Functional mapping of an entomocidal delta-endotoxin. Single amino acid changes produced by site-directed mutagenesis influence toxicity and specificity of the protein.
Mutagenesis has been used to investigate the toxicity and specificity of a larvicidal protein from Bacillus thuringiensis aizawai IC1 that is toxic to both lepidoptera and diptera and differs by only three residues from a monospecific lepidopteran toxin from B. thuringiensis berliner. Site-directed mutagenesis was used to investigate the contribution of these residues to the dual specificity of the aizawai protein. The results suggest that changes in the identity of residues adjacent to Arg544 and Arg567 on the C-terminal side may convert a monospecific toxin into a dual specificity toxin by altering the protease sensitivity of the arginyl peptide bond. A series of deletion mutants was constructed and their protein products analysed for toxicity in vitro and in vivo and for their ability to perturb phospholipid bilayers. The results indicate a different functional role for various protein segments in the toxin's mode of action and suggest that two separate regions close to the C terminus of the active toxin are important in conferring dual specificity on the aizawai IC1 toxin. A model suggesting a basis for the activity of monospecific and dual-specificity B. thuringiensis toxins is presented, which postulates that association of sequences at the C terminus of the active toxin with regions near the N terminus may be responsible for determining toxin specificity.
University of Cambridge, Department of Biochemistry, U.K.
M Z
MZ
Haider
D J
DJ
Ellar
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001413:Bacillus thuringiensis / Q000032:analysis*; D001426:Bacterial Proteins; D001483:Base Sequence; D002872:Chromosome Deletion; D004731:Endotoxins; D005798:Genes, Bacterial; D007306:Insecticides; D008969:Molecular Sequence Data; D009154:Mutation; D015183:Restriction Mapping
D000596:Amino Acids; D001426:Bacterial Proteins; D004731:Endotoxins; D007306:Insecticides