proteins and plants transformed with the genes. The insecticidal compositions and transformed plants are toxic to insects of the order Lepidoptera.
Background of the Invention B.t. 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 '0 91/16434 PC'I'/US91/02560 208'~~84
PROTEINS TOXIC TO LEPIDOPTERAN INSECTS
Field of the Invention The present invention relates to two genes isolated from Bacillus thuringiensis (hereinafter "B.t.") encoding insecticidal crystal proteins designated CryIF and CryIX, respectively, as well as insecticidal compositions containing the proteins and plants transformed with the genes. The insecticidal compositions and transformed plants are toxic to insects of the order Lepidoptera.
Background of the Invention B.t. 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 '0 91/16434 PC'I'/US91/02560 208'~~84
2 -acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms.
A number of genes er-coding crystal proteins have been cloned froin several strains of B.t. A good overview is set forth in H. H;fte et al., Microbiol'. Rev., 53, pp. 242-255 (1989), hereinafter "Hofte and Whiteley (1989)." While this reference is not prior art with respect to the present invention, it 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 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 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 H fte and Whiteley (1989), the majority of insecticidal B.t. strains are active against insects of the order T.apidoptera, i.e., caterpillar insects. These B.t. strains characteristically contain cryI-type genes that make Cryl crystal proteins. Other B.t. strains produce different classes of crystal proteins, e.g.,, Crylv protein, active against insects of the 'v0 91/16434 t'Cr/US91/02560
A number of genes er-coding crystal proteins have been cloned froin several strains of B.t. A good overview is set forth in H. H;fte et al., Microbiol'. Rev., 53, pp. 242-255 (1989), hereinafter "Hofte and Whiteley (1989)." While this reference is not prior art with respect to the present invention, it 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 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 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 H fte and Whiteley (1989), the majority of insecticidal B.t. strains are active against insects of the order T.apidoptera, i.e., caterpillar insects. These B.t. strains characteristically contain cryI-type genes that make Cryl crystal proteins. Other B.t. strains produce different classes of crystal proteins, e.g.,, Crylv protein, active against insects of the 'v0 91/16434 t'Cr/US91/02560
3 8 order Diptera, i.e., flies and mosquitoes, or CryII
= protein active against both lepidopteran and dipteran insects. in recent years, a few B.t.
strains have been reported as producing a new class of crystal protein, CryIII protein, that is insecticidal to insects of the order Coleoptera, i.e., beetles.
Lereclus et al., in Chapter 13 of Regulation of Procaryotic Development, I. Smith et al. (eds.), American Society for Microbiology, Washington, D.C., pp. 255-276 (1989), review the role, structure and molecular organization of crystal protein genes. A summary of toxin genes identified in B.t. is provided in Table 2 (p. 260) and amino acid sequence comparisons of the various crystal proteins reported in the literature are diagrammed in Figures 1 and 2. This reference is not prior art with respect to the present invention.
Schnepf et al., J. Biol. Chem., 260, pp. 6264-6272 (1985), report the complete nucleotide sequence for a toxin gene from B.t.
kurstaki HD-1 (Figure 2, pp. 6266-6267); this gene was subsequently classified as the crStlAa) gene by -H fte and Whiteley (1989). The published open reading frame extends 1176 amino acids and encodes a protein with a calculated M r of 133,500 daltons (Da).
= 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 crylA(b) by H6fte and Whiteley (1989). The molecular mass of the WO 91/16-434 i'CT/1JS91/0256Ã)
= protein active against both lepidopteran and dipteran insects. in recent years, a few B.t.
strains have been reported as producing a new class of crystal protein, CryIII protein, that is insecticidal to insects of the order Coleoptera, i.e., beetles.
Lereclus et al., in Chapter 13 of Regulation of Procaryotic Development, I. Smith et al. (eds.), American Society for Microbiology, Washington, D.C., pp. 255-276 (1989), review the role, structure and molecular organization of crystal protein genes. A summary of toxin genes identified in B.t. is provided in Table 2 (p. 260) and amino acid sequence comparisons of the various crystal proteins reported in the literature are diagrammed in Figures 1 and 2. This reference is not prior art with respect to the present invention.
Schnepf et al., J. Biol. Chem., 260, pp. 6264-6272 (1985), report the complete nucleotide sequence for a toxin gene from B.t.
kurstaki HD-1 (Figure 2, pp. 6266-6267); this gene was subsequently classified as the crStlAa) gene by -H fte and Whiteley (1989). The published open reading frame extends 1176 amino acids and encodes a protein with a calculated M r of 133,500 daltons (Da).
= 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 crylA(b) by H6fte and Whiteley (1989). The molecular mass of the WO 91/16-434 i'CT/1JS91/0256Ã)
-4-protean encoded is 130,615 Da and sequential deletions indicate that the NH2-terminal 612 amino acid polypeptide is toxic.
Hofte et al., Eur. J. Biochem., 161, pp. 273-280 (1986), describe the cloning and nucleotide sequencing of a crystal protein gene from B.t. subsp. berliner 1715, subsequently classified as cryl~,(b) by Hofte and Whiteley (1989). The cloned gene produces a 130 kilodalton (kDa) protein which coincides with the size of the major protein observed in the strain. A
restriction map of the cloned toxin gene is presented (p. 276, Figure 3A). Toxicity data for the cloned gene is shown in Table 1(p. 275). The complete nucleotide sequence for this gene is shown in Figure 3B (p. 276). It has an open reading frame of 3466 bases which would encode a protein 1155 amino acids in length having a molecular weight 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 on p. 278 and summarized in Figures 6 and 7 (pp. 279 and 280, respectively). Data identifying a minimal toxic fragment required for insecticidal activity is also presented (pp. 277 and 278, Figure 4, Table 2).
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 kb plasa-id of strain B.t subsp. kurstaki HD-73.
The restriction map (Figure 2, p. 292) identifies this gene as cryIA(c) under the current 'vO 91/16434 PC'i'/US91/02560
Hofte et al., Eur. J. Biochem., 161, pp. 273-280 (1986), describe the cloning and nucleotide sequencing of a crystal protein gene from B.t. subsp. berliner 1715, subsequently classified as cryl~,(b) by Hofte and Whiteley (1989). The cloned gene produces a 130 kilodalton (kDa) protein which coincides with the size of the major protein observed in the strain. A
restriction map of the cloned toxin gene is presented (p. 276, Figure 3A). Toxicity data for the cloned gene is shown in Table 1(p. 275). The complete nucleotide sequence for this gene is shown in Figure 3B (p. 276). It has an open reading frame of 3466 bases which would encode a protein 1155 amino acids in length having a molecular weight 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 on p. 278 and summarized in Figures 6 and 7 (pp. 279 and 280, respectively). Data identifying a minimal toxic fragment required for insecticidal activity is also presented (pp. 277 and 278, Figure 4, Table 2).
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 kb plasa-id of strain B.t subsp. kurstaki HD-73.
The restriction map (Figure 2, p. 292) identifies this gene as cryIA(c) under the current 'vO 91/16434 PC'i'/US91/02560
- 5 -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 Mr 133,330 Da, is shown in Figure 3 (p. 294).
Sequence comparisons of this gene and the published N-terminal sequence for the B<.t. kurstaki HD-1 Dipel gene reveal only 41 base pair differences, concentrated within the last 376 base pairs of the HD-1 sequence (p. 293). The 5' regulatory sequences are identical in both clones. A
schematic of this comparison is shown in Figure 4 (p. 295). Toxicity data against Manduca sexta for the protein made by the full length HD-73 gene are also presented (Table II, p. 296).
Brizzard et al., Nucleic Acids Res., 16
The complete sequence of the gene, spanning 3537 nucleotide base pairs (bp), coding for 1178 amino acids and potentially encoding a protein of Mr 133,330 Da, is shown in Figure 3 (p. 294).
Sequence comparisons of this gene and the published N-terminal sequence for the B<.t. kurstaki HD-1 Dipel gene reveal only 41 base pair differences, concentrated within the last 376 base pairs of the HD-1 sequence (p. 293). The 5' regulatory sequences are identical in both clones. A
schematic of this comparison is shown in Figure 4 (p. 295). Toxicity data against Manduca sexta for the protein made by the full length HD-73 gene are also presented (Table II, p. 296).
Brizzard et al., Nucleic Acids Res., 16
(6), pp. 2723-2724 (1988), describe the nucleotide sequence of crystal protein gene cryA4 (subsequently classified as crylH by Hofte and Whiteley (1989)) isolated from B.t. strain HD2.
This report makes a cursory statement distinguishing the amino-terminal region of the cryIB gene from those of the cryIA(a), cryTA(b) and cryIA(c) genes. The sequence of this gene is further differentiated from the three cryIA genes by virtue of its TTG translational start codon.
Honee et al., Nucleic Acids Res., 16 (13), p. 6240 (1988), describe the complete DNA
sequence for the BTVI crystal protein gene isolated from B.t. subsp. entomocidus 60.5 (termed cryIC by H fte and Whiteley (1989)). Extensive homology to the cryIA(a) crystal protein gene was evident downstream from the proteolytic cleavage site.
WO 91/16434 PCi'/US91/02560 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 (see Figure 2, p. 221). Two of these, BTIV and BTVIII, are crylA(a)-type genes according to the Hofte and Whiteley (1989) claassification scheme.
Two additional genes, BTVI and BTV, reside in opposite orientations on the same recombinant plasmid and are separated by approximately 3 kilobase (kb) of intervening DNA. BTVI is the crylC gene according to the Hofte and Whiteley (1989) classification scheme. The authors state that the restriction map for BTV closely resembles that identified for a gene isolated from B.t.
strain HD68 subsp. aizawai, now termed the cr ID
gene (p. 222). A fifth gene, BTVII, is also identified and its restriction map differs significantly from the other four genes described.
Toxicity data against S. exigua, S. littoralis, H.
virescens and P. brassicae are presented for each of the isolates (see Table 1, p. 223). BTVI is highly active against Spodoptera larvae. BTVII is toxic to P. brasicae.- The BTV gene product was inactive against all insects teste.d.
Sanchis et al., Mol. Microbiol., 2, pp. 393-404 (1988), describe the isolation of recombinant clones containing two novel B.t. toxin genes from strains aizawai 7.29 (plasmids pHTA4 and pHTA6) and entomocidus 601 (pHTE4 and pHTE6).
Toxin genes on pHTA6 and pHTE6 have been subsequently classified as crYIC according to the Hofte and bdhiteley (1989) classification scheme.
'VO 91/16434 PCf/US91/02560 2' 0 o
This report makes a cursory statement distinguishing the amino-terminal region of the cryIB gene from those of the cryIA(a), cryTA(b) and cryIA(c) genes. The sequence of this gene is further differentiated from the three cryIA genes by virtue of its TTG translational start codon.
Honee et al., Nucleic Acids Res., 16 (13), p. 6240 (1988), describe the complete DNA
sequence for the BTVI crystal protein gene isolated from B.t. subsp. entomocidus 60.5 (termed cryIC by H fte and Whiteley (1989)). Extensive homology to the cryIA(a) crystal protein gene was evident downstream from the proteolytic cleavage site.
WO 91/16434 PCi'/US91/02560 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 (see Figure 2, p. 221). Two of these, BTIV and BTVIII, are crylA(a)-type genes according to the Hofte and Whiteley (1989) claassification scheme.
Two additional genes, BTVI and BTV, reside in opposite orientations on the same recombinant plasmid and are separated by approximately 3 kilobase (kb) of intervening DNA. BTVI is the crylC gene according to the Hofte and Whiteley (1989) classification scheme. The authors state that the restriction map for BTV closely resembles that identified for a gene isolated from B.t.
strain HD68 subsp. aizawai, now termed the cr ID
gene (p. 222). A fifth gene, BTVII, is also identified and its restriction map differs significantly from the other four genes described.
Toxicity data against S. exigua, S. littoralis, H.
virescens and P. brassicae are presented for each of the isolates (see Table 1, p. 223). BTVI is highly active against Spodoptera larvae. BTVII is toxic to P. brasicae.- The BTV gene product was inactive against all insects teste.d.
Sanchis et al., Mol. Microbiol., 2, pp. 393-404 (1988), describe the isolation of recombinant clones containing two novel B.t. toxin genes from strains aizawai 7.29 (plasmids pHTA4 and pHTA6) and entomocidus 601 (pHTE4 and pHTE6).
Toxin genes on pHTA6 and pHTE6 have been subsequently classified as crYIC according to the Hofte and bdhiteley (1989) classification scheme.
'VO 91/16434 PCf/US91/02560 2' 0 o
- 7 -Restriction map data (see Figure 5, p. 398) indicate that the novel genes are in close proximity to each other (3 kb apart). The restriction maps and molecular organization for the novel aizawai genes are identical to the entomocidus genes, except foa: a 1.4 kb insert in the upstream entomocidus gene. Toxicity data are presented for Escherichia coli (E. coli) expression constructs of these novel geries (see Table 2, p. 400 and discussion on p. 401). The proteins produced by these cryiC genes are highly toxic to S. littoralis and P. brassicae. No significant toxicity was demonstrated for the protein of the novel gene on plasmid pHTA4.
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). The open reading frame encodes a polypeptide 824 amino acids long with a calculated molecular weight of 92,906 Da (see sequence, Figure 1, p. 231). Comparative analysis of this sequence to other known B.t. toxin genes indicates that for the N-terminal DNA sequence (amino acids 1-281), the CryIC prot$in has only 58% identity to other Pi proteins> The more homologous C-terminal region (amino acids 619-824) has less than 10%
variability. The authors report the identification of five N-terminal conserved domains present in lepidopterais-active, dipteran-active and coleopteran-active endotoxins.
WO 91/16434 PCd'/iJS91/02560
Sanchis et al., Mol. Microbiol., 3, pp. 229-238 (1989), report the nucleotide sequence for the N-terminal coding region (2470 nucleotides) and 5' flanking region of a gene from B.t. subsp.
aizawai 7.29 now classified as the cryIC gene under the classification system of Hofte and Whiteley (1989). The open reading frame encodes a polypeptide 824 amino acids long with a calculated molecular weight of 92,906 Da (see sequence, Figure 1, p. 231). Comparative analysis of this sequence to other known B.t. toxin genes indicates that for the N-terminal DNA sequence (amino acids 1-281), the CryIC prot$in has only 58% identity to other Pi proteins> The more homologous C-terminal region (amino acids 619-824) has less than 10%
variability. The authors report the identification of five N-terminal conserved domains present in lepidopterais-active, dipteran-active and coleopteran-active endotoxins.
WO 91/16434 PCd'/iJS91/02560
- 8 Sanchis et al., in European Patent Application Publication No. 0 295 156, published December 14, 1988, disclose the DNA and amino acid sequences of a truncated gene, the crylC gene, and encoded crystal protein isolated from B.t. subsp.
aizawai 7.29. The sequence revealed is the same as that in Sanchis et al., Molecular Microbiol., 3, pp. 229-238 (1989), described above.
Schnepf et al., in U.S. Patent 4,467,036, issued August 21, 1984, disclose a hybrid recombinant plasmid capable of replication in an E.
coli strain, and capable of expressing a polypeptide with the immunological properties of B.t. crystal protein, and being identifiable with a PvuII C DNA fragment probe derived from a gene, now known as crylA(a), of B.t. var. kurstaki strain HD-i. The following B.t. subspecies and strains are disclosed as sources of expressible heterologous DNA: tolworthi, darmstadiensis, sotto, thuringiensis, kurstaki, HD-290, HD-120, HD-2, HD-244, HD-73, HD-l, HD-4, HD-8, F-6, F-5 and F-9.
Generally, the sequences of B.t. genes encoding delta-endotoxin proteins active against different orders of insects are not well-conserved.
Rather, the sequences of genes responsible for a given crystal phenotype and active against the same insect order are significantly inore related. The homology of delta-endotoxin amino acid sequences, as well as similarities in insecticidal activity, have been used to define an ordered classification of genes encoding B.t. delta-endotoxin proteins (Hofte and Whiteley (1989)). The genes encoding WO 91/16434 PCF/bJS91/02560 ~~~068n g the 130-138 kDa, lepidopteran.-active delta-endotoxin proteins comprise the largest of these families, the cr I genes.
Within the cryl gene classification described by Hofte and Whiteley (1989), a subranking has been established based upon further refinement of sequence relationship. Thus, the crylA(a), crYIA(b) and cryIA c) gene sub-families embrace the previously designated 4.5 kb, 5.3 kb and 6.6 kb P1 genes, respectively. These genes originally were differentiated according to the size of a characteristic HindiIl fragment associated with the presence of the gene. The amino acid sequences of CryIA proteins are highly related (greater than 80% amino acid identity), with most of the sequence dissimilarity relegated to a short internal variable region (Whiteley et al., Ann. Rev. Microbiol., 40, pp. 549-576 (1986)).
It is believed that differences within this variable region account for the different insecticidal specificities exhibited by the proteins encoded by the crylA(a), crylA(b) and crylA(c) genes.
Recently, additional genes within the cryl family have been discovered, such as the cryIB
gene found in B.t. subsp. thuringiensis (Brizzard et al., (1988), supra), and the crylC and crylo genes found in subsp. aizawai (Sanchis et al., (1988), supra, Visser et a1. (1988), M.ra, Hofte and Whiteley (1989), European Patent Application Publication No. 0 358 557, published March 14, 1990, of Plant Genetic Systems, N.V.), and the crylE gene found in B.t. subsp. darmstadiensis WO 91/16434 FClf/US91/02560 2 f) C13 13 bi ;111 (EP 0 358 557 (1990), supra) and in B.t._ subsp.
kenyae (Visser et al., J. Bacteriol., 172, pp. 6783-6788 (1990)). Other cryI-type genes are disclosed in European Patent Application Publication No. 0 367 474, published May 9, 1991, of Mycogen Corporation, in European Patent Application Publication No. 0 401 979, published December 12, 1990 of Mycogen Corporation, and in PCT International Publication No. WO 90/13651, published November 15, 1990, of Imperial Chemical Industries PLC. Comparisons of the sequences for these cryz-type genes to the cryIA genes reveal significant sequence dissimilarities, particularly in the N-terminal protein domain.
The present invention is based on the discovery of at least one additional subgroup of crVl genes. The prototype of this subgroup, which the inventors have designated cryIF, was also isolated from a B.t. subsp. aizawai strain.
However, the sequence of the crylP gene and the insecticidal specificity of the CryIF protein it encodes, is distinctly different from the other cryI genes and their encoded Cryl proteins. This distinction is also true with respect to the crylC
gene and its encoded protein, even though a spontaneously cured derivative of a B.t. strain containing the cr XIC gene was used in the isolation of the crylF gene.
In addition, the present invention includes the identification and isolation of a second gene, designated cryz, which is located downstream from the novel cr IF gene. Of the cryI-type genes discussed above, the gryIX gene w0 91/16434 PC T/US91/02560 s 4Y Ci r"
) appears most closely related to the B.t. toxin gene disclosed in PCT International Publication No. WO 90/13651, published November 15, 1990, of Imperial Chemical Industries PLC.
Data are presented hereinafter concerning the identification, cloning, sec[uencing and expression of these novel cr IF and crylX toxin genes, as well as the insecticidlal activities of the CryIF and CryIX proteins against lepidopteran larvae.
Summary of the Invention One aspect of the present invention relates to a purified and isolated lepidopteran-active toxin gene having a nucleotide base sequence coding for the amino acid sequence illustrated in Figure 1 and hereinafter designated as the crylF
gene (SEQ ID NO:1). The cryIF gene (SEQ ID NO:1) has a coding region, i.e., open reading frame, extending from nucleotide bases 478 to 3999 shown in Figure 1.
Another aspect of the present invention relates to the insecticidal protein produced by the cryIF gene (SEQ ID NO:1). The CryIF protein (SEQ
ID NO:2) has the amino acid sequence, as deduced from the nucleotide sequence of the crylF gene from bases 478 to 3999, that is shown in Figure 1. The protein exhibits insecticidal activity against insects of the order Lepidoptera, in particular, Ostrinia nubilalis (European corn borer) and Spodootera exigua (beet armyworm).
Another aspect of the present invention relates to a purified and isolated insecticidal toxin gene hereinafter designated as the cryIX
WO 91/16434 PC?/US91/02560 ~~~~~?~
gene, a portion (SEQ ID No:3) of whose nucleotide 20base sequence is shown in Figure 2. The present invention also relates to the insecticidal protein produced by the cryIX gene and called the CryIX
protein, which protein has a molecular mass of about 81 kDa. A portion (SEQ ID N0:4) of the amino acid sequence for the 81 kDa CryIX protein, as deduced from the truncated portion (SEQ ID No:3) of the cryIX gene, is also shown in Figure 2. The 81 kDa CryIX protein exhibits insecticidal activity against lepidopteran insects, such as Plutella xylostella (diamondback moth) and Ostrinia nubilalis (European corn borer).
Still another aspect of the present invention relates to biologically pure cultures of B.t. and E. coli bacteria deposited with the NRRL
having Accession Nos. NRRL B-18633, B-18635, and B-18805 and being designated as B.t. strains EG6345 and EG1945, and E. coli strain EG1083, respectively. B.t. strains EG6345 and EG1945 carry the cryIF gene and produce the insecticidal CryIF
protein. E. coli strain EG1083 carries the crylX
gene and produces the CryIX protein. Biologically pure cultures of other B.t. bacteria carrying the crylF gene or of B.t. strains carrying the crylX
gene 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 CryIF or CryIX protein or fermentation cultures of a B.t. strain which has produced the CryIF protein or the CryIX protein.
91/16434 3'CT/US91/02560 ~~cl, 21 61 The invention also includes a method of controlling lepidopteran insects by applying to a host plant for such insects an insecticidally effective amount of the CryIF protein or the CryIX
protein or of a fermentation culture of a B.t.
strain that has made the CryIF protein or the CryIX
protein.
Still other aspects of the present invention relate to recombinazzt plasmids containing the cryIF gene and/or the crylX gene; biologically pure cultures of a bacterium transformed with such recombinant plasmids, the bacterium preferably being B.t., such as the aforementioned B.t. strain EG1945; as well as plants transformed with the cryiF gene and/or the cryIX gene.
Brief Description of the Drawings Figure 1 comprises Figures 1-A through 1-E and shows the partial nucleotide base sequence of DNA from a 5.7 kb fragment that contains the crylF gene inserted into plasmid pEG640. The DNA
sequence begins with the 51 Sau3A cloning site and extends 4020 bp in length. The open reading frame for the cryIF gene and the deduced amino acid sequence of the CryIF protein are indicated. The putative ribosome binding site (RBS) for the crylF
gene is indicated on Figure 1-A. Sites for the restriction enzymes Sau3A, BamHI and YRnI are also indicated.
Figure 2 comprises Figures 2-A and 2-B
and shows the partial nucleotide base sequence of DNA from the 5.7 kb fragment inserted into plasmid pEG640 that contains a portion of the cryIX gene.
WO 91/16434 PC?/US91/02560 -la-The DNA sequence begins with nucleotide base position 4021 in Figure 2-A, which is immediately adjacent to and downstream from position 4020 in Figure 1-E, and extends to nucleotide base position 5649 in Figure 2-B, ending at the 31 Sau3A cloning site. The open reading frame for the truncated crylX gene and the deduced amino acid sequence of the CryIX protein encoded by the cr-yIX gene fragment are indicated. The putative ribosome binding site (RBS) for the crylX gene is indicated.
Sites for the restriction enzymes KgnI and Sau3A
are indicated.
Figure 3 comprises a photocopy of a portion of an ethidium bromide stained agarose electrophoresis gel containing size fractionated native plasmids of B.t. subsp. aizawai strains EG6346 in the left lane and EG6345 in the right lane. The number to the right of Figure 3 indicates the approximately 45 MDa plasmid of B.t.
strain EG6345 which is absent in the cured B.t.
strain EG6346.
Figure 4 comprises Figures 4-A, 4-B and 4-C, which are photocopies of autoradiograms made by transferring total HindIII-digested DNA from B.t. strains EG6346 (lane 1 of each autoradiogram), EG6345 (lane 2 of each autoradiogram) and HD-1 (lane 3 of each autoradiogram) to nitrocellulose filters, hybridizing the filters with radioactively labeled probes and exposing the filter to X-ray film. The DNA in the autoradiogram labeled Figure 4-A follows hybridization of the DNA to a 32P-labeled 0.7 kb EcoRI probe from the cryIA(a) gene of B.t. strain HD-1. The autoradiogram "Y4) 91/16434 PE'T/US91/02560 labeled Figure 4-B follows hybridization of the DNA to a 32P-labeled intragenic 2.2 kb PvuII probe from the crylA(a) gene of B.t. strain HD-1. The autoradiogram labeled Figure 4-C follows hybridization of the DNA to a 32P-labeled plasmid pEG640 probe. The numbers to the left of Figure 4 indicate the sizes, in kb, of standard DNA
fragments of phage lambda.
Figure 5 shows a restriction map of plasmid pEG640. The locations and orientations of the cryIF gene and a gene designated the cryIX gene are indicated by arrows. The solid black line indicates the E. coli cloning vector pGEM"-3Z. The following letters designate the indicated restriction enzymes: B BamHI; B2 = BstEII;
C = Clal; E= EcoRI; H Hindill; K = KPnI,=
Pt1 = Pstl; Pv2 $ PvuII; S= SacI; X = XbaI.
Figure 6, based on the same scale as Figure 5, shows a restriction map of plasmid pEG642 which was created by inserting plasmid pEG640 into a HindIIl site on the Bacillus vector pEG434. The abbreviations and other indicators referred to with respect to Figure 5 apply with respect to Figure 6.
In addition, the crosshatched area of Figure 6 indicates vector pEG434 and the arrow labeled "tet"
indicates the direction of transcription of the tetracycline resistance gene encoded on plasmid vector pEG434.
Figure 7 is a photocopy of a Coomassie stained SDS-polyacrylamide gel showing gradient purified crystal protein from B.t. strain EG6345 (lane 1), B.t. strain EG6346 (lane 2) and recombinant B.t. strain EG1945 harboring the crylF
WO 91/16434 PC'T/US91/02560 - 16 - 2, 0G~
gene (lane 3). The unnumbered, extreme left lane adjacent to lane 1 contains molecular weight standards having the indicated sizes, in kDa.
Figure 8 comprises Figures 8-A, 8-B and 8-C. Figure 8-A is a photocopy of an ethidium bromide stained agarose electrophoresis gel containing size-fractionated plasmids of B.t.
strains HD-1 (lane 1), EG6345 (lane 2) and EG6346 (lane 3). Figure 8-B is a photocopy of an autoradiogram made by transferring the plasmids resolved by the gel shown in Figure 8-A to a nitrocellulose filter, hybridizing the filter with a 32P-labeled 2.2 kb PvuII intragenic fragment obtained from the crylA(a) gene of HD-1, where lanes 1 through 3 in Figure 8-B correspond to lanes 1 through 3 of Figure 8-A. Figure 8-C is a photocopy of an autoradiogram made by transferring the plasmids resolved by the gel shown in Figure 8-A to a nitrocellulose filter, hybridizing the filter with a 32 P-labeled 0.4 kb PstI-SacI
intragenic N-terminal fragment obtained from the crylF gene, where lanes I through 3 of Figure 8-C
correspond to lanes 1 through 3 of Figure 8-A. The numbers to the left of Figure 8-A indicate the sizes, in MDa, of various plasmids. The letter "L"
to the left of Figure 8-A indicates the linear degeneration fragments from the breakdown of the larger plasmids.
Figure 9 comprises a photocopy of an autoradiogram made by transferring total DNA from B.t. strain EG6346 digested with restriction enzymes (as described below) to a nitrocellulose filter, hybridizing the filter with a 32 P-labeled 0.6 kb K.nI-BamHI restrictiori fragment containing a portion of the cryrX gene, and exposing the filter to X-ray film. The digestiori of total DNA from B.t. strain EG6346 was carrie:d out with several restriction enzymes, as follows: As~718 (an isoschizomer of KpnI) in lane: 1, Clal in lane 2, Sphl in lane 3, Asp718 (KpnI) + S.hI in lane 4, Clal +SphI in lane 5, Sstl in lane 6, AsR718 (KpnI) + SstI in lane 7, Clal + SstI in lane 8.
The numbers to the left of Figure 9 indicate the sizes, in kb, of standard DNA fragments of phage lambda.
Figure 10 shows a circular restriction map of the 7.2 kb B.t.-E. coli cloning vector pEG854, originally described by Baum et al., ARpl.
Environ. Microbiol. 56, pp. 3420-3428 (1990). The open box represents the pT219u segment of the vector that contains an ampicillin resistance gene and a replication origin functional in E. coli.
The shaded box, designated ori 43, contains a 2.8 kb replication origin region derived from a native B.t. plasmid that is function in B.t.. The solid black arrow corresponds to a chloramphenicol acetyltransferase (cat) gene that confers chloramphenicol resistance on B.t.' strains transformed with pEG854 or its derivatives.
Cloning vector pEG854 contains a unique C1aI
restriction site within the multiple cloning site, designated MCS in the Figure. Restriction sites for Xbal, SfiI, and Notl restriction endonucleases are also shown.
`cf 6 Figure 11 shows a circular restriction map of the 11.8 kb recombinar-t plasmid pEG313 consisting of a 4.6 kb Clal restriction fragment isolated from total DNA of B.t. strain EG6346 inserted in the Clal site of cloning vector pEG854 (see Figure 10). The Clal sites flanking the 4.6 kb crylX-encoding fragment are indicated in bold type. An SstI restriction site, located downstream from the cryIX gene, is contained within the 4.6 kb Clal restriction fragment. The orientation and approximate length of the crylX coding region is indicated by the open boxed arrow. Other annotations are as described for Figure 10.
Figure 12 shows a circular restriction map of the 2.86 kb E. coli cloning vector pTZl9u, used to obtain expression of the cryIX gene in E. coli. A multiple cloning site region, containing unique restriction sites for Accl and Sstl (in bold type), is demarcated by unique HindIII and EcoRI restriction sites within the lacZ' gene. vector pTZ19u contains a beta-lactamase gene (bla) that confers resistance to ampicillin and also contains the replication region from an fl filamentous phage (1) used for the synthesis of single-stranded DNA. The lac promoter (Plac in bold type) is positioned upstream from the multiple cloning site region. Restriction sites for Nae2, Scal and g MI restriction endonucleases are also shown.
Figure 13 shows a circular restriction map of the 7.3 kb recombinant plasmid pEG314 consisting of a 4.4 kb Clal-SstI restriction fragment derived from pEG313 (see Figure 11) WO 91/16434 P('T/US91/02560 - 19 - `J~~0 'p) C~4 inserted into the AccI and SstI restriction sites of vector pTZ19u (see Figure.12). The orientation and approximate relative length of the cr IX coding region is indicated by the open arrow. Other annotations are as described for Figure 12.
Figure 14 is a photocopy of a Coomassie stained 10% SDS-polyacrylamide gel showing crude (in lane 1) and gradient purified (in lane 2) CryiX
crystal protein from E. coli strain EG1083. The numbers to the left of Figure 14 indicate the sizes, in kDa, of protein molecular weight (MW) standards displayed in the leftmost (unnumbered) lane.
Detailed Description of the Preferred Embodiments The isolation and purification of the cryIF gene (SEQ ID NO:1) and the lepidopteran-toxic CryIF crystal protein (SEQ ID NO:2), and the characterization of the native B.t. strain EG6345, the cured B.t. strain EG6346 derived from B.t.
strain EG6345, and the recombinant B.t. strain EG1945, both of which produce the CryIF protein, are described in the Examples. The utility of recombinant B.t. strain EG1945 and of the CryIF
crystal protein in insecticidal compositions and methods is also illustrated in the Examples.
Similarly, the isolation and purification of the cryIX gene and the characterization of its lepidopteran-toxic CryIX crystal protein are also illustrated in the Examples. The methods and procedures described in the Examples for the cr IF
W091 / 16434 PC'T/L'S91 /02560 and its Cr IF
gene y protein are also generally applicable to the cryIX gene and its insecticidal CryIX protein.
The cr I-type gene of this invention, the crylF gene (SEQ ID NO:1), has the nucleotide base sequence shown in Figure 1. The coding region of the crylF gene extends from nucleotide base position 478 to position 3999 shown in Figure 1.
A comparison of the nucleotide base pairs of the crylF gene coding region with the corresponding coding region of the prior art cr I
genes indicates significant differences between the new cryIF gene and the prior art cr I genes. The crylF gene is only about 67% to about 78%
homologous (positionally identical) with the cryIA(a), cryIA(b) and cryIA(c) genes and the crylB
and cryIC genes. There is even less homology with the cr II, crylli and cryIV genes, described in Hofte and Whiteley (1989). The homology is discussed in more detail hereinafter.
The CryI-type protein of this invention, the CryIF protein (SEQ ID NO:2) that is encoded by the cryIF gene, has the amino acid sequence shown in Figure 1. In this disclosure, references to the CryIF "protein" (and to the CryIX "protein") are synonymous with its description as a "crystal protein," "protein toxin," "insecticidal protein,"
"delta endotoxin" or the like, unless the context indicates otherwise.
30. The deduced size of the CryIF protein is 133,635 Da. The prior art Cryl-type proteins, encoded by the respective cryI genes, have similar deduced sizes. Despite the apparent size - 21 - ~~U~~ ~rJC}
similarity, comparison of the amino acid sequence of the CryIF protein with published sequences of the other prior art CryI-type proteins shows significant differences betweeri them and the CryIF
protein. The CryIF protein is only about 58% to about 72% identical with the other prior art Cryl-type proteins, even when considering the C-terminal regions which are more related than the N-terminal regions.
The cryIX gene of this invention contains approximately 2100-2200 basepairs in its coding region, of which approximately 1140 basepairs are shown for the truncated upstream portion (SEQ ID
NO:3) of the cryIX gene in Figure 2. The crylX
gene of this invention is contained in isolated form on a DNA fragment carried on a recombinant plasmid, in E. coli strain EG1083 which has been deposited in the NRRL under accession No. NRRL B-18805. The CryIX protein of this invention, produced by the cryIX gene, is about 81 kDa in size and exhibits insecticidal activity against insects of the order Lepidoptera. The amino acid sequence (SEQ ID NO:4) -for a portion of the CryIX protein, deduced from the truncated portion of the crylX
gene shown in Figure 2, is shown in Figure 2. The 380 amino acids of this initial portion (SEQ ID
NO:4) of the CryIX protein shown in Figure 2 represent approximately one-half of the CryIX
protein encoded by the cr IX gene.
The CryIF and CryIX proteins have been shown to be insecticidal against insects of the order Lepidoptera, as set forth in more detail in Examples 6 and 11, respectively.
The present invention is intended to cover mutants and recombinant or genetically engineered derivatives of the: crylF gene and crylX
gene that yield lepidopteran-toxic proteins with essentially the same properties as the respective CryIF and CryIX proteins.
The cryIF gene and cryIX gene are also useful as DNA hybridization probes, for discovering similar or closely related cryi-type genes in other B.t. strains. The crylF or cryzX gene, or portions or derivatives thereof, can be labeled for use as a hybridization probe, e.g., with a radioactive label, using conventional procedures. The labeled DNA hybridization probe may then be used in the manner described in the Examples.
The cryIF or cryIX gene 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 crylF or crylX gene, as the case may be. Suitable hosts that allow the er IF and cryIX
genes to be expressed and the respective CryIF and CryIX proteins to be produced include Bacillus thuringiensis and other Bacillus species such as B.
subtilis or B. megaterium. E. coli or Pseudomonas fluorescens are also suitable hosts for these genes. It should be evident that genetically altered or engineered microorganisms containing the crylF gene or crylX gene can also contain other toxin genes present in the same microorganism and WO 91/16434 FCI'/U591/02560 that these genes could concurrently produce insecticidal crystal proteins different from the cryIF and CryIX proteins.
The Bacillus and E. coli strains described in this disclosure may be cultured using conventional growth media and standard fermentation techniques. The B.t. strains harboring the cr IF
gene (or the cryIX gene) may be fermented, as described in the Examples, until the cultured B.t.
cells reach the stage of their growth cycle when CryIF crystal protein (or CryIX crystal protein) is formed. For sporogenous B.t. strains, fermentation is typically continued through the sporulation stage, when 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 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 crylF gene and the crylX gene also have 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 ,c1e IF or crylX gene) in this disclosure are intended to cover sporulated B.t. cultures, i.e., B.t. cultures containing the CryIF or CryIX crystal protein and spores, and sporogenous Bacillus strain cultures that have produced crystal protein during the vegetative stage, as well as asporogenous WO 9l/15434 P("T/US91/02560 24 - 208063d -Bacillus strains containing the cr IF or cr IX gene in which the culture has reached the growth stage where crystal protein is actually produced.
The separated fermentation solids are primarily CryIF or CryIX crystal protein, as the case may be, and B.t. spores, along with some cell debris, some intact cells, and residual fermentation medium solids. If desired, the crystal protein may be separa'ted from the other recovered solids via conventional methods, e.g., sucrose density gradient fractionation. Highly purified CryIF or CryIX protein may be obtained by solubilizing the recovered crystal protein and then reprecipitating the protein from solution.
The CryIF protein is an effective insecticidal compound against lepidopteran insects like the European cornborer, the beet armyworm, and the tobacco budworm, for example. Likewise, the CryIX protein is insecticidal to lepidopteran insect species. The CryIF protein or CryIX protein may be utilized as the active ingredient in insecticidal formulations useful for the control of lepidopteran insects. Such insecticidal formulations or compositions typically contain agriculturally acceptable carriers or adjuvants in addition to the active ingredient.
The CryZF protein or CryIX protein may be employed in insecticidal formulations in isolated or purified form, e.g., as the crystal protein itself. Alternatively, the CryIF protein or CryIX
protein may be present in the recovered fermentation solids, obtained from culturing of a Bacillus strain, e.g., Bacillus thuringiensis, or "'0 91/16434 4'CI'/US91/02560 other microorganism host carrying the crylF or cryIX gene and capable of producing the corresponding CryIF or CryIX protein. Preferred Bacillus hosts for the crylF gene include B.t.
strain EG6345 and genetically improved B.t. strains derived from B.t. strain EG6345, such as B.t.
strain EG6346. The derivative B.t. strains may be obtained via plasmid curing arad/or conjugation techniques and contain the native cryIF gene-containing plasmid from B.t. strain EG6345.
Genetically engineered or transformed B.t. strains or other host microorganisms, containing a recombinant plasmid that expresses the cloned cryIF
gene and obtained by recombinant DNA procedures, may also be used.
Examples of such transformants include B.t. strain EG1945 which contains the cloned cryIF
gene 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 CryIF or CryIX protein may be dried, if desired, prior to incorporation into the insecticidal formulation.
The formulations or compositions of this invention containing the insecticidal CryIF or CryIX protein as the active component are applied at an insect:tcidally effective amount which will vary depending on such factors as, for example, the specific lepidopteran insects to be controlled, the specific plarit or crop to be treated and the method WO 91/16434 PC3'/1J591/02560 - 26 208063d -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, 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 CryIF or CryIX protein 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 CryIF or CryIX 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.
WO 91/16434 JPCi'/US91/02560 - 27 - 2080~i~ 8iI
Other application techniques, e.g., dusting, sprinkling, soaking, soil injection, seed coating, seedling coating or spraying, or the like, are also feasible. These application procedures are well known in the art.
The crylF or crylX gene 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 cr IF gene results in the production of insecticidal CryIF crystal protein. Likewise, expression of the cryzx gene results in production of the insecticidal CryIX protein. Suitable hosts include B.t. and other species of Bacillus, such as B. subtilis or B. megaterium, for example. Other bacterial hosts such as E. coli and Pseudomonas fluorescens may also be used. Various procedures well known to those skilled in the art are available for introducing the crylF or crylX gene 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 ways, 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. Again, these techniques are standard procedures.
WO 91/16434 PG?/U591/02560 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 CryIF or CryIX
insecticidal protein in the host, and the presence of auxiliary genetic capabilities. The cellular host containing the insecticidal crylF or cryTX
gene may be grown in any convenient nutrient medium, where expression of the crZIF or crylX gene is obtained and corresponding CryIF or CryIX
protein produced, typically upon sporulation. The sporulated cells containing the crystal protein may then be harvested in accordance with conventional ways, e.g., centrifugation or filtration.
The cryaF and cryIX genes may also be incorporated into a plant which is capable of expressing the gene and producing CryIF or CryIX
protein, as the case may be, rendering the plant more resistant to insect attack. Genetic engineering of plants with the cryIF or cryIX gene 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 know to those skilled in plant genetic engineering. An example of a technique for 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 crylE or crylX gene or a modified crylF or crylX gene capable of producing the corresponding CryIF or CryIX protein may be '/0 91/16434 PCT/YJ591/02560 _ 29 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.
Slight variations may be made in the crXIF or crYIX gene nucleotide base sequences, since the various amino acids forming the proteins encoded by the respective genes 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 region of the cryIF and cryIX nucleotide base sequences which allow expression of the gene and production of functionally equivalent forms of the corresponding CryIF and CryIX insecticidal proteins. 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.
The.present invention will now be described in more detail with reference to the following specific, non-limiting examples. The examples relate to work which was actually done based on techniques generally known in the art and using commercially available equipment.
WO 91/16434 F'GT/US91/02560 The novel B.t. strain EG6345 and a cured derivative B.t. strain EG6346 were isolated following the procedures described in Example 1.
Examlale 1 Isolation of B.t. Strainst EG634S and EG6346 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'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 extensive screening of crop dust samples, a B.t. subsp. aizawai strain, designated B.t. strain EG6345, was isolated from a maize grain dust sample. A sporulated culture of B.t. strain EG6345 was spread for the growth of individual colonies on a nutrient salts agar plate and incubated for 3 days at 306C. After incubation, one colony was noted on this plate which displayed a different colony morphology (i.e., shinier) than the parent B.t. strain EG6345. The colony, designated B.t. strain EG6346, was isolated as an individual colony.
A sample of the isolated B~t. strain EG6346 was further purified by streaking on an agar plate containing Spizizen's glucose peptone beef '0 91/16434 PCT/US91/02560
aizawai 7.29. The sequence revealed is the same as that in Sanchis et al., Molecular Microbiol., 3, pp. 229-238 (1989), described above.
Schnepf et al., in U.S. Patent 4,467,036, issued August 21, 1984, disclose a hybrid recombinant plasmid capable of replication in an E.
coli strain, and capable of expressing a polypeptide with the immunological properties of B.t. crystal protein, and being identifiable with a PvuII C DNA fragment probe derived from a gene, now known as crylA(a), of B.t. var. kurstaki strain HD-i. The following B.t. subspecies and strains are disclosed as sources of expressible heterologous DNA: tolworthi, darmstadiensis, sotto, thuringiensis, kurstaki, HD-290, HD-120, HD-2, HD-244, HD-73, HD-l, HD-4, HD-8, F-6, F-5 and F-9.
Generally, the sequences of B.t. genes encoding delta-endotoxin proteins active against different orders of insects are not well-conserved.
Rather, the sequences of genes responsible for a given crystal phenotype and active against the same insect order are significantly inore related. The homology of delta-endotoxin amino acid sequences, as well as similarities in insecticidal activity, have been used to define an ordered classification of genes encoding B.t. delta-endotoxin proteins (Hofte and Whiteley (1989)). The genes encoding WO 91/16434 PCF/bJS91/02560 ~~~068n g the 130-138 kDa, lepidopteran.-active delta-endotoxin proteins comprise the largest of these families, the cr I genes.
Within the cryl gene classification described by Hofte and Whiteley (1989), a subranking has been established based upon further refinement of sequence relationship. Thus, the crylA(a), crYIA(b) and cryIA c) gene sub-families embrace the previously designated 4.5 kb, 5.3 kb and 6.6 kb P1 genes, respectively. These genes originally were differentiated according to the size of a characteristic HindiIl fragment associated with the presence of the gene. The amino acid sequences of CryIA proteins are highly related (greater than 80% amino acid identity), with most of the sequence dissimilarity relegated to a short internal variable region (Whiteley et al., Ann. Rev. Microbiol., 40, pp. 549-576 (1986)).
It is believed that differences within this variable region account for the different insecticidal specificities exhibited by the proteins encoded by the crylA(a), crylA(b) and crylA(c) genes.
Recently, additional genes within the cryl family have been discovered, such as the cryIB
gene found in B.t. subsp. thuringiensis (Brizzard et al., (1988), supra), and the crylC and crylo genes found in subsp. aizawai (Sanchis et al., (1988), supra, Visser et a1. (1988), M.ra, Hofte and Whiteley (1989), European Patent Application Publication No. 0 358 557, published March 14, 1990, of Plant Genetic Systems, N.V.), and the crylE gene found in B.t. subsp. darmstadiensis WO 91/16434 FClf/US91/02560 2 f) C13 13 bi ;111 (EP 0 358 557 (1990), supra) and in B.t._ subsp.
kenyae (Visser et al., J. Bacteriol., 172, pp. 6783-6788 (1990)). Other cryI-type genes are disclosed in European Patent Application Publication No. 0 367 474, published May 9, 1991, of Mycogen Corporation, in European Patent Application Publication No. 0 401 979, published December 12, 1990 of Mycogen Corporation, and in PCT International Publication No. WO 90/13651, published November 15, 1990, of Imperial Chemical Industries PLC. Comparisons of the sequences for these cryz-type genes to the cryIA genes reveal significant sequence dissimilarities, particularly in the N-terminal protein domain.
The present invention is based on the discovery of at least one additional subgroup of crVl genes. The prototype of this subgroup, which the inventors have designated cryIF, was also isolated from a B.t. subsp. aizawai strain.
However, the sequence of the crylP gene and the insecticidal specificity of the CryIF protein it encodes, is distinctly different from the other cryI genes and their encoded Cryl proteins. This distinction is also true with respect to the crylC
gene and its encoded protein, even though a spontaneously cured derivative of a B.t. strain containing the cr XIC gene was used in the isolation of the crylF gene.
In addition, the present invention includes the identification and isolation of a second gene, designated cryz, which is located downstream from the novel cr IF gene. Of the cryI-type genes discussed above, the gryIX gene w0 91/16434 PC T/US91/02560 s 4Y Ci r"
) appears most closely related to the B.t. toxin gene disclosed in PCT International Publication No. WO 90/13651, published November 15, 1990, of Imperial Chemical Industries PLC.
Data are presented hereinafter concerning the identification, cloning, sec[uencing and expression of these novel cr IF and crylX toxin genes, as well as the insecticidlal activities of the CryIF and CryIX proteins against lepidopteran larvae.
Summary of the Invention One aspect of the present invention relates to a purified and isolated lepidopteran-active toxin gene having a nucleotide base sequence coding for the amino acid sequence illustrated in Figure 1 and hereinafter designated as the crylF
gene (SEQ ID NO:1). The cryIF gene (SEQ ID NO:1) has a coding region, i.e., open reading frame, extending from nucleotide bases 478 to 3999 shown in Figure 1.
Another aspect of the present invention relates to the insecticidal protein produced by the cryIF gene (SEQ ID NO:1). The CryIF protein (SEQ
ID NO:2) has the amino acid sequence, as deduced from the nucleotide sequence of the crylF gene from bases 478 to 3999, that is shown in Figure 1. The protein exhibits insecticidal activity against insects of the order Lepidoptera, in particular, Ostrinia nubilalis (European corn borer) and Spodootera exigua (beet armyworm).
Another aspect of the present invention relates to a purified and isolated insecticidal toxin gene hereinafter designated as the cryIX
WO 91/16434 PC?/US91/02560 ~~~~~?~
gene, a portion (SEQ ID No:3) of whose nucleotide 20base sequence is shown in Figure 2. The present invention also relates to the insecticidal protein produced by the cryIX gene and called the CryIX
protein, which protein has a molecular mass of about 81 kDa. A portion (SEQ ID N0:4) of the amino acid sequence for the 81 kDa CryIX protein, as deduced from the truncated portion (SEQ ID No:3) of the cryIX gene, is also shown in Figure 2. The 81 kDa CryIX protein exhibits insecticidal activity against lepidopteran insects, such as Plutella xylostella (diamondback moth) and Ostrinia nubilalis (European corn borer).
Still another aspect of the present invention relates to biologically pure cultures of B.t. and E. coli bacteria deposited with the NRRL
having Accession Nos. NRRL B-18633, B-18635, and B-18805 and being designated as B.t. strains EG6345 and EG1945, and E. coli strain EG1083, respectively. B.t. strains EG6345 and EG1945 carry the cryIF gene and produce the insecticidal CryIF
protein. E. coli strain EG1083 carries the crylX
gene and produces the CryIX protein. Biologically pure cultures of other B.t. bacteria carrying the crylF gene or of B.t. strains carrying the crylX
gene 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 CryIF or CryIX protein or fermentation cultures of a B.t. strain which has produced the CryIF protein or the CryIX protein.
91/16434 3'CT/US91/02560 ~~cl, 21 61 The invention also includes a method of controlling lepidopteran insects by applying to a host plant for such insects an insecticidally effective amount of the CryIF protein or the CryIX
protein or of a fermentation culture of a B.t.
strain that has made the CryIF protein or the CryIX
protein.
Still other aspects of the present invention relate to recombinazzt plasmids containing the cryIF gene and/or the crylX gene; biologically pure cultures of a bacterium transformed with such recombinant plasmids, the bacterium preferably being B.t., such as the aforementioned B.t. strain EG1945; as well as plants transformed with the cryiF gene and/or the cryIX gene.
Brief Description of the Drawings Figure 1 comprises Figures 1-A through 1-E and shows the partial nucleotide base sequence of DNA from a 5.7 kb fragment that contains the crylF gene inserted into plasmid pEG640. The DNA
sequence begins with the 51 Sau3A cloning site and extends 4020 bp in length. The open reading frame for the cryIF gene and the deduced amino acid sequence of the CryIF protein are indicated. The putative ribosome binding site (RBS) for the crylF
gene is indicated on Figure 1-A. Sites for the restriction enzymes Sau3A, BamHI and YRnI are also indicated.
Figure 2 comprises Figures 2-A and 2-B
and shows the partial nucleotide base sequence of DNA from the 5.7 kb fragment inserted into plasmid pEG640 that contains a portion of the cryIX gene.
WO 91/16434 PC?/US91/02560 -la-The DNA sequence begins with nucleotide base position 4021 in Figure 2-A, which is immediately adjacent to and downstream from position 4020 in Figure 1-E, and extends to nucleotide base position 5649 in Figure 2-B, ending at the 31 Sau3A cloning site. The open reading frame for the truncated crylX gene and the deduced amino acid sequence of the CryIX protein encoded by the cr-yIX gene fragment are indicated. The putative ribosome binding site (RBS) for the crylX gene is indicated.
Sites for the restriction enzymes KgnI and Sau3A
are indicated.
Figure 3 comprises a photocopy of a portion of an ethidium bromide stained agarose electrophoresis gel containing size fractionated native plasmids of B.t. subsp. aizawai strains EG6346 in the left lane and EG6345 in the right lane. The number to the right of Figure 3 indicates the approximately 45 MDa plasmid of B.t.
strain EG6345 which is absent in the cured B.t.
strain EG6346.
Figure 4 comprises Figures 4-A, 4-B and 4-C, which are photocopies of autoradiograms made by transferring total HindIII-digested DNA from B.t. strains EG6346 (lane 1 of each autoradiogram), EG6345 (lane 2 of each autoradiogram) and HD-1 (lane 3 of each autoradiogram) to nitrocellulose filters, hybridizing the filters with radioactively labeled probes and exposing the filter to X-ray film. The DNA in the autoradiogram labeled Figure 4-A follows hybridization of the DNA to a 32P-labeled 0.7 kb EcoRI probe from the cryIA(a) gene of B.t. strain HD-1. The autoradiogram "Y4) 91/16434 PE'T/US91/02560 labeled Figure 4-B follows hybridization of the DNA to a 32P-labeled intragenic 2.2 kb PvuII probe from the crylA(a) gene of B.t. strain HD-1. The autoradiogram labeled Figure 4-C follows hybridization of the DNA to a 32P-labeled plasmid pEG640 probe. The numbers to the left of Figure 4 indicate the sizes, in kb, of standard DNA
fragments of phage lambda.
Figure 5 shows a restriction map of plasmid pEG640. The locations and orientations of the cryIF gene and a gene designated the cryIX gene are indicated by arrows. The solid black line indicates the E. coli cloning vector pGEM"-3Z. The following letters designate the indicated restriction enzymes: B BamHI; B2 = BstEII;
C = Clal; E= EcoRI; H Hindill; K = KPnI,=
Pt1 = Pstl; Pv2 $ PvuII; S= SacI; X = XbaI.
Figure 6, based on the same scale as Figure 5, shows a restriction map of plasmid pEG642 which was created by inserting plasmid pEG640 into a HindIIl site on the Bacillus vector pEG434. The abbreviations and other indicators referred to with respect to Figure 5 apply with respect to Figure 6.
In addition, the crosshatched area of Figure 6 indicates vector pEG434 and the arrow labeled "tet"
indicates the direction of transcription of the tetracycline resistance gene encoded on plasmid vector pEG434.
Figure 7 is a photocopy of a Coomassie stained SDS-polyacrylamide gel showing gradient purified crystal protein from B.t. strain EG6345 (lane 1), B.t. strain EG6346 (lane 2) and recombinant B.t. strain EG1945 harboring the crylF
WO 91/16434 PC'T/US91/02560 - 16 - 2, 0G~
gene (lane 3). The unnumbered, extreme left lane adjacent to lane 1 contains molecular weight standards having the indicated sizes, in kDa.
Figure 8 comprises Figures 8-A, 8-B and 8-C. Figure 8-A is a photocopy of an ethidium bromide stained agarose electrophoresis gel containing size-fractionated plasmids of B.t.
strains HD-1 (lane 1), EG6345 (lane 2) and EG6346 (lane 3). Figure 8-B is a photocopy of an autoradiogram made by transferring the plasmids resolved by the gel shown in Figure 8-A to a nitrocellulose filter, hybridizing the filter with a 32P-labeled 2.2 kb PvuII intragenic fragment obtained from the crylA(a) gene of HD-1, where lanes 1 through 3 in Figure 8-B correspond to lanes 1 through 3 of Figure 8-A. Figure 8-C is a photocopy of an autoradiogram made by transferring the plasmids resolved by the gel shown in Figure 8-A to a nitrocellulose filter, hybridizing the filter with a 32 P-labeled 0.4 kb PstI-SacI
intragenic N-terminal fragment obtained from the crylF gene, where lanes I through 3 of Figure 8-C
correspond to lanes 1 through 3 of Figure 8-A. The numbers to the left of Figure 8-A indicate the sizes, in MDa, of various plasmids. The letter "L"
to the left of Figure 8-A indicates the linear degeneration fragments from the breakdown of the larger plasmids.
Figure 9 comprises a photocopy of an autoradiogram made by transferring total DNA from B.t. strain EG6346 digested with restriction enzymes (as described below) to a nitrocellulose filter, hybridizing the filter with a 32 P-labeled 0.6 kb K.nI-BamHI restrictiori fragment containing a portion of the cryrX gene, and exposing the filter to X-ray film. The digestiori of total DNA from B.t. strain EG6346 was carrie:d out with several restriction enzymes, as follows: As~718 (an isoschizomer of KpnI) in lane: 1, Clal in lane 2, Sphl in lane 3, Asp718 (KpnI) + S.hI in lane 4, Clal +SphI in lane 5, Sstl in lane 6, AsR718 (KpnI) + SstI in lane 7, Clal + SstI in lane 8.
The numbers to the left of Figure 9 indicate the sizes, in kb, of standard DNA fragments of phage lambda.
Figure 10 shows a circular restriction map of the 7.2 kb B.t.-E. coli cloning vector pEG854, originally described by Baum et al., ARpl.
Environ. Microbiol. 56, pp. 3420-3428 (1990). The open box represents the pT219u segment of the vector that contains an ampicillin resistance gene and a replication origin functional in E. coli.
The shaded box, designated ori 43, contains a 2.8 kb replication origin region derived from a native B.t. plasmid that is function in B.t.. The solid black arrow corresponds to a chloramphenicol acetyltransferase (cat) gene that confers chloramphenicol resistance on B.t.' strains transformed with pEG854 or its derivatives.
Cloning vector pEG854 contains a unique C1aI
restriction site within the multiple cloning site, designated MCS in the Figure. Restriction sites for Xbal, SfiI, and Notl restriction endonucleases are also shown.
`cf 6 Figure 11 shows a circular restriction map of the 11.8 kb recombinar-t plasmid pEG313 consisting of a 4.6 kb Clal restriction fragment isolated from total DNA of B.t. strain EG6346 inserted in the Clal site of cloning vector pEG854 (see Figure 10). The Clal sites flanking the 4.6 kb crylX-encoding fragment are indicated in bold type. An SstI restriction site, located downstream from the cryIX gene, is contained within the 4.6 kb Clal restriction fragment. The orientation and approximate length of the crylX coding region is indicated by the open boxed arrow. Other annotations are as described for Figure 10.
Figure 12 shows a circular restriction map of the 2.86 kb E. coli cloning vector pTZl9u, used to obtain expression of the cryIX gene in E. coli. A multiple cloning site region, containing unique restriction sites for Accl and Sstl (in bold type), is demarcated by unique HindIII and EcoRI restriction sites within the lacZ' gene. vector pTZ19u contains a beta-lactamase gene (bla) that confers resistance to ampicillin and also contains the replication region from an fl filamentous phage (1) used for the synthesis of single-stranded DNA. The lac promoter (Plac in bold type) is positioned upstream from the multiple cloning site region. Restriction sites for Nae2, Scal and g MI restriction endonucleases are also shown.
Figure 13 shows a circular restriction map of the 7.3 kb recombinant plasmid pEG314 consisting of a 4.4 kb Clal-SstI restriction fragment derived from pEG313 (see Figure 11) WO 91/16434 P('T/US91/02560 - 19 - `J~~0 'p) C~4 inserted into the AccI and SstI restriction sites of vector pTZ19u (see Figure.12). The orientation and approximate relative length of the cr IX coding region is indicated by the open arrow. Other annotations are as described for Figure 12.
Figure 14 is a photocopy of a Coomassie stained 10% SDS-polyacrylamide gel showing crude (in lane 1) and gradient purified (in lane 2) CryiX
crystal protein from E. coli strain EG1083. The numbers to the left of Figure 14 indicate the sizes, in kDa, of protein molecular weight (MW) standards displayed in the leftmost (unnumbered) lane.
Detailed Description of the Preferred Embodiments The isolation and purification of the cryIF gene (SEQ ID NO:1) and the lepidopteran-toxic CryIF crystal protein (SEQ ID NO:2), and the characterization of the native B.t. strain EG6345, the cured B.t. strain EG6346 derived from B.t.
strain EG6345, and the recombinant B.t. strain EG1945, both of which produce the CryIF protein, are described in the Examples. The utility of recombinant B.t. strain EG1945 and of the CryIF
crystal protein in insecticidal compositions and methods is also illustrated in the Examples.
Similarly, the isolation and purification of the cryIX gene and the characterization of its lepidopteran-toxic CryIX crystal protein are also illustrated in the Examples. The methods and procedures described in the Examples for the cr IF
W091 / 16434 PC'T/L'S91 /02560 and its Cr IF
gene y protein are also generally applicable to the cryIX gene and its insecticidal CryIX protein.
The cr I-type gene of this invention, the crylF gene (SEQ ID NO:1), has the nucleotide base sequence shown in Figure 1. The coding region of the crylF gene extends from nucleotide base position 478 to position 3999 shown in Figure 1.
A comparison of the nucleotide base pairs of the crylF gene coding region with the corresponding coding region of the prior art cr I
genes indicates significant differences between the new cryIF gene and the prior art cr I genes. The crylF gene is only about 67% to about 78%
homologous (positionally identical) with the cryIA(a), cryIA(b) and cryIA(c) genes and the crylB
and cryIC genes. There is even less homology with the cr II, crylli and cryIV genes, described in Hofte and Whiteley (1989). The homology is discussed in more detail hereinafter.
The CryI-type protein of this invention, the CryIF protein (SEQ ID NO:2) that is encoded by the cryIF gene, has the amino acid sequence shown in Figure 1. In this disclosure, references to the CryIF "protein" (and to the CryIX "protein") are synonymous with its description as a "crystal protein," "protein toxin," "insecticidal protein,"
"delta endotoxin" or the like, unless the context indicates otherwise.
30. The deduced size of the CryIF protein is 133,635 Da. The prior art Cryl-type proteins, encoded by the respective cryI genes, have similar deduced sizes. Despite the apparent size - 21 - ~~U~~ ~rJC}
similarity, comparison of the amino acid sequence of the CryIF protein with published sequences of the other prior art CryI-type proteins shows significant differences betweeri them and the CryIF
protein. The CryIF protein is only about 58% to about 72% identical with the other prior art Cryl-type proteins, even when considering the C-terminal regions which are more related than the N-terminal regions.
The cryIX gene of this invention contains approximately 2100-2200 basepairs in its coding region, of which approximately 1140 basepairs are shown for the truncated upstream portion (SEQ ID
NO:3) of the cryIX gene in Figure 2. The crylX
gene of this invention is contained in isolated form on a DNA fragment carried on a recombinant plasmid, in E. coli strain EG1083 which has been deposited in the NRRL under accession No. NRRL B-18805. The CryIX protein of this invention, produced by the cryIX gene, is about 81 kDa in size and exhibits insecticidal activity against insects of the order Lepidoptera. The amino acid sequence (SEQ ID NO:4) -for a portion of the CryIX protein, deduced from the truncated portion of the crylX
gene shown in Figure 2, is shown in Figure 2. The 380 amino acids of this initial portion (SEQ ID
NO:4) of the CryIX protein shown in Figure 2 represent approximately one-half of the CryIX
protein encoded by the cr IX gene.
The CryIF and CryIX proteins have been shown to be insecticidal against insects of the order Lepidoptera, as set forth in more detail in Examples 6 and 11, respectively.
The present invention is intended to cover mutants and recombinant or genetically engineered derivatives of the: crylF gene and crylX
gene that yield lepidopteran-toxic proteins with essentially the same properties as the respective CryIF and CryIX proteins.
The cryIF gene and cryIX gene are also useful as DNA hybridization probes, for discovering similar or closely related cryi-type genes in other B.t. strains. The crylF or cryzX gene, or portions or derivatives thereof, can be labeled for use as a hybridization probe, e.g., with a radioactive label, using conventional procedures. The labeled DNA hybridization probe may then be used in the manner described in the Examples.
The cryIF or cryIX gene 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 crylF or crylX gene, as the case may be. Suitable hosts that allow the er IF and cryIX
genes to be expressed and the respective CryIF and CryIX proteins to be produced include Bacillus thuringiensis and other Bacillus species such as B.
subtilis or B. megaterium. E. coli or Pseudomonas fluorescens are also suitable hosts for these genes. It should be evident that genetically altered or engineered microorganisms containing the crylF gene or crylX gene can also contain other toxin genes present in the same microorganism and WO 91/16434 FCI'/U591/02560 that these genes could concurrently produce insecticidal crystal proteins different from the cryIF and CryIX proteins.
The Bacillus and E. coli strains described in this disclosure may be cultured using conventional growth media and standard fermentation techniques. The B.t. strains harboring the cr IF
gene (or the cryIX gene) may be fermented, as described in the Examples, until the cultured B.t.
cells reach the stage of their growth cycle when CryIF crystal protein (or CryIX crystal protein) is formed. For sporogenous B.t. strains, fermentation is typically continued through the sporulation stage, when 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 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 crylF gene and the crylX gene also have 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 ,c1e IF or crylX gene) in this disclosure are intended to cover sporulated B.t. cultures, i.e., B.t. cultures containing the CryIF or CryIX crystal protein and spores, and sporogenous Bacillus strain cultures that have produced crystal protein during the vegetative stage, as well as asporogenous WO 9l/15434 P("T/US91/02560 24 - 208063d -Bacillus strains containing the cr IF or cr IX gene in which the culture has reached the growth stage where crystal protein is actually produced.
The separated fermentation solids are primarily CryIF or CryIX crystal protein, as the case may be, and B.t. spores, along with some cell debris, some intact cells, and residual fermentation medium solids. If desired, the crystal protein may be separa'ted from the other recovered solids via conventional methods, e.g., sucrose density gradient fractionation. Highly purified CryIF or CryIX protein may be obtained by solubilizing the recovered crystal protein and then reprecipitating the protein from solution.
The CryIF protein is an effective insecticidal compound against lepidopteran insects like the European cornborer, the beet armyworm, and the tobacco budworm, for example. Likewise, the CryIX protein is insecticidal to lepidopteran insect species. The CryIF protein or CryIX protein may be utilized as the active ingredient in insecticidal formulations useful for the control of lepidopteran insects. Such insecticidal formulations or compositions typically contain agriculturally acceptable carriers or adjuvants in addition to the active ingredient.
The CryZF protein or CryIX protein may be employed in insecticidal formulations in isolated or purified form, e.g., as the crystal protein itself. Alternatively, the CryIF protein or CryIX
protein may be present in the recovered fermentation solids, obtained from culturing of a Bacillus strain, e.g., Bacillus thuringiensis, or "'0 91/16434 4'CI'/US91/02560 other microorganism host carrying the crylF or cryIX gene and capable of producing the corresponding CryIF or CryIX protein. Preferred Bacillus hosts for the crylF gene include B.t.
strain EG6345 and genetically improved B.t. strains derived from B.t. strain EG6345, such as B.t.
strain EG6346. The derivative B.t. strains may be obtained via plasmid curing arad/or conjugation techniques and contain the native cryIF gene-containing plasmid from B.t. strain EG6345.
Genetically engineered or transformed B.t. strains or other host microorganisms, containing a recombinant plasmid that expresses the cloned cryIF
gene and obtained by recombinant DNA procedures, may also be used.
Examples of such transformants include B.t. strain EG1945 which contains the cloned cryIF
gene 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 CryIF or CryIX protein may be dried, if desired, prior to incorporation into the insecticidal formulation.
The formulations or compositions of this invention containing the insecticidal CryIF or CryIX protein as the active component are applied at an insect:tcidally effective amount which will vary depending on such factors as, for example, the specific lepidopteran insects to be controlled, the specific plarit or crop to be treated and the method WO 91/16434 PC3'/1J591/02560 - 26 208063d -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, 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 CryIF or CryIX protein 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 CryIF or CryIX 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.
WO 91/16434 JPCi'/US91/02560 - 27 - 2080~i~ 8iI
Other application techniques, e.g., dusting, sprinkling, soaking, soil injection, seed coating, seedling coating or spraying, or the like, are also feasible. These application procedures are well known in the art.
The crylF or crylX gene 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 cr IF gene results in the production of insecticidal CryIF crystal protein. Likewise, expression of the cryzx gene results in production of the insecticidal CryIX protein. Suitable hosts include B.t. and other species of Bacillus, such as B. subtilis or B. megaterium, for example. Other bacterial hosts such as E. coli and Pseudomonas fluorescens may also be used. Various procedures well known to those skilled in the art are available for introducing the crylF or crylX gene 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 ways, 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. Again, these techniques are standard procedures.
WO 91/16434 PG?/U591/02560 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 CryIF or CryIX
insecticidal protein in the host, and the presence of auxiliary genetic capabilities. The cellular host containing the insecticidal crylF or cryTX
gene may be grown in any convenient nutrient medium, where expression of the crZIF or crylX gene is obtained and corresponding CryIF or CryIX
protein produced, typically upon sporulation. The sporulated cells containing the crystal protein may then be harvested in accordance with conventional ways, e.g., centrifugation or filtration.
The cryaF and cryIX genes may also be incorporated into a plant which is capable of expressing the gene and producing CryIF or CryIX
protein, as the case may be, rendering the plant more resistant to insect attack. Genetic engineering of plants with the cryIF or cryIX gene 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 know to those skilled in plant genetic engineering. An example of a technique for 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 crylE or crylX gene or a modified crylF or crylX gene capable of producing the corresponding CryIF or CryIX protein may be '/0 91/16434 PCT/YJ591/02560 _ 29 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.
Slight variations may be made in the crXIF or crYIX gene nucleotide base sequences, since the various amino acids forming the proteins encoded by the respective genes 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 region of the cryIF and cryIX nucleotide base sequences which allow expression of the gene and production of functionally equivalent forms of the corresponding CryIF and CryIX insecticidal proteins. 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.
The.present invention will now be described in more detail with reference to the following specific, non-limiting examples. The examples relate to work which was actually done based on techniques generally known in the art and using commercially available equipment.
WO 91/16434 F'GT/US91/02560 The novel B.t. strain EG6345 and a cured derivative B.t. strain EG6346 were isolated following the procedures described in Example 1.
Examlale 1 Isolation of B.t. Strainst EG634S and EG6346 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'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 extensive screening of crop dust samples, a B.t. subsp. aizawai strain, designated B.t. strain EG6345, was isolated from a maize grain dust sample. A sporulated culture of B.t. strain EG6345 was spread for the growth of individual colonies on a nutrient salts agar plate and incubated for 3 days at 306C. After incubation, one colony was noted on this plate which displayed a different colony morphology (i.e., shinier) than the parent B.t. strain EG6345. The colony, designated B.t. strain EG6346, was isolated as an individual colony.
A sample of the isolated B~t. strain EG6346 was further purified by streaking on an agar plate containing Spizizen's glucose peptone beef '0 91/16434 PCT/US91/02560
9 ON "ul extract (SGPB). A sample of this SGPB agar plate culture was used for agarose gel electrophoresis analysis of plasmid DNA using the standard Gonzalez technique (Gonzalez et al., Proc. Natl. Acad. Sci.
U.S.A., 79, pp. 6951-6955 (1982)). The agarose gel electrophoretic analysis was coupled with standard plasmid curing (ie., plasmid loss) and conjugation (ie., plasmid transfer) studies. The plasmid array of the new isolate of B.t. strain EG6346 was compared to that of B.t. strain EG6345 using agarose gel electrophoresis of plasmid DNA.
The agarose gel electrophoresis analyses of plasmid DNA, coupled with the plasmid curing and conjugation studies, indicated that B.t. strain EG6345 contained two plasmids of approximately 115 MDa and 45 MDa that encoded crystal protein. B.t.
strain EG6346 was identified as a spontaneously cured derivative of B.t. strain EG6345 which contained the plasmid of approximately 115 MDa, but which lacked the approximately 45 MDa plasmid.
Figure 3 is a photograph of a portion of an ethidium bromide stained agarose gel containing size-fractionated plasmids of B.t. strains EG6346 (left lane) and EG6345 (right lane). As illustrated in Figure 3, B.t. strain EG6346 does not contain the approximately 45 MDa plasmid contained in B.t. strain EG6345. Both B.t. strain EG6345 and the cured derivative B.t. strain EG6346 produced large bipyramidal inclusions during sporulation, as detected by phase contrast microscopy of sporulated cultures.
",0 91/16434 PC,T/U591/02560 Following the Southern blot technique (E.M. Southern, J. Mol. Biol., 98, pp. 503-517 (1975)), total DNA, prepared from both B.t. strains EG6345 and EG6346, was digested with Fiindill, electrophoresed through a 0.7% agarose gel, transferred to a nitrocellulose filter and hybridized at 50'C overnight to either a 32P-labeled 0.7 kb EcoRT N-terminal probe isolated from the B.t. strain HD-l crYIA(a) gene or a similarly labeled 2.2 kb intragenic PvuII probe also isolated from the HD-1 cryIA(a) gene. Digested DNA from B.t. subsp. kurstaki HD-1, which harbors the crylA(a), cryIA(b) and crylA(c) genes, was included as a control. The results of the Southern blot analyses are illustrated in Figure 4-A and 4-B.
Figure 4-A is the Southern blot of the agarose gel containing the total HindIiI-digested DNA from B.t.
strains EG6346 (lane 1), EG6345 (lane 2) and HD-1 (lane 3), following hybridization to the radiolabeled EcoRI probe. Figure 4-B shows the Southern blot of total Hindill-digested DNA from the B.t. strains indicated with respect to Figure 4-A, and in the same order, following hybridization to the radiolabeled PvuIS probe.
As shown in Figure 4-A, the 0.7 kb EcoRI
probe detected the expected 4.5, 5.3 and 6.6 kb fragments in HD-1 DNA (lane 3) corresponding to the previously described characteristic HindIIl fragments for the crylA(a), cUIA(b) and cryIA(c) genes, respectively. This probe also detected a prominent 5.3 kb band in B.t. strain EG6345 (lane 2) which was absent in the cured derivative B.t. strain EG6346 (lane 1). This result indicated WO 97/16434 P+L .T'/US91/02560 that the 45 MDa plasmid of EG6345 harbored at least one crylA(b) gene. The N-terminal 0.7 kb EcoRI
probe also hybridized to a 1.4 kb Hindlll fragment of unknown origin in both B.t. strains EG6345 and 5. EG6346.
The hybridization pattern obtained with the radiolabeled intragenic PvuII probe was more complex as can be seen in Figure 4-B. This probe, as expected, also hybridized to the 4.5, 5.3 and 6.6 kb fragments in HD-1 (lane 3) confirming the presence of the respective crylA(a), crylA(b~ and crylA(c) genes in this strain. Internal HindIIl fragments of 1.1 kb and 0.9 kb, derived from the resident crylA(a) and cryIA(b) genes in HD-1, respectively, were also detected with the PvuII
probe.
The 5.3, 2.8 and 0.9 kb fragments were also-detected by the PvuII probe in the DNA of B.t.
strain EG6345, indicating the presence of the crylA(b) gene in this strain (lane 2). These bands were not detected in B.t. strain EG6346 (lane 1).
However, the 1.4 kb Hindlll fragment, detected in both B,t. strains EG6345 and EG6346 by the EcoRI
probe, was similarly detected in both strains by the PvuII probe. A faintly hybridizing 2.5 kb Hindlll fragment was also detected with the PvuII
probe in both S.t. strains EG6345 and EG6346. This band corresponds in size to the characteristic HindIIl fragment of the cgYIC gene detected in other B.t. subsp. aizawai strains.
The Pvuil probe also hybridized to two large Hindlll fragments present in both B.t.
strains EG63465 and EG6346. These fragments, WO 91/16434 PCI'/IUS91/02560 - 34 - vI]
approximating 8.2 and 10.4 kb in size, were not detected by the EcoRI probe in either of B.t.
strains EG6345 or EG6346, nor were they observed with either probe in HD-i DNA. The unusual size of the hybridizing fragments, along with the production of large, bi-)yrami.dal crystal protein inclusions by B.t. strain EG6346, indicated the presence of one or more novel. toxin genes in B.t.
strains EG6345 and EG6346.
Ex!jmpls 2 Isolation of the crylP Gane in E. coli A genomic library was constructed for B.t. strain EG6346 and was screened at low stringency conditions with the intragenic 2.2 kb PvuII probe obtained from the cryIA(a) toxin gene.
DNA from B.t. strain EG6346 was chosen as the substrate DNA due to its apparent lack of cryIA-type toxin genes, whose presence could potentially increase the difficulty in screening the library at low stringency with the PvuII probe.
More specifically, high molecular weight DNA, obtained from B.t. strain EG6346, was partially digested with Sau3A and size-fractionated on a 10% to 40% sucrose gradient in 100 mM NaCl-10mM Tris hydrochloride (pH 7.4)-imM EDTA (TE).
Gradient fractions, containing DNA ranging in size from 5 to 10 kb, were pooled, dialyzed against TE
U.S.A., 79, pp. 6951-6955 (1982)). The agarose gel electrophoretic analysis was coupled with standard plasmid curing (ie., plasmid loss) and conjugation (ie., plasmid transfer) studies. The plasmid array of the new isolate of B.t. strain EG6346 was compared to that of B.t. strain EG6345 using agarose gel electrophoresis of plasmid DNA.
The agarose gel electrophoresis analyses of plasmid DNA, coupled with the plasmid curing and conjugation studies, indicated that B.t. strain EG6345 contained two plasmids of approximately 115 MDa and 45 MDa that encoded crystal protein. B.t.
strain EG6346 was identified as a spontaneously cured derivative of B.t. strain EG6345 which contained the plasmid of approximately 115 MDa, but which lacked the approximately 45 MDa plasmid.
Figure 3 is a photograph of a portion of an ethidium bromide stained agarose gel containing size-fractionated plasmids of B.t. strains EG6346 (left lane) and EG6345 (right lane). As illustrated in Figure 3, B.t. strain EG6346 does not contain the approximately 45 MDa plasmid contained in B.t. strain EG6345. Both B.t. strain EG6345 and the cured derivative B.t. strain EG6346 produced large bipyramidal inclusions during sporulation, as detected by phase contrast microscopy of sporulated cultures.
",0 91/16434 PC,T/U591/02560 Following the Southern blot technique (E.M. Southern, J. Mol. Biol., 98, pp. 503-517 (1975)), total DNA, prepared from both B.t. strains EG6345 and EG6346, was digested with Fiindill, electrophoresed through a 0.7% agarose gel, transferred to a nitrocellulose filter and hybridized at 50'C overnight to either a 32P-labeled 0.7 kb EcoRT N-terminal probe isolated from the B.t. strain HD-l crYIA(a) gene or a similarly labeled 2.2 kb intragenic PvuII probe also isolated from the HD-1 cryIA(a) gene. Digested DNA from B.t. subsp. kurstaki HD-1, which harbors the crylA(a), cryIA(b) and crylA(c) genes, was included as a control. The results of the Southern blot analyses are illustrated in Figure 4-A and 4-B.
Figure 4-A is the Southern blot of the agarose gel containing the total HindIiI-digested DNA from B.t.
strains EG6346 (lane 1), EG6345 (lane 2) and HD-1 (lane 3), following hybridization to the radiolabeled EcoRI probe. Figure 4-B shows the Southern blot of total Hindill-digested DNA from the B.t. strains indicated with respect to Figure 4-A, and in the same order, following hybridization to the radiolabeled PvuIS probe.
As shown in Figure 4-A, the 0.7 kb EcoRI
probe detected the expected 4.5, 5.3 and 6.6 kb fragments in HD-1 DNA (lane 3) corresponding to the previously described characteristic HindIIl fragments for the crylA(a), cUIA(b) and cryIA(c) genes, respectively. This probe also detected a prominent 5.3 kb band in B.t. strain EG6345 (lane 2) which was absent in the cured derivative B.t. strain EG6346 (lane 1). This result indicated WO 97/16434 P+L .T'/US91/02560 that the 45 MDa plasmid of EG6345 harbored at least one crylA(b) gene. The N-terminal 0.7 kb EcoRI
probe also hybridized to a 1.4 kb Hindlll fragment of unknown origin in both B.t. strains EG6345 and 5. EG6346.
The hybridization pattern obtained with the radiolabeled intragenic PvuII probe was more complex as can be seen in Figure 4-B. This probe, as expected, also hybridized to the 4.5, 5.3 and 6.6 kb fragments in HD-1 (lane 3) confirming the presence of the respective crylA(a), crylA(b~ and crylA(c) genes in this strain. Internal HindIIl fragments of 1.1 kb and 0.9 kb, derived from the resident crylA(a) and cryIA(b) genes in HD-1, respectively, were also detected with the PvuII
probe.
The 5.3, 2.8 and 0.9 kb fragments were also-detected by the PvuII probe in the DNA of B.t.
strain EG6345, indicating the presence of the crylA(b) gene in this strain (lane 2). These bands were not detected in B.t. strain EG6346 (lane 1).
However, the 1.4 kb Hindlll fragment, detected in both B,t. strains EG6345 and EG6346 by the EcoRI
probe, was similarly detected in both strains by the PvuII probe. A faintly hybridizing 2.5 kb Hindlll fragment was also detected with the PvuII
probe in both S.t. strains EG6345 and EG6346. This band corresponds in size to the characteristic HindIIl fragment of the cgYIC gene detected in other B.t. subsp. aizawai strains.
The Pvuil probe also hybridized to two large Hindlll fragments present in both B.t.
strains EG63465 and EG6346. These fragments, WO 91/16434 PCI'/IUS91/02560 - 34 - vI]
approximating 8.2 and 10.4 kb in size, were not detected by the EcoRI probe in either of B.t.
strains EG6345 or EG6346, nor were they observed with either probe in HD-i DNA. The unusual size of the hybridizing fragments, along with the production of large, bi-)yrami.dal crystal protein inclusions by B.t. strain EG6346, indicated the presence of one or more novel. toxin genes in B.t.
strains EG6345 and EG6346.
Ex!jmpls 2 Isolation of the crylP Gane in E. coli A genomic library was constructed for B.t. strain EG6346 and was screened at low stringency conditions with the intragenic 2.2 kb PvuII probe obtained from the cryIA(a) toxin gene.
DNA from B.t. strain EG6346 was chosen as the substrate DNA due to its apparent lack of cryIA-type toxin genes, whose presence could potentially increase the difficulty in screening the library at low stringency with the PvuII probe.
More specifically, high molecular weight DNA, obtained from B.t. strain EG6346, was partially digested with Sau3A and size-fractionated on a 10% to 40% sucrose gradient in 100 mM NaCl-10mM Tris hydrochloride (pH 7.4)-imM EDTA (TE).
Gradient fractions, containing DNA ranging in size from 5 to 10 kb, were pooled, dialyzed against TE
10:1 (pH 7.4), extracted with 2-butanol to reduce the volume and ethanol precipitated. The purified insert DNA was ligated to E. coli plasmid vector pGEM'"-32 digested with BamHI at a 1:2 molar ratio '10 91/16434 P'CT/US91/02560 - 35 - ( QU 0634~
of vector to insert and at a final DNA
concentration of 20 ,ug/m1, using T4 DNA ligase available from Promega Corp. Transformation of E. coli DH5al cells was based on the Hanahan procedure (Hanahan, J. Aiol. Biol., 166, pp. 557-580 (1983)) and transformed colonies were plated on agar plates of standard LB medium containing 100 jug/ml ampicillin and 50 )ag/ml X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside).
Approximately 3.3 x 106 colonies were screened for the presence of cryI-related toxin gene sequences under low stringency conditions, using as a probe the 32P-labeled 2.2 kb PvuII
intragenic fragment obtained from a crylA(a) gene present within B.t. strain HD-1. The low stringency conditions include hybridization conducted at 50-55'C overnight in 3X SSC (1X SSC
comprises 0.15 M NaC1, 0.015M sodium citrate), 10X
Denhardt's solution (1X Denhardt's solution comprises 0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone), 200 pg/ml heparin and 0.1% SDS. The probe hybridized strongly to one E. coli recombinant colony, designated E. coli strain EG1943, which contained an 8.4 kb recombinant plasmid, designated pE.G640, that consisted of plasmid pGEM%-3Z ligated to a 5.7 kb Sau3A insert of DNA from B.t. strain EG6346.
A restriction map for the pEG640 plasmid was generated as shown in Figure 5 using those restriction enzymes indicated above in the Brief Description of the Drawings and methods well known to those skilled in the art. The relative positions of restriction sites and localization of - 36 (yd toxin gene sequences within the map were initially accomplished by low stringency hybridization of Southern blots containing digested pEG640 DNA to the radiolabeled EcoRI and PvuII toxin gene probes as set forth above in Example 1.
Initial mapping data identified two regions on the pEG640 insert which reacted with varying intensity to the toxin gene probes. The larger region, spanning over 3 kb in length, hybridized strongly to the PvuII probe at low and high stringency hybridization conditions. The high stringency conditions are the same as the above-identified low stringency conditions, except that the temperature is increased to 65'C. The larger 3 kb region on the 5.7 kb insert of the pEG640 plasmid also reacted well with the EcoRI probe at low stringency hybridization conditions. A smaller region, positioned in close proximity to the vector, weakly hybridized to the EcoRI probe at low stringency conditions only. These data indicated the presence of two different toxin genes on the 5.7 kb insert of the pEG640 plasmid.
Pthen purified 32P-labeled pEG640 DNA was used to probe HindliI genomic digests, a single 10.4 kb hybridizing band was detected in B.t.
strains EG6345.and EG6346, as illustrated in lanes 1 and 2 of Figure 4-C, respectively. This 10.4 kb fragment was also detected in both B.t.
strains EG6345 and EG6346 with the PvuII probe as can be seen in Figure 4-B, lanes 1 and 2, respectively. No hybridizable bands were detected in the DNA from B.t. strain HD-i, as evidenced by WO 91/16434 PCT/L'S91/02560 lane 3 in Figure 4-C, which is consistent with the 2 absence of these novel gene sequences in this strain.
Example 3 Sequence Analyses of crylF and crylX Genes Standard dideoxy sequencing procedures (Sanger et al., Proc. Natl. Acaci. Sci. U.S.A., 74, pp. 5463-5467 (1977), with Sequenase'", available from United States Biochemical C:orp., were used to determine the DNA sequence of the 5.7 kb pEG640 insert from the recombinant E. coli strain EG1943.
Sequencing of the insert was initiated in both directions and on both strands from the SP6 and T7 promoters present on vector pGEM"'-3Z and utilized the specific primers supplied by Promega Corp.
Preparation and denaturation of the double stranded template was also according to manufacturers' directions (Promega Corp. and United States Biochemical Corp.). Subsequent 17mer oligonucleotide primers were synthesized on an Applied Biosystems, Inc. DNA synthesizer, Model 380B.
The DNA sequence, which is flanked by Sau3A cloning sites (GATC) extends 5649 nucleotide bases in length and is shown in Figures 1 and 2.
Translation of the sequence revealed the presence of two open reading frames which are separated by approximately 500 bases of non-coding DNA sequence and which are out of frame with respect to one another. The genes potentially encoded by these open reading frames have been designated cr'yIF (SEQ
ID NO:l) and crylX (SEQ ID NO:3). Justification for this designation derives from sequence WO 91/16434 F'Cr/U591/02560 comparisons to other toxin genes and is discussed below. The partial DNA sequence for the portion of the 5.7 kb insert of pEG640 including the cr IF
gene (SEQ ID NO:l) and the deduced amino acid sequence of the crystal protein encoded by the cryIF gene, designated the CryIF protein (SEQ ID
NO:2), are illustrated in Figure 1. The partial, truncated DNA sequence (SEQ ID NO:3) for the portion of the 5.7 kb insert of pEG640 including the truncated crylX gene, and the deduced, truncated amino acid sequence (SEQ ID NO:4) of the crystal protein encoded by the crylX gene, designated the CryIX protein are illustrated in Figure 2. The beginning of the sequences in Figure 2 follow immediately after the end of the sequences illustrated in Figure 1, and two figures are used merely for the sake of convenience.
The crylF open reading frame, which is the larger of the two, encodes a CryIF protein consisting of 1174 amino acids and having a deduced size of 133,635 Da. The position of the cryIF gene within pEG640 and its relationship to the position of the crylX gene is schematically represented in Figure 5. An NH2-terminal methionine translational start site was identified for the crylF gene at nucleotide base position 478 of the sequence. It was immediately preceded by a putative ribosome binding site (RES). The cryIF gene open reading frame terminates at nucleotide base position 3999.
A putative promoter sequence was identified for the cryIF gene 53 nucleotide bases upstream of the ribosome binding site. The nucleotide base sequence as counted from both base pairs 10 and 35 positions upstream of the methionine start is wO 91/16434 P4'T/[JS91/02560 exactly homologous to that identified for the HD-1 cryIA(a) gene promoter (Wong et al., J. Biol.
Chem., 258, pp. 1960-1967 (1983)).
As indicated in Figure 2, an NH2-terminal methionine codon, signifying the translational start site of the cr IX open reading frame, was identified at nucleotide base position 4508. The crylX open reading frame continued an additional 1141 nucleotides, encoding 380 amino acids, and terminated with the GATC cloning site delimiting the insert DNA. The sequence presented here for the crylX gene represents an artificially truncated version of the native gene present within B.t.
strain EG6346. Although a putative ribosome binding site has been identified upstream of the cryI7t sequence, it was not possible to identify promoter regions located 10 and 35 base pairs upstream from the methionine start for the cryzX
gene within the intervening DNA sequence between the cryIF and crylX open reading frames by sequence inspection. Tnspection of the intervening DNA
sequence between the cr IF and cryzl{ genes has identified a stem-loop termination structure at positions 4090-4132 (see Figure 2) that is nearly identical to that described downstream of the HD-1-Dipel crylA(a) gene (Wong et al., J. Biol. Chem., 258, pp. 1960-1967 (1983)).
The sequence analysis program of Queen and Korn was used to compare the sequences of the crylF and crYIX genes to the published sequences of other B.t. toxin genes (Queen et al., Nucleic Acids Res., 12, pp. 581-599 (1984)). The nucleotide base sequences anei deduced amino acid sequences of the WO 91/16434 PC'd'/US91/02560 cryIF and cr IX genes were aligned with the published sequences of various delta-endotoxin genes and the results of the comparisons are tabulated in Table 1. As shoi,rn in Table 1, the amino acid sequence of the N-terminal region (amino acids 1-618) of the cr yIF-encoded protein differs significantly from the N-terminal region of other CryI-type encoded proteins (about 40%-50%
identity). These sequence differences are likely responsible for the unique insecticidal activity spectrum of the CryIF protein (see Example 5 below), since previous studies of truncated cryI
genes indicate that it is the N-terminal region of the protein that determines insecticidal activity (Schnepf et al., J. Biol. Chem., 260, pp. 6273-6278 (1985); Hofte et al., Eur. J. Biochem., 161, pp.
273-280 (1986).
"'0 91/16434 PCt'/U591/02560 Amino Acid (aa) Comparisons of the N-terminal Region of CryIF Proteina Protein Class (Bd-terYainal region) ~ Similarity CryIA(a) (aa 1-608) 51Ø
CryIA(b) (aa 1-609) 52.0 cryIA(c) (aa 1-610) 49.0 CryIB (aa 1-637) 40.1 CryIC (aa 1-617) 48.8 CryID (aa 1-593) 52.0 CrylE (aa 1-602) 48.1 a Amino acids 1-602 of the CryIF protein were compared to the N-terminal regions of CryIA(a) (Schnepf et al., (1985), su ra), CryIA(b) (Hofte et al., (1986), supra), CryIA(c) (Adang et al., (1985), supra), CryIB (Brizzard et al. (1988), supra), CryIC (Ho6ee et al., (1988), supra), CryID and CryIE (both in EP 0 358 557 (1990), supra).
The nucleotide base sequence of the entire crYIF gene and the amino acid sequences of the CryIF protein were also compared to other crystal protein genes and their respectively encoded proteins. The comparisons were tabulated in Table 2.
WO 91/16434 PC1(/US91/02560 8equence Comparisons of cryYF and crylX
Ganes and Encoded Proteins With Other B.t. Genes and Proteins crylF CryIF crylX CryX
DNA aaa DNA aa Gene tXpe crylA(a) 77.6b 71.7b 52.5 35.2 crylA(b) 75.8 70.4 53.4 36.4 crylA(c) 75.8 69.9 53.4 36.7 crylB 66.6 58.3 70.4 62.9 cryiC 75.3 70.0 51.9 36.7 crylD 75.6 71.5 43.1 31.8 cryIE 77.2 69.8 51.2 34.8 cryllA 43.9 24.6 47.9 26.2 cryIIIA 53.0 35.6 55.3 38.8 cryIVD 44.5 20.8 45.0 22.9 a aa means amino acid.
b g Identity, i.e., positional identity.
Comparisons of the complete DNA sequence indicate the crylF gene was related to, but distinct from, the crYxA class og toxin genes (about 76-78% identity) (Table 2). Of the three crylA genes compared to the cryIF gene, cryIF was most related to the HD-i crylA(a) nucleotide sequence with about 78% of the nucleotides conserved between the two genes.
Table 2 indicates that the CryIF protein is significantly more related to other Cryl proteins than to the cryllA, CryIIiA or CryIVD
proteins. Amino acid identity ranged from about 70-72% for the CryIF protein and the CryIA, CryIC, CryID and CryIE proteins.
The crylF gene sequence was less related to crylB (about 67%) and, as expected, much less related to dipteran and coleopteran toxin genes (cryll, cryIII and cr IV genes).
The crystal protein genes thus far disclosed in the previously cited references have been divided into four major classes and several subclasses characterized by both structural similarities and the insecticidal spectrum of the encoded crystal proteins (Hofte and Whiteley (1989) p. 242). The four major classes, I, II, 111 and IV, encode lepidopteran-specific, lepidopteran- and dipteran-specific, coleopteran-specific and dipteran-specific proteins, respectively. Table 1 of Hofte and Whiteley (1989) at p. 243 lists the genes presently assigned to these four major classes.
The cryl genes can be distinguished from the other crystal protein genes by sequence homolocgy. The amino acid sequences encoded by the cryl genes exhibit greater than 50% identity (Table 3, H fte and Whiteley (1989) at p. 245).
The amino acid sequences of three Cryl-encoded proteins (CryIA(a), CryIA(b) and CryYA(c)) show greater than 80% identity, and thus they are considered members of the same subgroup (CryIA).
There is ample justification for designating the novel toxin gene identified in B.t.
strain EG6345 as cryIF. The CryIF protein exhibits greater than 50% amino acid identity to the other 1*/091/16434 PC'V'/US91/02560 - 44 - rl~~~06U4 Cryl proteins. More specifically, the CryIF
protein is about 70-72% identical to the CrYIA
subgroup proteins, about 58% identical to the CryIB
protein and about 70% identical to the CryIC and CryID proteins. The CryIF protein is less related to the crystal proteins encoded by the other crystal protein gene classes c:r II, cryIll and cryIV (see Table 2).
However, the CryIF protein is not greater than about 80% identical to the crystal proteins encoded by the crylA subgroup of genes, and thus the cryIF gene does not belong to the crylA
subgroup. The CryIF protein is only somewhat related to the CryIB, CryIC, CryID and CrylE
proteins, and thus, the cr IF gene is not a member of a new subgroup including any of the cr IB, cryIC, cryID or cryIE genes.
Further substantiation of the crylF
designation, i.e., its categorization as a cryl-type gene, is that the CryIB protein is about 55-56% identical to the proteins encoded by the crylA
subgroup of genes and the CryID protein is about 70-71% identical to the CryIA subgroup and CryIC
proteins (see Hofte and Whiteley (1989) Table 3).
The cryIX truncated nucleotide base sequence (SEQ ID NO:3) and the deduced amino acid sequence (SEQ ID NO:4) were similarly compared to other toxin gene sequences, as shown in Table 2.
The cryIX nucleotide base sequence is also distinct from, but related to, the other cryl genes in Table 2, such as that of the cr IB gene (about 70%
identical).
~ '!O 91/1643d PCr/IJS91/02560 2080t~84 EXample 4 Expression of the Cloned arylP Gene Studies were conducted to demonstrate the production of the CryIF protein by the crylF gene.
Table 3 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); Cry (crystalliferous);
Tc (tetracycline).
Table 3 Strain or Plasmid Relevant Characteristics B. thuringiensis HD73-26 Cry EG1078 HD73-26 harboring pEG310 (cr IF- c yIX
+
EG1945 HD73-26 harboring pEG642 (crylF+ cr IX+
EG6345 crylF+ cryIX+
EG6346 cr IF+ crySX+, derivative of EG6345 cured of 45 MDa plasmid E. coli DH5 cl, Cry , Amps GM2163 Cry-, ,Amps EG1943 DH50~. harboring pEG640(crylF+ cryIX+) Plasmids pEG310 crylF cryIX+ deletion mutant plasmid of pEG642 pEG434 Tcr Bacillus vector pGEM"-3Z Ampr E. coli vector pEG640 Fmpr pGEIV-3Z with 5.7 kb insert (c IF+ cryIX+) pEG642 Tcr, E. coli-Bacillus shuttle vector consisting of pEG640 lTgateT into the Hindll% site of pEG434 It has been reported that E. coli cells harboring cloned B.t. toxin genes fail to produce significant amounts of the toxin protein required for critical evaluations of insecticidal activity (Donovan et al., Mol. Gen. Genet., 214, pp. 365-372 (1988)). Returning the cloned B.t. toxin gene to a Bacillus species, and ideally to a B.t. host, EV 91/16434 PC'f/U591/02560 maximizes toxin gene expression from its native promoter. Accordingly, the cloned crylF gene was introduced into the Cry recipient B.t. strain HD73-26, as described below.
The pEG640 plasmid construct was ligated to the vector pEG434 (Mettus et al., Applied and Environ. Microbiol., 192, pp. 288-289 (1990)) at the unique Hindill site present on both pEG640 and pEG434 and the ligation mixture used to transform E. coli strain GM2163, which is defective for both adenine and cytosine methylation (Marinus et al., Mol. Gen. Genet., 56, pp. 1128-1134 (1983)). The resulting 11.4 kb recombinant plasmid, designated pEG642 and having a restriction map as shown in Figure 6, possessed both E. coli and Bacillus replication origins and a selectable marker for tetracycline resistance (Tcr) that could function in a B~t. host. Plasmid pEG642 DNA was isolated from E. coli strain GM2163 by alkaline/SDS lysis followed by ethanol precipitation using standard procedures. Plasmid DNA was then used to transform the B.t. Cry- recipient strain HD73-26 by electroporation. A single Tcr HD73-26 trafasformant containing pEG642, designated B.t. strain EG1945, was chosen for further study. Microscopic examination of sporulated cultures of B.t. strain EG1945 revealed the presence of crystalline inclusions (large, irregularly shaped rods and bipyramidal shapes).
Renografin density gradient purified crystal protein from B.t. strain EG1945 was used for SDS-PAGE analyses of the cr1rIF gene product.
The purified CryIF protein from the recombinant WO 91/16434 PC'1'/iJa91/02560 B.t. strain EG1945 was compared to similarly purified proteins obtained from the native B.t.
strains EG6345 and EG6346 harboring the cryIF gene.
Crystal protein preparations (2.8 j.tg of EG6345, 0.7 pg of EG6346 and 0.70 )ig of EG1945) were loaded onto a 5-20% gradient SD5-polyacrylamide gel and electrophoresed. Figure 7 is a photograph of the resulting Coomassie stained SDS-polyacrylamide gel, in which lanes 1, 2 and 3 contain proteins from native B.t. strains EG6345 and EG6346 and recombinant B.t. strain EG1945, respectively.
As indicated in lane 3, a single high molecular weight protein, approximating 135 kDa in size, was observed in recombinant B.t. strain EG1945, consistent with expression of the single crylF gene. The size of the observed protein correlates well with the predicted molecular weight of 133,635 Da deduced from the amino acid sequence.
At least three distinct protein species were observed in lane 1 of Figure 7, from B.t.
strain EG6345, which confirms the DNA hybridization result shown in Figure 4, verifying the presence of the crylA(b), cryIC and cryIF genes in this strain.
It is possible, however, that other similarly sized proteins encoded by additional toxin genes are also present in B.t. strain EG6345, e.g., the crylX
gene.
Similarly, B.t. strain EG6346, which was used to construct the library from which crylF was cloned, contains at least two crystal proteins, the largest of which appears to co-migrate with the approximately 135 kDa recombinant czylF protein in B.t. strain EG1945. The smaller protein present in 2 'S`~'~ 8 ~y~
B.t. strain EG6346, also evident in B.t. strain EG6345, is believed to represent the protein encoded by the crylC gene which has been identified in each of these strains by DNA hybridization analysis with a cr IC specific oligonucleotide probe.
Exaatple 3 Plasmid Localization of the cryZE' Gene To determine the location of the cryIF
gene in B.t. strains EG6345 and EG6346 and to compare its location to that of the crylA(b) gene present in B.t. strain EG6345, plasmid DNAs of B.t.
strains EG6345 and EG6346 were resolved by agarose gel electrophoresis. The resulting ethidium bromide stained gel is illustrated in Figure 8-A.
Plasmids from strain HD-1 (lane 1) were included as controls and were used as size standards. Lane 2 shows the plasmids from B.t. strain EG6345, while lane 3 shows the plasmids from B.t. strain EG6346.
Plasmid DNAs resolved by the gel of Figure 8-A were transferred to nitrocellulose and hybridized to either the intragenic radiolabeled 2.2 kb PvuII cryIA(a) probe or to a cryIF gene-specific probe consisting of a radiolabeled gel-purified 0.4 kb PstI-SacI fragment isolated from the N-terminal region of the cryIF gene on pEG640.
Hybridizations were conducted at 65"C overnight to assure specificity of the reaction with each probe.
As shown in the autoradiogram of Figure 8-B, the PvuII intragenic cryA(a) probe hybridized strongly to the 44 AiDa plasmid present in HD-1 (lane 1) which harbors a c IA b gene. Hybridization of WO 91 / 16434 1'Cf/1JS91 /02560 - 5o - 2 0 ~'10 `'18 the PvuII probe to this plasmid was expected, since the nucleotide base sequence of the probe is highly conserved among all three crYIA genes. Similarly, the PvuII probe also hybridized to the large 110 MDa plasmid in strain HD-1 containing the cryIA (a) and crYIA(c) toxin genes.
The PvuII probe also hybridized to the 45 MDa plasmid containing the cr IA b gene present within B.t. strain EG6345 (lane 2). Differences in the hybridization signal intensity of the PvuII
probe in detecting the cryIA(b) gene in B.t.
strains HD-1 and EG6345 may be attributed to different amounts of DNA loaded onto the gel shown in Figure 8-A. Lack of a hybridization band from the PvuII probe in strain EG6346 (lane 3) was entirely consistent with the classification of this strain as a cured derivative of B.t. strain EG6345 not containing the 45 MDa plasmid. The 115 MDa plasmid present within B.t. strains EG6345 and EG6346 was weakly detected by the PvuII probe. The reduced hybridization signal observed in each of these strains, as compared to strain HD-1, may be attributed to quantitative differences in the amounts of DNA loaded, as we11 as to the reduced sequence homology between the PvuII probe and the novel toxin genes present on this large plasmid.
Hybridization of the crylF PstI-SacI
intragenic probe to plasmid DNAs from B.t. strains HD-1 (lane 1), EG6345 (lane 2) and EG6346 (lane 3) is shown in the autoradiogram of Figure 8-C. The specificity of this probe for the crylF gene is confirmed by the lack of hybridization to plasmids harboring cry.IA genes in B.t. strains FiD-1 or 'VO 91/16434 ACI'/US91/02560 - 51 - 2 EG6345, and by its hybridization to the 115 MDa plasmid present in B.t. strains EG6345 and EG6346.
Both the PvuII and the PstI-SacI probes hybridized to a low molecular weight smear, identified as "L"
in Figure 8-A, which represents linear fragments of sheared larger toxin plasmids.
le ~
Insect Toxicity of the cry,IF Protein The insecticidal activity of CryIF
protein was determined against several lepidopteran larvae including Ostrinia nubilalis (European cornborer), Spodoptera exi a(beet armyworm), Heliothis virescens (tobacco budworm), Heliothis zea (bollworm) and Lymantria dispar (gypsy moth), using Renografin' density gradient purified CryIF
crystal protein from recombinant B.t. strain EG1945, which harbors the crylF gene on plasmid pEG642.
Activity was measured using a diet-surface overlay technique where the surface of an agar-based artificial diet was covered with an aliquot suspension containing CryIF protein crystals. After delivery of the aliquot to the diet surface, the diluent was allowed to evaporate, at which time one larva of the test species was placed in each cup. Each 2 ml well (cup) contained 1 ml diet having a surface area of 175 mm2 Bioassays were held at 280C for 7 days, at which time mortality was scored. Bioassays were first conducted at three doses with 1 to 10 dilutions.
If the CryIF protein demonstrated sufficient activity, eight dose assays (1 to 2 dilutions) were conducted to determine LC50 values via the well-known technique of probit analysis (Daum, Bull.
Entomol. Soc. Am., 16, pp. 10-15 (1970)). Each dose was tested against 32 insects. The diluent, 0.005% Triton' X-100, served as a control treatment. All insects were tested as newly hatched first-stage larvae. The results of effective insecticidal activity are set forth in Table 4 in comparison with the results of insecticidal bioassays using other CrylA crystal proteins.
Table 4 Insecticidal Activitp of CryI Crystal Proteins LC50 in ng protein/mm2 Heliothis Spodoptera Ostrinia Protein virescens exiqua nubilalis CryIA(a) 2.2 >57 0.27 CryIA(b) 0.7 33 0.17 CryIA(c) 0.03 >57 0.08 CryiF 0.31 26 0.17 The CryIF protein exhibited the greatest toxicity to Ostrinia nubilalis larvae as indicated in Table 4. The LC50 value obtained is similar to LC50 values obtained for the purified CryIA(b) crystal protein which is highly toxic to Ostrinia nubilalis larvae. In addition, the CryIF protein was toxic to Spodoptera exiqua larvae. CryIF
protein was considerably more toxic to Spodo tera VO 91/16434 Pt;T/US91/02560 exigua than purified CryIA(a) and CryIA(c) crystal proteins and slightly more toxic than purified CryIA(b) crystal protein. Purified CryIF crystal protein was also toxic to Heliothis virescens, with a toxicity between that of purified CryIA(c) and CryIA(b) crystal protein. CryIF crystal protein exhibited little toxicity to Iieliothis zea or Lymantria dispar at the doses tested.
Examp l e 7 Analysis of Insecticidal Activity of CryZX Fragment The sequence of the cryIX gene present on plasmid pEG642 (and likewise present on plasmid pEG640) does not encode a sufficient number of amino acids to constitute a "minimum toxic fragment" as defined by deletion analyses of cryTA
genes (Schnepf et al., J. Biol. Chem,, 260, pp. 6273-6278 (1985)), H fte et al., (1986) su ra).
Nonetheless, to assess the contribution of crylX, if any, to the overall toxicity of the pEG642 construct, the following study was performed.
Plasmid pEG310, containing a deletion in the cryIF gene, was constructed by restriction enzyme deletion from plasmid pEG642 of an N-terminal region of the cry%F gene which is flanked by BstEII sites (Figure 6). Following religation, plasmid pEG310 was introduced into the Cry B.t.
HD73-26 recipient via electroporation, resulting in a recombinant strain designated B.t. strain EG1078.
Fully sporulated cultures, containing the intact crylX gene sequence from plasmid pEG642, but not the cryTF gene which had been deleted, were assayed by the insect bioassay procedure previously WO 91/16434 FCI'/U591/02560 54 - 203O~~~,~~~
described in Example 6 for toxicity against Ostrinia nubilalis. B.t. strain EG1945, containing the intact crylF gene, was the positive control.
Thirty insect larvae were assayed, at a protein dose of 4.00 ng/550mm2. At this dose, the B.t. strain EG1945 was 100% toxic to larvae of Ostrinia nubilalis. However, B.t. strain EG1078, containing the cryIF deletion mutant, exhibited 0t mortality for Ostrinia nubilalis larvae. Thus, it was concluded that the sequence of the cr IX gene present on plasmid pEG642 does not contribute to the observed toxicity of B.t. strain EG1945 and that the cryIF gene product is the active insecticide in the strain.
Examplt 8 Southern Blot Analysis of the crvTX G ne in B.t. Strain Following the Southern blot technique cited in Example 1, total DNA was obtained from B.t. strain EG6346, digested to completion with the restriction endonucleases As~718 (an isoschizomer of Ksnl), C1aI, Sstl, and SshI both individually and in combination, electrophoresed through a 0.8%
agarose gel, transferred to a nitrocellulose filter, and hybridized at 65 C overnight to a 32p labgled 0.6 kb EpnIaBamHl probe that was isolated from pEG640 (previously described in Example 2) and that contained a portion of the cr IX coding region. The positions of the gi and BamFil sites flanking the crylX probe are shown in Figure 5.
The results of the Southern blot analysis are shown in Figure 9. Total DNA from B.t. strain EG6346 digested with restriction endonucleases exhibited, in each instance, a single DNA fragment hybridizing to the cr IX probe. Most importantly, B.t. strain EG6346 DNA digested with Clal (lane 2) yielded a 4.6 kb restriction jEragment that hybridized to the probe. In addition, B.t. strain EG6346 DNA digested with both ClaI and SstI
(lane 8) yielded a 4.4 kb restriction fragment that was detected by the cr IX probe. Since a ClaI
restriction site was present only 309 bp upstream from the cryIX open reading frame shown in Figure 2, these results indicated that the entire cryiX gene was likely to be contained on the 4.6 kb Clal restriction fragment. This assumption was shown to be correct by the fact that the CryIX
protein is only 81 kDa, which corresponds to a gene of about 2.1-2.2 kb in length. In addition, the absence of an SstI site immediately upstream to or within the sequenced portion of the cryIX gene displayed in Figure 2-A indicated that the SstI
restriction site detected by Southern blot analysis was located downstream from crylX.
Exam2l !
gaolatioa of the Entire cs~gt$X ene in B. coli A genomic library was constructed from Clag-digested DNA of B.t. strain EG6346 and screened under moderate stringency conditions with the 0.6 kb nDnI-BaaaFiI cryIX probe derived from pEG640 to ida:ntify recombinant E. coli colonies containing cr.ylX gene sequences.
More specifically, total DNA obtained from B.t. strain EG6346 was digested to completion with Cla2, electrophoresed th:rough a 0.8% agarose gel, and DNA fragments in the 4.3-5.0 kb range excised from the gel with a clean razor blade. DNA
fragments within the agarose gel slice were purified using the GeneClean II kit and procedure available from Bio 101, Inc. of La Jolla, CA.
The E. coli-B.t. cloning vector pEG854, depicted as a circular restriction map in Figure 10 and described by Baum et al., Appl. Environ.
Microbiol., 56, pp. 3420-3428 (1990), was used to clone the crylX gene on the Clal restriction fragments. The C1aI restriction fragments were ligated to C1aI-digested pEG854 vector DNA
pretreated with calf intestinal alkaline phosphatase to prevent self-ligation.
Transformation of E. coli HB101 cells with the ligation mixture was achieved by electroporation using the high-efficiency transformation procedure of Dower et al., Nucleic Acids Res., 16, pp. 6127-6145. Transformed cells were plated on agar plates of standard LB rmedium containing 50 pg/mi ampiciliin. Colonies were screened under moderaie stringency conditions for the presence of the cryIX
gene sequence using the colony blot hybridization procedure outlined in Example 2. The hybridization step was performed at 65'C, rather than at 50-55 C
as in Example 2, using the 0.6 kb KpnI-BamHI cryIX
probe described in Example 9. Filter washes were performed at 65=C in 3X SSC, 0.1% SDS. The crylX
probe hybridized strongly to one E. coli recombinant colony, designated E. coli strain '0 91/16434 PCT/U591/02560 EG1082, that contained an 11.8 kb recombinant plasmid, designated pEG313, that consisted of a 4.6 kb C1aI restriction fragment from B.t. strain EG6346 inserted into the C1aI restriction site of cloning vector pEG854.
A circular restriction map of recombinant plasmid pEG313 is depicted in F'igure 11. The orientation of the 4.6 kb ClaI restriction fragment was determined by restriction endonuclease mapping using methods well known to those skilled in the art.
Example 10 Expression of the cryIR G ne in E. coli and Production of Cry%8 ProtaTn To achieve expression of the crylX gene in E. coli and to characterize its encoded crystal protein, a 4.4. kb DNA fragment containing the crylX gene was inserted into the E. coli cloning vector pTZ19u, obtained from U.S. Biochemical Corporation. A circular restriction map of cloning vector pTZ19u, designated Plac in Figure 12, can be used to direct the transcription of cloned genes inserted into the multiple cloning site region demarcated by the unique HindIIl and EcoRI
restriction sites within the lacZ' gene.
Accordingly, a 4.4 kb ClaI-SstI restriction fragment containing the entire cryIX gene, as indicated by the Southern blot analysis in Example 8, was isolated from the recombinant plasmid pEG313 (see Figure 11) and ligated to pTZ19u DNA digested with Accl and SstI, two restriction endonucleases with cleavage sites within the multiple cloning site region of the WO 91/16434 1'CI'/tJS91/02560 cloning vector. Note that the Accl restriction site is compatible with that of C1aI, thereby allowing for efficient ligaticin of the cryIX gene fragment and orienting the cryIX gene in the same direction as the lac promoter. The ligation mixture was used to transform E. coli DH5;yL cells as described in Example 2. Using the X-gal screening procedure, a recombinant E. coli colony, designated EG1083, was recovered that contained a 7.3 kb recombinant plasmid, designated pEG314, that consisted of a 4.4 kb C1ai-Sstl restriction fragment derived from pEG313 inserted into the AccI
and Sstl sites of vector pTZ19u. A circular restriction map of recombinant plasmid pEG314, containing the cryIX gene inserted downstream from the lac promoter of pTZ19u, is depicted in Figure 13.
E. coli strain EG1083, containing pEG314 which carried the crylX gene, was grown in Luria broth containing 50 pg/ml ampicillin and 1mM
isopropyl-beta-D-thiogalactopyranoside (IPTG) at 376C overnight. IPTG is an inducer of the lac promoter and is commonly used to optimize transcription from that promoter in E. coli. After overnight growth, cells were examined by phase-contrast microscopy. E. coli strain EG1083 cells, but not E. coli strain DR5& cells containing pTZ19u, contained multiple phase-bright inclusions.
Subsequent lysis of the recombinant cells with lysozyme released the large inclusions, some of which appeared rhomboid in shape. The inclusions were purifieci from E. coli strain EG1083 by Renografin" density .gradient centrifugation and WO 91/16434 1'C"T/US91/02560 examined by SDS-polyacrylamide gel electrophoresis.
Figure 14 is.a photocopy of the resulting Coomassie-stained 10% SDS-polyacrylamide gel, in which lanes 1 and 2 contain CryIX crystal protein from E. coli strain EG1083 before and after Renografin' density gradient centrifugation, respectively. Protein molecular weight standards are displayed in the leftmost lane. Based on these standards, the CryIX crystal protein migrates with an apparent molecular mass of 81 kDa.
Example 11 Insect Toxicity of the CryIR Protein The insecticidal activity of the 81 kDa CryIX protein was determined against lepidopteran species, using Renografin'o density gradient purified CryIX crystal protein from recombinant E.
coli strain EG1083, which harbors the cr.xX gene on plasmid pEG314.
Activity was measured using a diet-surface overlay technique where the surface of an agar-based artificial diet was covered with an aqueous suspension containing CryIX protein crystals. Insect larvae were placed on the diet.
surface after the diluent had evaporated and held at 28'C for seven days, at which time mortality was scored.
In this bioassay screening.procedure, the purified CryIX protein exhibited insecticidal activity against larvae of Plutella arylostella (diamondback moth).
WO 91/16434 PCI'/L1S91/02560 In another bioassay screening procedure, using a cell paste of E. coli strain EG1083 that had produced CryIX protein instead of the purified CryIX protein, insecticidal activity was exhibited against larvae of ostrinia nubilalis (European corn borer).
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 61064, as indicated in the following Table 5:
Table 5 Bacterial Strain NRRL Accession No. Date of Deposit B.thuringiensis HD73-26 B-18508 June 12, 1989 B.thuringiensis EG6345 B-18633 March 27, 1990 B.thurinqiensis EG1945 B-18635 March 27, 1990 E. coli EG1943 B-18634 March 27, 1990 E. coli EG1083 B-18805 March 29, 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".
WO 91/16434 I'CI'/US91/02560 The present invention may be embodied in other specific forms without d+eparting 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.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Gawron-Burke, Cynthia Chambers, Judith A.
Gonzalez Jr., Jose M.
(ii) TITLE OF INVENTION: BACILLUS TF3I:JRINGIENSIS crylF and crylX
GENES AND PROTEINS TOXIC TO LEPIDOPTERAN INSECTS
(iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Panitch Schwarze et al. c/o A.S. Nadel (B) STREET: 1601 Market Street, 36th Floor (C) CITY: Philadelphia (D) STATE: PA
(E) COUNTRY: U.S.A.
(F) ZIP: 19103 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/510327 (B) FILING DATE: 16-APR-1990 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME:- Eqol, Christopher (B) REGISTRATION NAMBER: 27633 (C) REFERENCE/DOCKET NUMBER: 7205-27 U1 (2) INFORMATION FOR SEQ ID NO:1:' (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4020 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 478..4002 (xi) SEQUENCE DESCRIPTION: SEQ I?) NO:l:
- 63 - 2 0 Pior, S, d GATCTTCAAA TGAGAAAATA AGGGTATTCC GTATGGGATG CCTTTA'TTT GGTTGGGAAG 60 .GTATATTAAA AATAATGTTC TTATAACATA TATGTTGATT TTAAGAAAAT ATTTTGTTTA 180 TAAGCACACT ATTAACATAT TAGGTCTATT TAAATTAAGG: GCATATAGTG ATATTTTATA 360 Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe Va1 Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu I1e Tyr Ile Glu Ala Leu =AGA GAG TGG GAA GCA AAT CCT AAT AAT GCA CAA TTA AGG GAA GAT GTG 861 Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val CGT ATT CGA TTT GCT AAT ACA GAC GAC GC'T TTA ATA ACA GCA ATA AAT 909 Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asrn Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Lau Ser Leu Leu Arg Asp Ala Val Ser Phe WO 91/16434 PC'T/U591/02560 - 64 - 2, 0 89634 Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asri His Tyr Asn AGA TTA ATA AAT CTT ATT CAT AGA TAT ACG AA.A, CAT TGT TTG GAC ACA 1101 Arg Leu I1e Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Va1 Leu Asp Ile Va1 Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro 21e Gln Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile G1u Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu Va1 Ser Ser Arg Asn Thr Ala G1y Asn Arg Ile Asn Phe Pro Ser Tyr Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln Gln Thr Gly Thr Asn His Tllr Arg Thr Phe Arg Asn Ser Gly Thr Ile Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp WO 91/16434 POff'/US91/02560 - 65 - 2 ~3 ~i ~j~~'j ~~~
'U~(1 rc Asn Asp Tyr Ser His Val Leu Asn His Va]. Thr Phe Val Arg Trp Pro Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile Thr Gin Ile Pro Leu Val Lys Ala His Thr Lelu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gin Leu Pro Gin Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gin Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile .GAC AGA TTT GAA TTG ATT CCA GTT ACT GCA ACA TTT GAA GCA GPdA TAT 2301 Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala G1u Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln GTA TCC AAT TTA GTG GAT TGT TTA TCA GAT GAA,TTT TGT CTG GAT GAA 2445 Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu WO 91/16434 POt'/lJ591 /02560 Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg G1y Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp 705 710 71:5 720 Giu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Giu Ser Lys Leu Lys Pro Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Leu Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Gin Ser Pro ATC AGA A.AG TGT GGA GAA CCG AAT CGA TGC GCG CCA CAC CTT GAA TGG 2877 Ile Arg Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp A.AT CCT GAT CTA GAT TGT TCC TGC AGA GAC'GGG GAA AAA TGT GCA CAT 2925 Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Asp Val Trp Val Ile Phe Lys Ile Lys Thr Gin Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Giu Leu Giu Thr Asn Ile Val Tyr Lys Glu ~~090684 Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu A1. 'yr Leu Pro Glu Leu Ser Val Ili:~ Pro Gly Val Asn Val Asp Ile Phe Glu Glu Leu Lys Gly Arg Ile Phe Thr Ala Phe Phe Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Va1 Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala Asn G1n Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly TAT GAC GAA ACT TAT GGA AGC AAT TCT TCT GTA CCAi GCT GAT TAT GCG 3789 Tyr Asp Glu Thr Tyr Gly f3er Asn Ser Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys .~er Tyr Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly WO 91/16434 PC'I'/US91/02560 - 68 - 2 008 ~~
Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lyn Val T:p Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1174 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe Va1 Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu G1n Ile Glu Gln Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn WO'91/16434 I'CT/US91/02560 - 69 - lti~0,,/ ~ ~
06{.~d">':~
Arg Leu Ile Asn Leu I1e His Arg Tyr Thr Lys His Cys Leu Asp Thr Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile Va1 Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val I1e Glu Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365' Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe G1n Gin Thr Gly Thr Asn His Thr Arg Thr Phe Arg.Asn,Ser Gly Thr Ile Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro Leu Vai Lys Ala His Thr Leu Gin Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr I1e Val Asn Ile Asn Gly Gln Leu WO 91/16434 PC'T/US91/02560 - 70 - ~~ ~ ~ a n l~c~~~
~~y~ t~
Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phie Pro Met Ser Gln Ser Ser Phe Thr Va1 Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr I1e Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Va1 Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly I1e Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys Ile Asp Glu Ser Lys Leu Lys Pro Tyr Thr Arg Tyr Gin Leu Arg Gly Tyr Ile Glu Asp Ser G1n Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Leu Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Gln Ser Pro Yle Arg Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Gys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile AspVal Gly Cys Thr Asp Leu WO 91/16434 PfT/US91/02560 s ~~f,r ~ i'~ ~ a {
- ~
Asn Glu Asp Leu Asp Val Trp Val I1e Phe Lys I].e Lys Thr G1n Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Arg 21e Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Val Asp Ile Phe Glu Glu Leu Lys Gly Arg Tie Phe Thr Ala Phe Phe Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu I1e Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Glu Thr Tyr Gly Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu I,ys Ser Tyr Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Va1 Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp ~~~~0 ('3 Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe I1e Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO:3:
( i ) SEQUENCE CFiARACTERISTICS :
(A) LENGTH: 1629 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/REY: CDS
(B) LOCATION: 488..1629 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
=. TTGTAATGAA AAACGGACAT CACCTCCATT GAAACGGTGA GATGTCCGTT TTACTATGTT 120 Met Lys Leu Lys Asn Pro Asp Lys His G1n Ser Phe Ser Ser AAT GCG AAA GTA GAT AAA ATC TCT ACG GAT TCA CTA AAA AP.T GAA ACA 577 Asn Ala Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile Glu Leu Gin Asn I].e Asn His Glu Asp Cys Leu Lys Ile Ser GAG TAT GAA AAT GTA GAG CC:G TTT GTT AGT GCA TCA ACA ATT CAA AC.11. 673 Glu Tyr Giu Asn Val Glu Pro Phe Val Ser Ala Ser Thr Ile Gin Thr GGT ATT AGT ATT GCG GGT AAA ATA C.TT GGC ACC CTA GGC GTT CCT TTT 721 Gly Ile Ser Ile Ala Gly Lys Ile Leu Gly Thr Leu Gly Va1 Pro Phe 65 70 75 cp080639~~
GCA GGA yA GTA GCT AGT CTT TAT AGT TTT ATC TTA GGT GAG CTA TGG( 769 Ala Gly Gln Val Ala Ser Leu Tyr Ser Phe Ile Leu Gly Glu Leu Trp Pro Lys Gly Lys Asn Gln Trp Glu Ile Phe Met Glu His Val Glu Glu Ile Ile Asn Gln Lys Ile Ser Thr Tyr Ala Arg Asn Lys Ala Leu Thr Asp Leu Lys Gly Leu Gly Asp Ala Leu Ala Val Tyr His Glu Ser Leu Glu Ser Trp Val Gly Asn Arg Lys Asn Thr Arg Ala Arg Ser Val Val Lys Ser Gln Tyr Ile Ala Leu Glu Leu Met Phe Val Gln Lys Leu Pro Ser Phe Ala Val Ser Gly Glu Glu Val Pro Leu Leu Pro Ile Tyr Ala Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Ile Phe Gly Lys Glu Trp Gly Leu Ser Ser Ser Glu Ile Ser Thr Phe Tyr Asn Arg G1n Val Glu Arg Ala Gly Asp Tyr Ser Asp His Cys Val Lys Trp Tyr Ser Thr Gly Leu Asn Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Val Arg Tyr Asn Gln Phe Arg Lys Asp Met Thr Leu Met Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Leu Val Tyr Pro Ile Lys Thr Thr Ser Gin Leu Thr ls,rg Glu Val Tyr Thr Asp Ala Ile Gly Thr Val His Pro Asn Ala Ser Phe Ala Ser Thr Thr Trp Tyr Asn Asn Asn WO 91/16434 PCf/US91/02560 Ala Pro Ser Phe Ser Thr 21e Glu Ser Ala Val Val Arg Asn Pro His Leu Leu Asp Phe Leu Glu Gln Val Thr Ile ",Cyr Ser Leu Leu Ser Arg 335 340 :345 350 Trp Ser Asn Thr Gln Tyr Met Asn Met Trp Gly Gly His Arg Leu Glu Phe Arg Thr I1e Gly Gly Met Leu Asn Thr Ser Thr Gln Gly (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 380 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Lys Leu Lys Asn Pro Asp Lys His Gin Ser Phe Ser Ser Asn Ala Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile Glu Leu Gln Asn Ile Asn His Giu Asp Cys Leu Lys 21e Ser Glu Tyr Glu Asn Val Glu Pro Phe Val Ser Ala Ser Thr I1e Gin Thr Gly Ile Ser Ile Ala Gly Lys Ile Leu Gly Thr Leu Gly Val Pro Phe Ala Gly Gln Val Ala Ser Leu Tyr Ser Phe Ile Leu Gly Glu Leu Trp Pro Lys Gly Lys Asn Gln Trp Glu I1e Phe Met Glu His Val Glu Glu Ile Ile Asn Gin Lys Ile Ser Thr Tyr Ala Arg Asn Lys Ala Leu Thr Asp Leu Lys Gly Leu Gly Asp Ala Leu Ala Val Tyr His Glu Ser Leu Glu Ser Trp Val Gly Asn Arg Lys Asn Thr Arg Ala Arg Ser Val Val Lys Ser WO 91/16434 PCT/[!S91/02560 ~~ ~~
~+J ~ ~Jd Gln Tyr Ile Ala Leu Glu Leu Met Phe Val Gln Lys Leu Pro Ser Phe Ala Val Ser Gly Glu Glu Val Pro Leu Leu Pro Ile Tyr Ala Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Ile Phe Gly Lys Glu Trp Gly Leu Ser Ser Ser Glu I1e Ser Thr Phe Tyr Asn Arg Gln Val Glu Arg Ala Gly Asp Tyr Ser Asp His Cys Val Lys Trp Tyr Ser Thr Gly Leu Asn Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Val Arg Tyr Asn Gln Phe Arg Lys Asp Met Thr Leu Met Va1 Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Leu Val Tyr Pro Ile Lys Thr Thr Ser Gln Leu Thr Arg Glu Val Tyr Thr Asp Ala Ile Gly Thr Val His Pro Asn Ala Ser Phe Ala Ser Thr Thr Trp Tyr Asn Asn Asn Ala Pro Ser Phe Ser Thr Ile Glu Ser Ala Val Val Arg Asn Pro His Leu Leu Asp Phe Leu Glu Gin Val Thr Ile Tyr Ser Leu Leu Ser Arg Trp Ser Asn Thr Gln 'Tyr Met Asn Met Trp Gly Gly His Arg Leu Glu Phe Arg Thr Ile Gly Gly Met Leu Asn Thr Ser Thr Gln Gly
of vector to insert and at a final DNA
concentration of 20 ,ug/m1, using T4 DNA ligase available from Promega Corp. Transformation of E. coli DH5al cells was based on the Hanahan procedure (Hanahan, J. Aiol. Biol., 166, pp. 557-580 (1983)) and transformed colonies were plated on agar plates of standard LB medium containing 100 jug/ml ampicillin and 50 )ag/ml X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside).
Approximately 3.3 x 106 colonies were screened for the presence of cryI-related toxin gene sequences under low stringency conditions, using as a probe the 32P-labeled 2.2 kb PvuII
intragenic fragment obtained from a crylA(a) gene present within B.t. strain HD-1. The low stringency conditions include hybridization conducted at 50-55'C overnight in 3X SSC (1X SSC
comprises 0.15 M NaC1, 0.015M sodium citrate), 10X
Denhardt's solution (1X Denhardt's solution comprises 0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone), 200 pg/ml heparin and 0.1% SDS. The probe hybridized strongly to one E. coli recombinant colony, designated E. coli strain EG1943, which contained an 8.4 kb recombinant plasmid, designated pE.G640, that consisted of plasmid pGEM%-3Z ligated to a 5.7 kb Sau3A insert of DNA from B.t. strain EG6346.
A restriction map for the pEG640 plasmid was generated as shown in Figure 5 using those restriction enzymes indicated above in the Brief Description of the Drawings and methods well known to those skilled in the art. The relative positions of restriction sites and localization of - 36 (yd toxin gene sequences within the map were initially accomplished by low stringency hybridization of Southern blots containing digested pEG640 DNA to the radiolabeled EcoRI and PvuII toxin gene probes as set forth above in Example 1.
Initial mapping data identified two regions on the pEG640 insert which reacted with varying intensity to the toxin gene probes. The larger region, spanning over 3 kb in length, hybridized strongly to the PvuII probe at low and high stringency hybridization conditions. The high stringency conditions are the same as the above-identified low stringency conditions, except that the temperature is increased to 65'C. The larger 3 kb region on the 5.7 kb insert of the pEG640 plasmid also reacted well with the EcoRI probe at low stringency hybridization conditions. A smaller region, positioned in close proximity to the vector, weakly hybridized to the EcoRI probe at low stringency conditions only. These data indicated the presence of two different toxin genes on the 5.7 kb insert of the pEG640 plasmid.
Pthen purified 32P-labeled pEG640 DNA was used to probe HindliI genomic digests, a single 10.4 kb hybridizing band was detected in B.t.
strains EG6345.and EG6346, as illustrated in lanes 1 and 2 of Figure 4-C, respectively. This 10.4 kb fragment was also detected in both B.t.
strains EG6345 and EG6346 with the PvuII probe as can be seen in Figure 4-B, lanes 1 and 2, respectively. No hybridizable bands were detected in the DNA from B.t. strain HD-i, as evidenced by WO 91/16434 PCT/L'S91/02560 lane 3 in Figure 4-C, which is consistent with the 2 absence of these novel gene sequences in this strain.
Example 3 Sequence Analyses of crylF and crylX Genes Standard dideoxy sequencing procedures (Sanger et al., Proc. Natl. Acaci. Sci. U.S.A., 74, pp. 5463-5467 (1977), with Sequenase'", available from United States Biochemical C:orp., were used to determine the DNA sequence of the 5.7 kb pEG640 insert from the recombinant E. coli strain EG1943.
Sequencing of the insert was initiated in both directions and on both strands from the SP6 and T7 promoters present on vector pGEM"'-3Z and utilized the specific primers supplied by Promega Corp.
Preparation and denaturation of the double stranded template was also according to manufacturers' directions (Promega Corp. and United States Biochemical Corp.). Subsequent 17mer oligonucleotide primers were synthesized on an Applied Biosystems, Inc. DNA synthesizer, Model 380B.
The DNA sequence, which is flanked by Sau3A cloning sites (GATC) extends 5649 nucleotide bases in length and is shown in Figures 1 and 2.
Translation of the sequence revealed the presence of two open reading frames which are separated by approximately 500 bases of non-coding DNA sequence and which are out of frame with respect to one another. The genes potentially encoded by these open reading frames have been designated cr'yIF (SEQ
ID NO:l) and crylX (SEQ ID NO:3). Justification for this designation derives from sequence WO 91/16434 F'Cr/U591/02560 comparisons to other toxin genes and is discussed below. The partial DNA sequence for the portion of the 5.7 kb insert of pEG640 including the cr IF
gene (SEQ ID NO:l) and the deduced amino acid sequence of the crystal protein encoded by the cryIF gene, designated the CryIF protein (SEQ ID
NO:2), are illustrated in Figure 1. The partial, truncated DNA sequence (SEQ ID NO:3) for the portion of the 5.7 kb insert of pEG640 including the truncated crylX gene, and the deduced, truncated amino acid sequence (SEQ ID NO:4) of the crystal protein encoded by the crylX gene, designated the CryIX protein are illustrated in Figure 2. The beginning of the sequences in Figure 2 follow immediately after the end of the sequences illustrated in Figure 1, and two figures are used merely for the sake of convenience.
The crylF open reading frame, which is the larger of the two, encodes a CryIF protein consisting of 1174 amino acids and having a deduced size of 133,635 Da. The position of the cryIF gene within pEG640 and its relationship to the position of the crylX gene is schematically represented in Figure 5. An NH2-terminal methionine translational start site was identified for the crylF gene at nucleotide base position 478 of the sequence. It was immediately preceded by a putative ribosome binding site (RES). The cryIF gene open reading frame terminates at nucleotide base position 3999.
A putative promoter sequence was identified for the cryIF gene 53 nucleotide bases upstream of the ribosome binding site. The nucleotide base sequence as counted from both base pairs 10 and 35 positions upstream of the methionine start is wO 91/16434 P4'T/[JS91/02560 exactly homologous to that identified for the HD-1 cryIA(a) gene promoter (Wong et al., J. Biol.
Chem., 258, pp. 1960-1967 (1983)).
As indicated in Figure 2, an NH2-terminal methionine codon, signifying the translational start site of the cr IX open reading frame, was identified at nucleotide base position 4508. The crylX open reading frame continued an additional 1141 nucleotides, encoding 380 amino acids, and terminated with the GATC cloning site delimiting the insert DNA. The sequence presented here for the crylX gene represents an artificially truncated version of the native gene present within B.t.
strain EG6346. Although a putative ribosome binding site has been identified upstream of the cryI7t sequence, it was not possible to identify promoter regions located 10 and 35 base pairs upstream from the methionine start for the cryzX
gene within the intervening DNA sequence between the cryIF and crylX open reading frames by sequence inspection. Tnspection of the intervening DNA
sequence between the cr IF and cryzl{ genes has identified a stem-loop termination structure at positions 4090-4132 (see Figure 2) that is nearly identical to that described downstream of the HD-1-Dipel crylA(a) gene (Wong et al., J. Biol. Chem., 258, pp. 1960-1967 (1983)).
The sequence analysis program of Queen and Korn was used to compare the sequences of the crylF and crYIX genes to the published sequences of other B.t. toxin genes (Queen et al., Nucleic Acids Res., 12, pp. 581-599 (1984)). The nucleotide base sequences anei deduced amino acid sequences of the WO 91/16434 PC'd'/US91/02560 cryIF and cr IX genes were aligned with the published sequences of various delta-endotoxin genes and the results of the comparisons are tabulated in Table 1. As shoi,rn in Table 1, the amino acid sequence of the N-terminal region (amino acids 1-618) of the cr yIF-encoded protein differs significantly from the N-terminal region of other CryI-type encoded proteins (about 40%-50%
identity). These sequence differences are likely responsible for the unique insecticidal activity spectrum of the CryIF protein (see Example 5 below), since previous studies of truncated cryI
genes indicate that it is the N-terminal region of the protein that determines insecticidal activity (Schnepf et al., J. Biol. Chem., 260, pp. 6273-6278 (1985); Hofte et al., Eur. J. Biochem., 161, pp.
273-280 (1986).
"'0 91/16434 PCt'/U591/02560 Amino Acid (aa) Comparisons of the N-terminal Region of CryIF Proteina Protein Class (Bd-terYainal region) ~ Similarity CryIA(a) (aa 1-608) 51Ø
CryIA(b) (aa 1-609) 52.0 cryIA(c) (aa 1-610) 49.0 CryIB (aa 1-637) 40.1 CryIC (aa 1-617) 48.8 CryID (aa 1-593) 52.0 CrylE (aa 1-602) 48.1 a Amino acids 1-602 of the CryIF protein were compared to the N-terminal regions of CryIA(a) (Schnepf et al., (1985), su ra), CryIA(b) (Hofte et al., (1986), supra), CryIA(c) (Adang et al., (1985), supra), CryIB (Brizzard et al. (1988), supra), CryIC (Ho6ee et al., (1988), supra), CryID and CryIE (both in EP 0 358 557 (1990), supra).
The nucleotide base sequence of the entire crYIF gene and the amino acid sequences of the CryIF protein were also compared to other crystal protein genes and their respectively encoded proteins. The comparisons were tabulated in Table 2.
WO 91/16434 PC1(/US91/02560 8equence Comparisons of cryYF and crylX
Ganes and Encoded Proteins With Other B.t. Genes and Proteins crylF CryIF crylX CryX
DNA aaa DNA aa Gene tXpe crylA(a) 77.6b 71.7b 52.5 35.2 crylA(b) 75.8 70.4 53.4 36.4 crylA(c) 75.8 69.9 53.4 36.7 crylB 66.6 58.3 70.4 62.9 cryiC 75.3 70.0 51.9 36.7 crylD 75.6 71.5 43.1 31.8 cryIE 77.2 69.8 51.2 34.8 cryllA 43.9 24.6 47.9 26.2 cryIIIA 53.0 35.6 55.3 38.8 cryIVD 44.5 20.8 45.0 22.9 a aa means amino acid.
b g Identity, i.e., positional identity.
Comparisons of the complete DNA sequence indicate the crylF gene was related to, but distinct from, the crYxA class og toxin genes (about 76-78% identity) (Table 2). Of the three crylA genes compared to the cryIF gene, cryIF was most related to the HD-i crylA(a) nucleotide sequence with about 78% of the nucleotides conserved between the two genes.
Table 2 indicates that the CryIF protein is significantly more related to other Cryl proteins than to the cryllA, CryIIiA or CryIVD
proteins. Amino acid identity ranged from about 70-72% for the CryIF protein and the CryIA, CryIC, CryID and CryIE proteins.
The crylF gene sequence was less related to crylB (about 67%) and, as expected, much less related to dipteran and coleopteran toxin genes (cryll, cryIII and cr IV genes).
The crystal protein genes thus far disclosed in the previously cited references have been divided into four major classes and several subclasses characterized by both structural similarities and the insecticidal spectrum of the encoded crystal proteins (Hofte and Whiteley (1989) p. 242). The four major classes, I, II, 111 and IV, encode lepidopteran-specific, lepidopteran- and dipteran-specific, coleopteran-specific and dipteran-specific proteins, respectively. Table 1 of Hofte and Whiteley (1989) at p. 243 lists the genes presently assigned to these four major classes.
The cryl genes can be distinguished from the other crystal protein genes by sequence homolocgy. The amino acid sequences encoded by the cryl genes exhibit greater than 50% identity (Table 3, H fte and Whiteley (1989) at p. 245).
The amino acid sequences of three Cryl-encoded proteins (CryIA(a), CryIA(b) and CryYA(c)) show greater than 80% identity, and thus they are considered members of the same subgroup (CryIA).
There is ample justification for designating the novel toxin gene identified in B.t.
strain EG6345 as cryIF. The CryIF protein exhibits greater than 50% amino acid identity to the other 1*/091/16434 PC'V'/US91/02560 - 44 - rl~~~06U4 Cryl proteins. More specifically, the CryIF
protein is about 70-72% identical to the CrYIA
subgroup proteins, about 58% identical to the CryIB
protein and about 70% identical to the CryIC and CryID proteins. The CryIF protein is less related to the crystal proteins encoded by the other crystal protein gene classes c:r II, cryIll and cryIV (see Table 2).
However, the CryIF protein is not greater than about 80% identical to the crystal proteins encoded by the crylA subgroup of genes, and thus the cryIF gene does not belong to the crylA
subgroup. The CryIF protein is only somewhat related to the CryIB, CryIC, CryID and CrylE
proteins, and thus, the cr IF gene is not a member of a new subgroup including any of the cr IB, cryIC, cryID or cryIE genes.
Further substantiation of the crylF
designation, i.e., its categorization as a cryl-type gene, is that the CryIB protein is about 55-56% identical to the proteins encoded by the crylA
subgroup of genes and the CryID protein is about 70-71% identical to the CryIA subgroup and CryIC
proteins (see Hofte and Whiteley (1989) Table 3).
The cryIX truncated nucleotide base sequence (SEQ ID NO:3) and the deduced amino acid sequence (SEQ ID NO:4) were similarly compared to other toxin gene sequences, as shown in Table 2.
The cryIX nucleotide base sequence is also distinct from, but related to, the other cryl genes in Table 2, such as that of the cr IB gene (about 70%
identical).
~ '!O 91/1643d PCr/IJS91/02560 2080t~84 EXample 4 Expression of the Cloned arylP Gene Studies were conducted to demonstrate the production of the CryIF protein by the crylF gene.
Table 3 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); Cry (crystalliferous);
Tc (tetracycline).
Table 3 Strain or Plasmid Relevant Characteristics B. thuringiensis HD73-26 Cry EG1078 HD73-26 harboring pEG310 (cr IF- c yIX
+
EG1945 HD73-26 harboring pEG642 (crylF+ cr IX+
EG6345 crylF+ cryIX+
EG6346 cr IF+ crySX+, derivative of EG6345 cured of 45 MDa plasmid E. coli DH5 cl, Cry , Amps GM2163 Cry-, ,Amps EG1943 DH50~. harboring pEG640(crylF+ cryIX+) Plasmids pEG310 crylF cryIX+ deletion mutant plasmid of pEG642 pEG434 Tcr Bacillus vector pGEM"-3Z Ampr E. coli vector pEG640 Fmpr pGEIV-3Z with 5.7 kb insert (c IF+ cryIX+) pEG642 Tcr, E. coli-Bacillus shuttle vector consisting of pEG640 lTgateT into the Hindll% site of pEG434 It has been reported that E. coli cells harboring cloned B.t. toxin genes fail to produce significant amounts of the toxin protein required for critical evaluations of insecticidal activity (Donovan et al., Mol. Gen. Genet., 214, pp. 365-372 (1988)). Returning the cloned B.t. toxin gene to a Bacillus species, and ideally to a B.t. host, EV 91/16434 PC'f/U591/02560 maximizes toxin gene expression from its native promoter. Accordingly, the cloned crylF gene was introduced into the Cry recipient B.t. strain HD73-26, as described below.
The pEG640 plasmid construct was ligated to the vector pEG434 (Mettus et al., Applied and Environ. Microbiol., 192, pp. 288-289 (1990)) at the unique Hindill site present on both pEG640 and pEG434 and the ligation mixture used to transform E. coli strain GM2163, which is defective for both adenine and cytosine methylation (Marinus et al., Mol. Gen. Genet., 56, pp. 1128-1134 (1983)). The resulting 11.4 kb recombinant plasmid, designated pEG642 and having a restriction map as shown in Figure 6, possessed both E. coli and Bacillus replication origins and a selectable marker for tetracycline resistance (Tcr) that could function in a B~t. host. Plasmid pEG642 DNA was isolated from E. coli strain GM2163 by alkaline/SDS lysis followed by ethanol precipitation using standard procedures. Plasmid DNA was then used to transform the B.t. Cry- recipient strain HD73-26 by electroporation. A single Tcr HD73-26 trafasformant containing pEG642, designated B.t. strain EG1945, was chosen for further study. Microscopic examination of sporulated cultures of B.t. strain EG1945 revealed the presence of crystalline inclusions (large, irregularly shaped rods and bipyramidal shapes).
Renografin density gradient purified crystal protein from B.t. strain EG1945 was used for SDS-PAGE analyses of the cr1rIF gene product.
The purified CryIF protein from the recombinant WO 91/16434 PC'1'/iJa91/02560 B.t. strain EG1945 was compared to similarly purified proteins obtained from the native B.t.
strains EG6345 and EG6346 harboring the cryIF gene.
Crystal protein preparations (2.8 j.tg of EG6345, 0.7 pg of EG6346 and 0.70 )ig of EG1945) were loaded onto a 5-20% gradient SD5-polyacrylamide gel and electrophoresed. Figure 7 is a photograph of the resulting Coomassie stained SDS-polyacrylamide gel, in which lanes 1, 2 and 3 contain proteins from native B.t. strains EG6345 and EG6346 and recombinant B.t. strain EG1945, respectively.
As indicated in lane 3, a single high molecular weight protein, approximating 135 kDa in size, was observed in recombinant B.t. strain EG1945, consistent with expression of the single crylF gene. The size of the observed protein correlates well with the predicted molecular weight of 133,635 Da deduced from the amino acid sequence.
At least three distinct protein species were observed in lane 1 of Figure 7, from B.t.
strain EG6345, which confirms the DNA hybridization result shown in Figure 4, verifying the presence of the crylA(b), cryIC and cryIF genes in this strain.
It is possible, however, that other similarly sized proteins encoded by additional toxin genes are also present in B.t. strain EG6345, e.g., the crylX
gene.
Similarly, B.t. strain EG6346, which was used to construct the library from which crylF was cloned, contains at least two crystal proteins, the largest of which appears to co-migrate with the approximately 135 kDa recombinant czylF protein in B.t. strain EG1945. The smaller protein present in 2 'S`~'~ 8 ~y~
B.t. strain EG6346, also evident in B.t. strain EG6345, is believed to represent the protein encoded by the crylC gene which has been identified in each of these strains by DNA hybridization analysis with a cr IC specific oligonucleotide probe.
Exaatple 3 Plasmid Localization of the cryZE' Gene To determine the location of the cryIF
gene in B.t. strains EG6345 and EG6346 and to compare its location to that of the crylA(b) gene present in B.t. strain EG6345, plasmid DNAs of B.t.
strains EG6345 and EG6346 were resolved by agarose gel electrophoresis. The resulting ethidium bromide stained gel is illustrated in Figure 8-A.
Plasmids from strain HD-1 (lane 1) were included as controls and were used as size standards. Lane 2 shows the plasmids from B.t. strain EG6345, while lane 3 shows the plasmids from B.t. strain EG6346.
Plasmid DNAs resolved by the gel of Figure 8-A were transferred to nitrocellulose and hybridized to either the intragenic radiolabeled 2.2 kb PvuII cryIA(a) probe or to a cryIF gene-specific probe consisting of a radiolabeled gel-purified 0.4 kb PstI-SacI fragment isolated from the N-terminal region of the cryIF gene on pEG640.
Hybridizations were conducted at 65"C overnight to assure specificity of the reaction with each probe.
As shown in the autoradiogram of Figure 8-B, the PvuII intragenic cryA(a) probe hybridized strongly to the 44 AiDa plasmid present in HD-1 (lane 1) which harbors a c IA b gene. Hybridization of WO 91 / 16434 1'Cf/1JS91 /02560 - 5o - 2 0 ~'10 `'18 the PvuII probe to this plasmid was expected, since the nucleotide base sequence of the probe is highly conserved among all three crYIA genes. Similarly, the PvuII probe also hybridized to the large 110 MDa plasmid in strain HD-1 containing the cryIA (a) and crYIA(c) toxin genes.
The PvuII probe also hybridized to the 45 MDa plasmid containing the cr IA b gene present within B.t. strain EG6345 (lane 2). Differences in the hybridization signal intensity of the PvuII
probe in detecting the cryIA(b) gene in B.t.
strains HD-1 and EG6345 may be attributed to different amounts of DNA loaded onto the gel shown in Figure 8-A. Lack of a hybridization band from the PvuII probe in strain EG6346 (lane 3) was entirely consistent with the classification of this strain as a cured derivative of B.t. strain EG6345 not containing the 45 MDa plasmid. The 115 MDa plasmid present within B.t. strains EG6345 and EG6346 was weakly detected by the PvuII probe. The reduced hybridization signal observed in each of these strains, as compared to strain HD-1, may be attributed to quantitative differences in the amounts of DNA loaded, as we11 as to the reduced sequence homology between the PvuII probe and the novel toxin genes present on this large plasmid.
Hybridization of the crylF PstI-SacI
intragenic probe to plasmid DNAs from B.t. strains HD-1 (lane 1), EG6345 (lane 2) and EG6346 (lane 3) is shown in the autoradiogram of Figure 8-C. The specificity of this probe for the crylF gene is confirmed by the lack of hybridization to plasmids harboring cry.IA genes in B.t. strains FiD-1 or 'VO 91/16434 ACI'/US91/02560 - 51 - 2 EG6345, and by its hybridization to the 115 MDa plasmid present in B.t. strains EG6345 and EG6346.
Both the PvuII and the PstI-SacI probes hybridized to a low molecular weight smear, identified as "L"
in Figure 8-A, which represents linear fragments of sheared larger toxin plasmids.
le ~
Insect Toxicity of the cry,IF Protein The insecticidal activity of CryIF
protein was determined against several lepidopteran larvae including Ostrinia nubilalis (European cornborer), Spodoptera exi a(beet armyworm), Heliothis virescens (tobacco budworm), Heliothis zea (bollworm) and Lymantria dispar (gypsy moth), using Renografin' density gradient purified CryIF
crystal protein from recombinant B.t. strain EG1945, which harbors the crylF gene on plasmid pEG642.
Activity was measured using a diet-surface overlay technique where the surface of an agar-based artificial diet was covered with an aliquot suspension containing CryIF protein crystals. After delivery of the aliquot to the diet surface, the diluent was allowed to evaporate, at which time one larva of the test species was placed in each cup. Each 2 ml well (cup) contained 1 ml diet having a surface area of 175 mm2 Bioassays were held at 280C for 7 days, at which time mortality was scored. Bioassays were first conducted at three doses with 1 to 10 dilutions.
If the CryIF protein demonstrated sufficient activity, eight dose assays (1 to 2 dilutions) were conducted to determine LC50 values via the well-known technique of probit analysis (Daum, Bull.
Entomol. Soc. Am., 16, pp. 10-15 (1970)). Each dose was tested against 32 insects. The diluent, 0.005% Triton' X-100, served as a control treatment. All insects were tested as newly hatched first-stage larvae. The results of effective insecticidal activity are set forth in Table 4 in comparison with the results of insecticidal bioassays using other CrylA crystal proteins.
Table 4 Insecticidal Activitp of CryI Crystal Proteins LC50 in ng protein/mm2 Heliothis Spodoptera Ostrinia Protein virescens exiqua nubilalis CryIA(a) 2.2 >57 0.27 CryIA(b) 0.7 33 0.17 CryIA(c) 0.03 >57 0.08 CryiF 0.31 26 0.17 The CryIF protein exhibited the greatest toxicity to Ostrinia nubilalis larvae as indicated in Table 4. The LC50 value obtained is similar to LC50 values obtained for the purified CryIA(b) crystal protein which is highly toxic to Ostrinia nubilalis larvae. In addition, the CryIF protein was toxic to Spodoptera exiqua larvae. CryIF
protein was considerably more toxic to Spodo tera VO 91/16434 Pt;T/US91/02560 exigua than purified CryIA(a) and CryIA(c) crystal proteins and slightly more toxic than purified CryIA(b) crystal protein. Purified CryIF crystal protein was also toxic to Heliothis virescens, with a toxicity between that of purified CryIA(c) and CryIA(b) crystal protein. CryIF crystal protein exhibited little toxicity to Iieliothis zea or Lymantria dispar at the doses tested.
Examp l e 7 Analysis of Insecticidal Activity of CryZX Fragment The sequence of the cryIX gene present on plasmid pEG642 (and likewise present on plasmid pEG640) does not encode a sufficient number of amino acids to constitute a "minimum toxic fragment" as defined by deletion analyses of cryTA
genes (Schnepf et al., J. Biol. Chem,, 260, pp. 6273-6278 (1985)), H fte et al., (1986) su ra).
Nonetheless, to assess the contribution of crylX, if any, to the overall toxicity of the pEG642 construct, the following study was performed.
Plasmid pEG310, containing a deletion in the cryIF gene, was constructed by restriction enzyme deletion from plasmid pEG642 of an N-terminal region of the cry%F gene which is flanked by BstEII sites (Figure 6). Following religation, plasmid pEG310 was introduced into the Cry B.t.
HD73-26 recipient via electroporation, resulting in a recombinant strain designated B.t. strain EG1078.
Fully sporulated cultures, containing the intact crylX gene sequence from plasmid pEG642, but not the cryTF gene which had been deleted, were assayed by the insect bioassay procedure previously WO 91/16434 FCI'/U591/02560 54 - 203O~~~,~~~
described in Example 6 for toxicity against Ostrinia nubilalis. B.t. strain EG1945, containing the intact crylF gene, was the positive control.
Thirty insect larvae were assayed, at a protein dose of 4.00 ng/550mm2. At this dose, the B.t. strain EG1945 was 100% toxic to larvae of Ostrinia nubilalis. However, B.t. strain EG1078, containing the cryIF deletion mutant, exhibited 0t mortality for Ostrinia nubilalis larvae. Thus, it was concluded that the sequence of the cr IX gene present on plasmid pEG642 does not contribute to the observed toxicity of B.t. strain EG1945 and that the cryIF gene product is the active insecticide in the strain.
Examplt 8 Southern Blot Analysis of the crvTX G ne in B.t. Strain Following the Southern blot technique cited in Example 1, total DNA was obtained from B.t. strain EG6346, digested to completion with the restriction endonucleases As~718 (an isoschizomer of Ksnl), C1aI, Sstl, and SshI both individually and in combination, electrophoresed through a 0.8%
agarose gel, transferred to a nitrocellulose filter, and hybridized at 65 C overnight to a 32p labgled 0.6 kb EpnIaBamHl probe that was isolated from pEG640 (previously described in Example 2) and that contained a portion of the cr IX coding region. The positions of the gi and BamFil sites flanking the crylX probe are shown in Figure 5.
The results of the Southern blot analysis are shown in Figure 9. Total DNA from B.t. strain EG6346 digested with restriction endonucleases exhibited, in each instance, a single DNA fragment hybridizing to the cr IX probe. Most importantly, B.t. strain EG6346 DNA digested with Clal (lane 2) yielded a 4.6 kb restriction jEragment that hybridized to the probe. In addition, B.t. strain EG6346 DNA digested with both ClaI and SstI
(lane 8) yielded a 4.4 kb restriction fragment that was detected by the cr IX probe. Since a ClaI
restriction site was present only 309 bp upstream from the cryIX open reading frame shown in Figure 2, these results indicated that the entire cryiX gene was likely to be contained on the 4.6 kb Clal restriction fragment. This assumption was shown to be correct by the fact that the CryIX
protein is only 81 kDa, which corresponds to a gene of about 2.1-2.2 kb in length. In addition, the absence of an SstI site immediately upstream to or within the sequenced portion of the cryIX gene displayed in Figure 2-A indicated that the SstI
restriction site detected by Southern blot analysis was located downstream from crylX.
Exam2l !
gaolatioa of the Entire cs~gt$X ene in B. coli A genomic library was constructed from Clag-digested DNA of B.t. strain EG6346 and screened under moderate stringency conditions with the 0.6 kb nDnI-BaaaFiI cryIX probe derived from pEG640 to ida:ntify recombinant E. coli colonies containing cr.ylX gene sequences.
More specifically, total DNA obtained from B.t. strain EG6346 was digested to completion with Cla2, electrophoresed th:rough a 0.8% agarose gel, and DNA fragments in the 4.3-5.0 kb range excised from the gel with a clean razor blade. DNA
fragments within the agarose gel slice were purified using the GeneClean II kit and procedure available from Bio 101, Inc. of La Jolla, CA.
The E. coli-B.t. cloning vector pEG854, depicted as a circular restriction map in Figure 10 and described by Baum et al., Appl. Environ.
Microbiol., 56, pp. 3420-3428 (1990), was used to clone the crylX gene on the Clal restriction fragments. The C1aI restriction fragments were ligated to C1aI-digested pEG854 vector DNA
pretreated with calf intestinal alkaline phosphatase to prevent self-ligation.
Transformation of E. coli HB101 cells with the ligation mixture was achieved by electroporation using the high-efficiency transformation procedure of Dower et al., Nucleic Acids Res., 16, pp. 6127-6145. Transformed cells were plated on agar plates of standard LB rmedium containing 50 pg/mi ampiciliin. Colonies were screened under moderaie stringency conditions for the presence of the cryIX
gene sequence using the colony blot hybridization procedure outlined in Example 2. The hybridization step was performed at 65'C, rather than at 50-55 C
as in Example 2, using the 0.6 kb KpnI-BamHI cryIX
probe described in Example 9. Filter washes were performed at 65=C in 3X SSC, 0.1% SDS. The crylX
probe hybridized strongly to one E. coli recombinant colony, designated E. coli strain '0 91/16434 PCT/U591/02560 EG1082, that contained an 11.8 kb recombinant plasmid, designated pEG313, that consisted of a 4.6 kb C1aI restriction fragment from B.t. strain EG6346 inserted into the C1aI restriction site of cloning vector pEG854.
A circular restriction map of recombinant plasmid pEG313 is depicted in F'igure 11. The orientation of the 4.6 kb ClaI restriction fragment was determined by restriction endonuclease mapping using methods well known to those skilled in the art.
Example 10 Expression of the cryIR G ne in E. coli and Production of Cry%8 ProtaTn To achieve expression of the crylX gene in E. coli and to characterize its encoded crystal protein, a 4.4. kb DNA fragment containing the crylX gene was inserted into the E. coli cloning vector pTZ19u, obtained from U.S. Biochemical Corporation. A circular restriction map of cloning vector pTZ19u, designated Plac in Figure 12, can be used to direct the transcription of cloned genes inserted into the multiple cloning site region demarcated by the unique HindIIl and EcoRI
restriction sites within the lacZ' gene.
Accordingly, a 4.4 kb ClaI-SstI restriction fragment containing the entire cryIX gene, as indicated by the Southern blot analysis in Example 8, was isolated from the recombinant plasmid pEG313 (see Figure 11) and ligated to pTZ19u DNA digested with Accl and SstI, two restriction endonucleases with cleavage sites within the multiple cloning site region of the WO 91/16434 1'CI'/tJS91/02560 cloning vector. Note that the Accl restriction site is compatible with that of C1aI, thereby allowing for efficient ligaticin of the cryIX gene fragment and orienting the cryIX gene in the same direction as the lac promoter. The ligation mixture was used to transform E. coli DH5;yL cells as described in Example 2. Using the X-gal screening procedure, a recombinant E. coli colony, designated EG1083, was recovered that contained a 7.3 kb recombinant plasmid, designated pEG314, that consisted of a 4.4 kb C1ai-Sstl restriction fragment derived from pEG313 inserted into the AccI
and Sstl sites of vector pTZ19u. A circular restriction map of recombinant plasmid pEG314, containing the cryIX gene inserted downstream from the lac promoter of pTZ19u, is depicted in Figure 13.
E. coli strain EG1083, containing pEG314 which carried the crylX gene, was grown in Luria broth containing 50 pg/ml ampicillin and 1mM
isopropyl-beta-D-thiogalactopyranoside (IPTG) at 376C overnight. IPTG is an inducer of the lac promoter and is commonly used to optimize transcription from that promoter in E. coli. After overnight growth, cells were examined by phase-contrast microscopy. E. coli strain EG1083 cells, but not E. coli strain DR5& cells containing pTZ19u, contained multiple phase-bright inclusions.
Subsequent lysis of the recombinant cells with lysozyme released the large inclusions, some of which appeared rhomboid in shape. The inclusions were purifieci from E. coli strain EG1083 by Renografin" density .gradient centrifugation and WO 91/16434 1'C"T/US91/02560 examined by SDS-polyacrylamide gel electrophoresis.
Figure 14 is.a photocopy of the resulting Coomassie-stained 10% SDS-polyacrylamide gel, in which lanes 1 and 2 contain CryIX crystal protein from E. coli strain EG1083 before and after Renografin' density gradient centrifugation, respectively. Protein molecular weight standards are displayed in the leftmost lane. Based on these standards, the CryIX crystal protein migrates with an apparent molecular mass of 81 kDa.
Example 11 Insect Toxicity of the CryIR Protein The insecticidal activity of the 81 kDa CryIX protein was determined against lepidopteran species, using Renografin'o density gradient purified CryIX crystal protein from recombinant E.
coli strain EG1083, which harbors the cr.xX gene on plasmid pEG314.
Activity was measured using a diet-surface overlay technique where the surface of an agar-based artificial diet was covered with an aqueous suspension containing CryIX protein crystals. Insect larvae were placed on the diet.
surface after the diluent had evaporated and held at 28'C for seven days, at which time mortality was scored.
In this bioassay screening.procedure, the purified CryIX protein exhibited insecticidal activity against larvae of Plutella arylostella (diamondback moth).
WO 91/16434 PCI'/L1S91/02560 In another bioassay screening procedure, using a cell paste of E. coli strain EG1083 that had produced CryIX protein instead of the purified CryIX protein, insecticidal activity was exhibited against larvae of ostrinia nubilalis (European corn borer).
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 61064, as indicated in the following Table 5:
Table 5 Bacterial Strain NRRL Accession No. Date of Deposit B.thuringiensis HD73-26 B-18508 June 12, 1989 B.thuringiensis EG6345 B-18633 March 27, 1990 B.thurinqiensis EG1945 B-18635 March 27, 1990 E. coli EG1943 B-18634 March 27, 1990 E. coli EG1083 B-18805 March 29, 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".
WO 91/16434 I'CI'/US91/02560 The present invention may be embodied in other specific forms without d+eparting 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.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Gawron-Burke, Cynthia Chambers, Judith A.
Gonzalez Jr., Jose M.
(ii) TITLE OF INVENTION: BACILLUS TF3I:JRINGIENSIS crylF and crylX
GENES AND PROTEINS TOXIC TO LEPIDOPTERAN INSECTS
(iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Panitch Schwarze et al. c/o A.S. Nadel (B) STREET: 1601 Market Street, 36th Floor (C) CITY: Philadelphia (D) STATE: PA
(E) COUNTRY: U.S.A.
(F) ZIP: 19103 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/510327 (B) FILING DATE: 16-APR-1990 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME:- Eqol, Christopher (B) REGISTRATION NAMBER: 27633 (C) REFERENCE/DOCKET NUMBER: 7205-27 U1 (2) INFORMATION FOR SEQ ID NO:1:' (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4020 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 478..4002 (xi) SEQUENCE DESCRIPTION: SEQ I?) NO:l:
- 63 - 2 0 Pior, S, d GATCTTCAAA TGAGAAAATA AGGGTATTCC GTATGGGATG CCTTTA'TTT GGTTGGGAAG 60 .GTATATTAAA AATAATGTTC TTATAACATA TATGTTGATT TTAAGAAAAT ATTTTGTTTA 180 TAAGCACACT ATTAACATAT TAGGTCTATT TAAATTAAGG: GCATATAGTG ATATTTTATA 360 Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe Va1 Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu I1e Tyr Ile Glu Ala Leu =AGA GAG TGG GAA GCA AAT CCT AAT AAT GCA CAA TTA AGG GAA GAT GTG 861 Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val CGT ATT CGA TTT GCT AAT ACA GAC GAC GC'T TTA ATA ACA GCA ATA AAT 909 Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asrn Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Lau Ser Leu Leu Arg Asp Ala Val Ser Phe WO 91/16434 PC'T/U591/02560 - 64 - 2, 0 89634 Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asri His Tyr Asn AGA TTA ATA AAT CTT ATT CAT AGA TAT ACG AA.A, CAT TGT TTG GAC ACA 1101 Arg Leu I1e Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Va1 Leu Asp Ile Va1 Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro 21e Gln Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile G1u Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu Va1 Ser Ser Arg Asn Thr Ala G1y Asn Arg Ile Asn Phe Pro Ser Tyr Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln Gln Thr Gly Thr Asn His Tllr Arg Thr Phe Arg Asn Ser Gly Thr Ile Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp WO 91/16434 POff'/US91/02560 - 65 - 2 ~3 ~i ~j~~'j ~~~
'U~(1 rc Asn Asp Tyr Ser His Val Leu Asn His Va]. Thr Phe Val Arg Trp Pro Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile Thr Gin Ile Pro Leu Val Lys Ala His Thr Lelu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gin Leu Pro Gin Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gin Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile .GAC AGA TTT GAA TTG ATT CCA GTT ACT GCA ACA TTT GAA GCA GPdA TAT 2301 Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala G1u Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln GTA TCC AAT TTA GTG GAT TGT TTA TCA GAT GAA,TTT TGT CTG GAT GAA 2445 Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu WO 91/16434 POt'/lJ591 /02560 Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg G1y Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp 705 710 71:5 720 Giu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Giu Ser Lys Leu Lys Pro Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Leu Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Gin Ser Pro ATC AGA A.AG TGT GGA GAA CCG AAT CGA TGC GCG CCA CAC CTT GAA TGG 2877 Ile Arg Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp A.AT CCT GAT CTA GAT TGT TCC TGC AGA GAC'GGG GAA AAA TGT GCA CAT 2925 Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Asp Val Trp Val Ile Phe Lys Ile Lys Thr Gin Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Giu Leu Giu Thr Asn Ile Val Tyr Lys Glu ~~090684 Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu A1. 'yr Leu Pro Glu Leu Ser Val Ili:~ Pro Gly Val Asn Val Asp Ile Phe Glu Glu Leu Lys Gly Arg Ile Phe Thr Ala Phe Phe Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Va1 Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala Asn G1n Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly TAT GAC GAA ACT TAT GGA AGC AAT TCT TCT GTA CCAi GCT GAT TAT GCG 3789 Tyr Asp Glu Thr Tyr Gly f3er Asn Ser Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys .~er Tyr Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly WO 91/16434 PC'I'/US91/02560 - 68 - 2 008 ~~
Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lyn Val T:p Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1174 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe Va1 Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu G1n Ile Glu Gln Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn WO'91/16434 I'CT/US91/02560 - 69 - lti~0,,/ ~ ~
06{.~d">':~
Arg Leu Ile Asn Leu I1e His Arg Tyr Thr Lys His Cys Leu Asp Thr Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile Va1 Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val I1e Glu Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365' Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe G1n Gin Thr Gly Thr Asn His Thr Arg Thr Phe Arg.Asn,Ser Gly Thr Ile Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro Leu Vai Lys Ala His Thr Leu Gin Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr I1e Val Asn Ile Asn Gly Gln Leu WO 91/16434 PC'T/US91/02560 - 70 - ~~ ~ ~ a n l~c~~~
~~y~ t~
Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phie Pro Met Ser Gln Ser Ser Phe Thr Va1 Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr I1e Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Va1 Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly I1e Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys Ile Asp Glu Ser Lys Leu Lys Pro Tyr Thr Arg Tyr Gin Leu Arg Gly Tyr Ile Glu Asp Ser G1n Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Leu Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Gln Ser Pro Yle Arg Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Gys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile AspVal Gly Cys Thr Asp Leu WO 91/16434 PfT/US91/02560 s ~~f,r ~ i'~ ~ a {
- ~
Asn Glu Asp Leu Asp Val Trp Val I1e Phe Lys I].e Lys Thr G1n Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Arg 21e Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Val Asp Ile Phe Glu Glu Leu Lys Gly Arg Tie Phe Thr Ala Phe Phe Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu I1e Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Glu Thr Tyr Gly Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu I,ys Ser Tyr Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Va1 Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp ~~~~0 ('3 Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe I1e Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO:3:
( i ) SEQUENCE CFiARACTERISTICS :
(A) LENGTH: 1629 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/REY: CDS
(B) LOCATION: 488..1629 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
=. TTGTAATGAA AAACGGACAT CACCTCCATT GAAACGGTGA GATGTCCGTT TTACTATGTT 120 Met Lys Leu Lys Asn Pro Asp Lys His G1n Ser Phe Ser Ser AAT GCG AAA GTA GAT AAA ATC TCT ACG GAT TCA CTA AAA AP.T GAA ACA 577 Asn Ala Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile Glu Leu Gin Asn I].e Asn His Glu Asp Cys Leu Lys Ile Ser GAG TAT GAA AAT GTA GAG CC:G TTT GTT AGT GCA TCA ACA ATT CAA AC.11. 673 Glu Tyr Giu Asn Val Glu Pro Phe Val Ser Ala Ser Thr Ile Gin Thr GGT ATT AGT ATT GCG GGT AAA ATA C.TT GGC ACC CTA GGC GTT CCT TTT 721 Gly Ile Ser Ile Ala Gly Lys Ile Leu Gly Thr Leu Gly Va1 Pro Phe 65 70 75 cp080639~~
GCA GGA yA GTA GCT AGT CTT TAT AGT TTT ATC TTA GGT GAG CTA TGG( 769 Ala Gly Gln Val Ala Ser Leu Tyr Ser Phe Ile Leu Gly Glu Leu Trp Pro Lys Gly Lys Asn Gln Trp Glu Ile Phe Met Glu His Val Glu Glu Ile Ile Asn Gln Lys Ile Ser Thr Tyr Ala Arg Asn Lys Ala Leu Thr Asp Leu Lys Gly Leu Gly Asp Ala Leu Ala Val Tyr His Glu Ser Leu Glu Ser Trp Val Gly Asn Arg Lys Asn Thr Arg Ala Arg Ser Val Val Lys Ser Gln Tyr Ile Ala Leu Glu Leu Met Phe Val Gln Lys Leu Pro Ser Phe Ala Val Ser Gly Glu Glu Val Pro Leu Leu Pro Ile Tyr Ala Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Ile Phe Gly Lys Glu Trp Gly Leu Ser Ser Ser Glu Ile Ser Thr Phe Tyr Asn Arg G1n Val Glu Arg Ala Gly Asp Tyr Ser Asp His Cys Val Lys Trp Tyr Ser Thr Gly Leu Asn Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Val Arg Tyr Asn Gln Phe Arg Lys Asp Met Thr Leu Met Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Leu Val Tyr Pro Ile Lys Thr Thr Ser Gin Leu Thr ls,rg Glu Val Tyr Thr Asp Ala Ile Gly Thr Val His Pro Asn Ala Ser Phe Ala Ser Thr Thr Trp Tyr Asn Asn Asn WO 91/16434 PCf/US91/02560 Ala Pro Ser Phe Ser Thr 21e Glu Ser Ala Val Val Arg Asn Pro His Leu Leu Asp Phe Leu Glu Gln Val Thr Ile ",Cyr Ser Leu Leu Ser Arg 335 340 :345 350 Trp Ser Asn Thr Gln Tyr Met Asn Met Trp Gly Gly His Arg Leu Glu Phe Arg Thr I1e Gly Gly Met Leu Asn Thr Ser Thr Gln Gly (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 380 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Lys Leu Lys Asn Pro Asp Lys His Gin Ser Phe Ser Ser Asn Ala Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile Glu Leu Gln Asn Ile Asn His Giu Asp Cys Leu Lys 21e Ser Glu Tyr Glu Asn Val Glu Pro Phe Val Ser Ala Ser Thr I1e Gin Thr Gly Ile Ser Ile Ala Gly Lys Ile Leu Gly Thr Leu Gly Val Pro Phe Ala Gly Gln Val Ala Ser Leu Tyr Ser Phe Ile Leu Gly Glu Leu Trp Pro Lys Gly Lys Asn Gln Trp Glu I1e Phe Met Glu His Val Glu Glu Ile Ile Asn Gin Lys Ile Ser Thr Tyr Ala Arg Asn Lys Ala Leu Thr Asp Leu Lys Gly Leu Gly Asp Ala Leu Ala Val Tyr His Glu Ser Leu Glu Ser Trp Val Gly Asn Arg Lys Asn Thr Arg Ala Arg Ser Val Val Lys Ser WO 91/16434 PCT/[!S91/02560 ~~ ~~
~+J ~ ~Jd Gln Tyr Ile Ala Leu Glu Leu Met Phe Val Gln Lys Leu Pro Ser Phe Ala Val Ser Gly Glu Glu Val Pro Leu Leu Pro Ile Tyr Ala Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Ile Phe Gly Lys Glu Trp Gly Leu Ser Ser Ser Glu I1e Ser Thr Phe Tyr Asn Arg Gln Val Glu Arg Ala Gly Asp Tyr Ser Asp His Cys Val Lys Trp Tyr Ser Thr Gly Leu Asn Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Val Arg Tyr Asn Gln Phe Arg Lys Asp Met Thr Leu Met Va1 Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Leu Val Tyr Pro Ile Lys Thr Thr Ser Gln Leu Thr Arg Glu Val Tyr Thr Asp Ala Ile Gly Thr Val His Pro Asn Ala Ser Phe Ala Ser Thr Thr Trp Tyr Asn Asn Asn Ala Pro Ser Phe Ser Thr Ile Glu Ser Ala Val Val Arg Asn Pro His Leu Leu Asp Phe Leu Glu Gin Val Thr Ile Tyr Ser Leu Leu Ser Arg Trp Ser Asn Thr Gln 'Tyr Met Asn Met Trp Gly Gly His Arg Leu Glu Phe Arg Thr Ile Gly Gly Met Leu Asn Thr Ser Thr Gln Gly
Claims (14)
1. A purified and isolated cryIF gene characterized by a nucleotide base sequence as illustrated in Figure 1 (SEQ ID NO:1).
2. A purified and isolated cryIF gene according to claim 1 further characterized in that the gene has a coding region extending from nucleotide bases 478 to 3999 in the nucleotide base sequence illustrated in Figure 1 (SEQ ID N0:1).
3. A biologically pure culture of a bacterium characterized in that the bacterium has been transformed to express a lepidopteran-toxic protein from the gene of claim 1 or claim 2 contained within a recombinant plasmid.
4. The bacterium of claim 3 further characterized in that the bacterium is E. coli.
5. The E. coli bacterium of claim 4 further characterized in that the bacterium is deposited with the NRRL with accession number NRRL B-18634.
6. The bacterium of claim 3 further characterized in that the bacterium is Bacillus thuringiensis.
7. The Bacillus thuringiensis bacterium of claim 6 further characterized in that the bacterium is deposited with the NRRL with accession number NRRL B-18635.
8. An insecticide composition characterized in that the composition contains the bacterium of claim 3 expressing said lepidopteran-toxic protein and an agriculturally acceptable carrier.
9. A genetically engineered polynucleotide encoding a lepidopteran-toxic protein that is a C-terminal truncated derivative of the protein coded by the cryIF
gene of claim 1, wherein said C-terminal truncated derivative comprises amino acids 1 to 618 as set forth in Figure 1 (SEQ ID NO: 2).
gene of claim 1, wherein said C-terminal truncated derivative comprises amino acids 1 to 618 as set forth in Figure 1 (SEQ ID NO: 2).
10. A biologically pure culture of a bacterium characterized in that the bacterium has been transformed to express said lepidopteran-toxic protein from the polynucleotide of claim 9.
11. An insecticide composition comprising the bacterium of claim 10 expressing said lepidopteran-toxic protein and an agriculturally-acceptable carrier.
12. A lepidopteran-toxic protein encoded by the polynucleotide of claim 9.
13. A plant cell that has been transformed to express a lepidopteran-toxic protein comprising amino acids 1 to 618 as set forth in Figure 1 (SEQ ID NO: 2) from a genetically engineered polynucleotide.
14. A plant cell that has been transformed to express a lepidopteran-toxic protein from the genetically engineered polynucleotide of claim 9.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US51032790A | 1990-04-16 | 1990-04-16 | |
| US510,327 | 1990-04-16 | ||
| PCT/US1991/002560 WO1991016434A2 (en) | 1990-04-16 | 1991-04-15 | BACILLUS THURINGIENSIS cryIF AND cryIX GENES AND PROTEINS TOXIC TO LEPIDOPTERAN INSECTS |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2080684A1 CA2080684A1 (en) | 1991-10-17 |
| CA2080684C true CA2080684C (en) | 2010-02-02 |
Family
ID=24030295
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002080684A Expired - Lifetime CA2080684C (en) | 1990-04-16 | 1991-04-15 | Bacillus thuringiensis cryif and cryix genes and proteins toxic to lepidopteran insects |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0528823A1 (en) |
| AU (1) | AU647121B2 (en) |
| CA (1) | CA2080684C (en) |
| TR (1) | TR26973A (en) |
| WO (1) | WO1991016434A2 (en) |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5686069A (en) * | 1990-10-15 | 1997-11-11 | Mycogen Corporation | Protein toxins active against lepidopteran pests |
| TW261517B (en) * | 1991-11-29 | 1995-11-01 | Mitsubishi Shozi Kk | |
| US5356623A (en) * | 1993-03-17 | 1994-10-18 | Ecogen Inc. | Bacillus thuringiensis cryET1 toxin gene and protein toxic to lepidopteran insects |
| US5877012A (en) * | 1993-03-25 | 1999-03-02 | Novartis Finance Corporation | Class of proteins for the control of plant pests |
| US5849870A (en) * | 1993-03-25 | 1998-12-15 | Novartis Finance Corporation | Pesticidal proteins and strains |
| HU220714B1 (en) * | 1993-03-25 | 2002-04-29 | Novartis Ag. | Novel pesticidal proteins and strains |
| US5322687A (en) * | 1993-07-29 | 1994-06-21 | Ecogen Inc. | Bacillus thuringiensis cryet4 and cryet5 toxin genes and proteins toxic to lepidopteran insects |
| US5508264A (en) * | 1994-12-06 | 1996-04-16 | Mycogen Corporation | Pesticidal compositions |
| ES2330168T3 (en) * | 1995-10-13 | 2009-12-04 | Dow Agrosciences Llc | MOPDIFIED GENE OF BACILLUS THURINGIENSIS TO COMBAT LEPIDOPTERS IN PLANTS. |
| EP0909324A2 (en) | 1996-07-01 | 1999-04-21 | Mycogen Corporation | Toxins active against pests |
| US6369213B1 (en) | 1996-07-01 | 2002-04-09 | Mycogen Corporation | Toxins active against pests |
| US5965428A (en) * | 1996-10-08 | 1999-10-12 | Ecogen, Inc. | Chimeric lepidopteran-toxic crystal proteins |
| IN1998CH00710A (en) | 1997-04-03 | 2005-03-04 | Novartis Ag | Plant pest control |
| AR025349A1 (en) * | 1999-08-23 | 2002-11-20 | Mycogen Corp | METHODS TO CONTROL GRAY WORM PESTS |
| US11130964B2 (en) | 2014-11-20 | 2021-09-28 | Monsanto Technology Llc | Insect inhibitory proteins |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU624349B2 (en) * | 1987-11-05 | 1992-06-11 | David M. Gjerdrum | Device for monitoring radon exposure |
| GB8800652D0 (en) * | 1988-01-13 | 1988-02-10 | Ici Plc | Bacterial strain |
| ATE242327T1 (en) * | 1988-09-06 | 2003-06-15 | Bayer Bioscience Nv | PLANTS TRANSFORMED WITH A LEPIDOPTER LETHAL DNA SEQUENCE FROM BACILLUS THURINGIENSIS |
| GB8910624D0 (en) * | 1989-05-09 | 1989-06-21 | Ici Plc | Bacterial strains |
-
1991
- 1991-04-10 TR TR00366/91A patent/TR26973A/en unknown
- 1991-04-15 AU AU76873/91A patent/AU647121B2/en not_active Expired
- 1991-04-15 EP EP91908010A patent/EP0528823A1/en not_active Withdrawn
- 1991-04-15 WO PCT/US1991/002560 patent/WO1991016434A2/en not_active Application Discontinuation
- 1991-04-15 CA CA002080684A patent/CA2080684C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| WO1991016434A3 (en) | 1991-12-12 |
| AU7687391A (en) | 1991-11-11 |
| AU647121B2 (en) | 1994-03-17 |
| WO1991016434A2 (en) | 1991-10-31 |
| EP0528823A1 (en) | 1993-03-03 |
| CA2080684A1 (en) | 1991-10-17 |
| TR26973A (en) | 1994-09-12 |
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| EEER | Examination request | ||
| MKEX | Expiry | ||
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Effective date: 20110415 |