Karim S, Dean D H
Department of Biochemistry, Ohio State University, Columbus, OH 43210, USA.
Curr Microbiol. 2000 Dec;41(6):430-40. doi: 10.1007/s002840010163.
Bacillus thuringiensis produces several larvicidal crystalline inclusions during sporulation. An understanding of their mechanisms of action is commercially important. In this study, two toxins, Cry1Ab and Cry1Ac, were compared that showed 98% amino acid identity in domain I and II, but differed significantly in domain III. Using site-directed mutagenesis techniques, two conserved loop 2 Arg's ((368)RR(369)) of Cry1Ab and Cry1Ac toxins were replaced with Ala ((368)AR(369), (368)RA(369), (368)AA(369)), Glu ((368)EE(369)), Phe ((368)FF(369)), His ((368)HH(369)), and Lys ((368)KK(369)). The effect of these mutants on structural stability, larvicidal potency, receptor binding, and ionic permeability towards two important cotton pests, pink bollworm (Pectinophora gossypiella) and bollworm (Helicoverpa zea) were analyzed. All seven mutants of Cry1Ab, excluding (368)AR(369), produced a stable protoxin, whereas for Cry1Ac all seven mutants yielded stable protoxin. Results showed that all the stable mutants behaved similarly to the wild type on incubation with trypsin and gut extract of both insect larvae. The Cry1Ab mutants, (368)AR(369), (368)AA(369), (368)FF(369), and (368)HH(369), lost toxicity; (368)EE(369) had reduced toxicity; whereas the more conserved change (368)KK(369) retained the toxicity similar to the wild type towards P. gossypiella. Double mutants of Cry1Ac, (368)AA(369) and (368)FF(369), abolished the toxicity. Double mutant (368)KK(369) of Cry1Ac retained its toxicity against P. gossypiella, whereas single mutants (368)AR(369), (368)RA(369), and (368)HH(369) retained only reduced toxicity. All the mutants of Cry1Ab lost their toxicity against H. zea except (368)KK(369). In Cry1Ac single mutants, (368)AR(369) and (368)RA(369), reduction in the toxicity was observed. A double mutant of Cry1Ac, (368)KK(369), also retained reduced toxicity. All the other double mutants lost their toxicity. Voltage clamping experiments on H. zea midguts provided an additional evidence about the insecticidal property and inhibition of I(sc) across the transepithelial membrane of the insect midgut.
