EFFECTS ON LARVICIDAL ACTIVITY OF SINGLE PROLINE SUBSTITUTIONS IN ALPHA-3 OR ALPHA-4 OF THE BACILLUS-THURINGIENSIS CRY4B TOXIN

The possible role of alpha-helices 3 and 4 in toxicity of the dipteran -active Bacillus thuringiensis Cry4B delta-endotoxin was investigated by employing proline substitutions via site-directed mutagenesis. Simi lar to the wild-type Cry4B, the mutant toxins were over-expressed in E scherichia coli as cytoplasmic inclusions and were structurally stable upon solubilization and trypsin activation. The substitution of gluta mine 149 by proline in the center of helix 4 (Q149P) resulted in a nea rly complete loss of toxicity against Aedes aegypti mosquito-larvae. H owever, single proline replacements near the center of helix 3 (V119P) and at the N-terminus of helix 4 (Q140P) did not decrease larvicidal activity. The toxicity of E. coli cells expressing the wild-type toxin was significantly reduced by two-hour preincubation with the non-toxi c mutant (Q149P), thus indicating that the primary binding step was no t affected by the proline substitution in helix 4. The results therefo re reveal a crucial role for helix 4 of the Cry4B toxin in toxicity, p ossibly in membrane insertion and pore formation rather than in recept or recognition.