Targeted mutagenesis of loop residues in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin affects larvicidal activity

Loop residues in domain II of Bacillus thuringiensis Cry δ-endotoxins have been demonstrated to be involved in insecticidal specificity. In this study, selected residues in loops β6-β7 (S387SPS390), β8-β9 (S410, N411, T413, T415, E417 and G418) and β10-β11 (D454YNS457) in domain II of the Cry4Ba mosquito-larvicidal protein were changed individually to alanine by PCR-based directed mutagenesis. All mutant toxins were expressed in Escherichia coli JM109 cells as 130-kDa protoxins at levels comparable to the wild type. Only E. coli cells that express the P389A, S410A, E417A, Y455A or N456A mutants exhibited a loss in toxicity against Aedes aegypti mosquito larvae of approximately 30% when compared to the wild type. In addition, E. coli cells expressing double mutants, S410A/E417A or Y455A/N456A, at wild-type levels revealed a significantly higher loss in larvicidal activity of approximately 70%. Similar to the wild-type protoxin, both double mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These results indicate that S410 and E417 in the β8-β9 loop, and Y455 and N456 in the β10-β11 loop are involved in larvicidal activity of the Cry4Ba toxin.