Targeted mutagenesis at charged residues in Bacillus sphaericus BinA toxin affects mosquito-larvicidal activity.

The mosquito-larvicidal binary toxin from Bacillus sphaericus is composed of two polypeptides called BinA and BinB with molecular masses of approximately 42 and 51 kDa. Both components are required for full activity, with BinB acting as a specificity determinant and BinA being responsible for toxic action. To investigate the role of the selected charged residues in BinA, four mutants were generated by replacing charged amino acids with alanine (R97A, E98A, R101A, and E114A). All mutant proteins were produced at high levels and formed inclusion bodies similar to that of the wild type. Mosquito-larvicidal assays against Culex quinquefasciatus larvae revealed that the mutant R97A completely lost its activity and mutants E98A, R101A, and E114A showed significantly reduced toxicity. Intrinsic fluorescence spectroscopy analysis indicated that alanine substitutions at these positions did not alter the overall structure of the toxin. Binding of the mutants to BinB was not different from that of the wild type, suggesting that these mutations did not affect BinA-BinB interaction. Results demonstrated that R97, E98, R101, and E114 neither play a direct role in maintenance of BinA structure nor are involved in BinA-BinB interaction. Since these residues are required for full activity, they may play an important role during toxin internalization and/or toxic action of BinA inside the target cells.