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Toxicity, binding, and permeability analyses of four Bacillus thuringiensis Cry1 delta-endotoxins using brush border membrane vesicles of Spodoptera exigua and Spodoptera frugiperda.

ABSTRACT: The binding and pore formation properties of four Bacillus thuringiensis Cry1 toxins were analyzed by using brush border membrane vesicles from Spodoptera exigua and Spodoptera frugiperda, and the results were compared to the results of toxicity bioassays. Cry1Fa was highly toxic and Cry1Ac was nontoxic to S. exigua and S. frugiperda larvae, while Cry1Ca was highly toxic to S. exigua and weakly toxic to S. frugiperda. In contrast, Cry1Bb was active against S. frugiperda but only marginally active against S. exigua. Bioassays performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed that the effects of iodination on toxin activity were different. The toxicities of I-labeled Cry1Bb and Cry1Fa against Spodoptera species were significantly less than the toxicities of the unlabeled toxins, while Cry1Ca retained its insecticidal activity when it was labeled with 125I. Binding assays showed that iodination prevented Cry1Fa from binding to Spodoptera brush border membrane vesicles. 125I-labeled Cry1Ac, Cry1Bb, and Cry1Ca bound with high-affinities to brush border membrane vesicles from S. exigua and S. frugiperda. Competition binding experiments performed with heterologous toxins revealed two major binding sites. Cry1Ac and Cry1Fa have a common binding site, and Cry1Bb, Cry1C, and Cry1Fa have a second common binding site. No obvious relationship between dissociation of bound toxins from brush border membrane vesicles and toxicity was detected. Cry1 toxins were also tested for the ability to alter the permeability of membrane vesicles, as measured by a light scattering assay. Cry1 proteins toxic to Spodoptera larvae permeabilized brush border membrane vesicles, but the extent of permeabilization did not necessarily correlate with in vivo toxicity.

SUBMITTER: Luo K

PROVIDER: S-EPMC91047 | biostudies-literature | 1999 Feb

REPOSITORIES: biostudies-literature

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Toxicity, binding, and permeability analyses of four Bacillus thuringiensis Cry1 delta-endotoxins using brush border membrane vesicles of Spodoptera exigua and Spodoptera frugiperda.

Luo K K Banks D D Adang M J MJ

Applied and environmental microbiology 19990201 2

The binding and pore formation properties of four Bacillus thuringiensis Cry1 toxins were analyzed by using brush border membrane vesicles from Spodoptera exigua and Spodoptera frugiperda, and the results were compared to the results of toxicity bioassays. Cry1Fa was highly toxic and Cry1Ac was nontoxic to S. exigua and S. frugiperda larvae, while Cry1Ca was highly toxic to S. exigua and weakly toxic to S. frugiperda. In contrast, Cry1Bb was active against S. frugiperda but only marginally activ ...[more]

PMID: 9925568

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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

Toxicity, binding, and permeability analyses of four Bacillus thuringiensis Cry1 delta-endotoxins using brush border membrane vesicles of Spodoptera exigua and Spodoptera frugiperda.

Publications

Toxicity, binding, and permeability analyses of four Bacillus thuringiensis Cry1 delta-endotoxins using brush border membrane vesicles of Spodoptera exigua and Spodoptera frugiperda.

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