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Cloning and expression of the first anaerobic toxin gene from Clostridium bifermentans subsp. malaysia, encoding a new mosquitocidal protein with homologies to Bacillus thuringiensis delta-endotoxins.

ABSTRACT: A gene (cbm71) encoding a 71,128-Da mosquitocidal protein (Cbm71) was obtained by screening a size-fractionated XbaI digest of total genomic DNA from Clostridium bifermentans subsp. malaysia CH18 with two gene-specific oligonucleotide probes. The sequence of the Cbm71 protein, as deduced from the sequence of cbm71, corresponds to that of the 66-kDa protein previously described as one of the mosquitocidal components of C. bifermentans subsp. malaysia. Cbm71 shows limited similarities with Bacillus thuringiensis delta-endotoxins, especially in the four first conserved blocks. However, Cbm71 was not immunologically related to any of the Cry toxins and thus belongs to a novel class of mosquitocidal protein. The cbm71 gene was expressed in a nontoxic strain of B. thuringiensis, and Cbm71 was produced during sporulation and secreted to the supernatant of culture. Trichloroacetic-precipitated supernatant preparations were toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi.

SUBMITTER: Barloy F

PROVIDER: S-EPMC178058 | biostudies-other | 1996 Jun

REPOSITORIES: biostudies-other

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Cloning and expression of the first anaerobic toxin gene from Clostridium bifermentans subsp. malaysia, encoding a new mosquitocidal protein with homologies to Bacillus thuringiensis delta-endotoxins.

Barloy F F Delécluse A A Nicolas L L Lecadet M M MM

Journal of bacteriology 19960601 11

A gene (cbm71) encoding a 71,128-Da mosquitocidal protein (Cbm71) was obtained by screening a size-fractionated XbaI digest of total genomic DNA from Clostridium bifermentans subsp. malaysia CH18 with two gene-specific oligonucleotide probes. The sequence of the Cbm71 protein, as deduced from the sequence of cbm71, corresponds to that of the 66-kDa protein previously described as one of the mosquitocidal components of C. bifermentans subsp. malaysia. Cbm71 shows limited similarities with Bacillu ...[more]

PMID: 8655486

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Project description:During sporulation, many Bacillus thuringiensis subspecies synthesize several related delta-endotoxins which are packaged into bipyramidal intracellular inclusions. These inclusions are solubilized in the alkaline, reducing conditions of the midguts of susceptible insect larvae and are converted by proteolysis to active toxins. The toxins insert into the membranes of cells lining the midgut and form cation-selective channels, which results in lethality. There are three delta-endotoxins, Cry1Ab3, Cry1Ca1, and Cry1Da1, present in the inclusions produced by a B. thuringiensis subsp. aizawai cell. While the ratio of the steady-state mRNAs for these three protoxins has been shown to differ (cry1Ab3/cry1Ca1/cry1Da1 mRNA ratio, 4:2:1), the half-lives of the cry1Da1 and cry1Ab3 mRNAs were found to be similar, indicating that there were differences in the transcription rates. The relative contents of these delta-endotoxins in purified inclusions from B. thuringiensis subsp. aizawai have been measured previously, and an even greater relative deficiency of the Cry1Da1 protoxin (ratio, 20:12:1) was found. In order to account for this deficiency, other steps which could be involved in inclusion formation, such as translation and packaging, were examined. The three cry genes have the same dual overlapping promoters, but the ribosome binding sequence for the cry1Da1 gene was not the consensus sequence. Translation was enhanced about fourfold by changing to the consensus sequence. In addition, the relative amount of Cry1Da1 protoxin in inclusions was twofold lower when cells were sporulated in Luria-Bertani (LB) medium than when cells were sporulated in a glucose-yeast extract medium. This difference was attributable to packaging since the relative amounts of Cry1Da1 antigen in cells sporulating in the two media were the same. Some factor(s) required for packaging of the Cry1Da1 protoxin in inclusions is apparently limiting in LB medium. Differences in the initial transcription rates, translation efficiencies, and packaging all contribute to the delta-endotoxin composition of an inclusion.

Dataset Information

Cloning and expression of the first anaerobic toxin gene from Clostridium bifermentans subsp. malaysia, encoding a new mosquitocidal protein with homologies to Bacillus thuringiensis delta-endotoxins.

Publications

Cloning and expression of the first anaerobic toxin gene from Clostridium bifermentans subsp. malaysia, encoding a new mosquitocidal protein with homologies to Bacillus thuringiensis delta-endotoxins.

Similar Datasets

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