Analyses of the Pore Forming Ability of Bacillus thuringiensis Cry1A Mutant Toxins Using a LightScattering Technique

The pore formation properties of Bacillus thuringiensis Cry1 wild type and domain I and II mutant toxins were studied on Manduca sexta brush border membrane vesicles (BBMV) using a light-scattering technique. Wild type Cry1Ac, Cry1Ab, and Cry1Ba; Cry1Ab mutant toxins A92E, Y153D, R368A/R369A, and F371A; and Cry1Ac mutant toxin A92D were analyzed. In a direct mixing assay the mutant toxins Y153D, R368A/R369A, F371A, and Cry1Ba did not elicit a response in a 1-min signalmonitoring period. Mutant toxins A92D and A92E elicited slight responses. After preincubation of toxin with BBMV, the signal recovery response increased for all toxins. The signal recoveries caused by A92D and A92E were greater than Y153D-, F371A-, and R368A/R369A-induced signal recoveries, which were slightly greater than Cry1Bainduced recoveries. By increasing the monitoring period to 3 min in direct mixing experiments, we observed greater pore formation by A92D. A92E had a response similar to, but lower than that of A92D. The response induced by both wild type and mutant toxins decreased when the hyperosmotic solution was changed from KCl to sucrose. However, in the presence of sucrose the responses induced by A92D and A92E were substantially reduced relative to KCl.