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Showing papers on "Bacillus thuringiensis published in 1986"



Patent
09 Dec 1986
TL;DR: A process for altering the insect host range (spectrum) of pesticidal toxins comprises recombining in vitro the variable regions(s) (nonhomologous) of two or more genes encoding a pesticidal toxin this article.
Abstract: A process for altering the insect host range (spectrum) of pesticidal toxins comprises recombining in vitro the variable region(s) (non-homologous) of two or more genes encoding a pesticidal toxin Specifically exemplified is the recombining of the variable regions of two genes obtained from well-known strains of Bacillus thurinaiensis var kurstaki The resulting products are chimeric toxins which are shown to have an expanded and/or amplified insect host range as compared to the parent toxins

421 citations


Journal ArticleDOI
TL;DR: The minimal portion of the Bt2 protein required for toxicity has been determined by analysing the polypeptides produced by deletion derivatives of the bt2 gene, which coincides with the 60-kDa protease-resistant Bt1 fragment.
Abstract: A plasmid-encoded crystal protein gene (bt2) has been cloned from Bacillus thuringiensis berliner 1715. In Escherichia coli, it directs the synthesis of the 130-kDa protein (Bt2) which is toxic to larvae of Pieris brassicae and Manduca sexta. Comparison of the deduced amino acid sequence of this Bt2 protein with the B. thuringiensis kurstaki HD1 Dipel, B. thuringiensis kurstaki HD73 and B. thuringiensis sotto crystal protein sequences suggests that homologous recombination between the different genes has occurred during evolution. Treatment of the Bt2 protein with trypsin or chymotrypsin yields a 60-kDa protease-resistant and fully toxic polypeptide. The minimal portion of the Bt2 protein required for toxicity has been determined by analysing the polypeptides produced by deletion derivatives of the bt2 gene. It coincides with the 60-kDa protease-resistant Bt2 fragment and it starts between amino acids 29 and 35 at the N-terminus and terminates between positions 599 and 607 at the C-terminus.

250 citations


Journal ArticleDOI
TL;DR: An isolate of Bacillus thuringiensis has been identified with toxic activity against coleopteran insects but not against lepidopteran or dipteran species, suggesting the possibility of commercial applications of this strain.
Abstract: An isolate of Bacillus thuringiensis has been identified with toxic activity against coleopteran insects but not against lepidopteran or dipteran species. Toxin crystals from this strain contain a polypeptide of approximately 64,000 molecular weight possessing insecticidal activity. This toxin differs in polypeptide composition from other reported Bacillus thuringiensis toxins and does not appear to be processed from a larger precursor form. Insecticidal activity against the Colorado potato beetle and the boll weevil suggests the possibility of commercial applications of this strain.

222 citations


Journal ArticleDOI
TL;DR: Data obtained using several experimental methods indicate that crystal protein genes in many subspecies of BT that are toxic to lepidopterans are located on one or more large plasmids; in some subspecies, the gene may be located on the chromosome.
Abstract: Data obtained using several experimental methods (curing, transconjugation, cloning, and hybridization) indicate that crystal protein genes in many subspecies of BT that are toxic to lepidopterans are located on one or more large plasmids; in some subspecies, the gene may be located on the chromosome. Detailed mapping has shown that in three plasmids (each from a different strain) the genes are surrounded by multiple copies of two repeated DNA elements; the arrangement of these elements is the same in the three plasmids. An analysis of the sequence of one of these repeated DNAs strongly suggests that it contains a transposase. Thus, transfer of crystal protein genes between plasmids and/or between plasmids and the chromosome would be possible either by transposition or by recombinational events mediated by the repeated DNAs. Crystal protein genes have been cloned from several plasmids and were expressed in E. coli and B. subtilis, whereas two genes cloned from chromosomal preparations were not expressed. Some of the factors that regulate expression of a plasmid-borne gene in E. coli and B. subtilis have been identified. Very little is known about the role of sporulation genes in regulating expression of the crystal protein gene in B. subtilis or BT. In BT, expression may also be affected by genes on other plasmids. Three homologous crystal protein genes have been identified and cloned from subsp. kurstaki and thuringiensis; different strains of these subspecies may contain one, two, or three of these genes. It seems probable that additional gene families will be found, since the crystals of different subspecies contain immunologically distinguishable proteins. The DNA sequences of the three homologous genes have been published as has the sequence of the crystal protein gene from subsp. sotto. These four genes have regions of identity (the promoter region) and similarity (the N-terminal approximately 280 amino acids, the C-terminal half of the protein, and the terminator). It is interesting that the divergent portions of the molecules are not in precisely the same positions and that all overlap the toxin-encoding portion of the gene. It would be worthwhile to determine if the differences in the amino acid sequence are related to differences in the toxicity and/or the host range of the cloned genes, and to establish how the complement of genes in a given strain contributes to the overall toxicity of that strain.(ABSTRACT TRUNCATED AT 400 WORDS)

215 citations


Journal ArticleDOI
TL;DR: The native crystal delta-endotoxin produced by Bacillus thuringiensis var.
Abstract: The native crystal delta-endotoxin produced by Bacillus thuringiensis var. colmeri, serotype 21, is toxic to both lepidopteran (Pieris brassicae) and dipteran (Aedes aegypti) larvae. Solubilization of the crystal delta-endotoxin in alkaline reducing conditions and activation with trypsin and gut extracts from susceptible insects yielded a preparation whose toxicity could be assayed in vitro against a range of insect cell lines. After activation with Aedes aegypti gut extract the preparation was toxic to all of the mosquito cell lines but only one lepidopteran line (Spodoptera frugiperda), whereas an activated preparation produced by treatment with P. brassicae gut enzymes or trypsin was toxic only to lepidopteran cell lines. These in vitro results were paralleled by the results of in vivo bioassays. Gel electrophoretic analysis of the products of these different activation regimes suggested that a 130-kDa protoxin in the native crystal is converted to a 55-kDa lepidopteran-specific toxin by trypsin or P. brassicae enzymes and to a 52-kDa dipteran toxin by A. aegypti enzymes. Two-step activation of the 130-kDa protoxin by successive treatment with trypsin and A. aegypti enzymes further suggested that the 52-kDa dipteran toxin is derived from the 55-kDa lepidopteran toxin by enzymes specific to the mosquito gut. Confirmation of this suggestion was obtained by peptide mapping of these two polypeptides. The native crystal 130 kDa delta-endotoxin and the two insect-specific toxins all cross-reacted with antiserum to B. thuringiensis var. kurstaki P1 lepidopteran toxin. Preincubation of the two activated colmeri toxins with P1 antiserum neutralized their cytotoxicity to both lepidopteran and dipteran cell lines.

175 citations


Journal ArticleDOI
TL;DR: Lutte biologique par des virus, des protozoaires, des champignons, des Championignons (Lagedinium giganteum, Culicinomyces clavisporus, Coelomomyces) andrology.
Abstract: Lutte biologique par des virus, des protozoaires, des champignons (Lagedinium giganteum, Culicinomyces clavisporus, Coelomomyces) des bacteries (Bacillus sphaericus, Bacillus thuringiensis)

172 citations


Journal ArticleDOI
TL;DR: A gene from Bacillus thuringiensis subsp.
Abstract: A gene from Bacillus thuringiensis subsp. "israelensis" was cloned from the large plasmids of this subspecies and was shown to code for a mosquitocidal polypeptide. The gene could be expressed in either Escherichia coli, Bacillus subtilis, or B. thuringiensis subsp. "israelensis" to produce the larvicidal activity. Similarly, a Lepidoptera-specific toxin gene from B. thuringiensis subsp. "kurstaki" was also cloned and expressed in E. coli and B. subtilis. Both cloned genes were sequenced and subjected to computer analysis. A long open translational reading frame coded for the B. thuringiensis subsp. "kurstaki" gene product. However, the B. thuringiensis subsp. "israelensis" clone was composed of two adjacent open reading frames oriented as if they were in a transcriptional operon. The products of the cloned genes retained their specificity for either Lepidoptera or Diptera. The control regions immediately preceding the toxin genes of both B. thuringiensis subspecies showed considerable DNA homology, most likely because both toxins are expressed only during sporulation. In addition, the deduced amino acid sequences from the two contiguous B. thuringiensis subsp. "israelensis" genes bore a striking resemblance to the deduced amino acid sequence from the single larger B. thuringiensis subsp. "kurstaki" gene, as if these two arrangements were evolutionarily related.

156 citations


Journal ArticleDOI
01 Jan 1986-Gene
TL;DR: One of the genes for the entomophatogenic crystal protein of Bacillus thuringiensis (subsp. kurstaki strain HD1) has been cloned in Escherichia coli, and its nucleotide sequence determined completely.

149 citations


Journal ArticleDOI
TL;DR: The results suggested that B. thuringiensis flora in soils was remarkably different from that in the sericultural environment.

143 citations


Journal ArticleDOI
TL;DR: Although 121 isolates of Bacillus thuringiensis producing parasporal inclusions produced PIs with various morphologies including typically bipyramidal ones, they did not demonstrate any toxicity against insects of three orders, suggesting that nontoxic PI-forming bacteria predominate in natural environments rather than toxic ones.

Journal ArticleDOI
01 Jan 1986-Gene
TL;DR: Four homologous genes encoding insecticidal crystal proteins from Bacillus thuringiensis have been cloned in Escherichia coli and an analysis of plasmid DNA flanking three of these genes revealed a complex pattern of two different inverted repeat (IR) sequences, IR2150 and IR1750.

Patent
08 Aug 1986
TL;DR: The toxin gene encoding a protein toxic to beetles of the order Coleoptera, named M-7, has been cloned and expressed as discussed by the authors, and a novel Bacillus thuringiensis strain which has been deposited with a recognized culture repository.
Abstract: The toxin gene encoding a protein toxic to beetles of the order Coleoptera, named M-7, has been cloned and expressed. M-7 is a novel Bacillus thuringiensis strain which has been deposited with a recognized culture repository.

Journal ArticleDOI
01 Jan 1986-Gene
TL;DR: The delta-endotoxin gene (tox) from Bacillus thuringiensis subsp.

Journal ArticleDOI
TL;DR: Transcriptional mapping of delta-endotoxin-specific mRNA has shown that the same region of initiation is used at a relatively low level in all three strains during vegetative growth.

Journal ArticleDOI
TL;DR: It is proposed that this glycoprotein is a good candidate for the cellular receptor of the lepidopteran-specific P1 delta-endotoxin of B. thuringiensis var.
Abstract: The lepidopteran-specific P1 delta-endotoxin of Bacillus thuringiensis var kurstaki HD-1 was activated in vitro using insect gut proteases and found to be highly specific for the lepidopteran cell line Choristoneura fumiferana CF1 among a wide range of lepidopteran and dipteran cell lines tested The toxicity of P1 against CF1 cells is inhibited by N-acetylgalactosamine (GalNAc), and the lectins soybean agglutinin (SBA) and wheat-germ agglutinin Protein blotting was used to identify a glycoprotein of 146 X 10(3) Mr in the plasma membrane of CF1 cells, capable of binding both the toxin and SBA, which is specific for GalNAc This glycoprotein was labelled using galactose oxidase and sodium boro-[3H]hydride and solubilized in Triton X-100 before partial purification by affinity chromatography on SBA-agarose We propose that this glycoprotein is a good candidate for the cellular receptor of the lepidopteran-specific P1 delta-endotoxin of B thuringiensis var kurstaki HD-1

Journal ArticleDOI
01 Aug 1986
TL;DR: A model whereby the protoxin molecule is divided into distinct structural and functional domains is proposed, which may be responsible for the differences in specific toxicities and spectra of insect host range among these subspecies.
Abstract: A 3778-bp DNA sequence of the insecticidal protoxin gene coding sequence and flanking regions from Bacillus thuringiensis subspecies berliner 1715 has been determined. The protoxin is composed of 1155 amino acids, deduced from the nucleotide sequence, and has a calculated molecular mass of 130,615 daltons. To determine the DNA portion that encodes toxicity, sequential deletions were constructed from the 3' end of the coding region using nuclease Bal-31. Using these mutants in an insect bioassay, we found that an amino-terminal 612-amino-acid peptide is toxic, whereas, a 603-amino-acid peptide is not toxic to insects. Ninety percent of the amino acid residues were homologous to the protoxins from closely related subspecies kurstaki HD-1-Dipel and sotto. The differences occurred both in the amino-terminal half, or toxic portion, and in the carboxy-terminal half. These differences were clustered in several regions. From comparative analysis of subspecies berliner and kurstaki, we propose a model whereby the protoxin molecule is divided into distinct structural and functional domains. These domains may be responsible for the differences in specific toxicities and spectra of insect host range among these subspecies.

Journal ArticleDOI
TL;DR: Interestingly, the highest level of delta-endotoxin mRNA is seen during mid-exponential growth of E. coli and the level appears to decrease as the cells enter the stationary phase of growth, consistent with a postulated cytolytic mechanism of action for the toxin.

Journal ArticleDOI
TL;DR: One laboratory and two wild populations of the yellow fever mosquito, Aedes aegypti, were used in an attempt to artificially select for resistance to Bacillus thuringiensis subsp.

Journal ArticleDOI
TL;DR: Results indicate that B. thuringiensis Serotype 8a:8b, which is generally considered to produce proteins toxic to lepidopterous insects, is capable of producing a protein toxin complement similar to B. israelensis Serotype 14.
Abstract: The mosquitocidal parasporal bodies of the PG-14 isolate of Bacillus thuringiensis ssp. morrisoni and B. thuringiensis ssp. israelensis were purified on sodium bromide gradients and compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electron microscopy and bioassays against mosquito larvae. The parasporal bodies of both subspecies were spherical/ovoidal, approx. 0.7–1.2 μm in diameter, and contained major proteins of 28, 65, 126 and 135 kDa. In addition to these, the parasporal body of B. thuringiensis ssp. morrisoni contained at least one other major protein, of 144 kDa, which correlated with the presence of a quasi-bi-pyramidal inclusion not present in the B. thuringiensis ssp. israelensis parasporal body. The LC50 for parasporal bodies of each subspecies was in the range of 3 ng/ml for fourth-instars of Aedes aegypti. These results indicate that B. thuringiensis Serotype 8a:8b, which is generally considered to produce proteins toxic to lepidopterous insects, is capable of producing a protein toxin complement similar to B. thuringiensis Serotype 14.

Journal ArticleDOI
TL;DR: The crystalline parasporal inclusions (crystals) of Bacillus thuringiensis israelensis (Bti), which are specifically toxic to mosquito and black fly larvae, contain three main polypeptides of 28, 68 and 130 kDa.
Abstract: The crystalline parasporal inclusions (crystals) of Bacillus thuringiensis israelensis (Bti), which are specifically toxic to mosquito and black fly larvae, contain three main polypeptides of 28 kDa, 68 kDa and 130 kDa. The genes encoding the 28 kDa protein and the 130 kDa protein have been cloned from a large plasmid of Bti. Escherichiacoli recombinant clones containing the 130 kDa protein gene were highly active against larvae of Aedes aegypti and Culex pipiens, while B. subtilis recombinant cells containing the 28 kDa protein gene were haemolytic for sheep red blood cells. A fragment of the Bti plasmid which is partially homologous to the 130 kDa protein gene was also isolated; it probably corresponds to part of a second type of mosquitocidal toxin gene. Furthermore, restriction enzyme analysis suggested that the 130 kDa protein gene is located on the same Bti EcoRI fragment as another kind of Bti mosquitocidal protein gene cloned by Thorne et al. (1986). Hybridization experiments conducted with the 28 kDa protein gene and the 230 kDa protein gene showed that these two Bti genes are probably present in the plasmid DNA of B. thuringiensis subsp. morrisoni (PG14), which is also highly active against mosquito larvae.

Journal ArticleDOI
TL;DR: Parasporal crystals of Bacillus thuringiensis var. tenebrionis are toxic for coleopteran larvae and are found to be soluble in aqueous solutions of NaBr at neutral pH or in water after titration to pH values above pH 10.0 as discussed by the authors.
Abstract: Parasporal crystals of the recently isolated Bacillus thuringiensis var. tenebrionis are toxic for coleopteran larvae. Unlike those of other strains they are soluble either in aqueous solutions of NaBr at neutral pH or in water after titration to pH values above pH 10.0. The dissolved crystal protein readily forms crystals after removal of the salt or neutralization. The crystal protein was not found to differ much in the amino acid composition from other crystal proteins. The parasporal crystals are composed of subunits of Mr 68 000 which are not linked by disulfide bridges.

Journal ArticleDOI
TL;DR: Gene replacement mediated by Tn5 sequences was used to integrate the Bacillus thuringiensis subsp.
Abstract: Gene replacement mediated by Tn5 sequences was used to integrate the Bacillus thuringiensis subsp. kurstaki HD-1 delta-endotoxin gene (tox) into the chromosome of two corn root-colonizing strains of Pseudomonas fluorescens. A Tn5 transposase deletion element containing the tox gene (delta Tn5-tox) was substituted for a Tn5 element previously present in the P. fluorescens chromosome. Two classes of delta Tn5-tox elements were made. The first class encodes kanamycin resistance in addition to the Tox protein, whereas the second class encodes only the Tox protein. Both classes of delta Tn5-tox elements can no longer transpose, owing to a 324-base-pair deletion in the transposase gene of IS50R, minimizing the potential for horizontal gene transfer of the tox gene to other bacterial species. A frameshift mutation in the transposase gene of IS50L was also constructed to eliminate the possibility of suppression or of a spontaneous reversion at the ochre termination codon that would create an active transposase. Expression of the Tox protein in P. fluorescens strains 112-12 and Ps3732-3-7 was demonstrated by an immunological assay (Western blot) and toxicity against larvae of the tobacco hornworm (Manduca sexta). Images

Journal ArticleDOI
TL;DR: Results suggest that toxin gene expression in B. subtilis is independent of sporulation, and a recombinant plasmid expressed a protein of 135,000 daltons that was toxic to caterpillars.
Abstract: Bacillus thuringiensis subsp. kurstaki total DNA was digested with BglII and cloned into the BamHI site of plasmid pUC9 in Escherichia coli. A recombinant plasmid, pHBHE, expressed a protein of 135,000 daltons that was toxic to caterpillars. A HincII-SmaI double digest of pHBHE was then ligated to BglII-cut plasmid pBD64 and introduced into Bacillus subtilis by transformation. The transformants were identified by colony hybridization and confirmed by Southern blot hybridization. A 135,000-dalton protein which bound to an antibody specific for the crystal protein of B. thuringiensis was detected from the B. subtilis clones containing the toxin gene insert in either orientation. A toxin gene insert cloned into a PvuII site distal from the two drug resistance genes of the pBD64 vector also expressed a 135,000-dalton protein. These results suggest that the toxin gene is transcribed from its own promoter. Western blotting of proteins expressed at various stages of growth revealed that the crystal protein expression in B. subtilis begins early in the vegetative phase, while in B. thuringiensis it is concomitant with the onset of sporulation. The cloned genes when transferred to a nonsporulating strain of B. subtilis also expressed a 135,000-dalton protein. These results suggest that toxin gene expression in B. subtilis is independent of sporulation. Another toxin gene encoding a 130,000- to 135,000-dalton protein was cloned in E. coli from a library of B. thuringiensis genes established in lambda 1059. This gene was then subcloned in B. subtilis. The cell extracts from both clones were toxic to caterpillars. Electron microscope studies revealed the presence of an irregular crystal inclusion in E. coli and a well-formed bipyramidal crystal in B. subtilis clones similar to the crystals found in B. thuringiensis. Images

Patent
21 Mar 1986
TL;DR: In this paper, a novel and useful insecticide with activity against insect pests of cotton, potato, alfalfa, and corn crops is described, which is a B. thuringiensis microbe given the designation strain san diego.
Abstract: The subject invention concerns a novel and useful insecticide with activity against insect pests of cotton, potato, alfalfa and corn crops. These pests do heavy damage to the crops. The insecticide of the subject invention is a B. thuringiensis microbe given the designation strain san diego. The spores or crystals of this microbe are useful to control the cotton boll weevil, the Colorado potato beetle, the alfalfa weevil, and the corn rootworm.

Patent
16 Jun 1986
TL;DR: The toxin gene encoding a protein toxic to beetles of the order Coleoptera, named M-7, has been cloned and expressed as discussed by the authors, and a novel Bacillus thuringiensis strain which has been deposited with a recognized culture repository.
Abstract: The toxin gene encoding a protein toxic to beetles of the order Coleoptera, named M-7, has been cloned and expressed. M-7 is a novel Bacillus thuringiensis strain which has been deposited with a recognized culture repository. The microbe is now known as B. thuringiensis strain san diego.

Journal ArticleDOI
TL;DR: N nondenaturing gel electrophoresis is used to show that the hemolytic activity resides in the 28-kilodalton protein, however, higher-molecular-weight proteins are required to achieve the level of toxicity observed in intact toxin.
Abstract: The immunological relationships among the proteins of the mosquito larvicidal toxin produced by Bacillus thuringiensis subsp. israelensis have been investigated by using polyclonal antisera specific for the 28-, 70-, and 135-kilodalton proteins. Each of these proteins was immunologically distinct. There was no cross-reaction among the three proteins and the two non-homologous antisera. Treatment of toxin proteins with larval gut enzymes for 20 h identified protease-resistant domains at approximately 65, 38, and 22 kilodaltons. Similar domains were generated by treatment with trypsin and chymotrypsin. Our immunological and kinetic data indicate that the 28-kilodalton protein is degraded successively to protein bands at 26, 25, 23, and 22 kilodaltons, the 70-kilodalton protein is degraded to a protein at 38 kilodaltons, and the 135-kilodalton protein is degraded successively to protein bands at 94, 72, and, probably, 65 kilodaltons. Solubilized toxin possesses two biological activities, larvicidal and general cytolytic (hemolytic). We used nondenaturing gel electrophoresis to show that the hemolytic activity resides in the 28-kilodalton protein. However, higher-molecular-weight proteins are required to achieve the level of toxicity observed in intact toxin.

Journal ArticleDOI
TL;DR: Crystals from Bacillus thuringiensis var.
Abstract: Crystals from Bacillus thuringiensis var. israelensis appeared to contain three major proteins of Mr 230 000, 130 000 and 28 000. These proteins were solubilized from the crystals by incubation in 10 mM DTT, pH 9.5, and purified by sucrose gradient centrifugation. The Mr 230 000 and 130 000 crystal proteins showed mosquitocidal properties, whereas the Mr 28 000 crystal protein contained haemolytic activity. Immobilization of these proteins on latex beads did not alter these properties. Partial proteolytic degradation showed that the Mr 130 000 and 28 000 proteins are structurally different.

Patent
03 Feb 1986
TL;DR: In this article, recombinant plasmids containing the larvicidal delta-endotoxin gene were constructed by inserting HindIII fragments of the Bacillus thuringiensis var. israelensis 72-75 Md plasmid into the Escherichia coli vector pUC12.
Abstract: Recombinant plasmids containing the larvicidal delta-endotoxin gene were constructed by inserting HindIII fragments of the Bacillus thuringiensis var. israelensis 72-75 Md plasmid into the Escherichia coli vector pUC12. Two recombinants producing a 27-kdal toxin (pIP173 and pIP174) were indentified by screening clones in an E. coli in vitro transcription-translation system. Both recombinants comprised pUC12 and common 9.7 kb HindIII fragment of the B. thuringiensis plasmid. The 27,340 Da polypeptide synthesized in vito from pIP174 transformed into E. coli JM101 and from B. subtilis 168 and spoOJ87 containing the 1.2 kb TaqI fragment from pIP173 was lethal to mosquito larvae.

Journal ArticleDOI
TL;DR: Certain bacteriophage-resistant mutants which showed decreased virulence in pupae of the cecropia moth showed resistance to seven or eight different phages, sensitivity to methicillin, and loss of flagella, while revertants were sensitive to phage resistance, virulent, and resistant to penicillin derivatives.
Abstract: Starting from a crystal-negative parental strain of Bacillus thuringiensis, we isolated certain bacteriophage-resistant mutants which showed decreased virulence in pupae of the cecropia moth (Hyalophora cecropia) These strains (class I mutants) were highly pleiotropic and showed resistance to seven or eight different phages, sensitivity to methicillin, and loss of flagella They were also more sensitive to cecropia immune hemolymph in vitro In addition, the export of at least three proteins was reduced Revertants (class II mutants) were sensitive to phages, virulent, and resistant to penicillin derivatives One class II mutant was a complete revertant in all properties examined The other class II mutant was an incomplete revertant still susceptible to immune hemolymph and with repressed export of proteins Virulence was not coupled to phage resistance as such or to lack of flagella because other mutants affected in these properties were virulent Other factors which could be excluded as causes of virulence were production of extracellular protease and hemolysin