38th Annual Meeting of the Society for Invertebrate Pathology

The proposed toxicity mechanism of the Bacillus thuringiensis Cry δ-endotoxins involves the penetration of α-helices 4 and 5 to form lytic pores in the target cell membrane. In this study, alanine substitutions of selected polar residues (Tyr-178, Gln-180, Asn-183, Asn-185 and Asn-195) in the relatively hydrophobic transmembrane α5 of the Cry4Ba mosquito-larvicidal protein were initially conducted via PCR-based directed mutagenesis. Upon IPTG induction, all mutant proteins were highly expressed in Escherichia coli as cytoplasmic inclusions, with yields similar to the wild-type protoxin. When E. coli cells expressing each mutant toxin were tested against Aedes aegypti mosquito larvae, the larvicidal activity was almost completely abolished for the N183A mutation, whereas the other four mutant toxins showed only a small reduction in toxicity. Further replacements of this critical residue with various amino acids revealed that the polarity at position 183 of the transmembrane α5 play a crucial role in larvicidal activity of the Cry4Ba toxin.