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Bacillus thuringiensis var. israelensis δ-endotoxin. Nucleotide sequence and characterization of the transcripts in Bacillus thuringiensis and Escherichia coli
No full textThe nucleotide sequence of a 1408 base-pair DNA fragment encoding the insecticidal 27,340 Mr δ-endotoxin of Bacillus thuringiensis var. israelensis has been determined by analysis of a recombinant plasmid from Escherichia coli. The hydropathy plot of the protein shows it to be highly hydrophobic, consistent with a postulated cytolytic mechanism of action for the toxin. In addition, the δ-endotoxin transcriptional start points that are used in B. thuringiensis and an E. coli recombinant have been determined. In B. thuringiensis var. israelensis, transcription initiates from a single start point, and gene-specific transcripts are not observed before stage II of sporulation. This is the stage at which δ-endotoxin antigen is first detected, indicating that control of expression is primarily at the transcriptional level for this protein. Analysis of gene-specific transcription in E. coli indicates that at least three start points are utilized in this organism. Interestingly, the highest level of δ-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.</p
Cloning and expression of two homologous genes of Bacillus thuringiensis subsp. israelensis which encode 130-kilodalton mosquitocidal proteins.
No full textTwo homologous genes encoding 130-kilodalton (kDa) mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis have been cloned and expressed in Escherichia coli or Bacillus subtilis or both. One of these genes, pPC130, was expressed as a lacZ transcriptional fusion in E. coli at a level sufficient to produce phase-bright inclusions, which were purified and shown to be toxic to Aedes aegypti larvae. The second gene, pCH130, was expressed at a low level in recombinant E. coli cells and was therefore cloned in B. subtilis as a transcriptional fusion of the promoter sequences corresponding to a B. thuringiensis subsp. israelensis 27-kDa delta-endotoxin (E. S. Ward, A. R. Ridley, D. J. Ellar, and J. A. Todd, J. Mol. Biol. 191:13-22, 1986) and the structural gene. Recombinant B. subtilis cells produced phase-bright inclusions during late sporulation; these were partially purified and shown to be toxic to A. aegypti larvae at an LC50 (concentration required to cause 50% mortality of larvae after 24 h of assay) which is significantly lower than that of the pPC130 protein. Neither 130-kDa protein was hemolytic under the assay conditions. Comparison of the nucleotide sequences of these two genes indicates that they share a high degree of homology in the C-terminal portions, but relatively little similarity in the N termini. In addition, significant homologies were found between the pCH130 gene and the HD-1 Dipel gene of B. thuringiensis subsp. kurstaki (H. E. Schnepf, H. C. Wong, and H. R. Whiteley, J. Biol. Chem. 260:6264-6272, 1985).</p
Cloning and heterologous expression of an insecticidal delta-endotoxin gene from Bacillus thuringiensis var. aizawai ICI toxic to both lepidoptera and diptera
No full textBacillus thuringiensis var. aizawai ICI synthesises an insecticidal protein δ-endotoxin (130-135 kDa) that is toxic to both lepidopteran and dipteran larvae, and cross-reacts immunologically with certain monospecific lepidopteran toxins. A 166-kb plasmid from this bacterium was found to hybridise with an intragenic probe derived from the cloned B. thuringiensis var. sotto lepidopteran-specific δ-endotoxin gene. A strongly hybridising 5.2-kb Sst I fragment from var. aizawai plasmid DNA was cloned in pUC18. After subcloning of this DNA in Escherichia coli, recombinants were obtained that synthesised large amounts of a 130-135-kDa protein. The protein was deposited in the cytoplasm as microscopically visible inclusion bodies and lysates of these cells were found to be toxic to both lepidopteran and dipteran larvae by comparison with controls. The structural basis for the dual specificity of this var. aizawai toxin is now amenable to further study.</p
New hope for the biological control of Musca domestica L
No full textThe housefly, Musca domestica L., is a pest of medical and veterinary importance because of its nuisance value and as a vector of enteric disease microrganisms. During a screening programme designed to isolate new entomopathogenic bacteria showing activity against fly pests, a new strain of Brevibacillus laterosporus (Laubach) was collected in a soil sample from Sardinia (Italy). Morphological and genetic observations, biomolecular analysis and bacterial fractionation were carried out to identify and characterize the new B. laterosporus isolate. Lethal and sub-lethal effects were recorded in laboratory experiments on both adults and juveniles, and spores were identified as the main source of toxicity.The results of experimental treatments in cattle farms with a B. laterosporus are promising and stimulate further experimentation to fully evaluate the potential use of B. laterosporus as a biocontrol agent for housefly control
Single amino acid changes in the Bacillus thuringiensis var. israelensis δ-endotoxin affect the toxicity and expression of the protein
No full textSite-directed mutagenesis has been used to change individual amino acids of the larvicidal 27,000 Mr, δ-endotoxin of Bacillus thuringiensis var. israelensis. Basic and acidic residues have been systematically replaced by alanine, and the resulting mutant polypeptides analysed for cytolytic and larvicidal activity, and binding to phosphatidyl choline liposomes. Replacement of residues at positions 154, 163, 164, 213 and 225 results in proteins which accumulate as inclusions in recombinant Bacillus subtilis cells similar to the wild-type, but have considerably reduced in-vitro and in-vivo toxicity. One mutant (Glu45 to Ala45) results in a protein that has reduced activity in vitro, but retains wild-type larvicidal toxicity. In addition, seven other mutations of charged residues result in proteins which form small or no inclusions in recombinant cells, despite being produced at levels similar to the wild-type in six out of seven cases. In most instances, the toxicity of these aberrantly expressed proteins is considerably less than the wild-type, although one (Lys124 to Ala124) results in a polypeptide with approximately threefold increased activity in vitro. A secondary structural model is proposed to explain these observations.</p
Toxicity of a Brevibacillus laterosporus strain lacking parasporal crystals against Musca domestica and Aedes aegypti
No full textAssignment of the δ-endotoxin gene of Bacillus thuringiensis var. israelensis to a specific plasmid by curing analysis
No full textAbstractPlasmid analysis of Bacillus thuringiensis var. israelensis revealed the presence of at least 9 plasmids. A high frequency of plasmid loss occurred when this organism was grown at elevated temperature (42°C). Analysis of over 100 isolates cured of one or more plasmids by this method revealed that loss of a 72 MDa plasmid was invariably accompanied by loss of the ability to synthesize the insecticidal δ-endotoxin protein. Deletion of any of the other plasmids had no effect on δ-endotoxin production. These results indicate the presence of a plasmid-coded copy of the structural gene for the insecticidal δ-endotoxin in B. thuringiensis var. israelensis
Bacillus thuringiensis var. israelensis δ-endotoxin. Cloning and expression of the toxin in sporogenic and asporogenic strains of Bacillus subtilis
No full textA plasmid-borne gene from Bacillus thuringiensis var. israelensis encoding a 27,340 Mr insecticidal δ-endotoxin has been cloned on a bifunctional multicopy plasmid in a wild-type sporogenic strain and two asporogenic mutants of Bacillus subtilis. The δ-endotoxin gene is expressed at a low level during vegetative growth in all three strains, but the synthesis of the toxin increases markedly during the third hour of stationary phase for both the sporogenic strain and an asporogenic mutant containing the OJ lesion. However, in a stage OA mutant, this increase in δ-endotoxin synthesis is not observed. In both the wild-type sporogenic B. subtilis and the asporogenic OJ strain, phase-bright inclusions, resembling the israelensis crystal in appearance, are visible during late stationary phase. The insoluble inclusions from the B. subtilis transformants, consisting solely of the 27,340 Mr polypeptide, were purified by density gradient centrifugation and found to be extremely toxic to Aedes aegypti larvae. After solubilization in alkaline buffer, this polypeptide was also shown to be haemolytic for human erythrocytes and to lyse Aedes albopictus cells with the same LC50 value as native israelensis δ-endotoxin crystals. During stationary phase, novel mRNA species appear in both the wild-type strain and the OJ mutant, but not in the OA mutant, and these appear to be the major gene-specific transcripts. Transcriptional mapping of δ-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.</p
Cloning and expression in Escherichia coli of the insecticidal δ-endotoxin gene of Bacillus thuringiensis var. israelensis
No full textAbstractRecombinant plasmids containing the mosquitocidal δ-endotoxin gene were constructed by inserting HindIII fragments of the Bacillus thuringiensis var. israelensis 72–75 Md plasmid in to the Escherichia coli vector pUC12. Two recombinants producing the 26000 Da δ-endotoxin (pIP173 and pIP174) were identified by screening clones in an E. coli in vitro transcription-translation system. Both recombinants were 12.4 kb chimaeric plasmids comprising pUC12 and a common 9.7 kb HindIII fragment of the B. thuringiensis plasmid. The 26000 Da polypeptide synthesis in vivo from pIP174 transformed into E. coli JM101 was lethal to mosquito larvae and cytotoxic to mosquito cells in vitro. The biological authenticity of the cloned product was further confirmed by demonstrating that the cytotoxicity of the polypeptide was neutralised by antiserum to the authentic δ-endotoxin or by preincubation with excess toxin receptor. Transcription of the recombinant δ-endotoxin gene in E. coli appears to utilise a Bacillus promotor sequence(s) rather than the pUC12 β-galactosidase promotor
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