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PubMed1985

Delta endotoxin of Bacillus thuringiensis subsp. israelensis.

From Bacillus thuringiensis subsp. israelensis, a proteinase-resistant protein was purified which exhibited toxicity to larval mosquitoes and cultured mosquito cells, lysed erythrocytes, and was lethal to mice. To extract the protein, a sporulating culture of B. thuringiensis subsp. israelensis was treated with alkali, neutralized, and incubated with trypsin and proteinase K. It was then purified by gel filtration and DEAE column chromatography. Up to 240 micrograms of toxic protein was purified from 1 g (wet weight) of culture pellet. Two closely related forms of toxic protein were obtained: the 25a and 25b proteins. The two forms comigrated near 25,000 daltons in a sodium dodecyl sulfate-polyacrylamide gel, were serologically related, and showed similar partial protease digestion profiles, but were distinguishable by DEAE chromatography and nondenaturing polyacrylamide gel electrophoresis. Protein sequencing data indicated the 25b protein lacked the two amino acids at the amino terminus of the 25a protein. A Western blot enzyme-linked immunosorbent assay of alkali-solubilized proteins that were not treated with proteases suggested the toxic 25a and 25b proteins were proteolytically derived from a larger molecule of about 28,000 daltons. Alkali-solubilized proteins from an acrystalliferous strain of B. thuringiensis subsp. israelensis and from B. thuringiensis subsp. kurstaki failed to cross-react with antibodies to the 25a protein.

Amino Acid Sequence↗
PubMed1997

Plasmid patterns of Bacillus thuringiensis strains and isolates.

Bacillus thuringiensis produces crystal proteins which are toxic to several orders of economically important insects and other invertebrates. The genes encoding these toxins reside mainly on plasmids. This report consists of a comparative analysis of the plasmid content of a number of B. thuringiensis strains and isolates which may facilitate the search for novel toxin genes and other important products of this organism.

Animals↗
PubMed1977

[Isolation and preliminary characteristics of the extrachromosomal elements of Bacillus thuringiensis DNA].

Centrifugation of Bacillus thuringiensis 351 DNA in CsCl-ethidium bromide density gradient has revealed, besides the main band of chromosomal DNA (p = 1.56 g/cm2), some additional bands with higher density, which usually correspond cicle superhelix DNAs, are revealed. 6 discrete bands are observed under electrophoresis of total fraction of minor DNA bands, which suggests, that the preparation contains cicle DNAs of different size. The treatment with DNAse results in the appearance of 3 bands corresponding to opened cicle DNA forms, their molecular weight being 12-10(6)--4-10(6) daltons. Cicle super-helix and opened cicle DNA forms are found in minor fraction by means of electrone microscopy. Statistical analysis has revealed the presence of at least three types of cicle molecules of different size. The variant 351-10, free of extrachromosomal elements, is isolated after ethidium bromide treatment at high temperature. Possible mechanisms of cicle DNAs formation are discussed. Biological function of DNA extrachromosomes fragments in Bacillus thuringiensis is still obscure.

Bacillus thuringiensis↗
PubMed2006

Cloning, characterization, and expression of a new cry2Ab gene from Bacillus thuringiensis strain 14-1.

Bacillus thuringiensis is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of B. thuringiensis are promising candidates for management of resistance development in insects owing to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. The cry2Ab gene was found to lack a functional promoter and, hence, is cryptic in nature. The cry2Ab7 gene was cloned from a new indigenous B. thuringiensis strain, 14-1. Nucleotide sequencing of the cry2Ab gene cloned from B. thuringiensis strain 14-1 revealed an open reading frame of 1902 bp. The deduced amino acid sequence of Cry2Ab of B. thuringiensis strain 14-1 showed a variation in three amino acid residues in comparison to the holotype sequence, Cry2Ab1. Expression of the newly cloned cry2Ab gene was studied in an acrystalliferous strain of B. thuringiensis (4Q7) by fusing the cry2Ab gene downstream of cry2Aa promoter and orf1 + orf2 sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a spore-crystal mixture obtained from transformants of B. thuringiensis strain 4Q7 showed production of Cry2Ab protein of about 65 kDa. Alkali solubilized Cry2Ab7 protein showed toxicity against Helicoverpa armigera neonates.

Amino Acid Sequence↗
PubMed1999

Introduction and expression of the cry1Ac gene of Bacillus thuringiensis in a cereal-associated bacterium, Bacillus polymyxa.

The abilities of Bacillus polymyxa and Bacillus thuringiensis to survive on the rice phyllospere were compared; it was found that B. polymyxa colonizes the crop better. This study also showed that B. polymyxa inoculation to rice plants increased the shoot and the root growth of the crop. Efforts were made to introduce the cry1Ac gene of B. thuringiensis subsp. kurstaki into B. polymyxa so that the application of such transgenic B. polymyxa strains would prove to be dually beneficial to rice crops both as a biopesticide and as a biofertilizer. Immunoblot analysis of the recombinant organism containing the cry1Ac gene, strain BP113, indicated efficient expression of this gene in the heterologous host. Bioassays with the first instar larvae of the yellow stem borer of rice (Scirpophaga incertulas) revealed that the protein preparations from BP113 were toxic.

Animals↗
PubMed1999

Updating the H-antigen classification of Bacillus thuringiensis.

The classification of Bacillus thuringiensis strains has been revised and updated based on flagellar antigens which have been in use for many years. Sixty-nine serotypes and 13 sub-antigenic groups have now been identified, giving 82 serovars among the 3500 B. thuringiensis isolates of the IEBC Collection. The number of serovars has gradually increased with the total number of strains. The biochemical characters used have also been investigated and their value assessed for identification of B. thuringiensis at the subspecies level. A crystal analysis was carried out in terms of morphology, delta-endotoxin profiles and larvicidal activity for the newly identified serovars. It was found that atypical crystals, some with novel components, are becoming more common. No insect susceptible to these serovars has been discovered among known target species. The number of cross-reacting H-antigens among B. cereus strains is increasing and may be of biological significance.

Antigens, Bacterial↗
PubMed2000

Incorporation of protease K into larval insect membrane vesicles does not result in disruption of integrity or function of the pore-forming Bacillus thuringiensis delta-endotoxin.

Bacillus thuringiensis delta-endotoxins insert into the brush border membranes of insect larval cells to form ion channels. A possible interaction of these toxins with a cytoplasmic component was examined by preloading vesicles from insect larval cells with protease K followed by incubation with toxin. There was no evidence for toxin antigens smaller than the intact toxin in extracts of solubilized vesicles, nor was there an effect of the inclusion of protease K on either of two functional properties, the formation of toxin aggregates or of ion pores. These toxins, physically and functionally, appear to be confined to the membrane.

Animals↗
PubMed2001

The role of a proline-induced broken-helix motif in alpha-helix 2 of Bacillus thuringiensis delta-endotoxins.

Bacillus thuringiensis delta-endotoxins (Cry proteins), are widely used for insect control and plant protection. They are water-soluble proteins that insert into membranes forming ion channels. In most Cry toxins alpha-helix 2 is broken by a highly conserved proline residue (Pro70 in Cry1Ab), generating a broken-helix motif. The flexibility of the motif was altered through site-directed mutagenesis. It was found that increasing the flexibility of the motif decreased the stability, the ion transport ability and the toxicity of the protein. By removing the broken-helix motif, the biological properties were restored to a wild type level.

Alanine↗
PubMed2003

Characterization of cry1, cry2, and cry9 genes in Bacillus thuringiensis isolates from China.

Bacillus thuringiensis isolates from different ecological regions and sources of China were analyzed to study the distribution and diversity of cry genes and to detect the presence of novel cry genes. Strains containing cry1-type genes were the most abundant and represent 237 of the 310 B. thuringiensis isolates (76.5%). About 70 and 15.5% of the isolates contained a cry2 gene or cry9 gene, respectively, while 10.0% of the strains did not contain a cry1, cry2, or cry9 gene. Among the cry1 containing isolates, cry1A (67.7%), cry1I (60.6%), cry1C (43.9%), and cry1D (39.4%) genes were the most abundant. Forty-three different cry1 gene profiles were detected in this collection. Several cry1 genes were associated at a high frequency, such as the cry1C-cry1D and cry1A-cry1I gene combination. The cry1A and cry2 amplicons were digested with selected restriction enzymes to examine sequence diversity. Based on this RFLP analysis, one novel cry1A-type gene was observed.

Bacillus thuringiensis↗
PubMed2006

Transcriptional analysis of the toxin-coding plasmid pBtoxis from Bacillus thuringiensis subsp. israelensis.

In Bacillus thuringiensis subsp. israelensis all of the insecticidal toxins are encoded on a single, large plasmid, pBtoxis. Sequencing of this plasmid revealed 125 potential coding sequences, many of which have predicted functions in gene regulation and physiological processes, such as germination. As a first step in understanding the possible role of pBtoxis in its host bacterium, a survey of the transcription of genes with predicted functions was carried out. Whereas many coding sequences, including those previously identified as probable pseudogenes, were not transcribed, mRNA was detected for 29 of the 40 sequences surveyed. Several of these sequences, including eight with similarities to the sequences of known transcriptional regulators, may influence wider gene regulation and thus may alter the phenotype of the host bacterium.

Bacillus thuringiensis↗
PubMed2002

[Characterization of cry gene and broad spectrum against lepidopteran of Bacillus thuringiensis subsp. colmeri 15A3].

Bacillus thuringiensis wild type strain 15A3 belongs to subspecies colmeri serotype H-21. RFLP and PCR analysis show that it contains six types of ICP genes: cry1Aa, cry1Ac, cry1Ca, cry1D, cry1I and cry. The sequence of the 1.45 kb N-terminal fragment of cry1Aa differed from that of published. SDS-PAGE showed that the crystal consists of proteins with molecular weight about 130, 79, 70, 65, 51 and 45 kD. Strain 15A3 didn't sysnthesize heat-stable beta-exotoxins according to test of house fly aberration. The 1.2 tons fermentative production exhibited high toxicity against three lepidopteran pests: H. armigera, S. exigua and H. cunea. It was proved that wild type strain can produce a broad specturm of ICP.

Animals↗
PubMed2006

Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produced by Bacillus thuringiensis.

Cry toxins from Bacillus thuringiensis are used for insect control. Their primary action is to lyse midgut epithelial cells. In this review we will summarize recent findings on the Cry toxin-receptor interaction and the role of receptor recognition in their mode of action. Cry toxins interact sequentially with multiple receptors. In lepidopteran insects, Cry1A monomeric toxins interact with the first receptor and this interaction triggers oligomerization of the toxins. The oligomer then interacts with second receptor inducing insertion into membrane microdomains and larval death. In the case of mosquitocidal toxins, Cry and Cyt toxins play a part. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin resistance. Recently, it was proposed that Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a membrane-bound receptor for Cry toxin.

Bacillus thuringiensis↗
PubMed2006

Conjugal transfer of a toxin-coding megaplasmid from Bacillus thuringiensis subsp. israelensis to mosquitocidal strains of Bacillus sphaericus.

Both Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis produce mosquitocidal toxins during sporulation and are extensively used in the field for control of mosquito populations. All the known toxins of the latter organism are known to be encoded on a large plasmid, pBtoxis. In an attempt to combine the best properties of the two bacteria, an erythromycin resistance-marked pBtoxis plasmid was transferred to B. sphaericus by a mating technique. The resulting transconjugant bacteria were significantly more toxic to Aedes aegypti mosquitoes and were able to overcome resistance to B. sphaericus in a resistant colony of Culex quinquefasciatus, apparently due to the production of Cry11A but not Cry4A or Cry4B. The stability of the plasmid in the B. sphaericus host was moderate during vegetative growth, but segregational instability was observed, which led to substantial rates of plasmid loss during sporulation.

Aedes↗
PubMed2000

Domain I plays an important role in the crystallization of Cry3A in Bacillus thuringiensis.

The insecticidal bacterium Bacillus thuringiensis synthesizes endotoxin Cry proteins of two size classes, 135 and 70 kDa, and both form crystalline inclusions in cells after synthesis. Crystallization of 135-kDa proteins is due to intermolecular attraction of regions in the C-terminal half of the molecule, and the N-terminal half fails to crystallize when synthesized in vivo. Alternatively, endotoxins of the 70-kDa class such as Cry2A and Cry3A, which correspond to the N-terminal half of 135-kDa molecules, crystallize readily after synthesis. Cry molecules of this size class consist of three principal domains, but the domains responsible for crystallization are not known. To identify these domains, chimeric proteins were constructed in which Cry3A Domains I or III, or I and III were substituted for the corresponding domains in truncated Cry1C molecules. Cry1C molecules with only Cry3A Domain III did not crystallize, whereas when Cry3A Domains I and III, or Domain I alone, were substituted, large inclusions were obtained. Except for the chimera consisting of Cry3A Domains I and III and Cry1C Domain II, most chimeras were not as stable as wild-type Cry3A or truncated Cry1C. These results show that Cry3A Domain I plays an important role in its crystallization in vivo.

Animals↗
PubMed2004

Analysis of glycan structures on the 120 kDa aminopeptidase N of Manduca sexta and their interactions with Bacillus thuringiensis Cry1Ac toxin.

The Bacillus thuringiensis Cry1Ac toxin specifically binds to a 120 kDa aminopeptidase N (APN) receptor in Manduca sexta. The binding interaction is mediated by GalNAc, presumably covalently attached to the APN as part of an undefined glycan structure. Here we detail a simple, rapid and specific chemical deglycosylation technique, applicable to glycoproteins immobilized on Western blots. We used the technique to directly and unambiguously demonstrate that carbohydrates attached to 120 kDA APN are in fact binding epitopes for Cry1Ac toxin. This technique is generally applicable to all putative Cry toxin/receptor combinations. We analyzed the various glycans on the 120 kDA APN using carbohydrate compositional analysis and lectin binding. The data indicate that in the average APN molecule, 2 of 4 possible N-glycosylation sites are occupied with fucosylated paucimannose [Man(2-3)(Fuc(1-2)GlcNAc(2)-peptide] type N-glycans. Additionally, we identified 13 probable O-glycosylation sites, 10 of which are located in the Thr/Pro rich C-terminal "stalk" region of the protein. It is likely that 5-6 of the 13 sites are occupied, probably with simple [GalNAc-peptide] type O-glycans. This O-glycosylated C-terminal stalk, being GalNAc-rich, is the most likely binding site for Cry1Ac.

Acetylgalactosamine↗
PubMed2003

Efficacy and efficiency of new Bacillus thuringiensis var israelensis and Bacillus sphaericus formulations against Afrotropical anophelines in Western Kenya.

We evaluated the efficacy of new water-dispersible granular (WDG) formulations of Bacillus thuringienis var. israelensis (Bti; VectoBac) and B. sphaericus (Bs; VectoLex), Valent BioScience Corp., Illinois, USA) for the control of larval Anopheles gambiae sensu lato Giles mosquitoes in a malaria-endemic area around Lake Victoria, Western Kenya. WDG and powder formulations were compared in laboratory bioassays and followed by efficiency and residual effect assessments of both WDG formulations in open field experiments. LC50 and LC95 values for the Bti/Bs strains and their formulations show high susceptibility of A. gambiae sensu stricto under laboratory conditions. The larvae proved more susceptible to Bs than to Bti and the WDG formulations were slightly superior to the powder formulations. High efficiency was also shown in the open field trials, and a minimum dosage of 200 g/ha Bti WDG, representing the LC95 of the laboratory tests, was sufficient to fully suppress emergence of mosquitoes when applied at weekly intervals. Bti WDG did not show a residual effect, irrespective of the concentration applied. The Bs WDG formulation, however, showed significant larval reductions up to 11 days post-treatment at application doses of either 1 or 5 kg/ha. We conclude that the main malaria vector in our study area is highly susceptible to these microbial control agents. Minimum effective dosages to achieve elimination of the larval population in a given habitat are extremely low and environmental impact is negligible. Microbial products for larval control have therefore great potential within Integrated Vector Management programmes and may augment control efforts against adult vector stages, such as the use of insecticide-treated bednets, in many parts of Africa.

Animals↗
PubMedDate not supplied

[The duration of action of Bacillus thuringiensis spp. israelensis and Bacillus sphaericus after encapsulation by infusoria Tetrahymena pyriformis].

The study of the duration of the insecticidal activity of a mixture of infusoria with each of these bacteria revealed differences in the pattern of their relations during their long contact. T. pyriformis may be used for the prolongation of its insecticidal action if Bti is applied. An increase in the duration of this action depends on the viability of tetrahymena in the experimental flasks and on the presence of free spores and crystals of Bti. The prolongation of the action may be associated with the multiplication of this type of bacteria in the digestive vacuoles of infusoria. The use of Bsph in the mixture with T. pyriformis caused no increase in the insecticidal activity of bacteria. Tetrahymena increased the duration of Bsph and even reduced the period of its larvicidal activity. Tetrahymena used a sporocrystalline complex of Bsph as food and reduced its water concentration.

Animals↗