38th Annual Meeting of the Society for Invertebrate Pathology

The detailed information of the highly conserved helix 7 in the pore-forming domain of the Bacillus thuringiensis Cry δ-endotoxins remains to be investigated. In the present study, alanine substitutions of three conserved aromatic residues in α7 (Phe-246, Tyr-249 and Phe-264) of the Cry4Ba mosquito-larvicidal protein were performed via PCR-based mutagenesis. All the mutant toxins were highly expressed in Escherichia coli as 130-kDa protoxins at levels comparable to the wild-type toxin. Bioassays against Aedes aegypti mosquito larvae revealed that E. coli cells expressing Y249A or F264A mutant toxins displayed a dramatic decrease in toxicity, but not for the F246A mutant. Further mutagenic analysis showed that only replacements with an aromatic residue, Y249F or Y249W and F264Y or F264W, still retained the high level in toxicity, while substitutions with Glu, Arg or His almost completely abolished larvicidal activity. These results suggested that aromatic side-chains of these two critical residues, Tyr-249 and Phe-264, within helix 7 of the Cry4Ba toxin play an important role in larvicidal activity.