Mutations of loop 2 and loop 3 residues in domain II of Bacillus thuringiensis Cry1C δ-endotoxin affect insecticidal specificity and initial binding to Spodoptera littoralis and Aedes aegypti midgut membranes

Site-directed mutagenesis was used to examine the role of predicted loops 2 ((374)QPWP(377)),and 3 ((436)QRSGTPF(442)) in, domain II of the Bacillus thuringiensis Cry1C delta endotoxin for insecticidal specificity and receptor binding. Q(374)E, (SF)-F-438,,d G(439)A substitutions resulted in near or complete loss of toxicity toward both Spodoptera littoralis and Aedes aegypti. (RK)-K-437, (RI)-I-437. and G(439)V mutants exhibited significantly reduced toxicity to S. littoralis and A. aegypti, while mutations of T-440, P-441 and F-442 showed only slight reductions in toxicity to both insects. Loop 2 mutations Q(374)N, p(375)A, (WY)-Y-376 and p(377)A did not significantly affect S. littoralis toxicity but exhibited reduced activity to A. aegypti. In contrast, the loop 3 mutations Q(436)K, Q(436)E, and (SY)-Y-438 had no effect on A. aegypti toxicity, but showed significantly decreased S. littoralis activity Heterologous competition binding assays with brush border membrane vesicles (BBMV) from both insects correlated well with the toxicity data with the exception of the R-437 mutants, where steps other than initial receptor binding appear to be involved. Overall we conclude that, while loops 2 and 3 play an important role in binding and toxicity to both insects, loop 2 appears to play the greater role in A. aegypti activity, while loop 3 is more important For S. littoralis toxicity.