Cytolytic activity and immunological similarity of the Bacillus thuringiensis subsp. israelensis and Bacillus thuringiensis subsp. morrisoni isolate PG-14 toxins.

The parasporal bodies of the mosquitocidal isolates of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 were compared with regard to their hemolytic and cytolytic activities and the immunological relatedness of the 28- and 65-kilodalton (kDa) proteins that occur in both subspecies. The alkali-solubilized parasporal bodies of B. thuringiensis subsp. israelensis caused 50% lysis of human erythrocytes at 1.14 micrograms/ml, whereas those of B. thuringiensis subsp. morrisoni caused similar lysis at 1.84 micrograms/ml. Preincubation of solubilized parasporal bodies with dioleolyl phosphatidylcholine significantly inhibited the hemolytic activity of both supspecies. In cytolytic assays against Aedes albopictus cells, the toxin concentrations causing 50% lysis for B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni were 1.87 and 11.98 micrograms/ml, respectively. Polyclonal antibodies raised separately against the 25-kDa protein (a tryptic digest of the 28-kDa protein) or the 65-kDa protein of B. thuringiensis subsp. israelensis cross-reacted, respectively, with the 28- and the 65-kDa proteins of B. thuringiensis subsp. morrisoni. However, neither of these antibodies cross-reacted with the 135-kDa protein of either subspecies. These results indicate that the mosquitocidal and hemolytic properties of B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 are probably due to the biologically related proteins that are present in the parasporal bodies of both subspecies. The lower hemolytic activity of the B. thuringiensis subsp. morrisoni may be due to the presence of lower levels of the 28-kDa protein in that subspecies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytolytic activity and immunological similarity of the Bacillus thuringiensis subsp. israelensis and Bacillus thuringiensis subsp. morrisoni isolate PG-14 toxins

The parasporal bodies of the mosquitocidal isolates of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 were compared with regard to their hemolytic and cytolytic activities and the immunological relatedness of the 28- and 65-kilodalton (kDa) proteins that occur in both subspecies. The alkali-solubilized parasporal bodies of B. thuringiensis subsp. israelensis caused 50% lysis of human erythrocytes at 1.14 micrograms/ml, whereas those of B. thuringiensis subsp. morrisoni caused similar lysis at 1.84 micrograms/ml. Preincubation of solubilized parasporal bodies with dioleolyl phosphatidylcholine significantly inhibited the hemolytic activity of both supspecies. In cytolytic assays against Aedes albopictus cells, the toxin concentrations causing 50% lysis for B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni were 1.87 and 11.98 micrograms/ml, respectively. Polyclonal antibodies raised separately against the 25-kDa protein (a tryptic digest of the 28-kDa protein) or the 65-kDa protein of B. thuringiensis subsp. israelensis cross-reacted, respectively, with the 28- and the 65-kDa proteins of B. thuringiensis subsp. morrisoni. However, neither of these antibodies cross-reacted with the 135-kDa protein of either subspecies. These results indicate that the mosquitocidal and hemolytic properties of B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 are probably due to the biologically related proteins that are present in the parasporal bodies of both subspecies. The lower hemolytic activity of the B. thuringiensis subsp. morrisoni may be due to the presence of lower levels of the 28-kDa protein in that subspecies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parasporal bodies of Bacillus thuringiensis subsp. morrisoni (PG-14) and Bacillus thuringiensis subsp. israelensis are similar in protein composition and toxicity

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.     

Isolation of Multiple Subspecies of Bacillus thuringiensis from a Population of the European Sunflower Moth, Homoeosoma nebulella

Five subspecies of Bacillus thuringiensis were isolated from dead and diseased larvae obtained from a laboratory colony of the European sunflower moth, Homoeosoma nebulella. The subspecies isolated were B. thuringiensis subspp. thuringiensis (H 1a), kurstaki (H 3a3b3c), aizawai (H 7), morrisoni (H 8a8b), and thompsoni (H 12). Most isolates produced typical bipyramidal crystals, but the B. thuringiensis subsp. thuringiensis isolate produced spherical crystals and the B. thuringiensis subsp. thompsoni isolate produced a pyramidal crystal. Analysis of the parasporal crystals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the crystals from the B. thuringiensis subsp. kurstaki and aizawai isolates contained a protein of 138 kDa whereas those from B. thuringiensis subsp. morrisoni contained a protein of 145 kDa. The crystals from B. thuringiensis subsp. thuringiensis contained proteins of 125, 128, and 138 kDa, whereas those from B. thuringiensis subsp. thompsoni were the most unusual, containing proteins of 37 and 42 kDa. Bioassays of purified crystals conducted against second-instar larvae of H. nebulella showed that the isolates of B. thuringiensis subspp. aizawai, kurstaki, and thuringiensis were the most toxic, with 50% lethal concentrations (LC(inf50)s) of 0.15, 0.17, and 0.26 (mu)g/ml, respectively. The isolates of B. thuringiensis subspp. morrisoni and thompsoni had LC(inf50)s of 2.62 and 37.5 (mu)g/ml, respectively. These results show that a single insect species can simultaneously host and be affected by a variety of subspecies of B. thuringiensis producing different insecticidal proteins.     

Bioassay for homogeneous parasporal crystal of Bacillus thuringiensis using the tobacco hornworm, Manduca sexta.

A method for determining the toxicity of Bacillus thuringiensis subsp. kurstaki parasporal crystal to the tabocco hornworm, Manduca sexta, is described. The use of both mortality and weight loss data have provided a highly sensitive and reproducible bioassay that can be used to compare relative toxicities of crystals from other subspecies as well as toxic components contained therein.     

Enzyme-linked immunosorbent assays for detection and quantitation of the entomocidal parasporal crystalline protein of Bacillus thuringiensis subspp. kurstaki and israelensis.

An enzyme-linked immunosorbent assay was used to detect and quantitate the parasporal crystal toxins of Bacillus thuringiensis subspp. kurstaki and israelensis. The assay method described is extremely sensitive, accurate, and highly specific. With this technique, crystalline insecticidal proteins from several subspecies of B. thuringiensis were compared. The dipteran crystal toxin produced by B. thuringiensis subsp. israelensis was shown to share few epitopes with the lepidopteran toxin from B. thuringiensis subspp. kurstaki, tolworthi, berliner, and alesti.     

Isolation and Identification of novel toxins from a new mosquitocidal isolate from Malaysia, Bacillus thuringiensis subsp. jegathesan.

A new mosquitocidal Bacillus thuringiensis subsp., jegathesan, has recently been isolated from Malaysia. Parasporal crystal inclusions were purified from this strain and bioassayed against fourth-instar larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis. The 50% lethal concentration of crystal inclusions for each species was 0.34, 8.08, 0.34, 17.59, 3.91, and 120 ng/ml, respectively. These values show that parasporal inclusions from this new subspecies have mosquitocidal toxicity comparable to that of inclusions isolated from B. thuringiensis subsp. israelensis. Solubilized and chymotrypsin-activated parasporal inclusions possessed low-level hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the crystals were composed of polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Analysis by Western blotting (immunoblotting) with polyclonal antisera raised against toxins purified from B. thuringiensis subsp. israelensis reveals that proteins in parasporal inclusions of subsp. jegathesan are distinct, because little cross-reactivity was shown. Analysis of the plasmid content of B. thuringiensis subsp. jegathesan indicates that the genes for toxin production may be located on 105- to 120-kb plasmids. Cry- clones that have been cured of these plasmids are nontoxic. Southern blot analysis of plasmid and chromosomal DNA from subsp. jegathesan showed little or low homology to the genes coding for CryIVA, CryIVB, and CryIVD from B. thuringiensis subsp. israelensis.     

A 54-kilodalton protein encoded by pBtoxis is required for parasporal body structural integrity in Bacillus thuringiensis subsp. israelensis

Strains of Bacillus thuringiensis such as B. thuringiensis subsp. israelensis (ONR-60A) and B. thuringiensis subsp. morrisoni (PG-14) pathogenic for mosquito larvae produce a complex parasporal body consisting of several protein endotoxins synthesized during sporulation that form an aggregate of crystalline inclusions bound together by a multilamellar fibrous matrix. Most studies of these strains focus on the molecular biology of the endotoxins, and although it is known that parasporal body structural integrity is important to achieving high toxicity, virtually nothing is known about the matrix that binds the toxin inclusions together. In the present study, we undertook a proteomic analysis of this matrix to identify proteins that potentially mediate assembly and stability of the parasporal body. In addition to fragments of their known major toxins, namely, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa, we identified peptides with 100% identity to regions of Bt152, a protein coded for by pBtoxis of B. thuringiensis subsp. israelensis, the plasmid that encodes all endotoxins of this subspecies. As it is known that the Bt152 gene is expressed in B. thuringiensis subsp. israelensis, we disrupted its function and showed that inactivation destabilized the parasporal body matrix and, concomitantly, inclusion aggregation. Using fluorescence microscopy, we further demonstrate that Bt152 localizes to the parasporal body in both strains, is absent in other structural or soluble components of the cell, including the endospore and cytoplasm, and in ligand blots binds to purified multilamellar fibrous matrix. Together, the data show that Bt152 is essential for stability of the parasporal body of these strains.     

Complete genome sequence of Bacillus thuringiensis serovar finitimus strain YBT-020

Bacillus thuringiensis is a gram-positive, spore-forming bacterium that forms parasporal crystals at the onset of the sporulation phase of its growth. Here, we report the complete genome sequence of B. thuringiensis serovar finitimus strain YBT-020, whose parasporal crystals consist of Cry26Aa and Cry28Aa crystal proteins and are located between the exosporium and the spore coat and remain adhering to the spore after sporulation.     

Toxicity of Bacillus thuringiensis subsp. israelensis to adult Aedes aegypti mosquitoes.

Adult female Aedes aegypti mosquitoes were killed by the parasporal crystals of Bacillus thuringiensis subsp. israelensis (ONR-60A) when the crystals were introduced into the insect midgut as an enema. The 50% lethal dose for intact parasporal crystals was 0.21 microgram/mg of mosquito (wet weight), and for solubilized crystals the 50% lethal dose was 0.04 microgram/mg. These values were compared with 50% lethal concentrations in a free-feeding larval mosquito bioassay of 0.018 and 1.28 microgram/ml for intact and solubilized crystals, respectively. Preparations from B. thuringiensis subsp. kurstaki were ineffective against both adult and larval mosquitoes. An adult mosquito bioassay is suggested as a direct means of screening potential mosquito control agents.     

Toxicity of Bacillus thuringiensis subsp. israelensis to adult Aedes aegypti mosquitoes

Adult female Aedes aegypti mosquitoes were killed by the parasporal crystals of Bacillus thuringiensis subsp. israelensis (ONR-60A) when the crystals were introduced into the insect midgut as an enema. The 50% lethal dose for intact parasporal crystals was 0.21 microgram/mg of mosquito (wet weight), and for solubilized crystals the 50% lethal dose was 0.04 microgram/mg. These values were compared with 50% lethal concentrations in a free-feeding larval mosquito bioassay of 0.018 and 1.28 microgram/ml for intact and solubilized crystals, respectively. Preparations from B. thuringiensis subsp. kurstaki were ineffective against both adult and larval mosquitoes. An adult mosquito bioassay is suggested as a direct means of screening potential mosquito control agents.     

Two different parasporal inclusions are produced by Bacillus thuringiensis subsp. finitimus.

Bacillus thuringiensis subsp. finitimus produced at least two parasporal inclusions. One inclusion was formed within the exosporium and remained with the spore after mother cell lysis. A second inclusion formed somewhat later exterior to the exosporium. Each inclusion contained a major polypeptide of about 135,000 daltons with unique antigenic determinants. This subspecies contained only two plasmids, of 98 and 77 megadaltons (MDa). Strains cured of these plasmids produced only the free inclusion. Since the plasmid-cured strains did not contain DNA sequences homologous to plasmid DNA, the gene for the free-inclusion protein must be encoded in the chromosome. In contrast, the enclosed parasporal inclusion was produced only when the plasmid of 98 MDa was present. In addition, transfer of the 98-MDa plasmid to Bacillus cereus resulted in transcipients that produced small inclusions enclosed within the exosporium, and the protein extracted from these inclusions reacted with antibody specific for enclosed inclusion protein of B. thuringiensis subsp. finitimus. Genes in both the chromosome and a plasmid function in the synthesis of distinct parasporal proteins in this subspecies.     

Antimicrobial activity of different proteins and their fragments from Bacillus thuringiensis parasporal crystals against clostridia and archaea

Proteins of parasporal crystals (Cry proteins) from entomopathogenic bacterium Bacillus thuringiensis (subspecies kurstaki, galleriae, tenebrionis) as well as some fragments of these proteins, obtained by limited proteolysis, are capable of antimicrobial action against anaerobic bacteria and archaea-Clostridium butyricum, Clostridium acetobutylicum and Methanosarcina barkeri. The MICs are 45-150 microg/mL. Electron microscopy showed that lysis of M. barkeri cells in the presence of 49kDa fragment of Cry3Aa toxin is generally similar to the bacterial cell lysis, which has been previously detected in the presence of Cry11A, Cry1Ab and other Cry proteins. The Cry1D-like toxin from crystals of B. thuringiensis subsp. galleriae has been put forward as an example of the supposition that cell wall and some of its components like teichoic acid and N-acetylgalactosamine have possible influence on Cry toxins, enhancing their antimicrobial activity. The possible ecological role of the antimicrobial activity of Cry proteins is also discussed.     

Toxicity of parasporal crystals of Bacillus thuringiensis subsp. israelensis to mosquitoes.

Toxicity of Bacillus thuringiensis subsp. israelensis (ONR-60A/WHO 1897) parasporal crystals to three medically important mosquito larvae is described. The numbers of larvae killed are in relation to crystal dry weight. The crystals are lethally toxic to Aedes aegypti Linnaeus (mean 50% lethal concentration [LC50] = 1.9 x 10(-4) micrograms/ml), Culex pipiens var. quinquefasciatus Say (LC50 = 3.7 x 10(-4) micrograms/ml), and Anopheles albimanus Wiedemann (LC50 = 8.0 x 10(-3) micrograms/ml). Purfied crystals of B. thuringiensis subsp. kurstaki, which are toxic to lepidopteran insects, are ineffective against the mosquito larvae. Likewise, B. thuringiensis subsp. israelensis parasporal crystals are not efficacious for larvae of the lepidopteran, Manduca sexta.     

Serotypes of verocytotoxigenic Escherichia coli isolated from cattle and buffalo calf diarrhoea

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 M _r 68 000 which are not linked by disulfide bridges.     

Toxicity of parasporal crystals of Bacillus thuringiensis subsp. israelensis to mosquitoes.

Toxicity of Bacillus thuringiensis subsp. israelensis (ONR-60A/WHO 1897) parasporal crystals to three medically important mosquito larvae is described. The numbers of larvae killed are in relation to crystal dry weight. The crystals are lethally toxic to Aedes aegypti Linnaeus (mean 50% lethal concentration [LC50] = 1.9 x 10(-4) micrograms/ml), Culex pipiens var. quinquefasciatus Say (LC50 = 3.7 x 10(-4) micrograms/ml), and Anopheles albimanus Wiedemann (LC50 = 8.0 x 10(-3) micrograms/ml). Purfied crystals of B. thuringiensis subsp. kurstaki, which are toxic to lepidopteran insects, are ineffective against the mosquito larvae. Likewise, B. thuringiensis subsp. israelensis parasporal crystals are not efficacious for larvae of the lepidopteran, Manduca sexta.     

苏云金芽胞杆菌CTC菌株的S-层蛋白可以形成伴胞晶体

苏云金芽胞杆菌(Bacillus thuringiensis)CTC菌株产生卵圆形伴胞晶体,晶体蛋白分子量为100kD;透射电子显微镜观察结果表明该菌株有S—层结构,而且在母细胞内可以形成伴胞晶体和S—层的初体结构;其蛋白基因导入苏云金芽胞杆菌无晶体突变株BMB171后,扫描电子显微镜观察结果表明转化子能形成晶体,而其形状与CTC菌株的相同;转化子晶体蛋白的分子量大小也与CTC菌株的相同,为100kD。以上实验结果结合以前晶体蛋白N—末端测序和基因核苦酸序列,表明苏云金芽胞杆菌CTC菌株的S—层蛋白可以形成伴胞晶体。     

Antagonism between Cry1Ac1 and Cyt1A1 toxins of bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC50s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC50s were obtained. All of these LC50s were significantly higher than the expected LC50s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC50 of Cry1Ac1 was 2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm2 of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Detection of chromosomally located and plasmid-borne genes on 20 kb DNA fragments in parasporal crystals from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The association of 20 kb heterologous DNA fragments with the parasporal crystals from native and recombinant Bacillus thuringiensis strains was analyzed, respectively. The cry2Aa10 gene cloned in plasmid pHC39 was transformed into B. thuringiensis subsp. kurstaki strains CryˉB and HD73, producing recombinant strains CryˉB(pHC39) and HD73(pHC39). SDS-PAGE and scanning electron microscopy analyses demonstrated that the recombinant CryˉB(pHC39) produced cuboidal crystals of Cry2Aa10 protoxin, while recombinant HD73(pHC39) produced both bipyramidal crystals of Cry1Ac1 protoxin and cuboidal crystals of Cry2Aa10 protoxin. Bioassay results proved that recombinant HD73(pHC39) showed higher insecticidal activity to Helicoverpa armigera than CryˉB(pHC39). It was found that 20 kb DNA fragments were present in bipyramidal and cuboidal crystals from both native and recombinant strains, and the 20 kb heterologous DNAs contained chromosome-specific and resident large plasmid-borne DNA fragments, suggesting the 20 kb heterologous DNA fragment embodied in crystals came randomly from the bacterial chromosomal and plasmid genome. This was the first investigation devoted exclusively on the origin of 20 kb DNA fragments in the parasporal crystals of B. thuringiensis. The data provides a basis for further investigation of the origin of 20 kb DNAs in the crystals and the interaction of DNA and protoxins.     

活性氧对苏云金芽孢杆菌伴孢晶体的损伤作用

用SDSPAGE电泳分析和生物测定方法研究了过氧化氢(H2O2)和羟自由基(·OH)对苏云金芽孢杆菌(Bacillus thuringiensis)伴孢晶体的损伤作用。结果表明,这两种活性氧对伴孢晶体均有一定程度的损伤作用,这种损伤作用与活性氧的浓度成正相关,并且·OH对伴孢晶体的损伤作用明显强于H2O2。