Identification of the functional site in the mosquito larvicidal binary toxin of Bacillus sphaericus 1593M by site-directed mutagenesis

To study the mode of action of the binary toxin (51- and 42-kDa) of Bacillu s sphaericus, amino acid residues were substituted at selected sites of the N- and C-terminal regions of both peptides. Bioassay results of the mutant binary toxins tested against mosquito larvae, Culex quinquefasciatus, reve aled that most of the substitutions made on both peptides led to either dec rease or total loss of the activity. Furthermore, receptor binding studies carried out for some of the mutants of the 42-kDa peptide showed mutations in N- and C-terminal regions of the 42-kDa peptide did not affect the bindi ng of the binary toxin to brush border membrane vesicles of mosquito larvae . One of the mutants having a single amino acid substitution at the C-termi nal region (R-312) Of the 42-kDa peptide completely abolished the biologica l activity, implicating the role of this residue in membrane pore formation . These results indicate the importance of the C-terminal region of the 42- kDa of binary toxin, in general, and particularly the residue R-312 for bio logical activity against mosquito larvae. (C) 2000 Academic Press.