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 Bacillus 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, revealed that most of the substitutions made on both peptides led to either decrease 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 binding 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-terminal region ((312)R) of the 42-kDa peptide completely abolished the biological 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 (312)R for biological activity against mosquito larvae.
Elangovan
G
G
Centre for Biotechnology, Anna University, Chennai, India.
Shanmugavelu
M
M
Rajamohan
F
F
Dean
D H
DH
Jayaraman
K
K
D000818:Animals; D001407:Bacillus / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D001667:Binding, Competitive; D001681:Biological Assay; D003465:Culex / Q000187:drug effects*; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D005816:Genetic Complementation Test; D007814:Larva / Q000166:cytology / Q000187:drug effects*; D008871:Microvilli / Q000378:metabolism; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D013816:Thermodynamics
D001427:Bacterial Toxins; D010446:Peptide Fragments