Single-site mutations in the conserved alternating-arginine region affect ionic channels formed by CryIAa, a Bacillus thuringiensis toxin

Schwartz, Jean-Louis; Potvin, L; Chen, X J; Brousseau, Roland; Laprade, Raynald; Dean, Donald H.

doi:10.1128/aem.63.10.3978-3984.1997

Cited by 62 publications

(25 citation statements)

References 28 publications

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“…A further effect worth considering is membrane potential. Schwartz et al (1997b) report the stimulating effect of a holding potential on insertion of toxin into artificial bilayers. In the present study, all cells were clamped to a holding potential of À40 mV and exposed to a protocol of variable potentials that may have enhanced pore formation.…”

Section: Discussionmentioning

confidence: 99%

Effects of the Bacillus thuringiensis Toxin Cry1Ab on Membrane Currents of Isolated Cells of the Ruminal Epithelium

et al. 2007

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A previous study has shown that Cry1Ab, a lepidopteran-specific toxin derived from Bacillus thuringiensis, does not affect the vitality of cultured cells of the ruminal epithelium of the sheep. While this may be due to lack of specific receptors for toxin action, other mechanisms of resistance should also be considered. In order to directly assess the pore-forming potential of Cry1Ab, we studied the interaction of this toxin with isolated, perfused cells of the ruminal epithelium using the whole-cell and single-channel configurations of the patch-clamp technique. At concentrations found in vivo in the rumen of cows (<10 ng/ml) and at a temperature of 37 degrees C, no significant effects of Cry1Ab could be observed. At 100 ng/ml, exposure of ruminal cells to Cry1Ab induced a significant rise in outward current in 16 of 34 cells, with a fourfold increase in the conductance for potassium. The cell membrane remained selective for potassium over sodium (p(K)/p(Na) = 1.8 + or - 0.3), with a considerable additional chloride conductance. In outside-out patches, exposure to high Cry1Ab concentrations induced channel-like events that reached levels of over 500 pS. We conclude that the unchanged vitality of intact ruminal epithelial cells exposed to Cry1Ab in vitro at high concentrations may be related to other factors besides the proposed absence of a specific receptor for the membrane insertion of this toxin.

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Section: Discussionmentioning

confidence: 99%

Effects of the Bacillus thuringiensis Toxin Cry1Ab on Membrane Currents of Isolated Cells of the Ruminal Epithelium

et al. 2007

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“…Studies with site‐directed mutations in domain II loop residues suggested that the loop regions are important in the initial, reversible binding of BBMV and receptors, as well as in irreversible binding of BBMV (reviewed by Dean et al ., 1996). Domain III, a β‐sheet sandwich with a jelly roll topology, was originally suggested to play a role in toxin stability, but recent data strongly suggest that domain III is important in ion conductivity (Chen et al ., 1993; Schwartz et al ., 1997a), and receptor binding and specificity (Lee et al ., 1995b; 1999; deMaagd et al ., 1996; 1999; Burton et al ., 1999; Jenkins et al ., 2000).…”

Section: Introductionmentioning

confidence: 99%

Role of two arginine residues in domain II, loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensisδ‐endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N

Lee

Rajamohan²,

Jenkins

et al. 2000

Molecular Microbiology

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SummaryTwo arginine residues (368±369) of Cry1Ab and Cry1Ac were mutated to alanine, glutamic acid and lysine by site-directed mutagenesis. Insecticidal activities of the mutant toxins on Manduca sexta and Lymantria dispar larvae were examined. Cry1Ac mutant toxins (c)RR-AA and (c)RR-EE and Cry1Ab mutant toxins (b)RR-AA and (b)RR-EE showed great reductions in toxicity against both insects. In contrast, conservatively changed (c)RR-KK and (b)RR-KK mutants did not alter toxicity to either insect. Binding assays with brush border membrane vesicles (BBMVs) prepared from L. dispar midguts demonstrated that (c)RR-AA, (c)RR-EE, (b)RR-AA and (b)RR-EE bound with lower affinities compared with their respective wild-type toxins. To M. sexta BBMVs, (c)RR-AA and (c)RR-EE showed great reductions in BBMV binding. However, (b)RR-AA and (b)RR-EE did not alter BBMV competition patterns, despite their reduced toxicity. Further binding assays were performed with aminopeptidase N (APN) purified from L. dispar and M. sexta BBMVs using surface plasmon resonance (BIAcore). Direct correlation between toxicity and APN binding was observed for the mutant toxins using this technique. The inconsistency between BBMV and APN binding data with Cry1Ab to M. sexta suggests the possibility of a different Cry1Ab toxin-binding mechanism or the importance of another receptor in M. sexta.

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“…The comparatively simple structure of the Cry3A toxin , which is remarkably similar to Cry1Aa makes it a useful model for exploring the structure and function relationship between ligand and receptor. The three‐dimensional structure of the toxins consists of three functional domains: (i) a cluster of seven K‐helices predicted to be involved in membrane interaction (ii) three antiparallel L‐sheets involved in receptor binding ; and (iii) a L‐sandwich implicated in receptor binding and ion channel activity in related Cry toxins.…”

Section: Discussionmentioning

confidence: 99%

Expression ofcry3Agene and its toxicity against Asian Gray WeevilMyllocerus undecimpustulatus undatusMarshall (Coleoptera: Curculionidae)

Swamy

Asokan

Thimmegowda

et al. 2013

J. Basic Microbiol.

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Coleopterans are the most damaging pests of many agricultural and forestry crops; there is an urgent need to develop effective biopesticides against these insects. Enhancers of Bt toxicity typify an opportunity to improve currently available commercial products into more effective control agents against diverse pests. A 1.9 kb DNA fragment, PCR amplified from native isolates of Bt using cry3A gene specific primers was cloned in expression vector pQE-80L and then used for transformation of Escherichia coli M15 cells. The sequence of the cloned crystal protein gene showed almost complete homology with a Coleopteran active Cry3A toxin gene with 117 mutations scattered in different domain regions encoding a protein of 645 amino acid residues in length, with a predicted molecular mass of 77.4 kDa. Phylogenetic analysis could be compulsive for new/novel Bacillus thuringiensis strains, allowing them to be grouped with related Cry proteins. The toxicity of Bt protein was determined against Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) LC50 152 ng cm(-2). Genes coding for Coleopteran active Cry3A proteins have been isolated and their efficient expression will provide the tools necessary to increase the efficacy of Cry-based biopesticide against economically important beetles.

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Single-site mutations in the conserved alternating-arginine region affect ionic channels formed by CryIAa, a Bacillus thuringiensis toxin

Cited by 62 publications

References 28 publications

Effects of the Bacillus thuringiensis Toxin Cry1Ab on Membrane Currents of Isolated Cells of the Ruminal Epithelium

Effects of the Bacillus thuringiensis Toxin Cry1Ab on Membrane Currents of Isolated Cells of the Ruminal Epithelium

Role of two arginine residues in domain II, loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensisδ‐endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N

Expression ofcry3Agene and its toxicity against Asian Gray WeevilMyllocerus undecimpustulatus undatusMarshall (Coleoptera: Curculionidae)

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