Evidence for intermolecular interaction as a necessary step for pore-formation activity and toxicity of Bacillus thuringiensis Cry1Ab toxin.

Based on the observation of large conductance states formed by Bacillus thuringiensis Cry toxins in synthetic planar lipid bilayers and the estimation of a pore size of 10-20 A, it has been proposed that the pore could be formed by an oligomer containing four to six Cry toxin monomers. However, there is a lack of information regarding the insertion of Cry toxins into the membrane and oligomer formation. Here we provide direct evidence showing that the intermolecular interaction between Cry1Ab toxin monomers is a necessary step for pore formation and toxicity. Two Cry1Ab mutant proteins affected in different steps of their mode of action (F371A in receptor binding and H168F in pore formation) were affected in toxicity against Manduca sexta larvae. Binding analysis showed that F371A protein bound more efficiently to M. sexta brush border membrane vesicles when mixed with H168F in a one to one ratio. These mutant proteins also recovered pore-formation activity, measured with a fluorescent dye with isolated brush border membrane vesicles, and toxicity against M. sexta larvae when mixed, showing that monomers affected in different steps of their mode of action can form functional hetero-oligomers.

Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection.

Mexico is located in a transition zone between the Nearctic and Neotropical biogeographical regions and contains a rich and unique biodiversity. A total of 496 Bacillus thuringiensis strains were isolated from 503 soil samples collected from the five macroregions of the country. The characterization of the strain collection provided useful information on the ecological patterns of distribution of B. thuringiensis and opportunities for the selection of strains to develop novel bioinsecticidal products. The analysis of the strains was based on multiplex PCR with novel general and specific primers that could detect the cry1, cry3, cry5, cry7, cry8, cry9, cry11, cry12, cry13, cry14, cry21, and cyt genes. The proteins belonging to the Cry1 and Cry9 groups are toxic for lepidopteran insects. The Cry3, Cry7, and Cry8 proteins are active against coleopteran insects. The Cry5, Cry12, Cry13, and Cry14 proteins are nematocidal. The Cry11, Cry21, and Cyt proteins are toxic for dipteran insects. Six pairs of general primers are used in this method. Strains for which unique PCR product profiles were obtained with the general primers were further characterized by additional PCRs with specific primers. Strains containing cry1 genes were the most abundant in our collection (49.5%). Thirty-three different cry1-type profiles were identified. B. thuringiensis strains harboring cry3 genes represented 21.5% of the strains, and 7.9% of the strains contained cry11 and cyt genes. cry7, cry8, and cry9 genes were found in 0.6, 2.4, and 2.6% of the strains, respectively. No strains carrying cry5, cry12, cry13, cry14, or cry21 genes were found. Finally, 14% of the strains did not give any PCR product and did not react with any polyclonal antisera. Our results indicate the presence of strains that may harbor potentially novel Cry proteins as well as strains with combinations of less frequently observed cry genes.

Isolation of Cry1Ab protein mutants of Bacillus thuringiensis by a highly efficient PCR site-directed mutagenesis system.

A site-directed mutagenesis method was designed and used to create Cry1Ab mutant proteins in two of the five highly conserved blocks present in the Cry protein family. Region 1 comprises the central alpha-helix 5 of domain I and has been implicated in the pore formation activity of the toxin. Substitution of arginine by serine at position 173 (R173S) affects neither structural integrity nor toxicity. Region 2 comprises the major part of the domain I/domain II interface, characterized by the presence of numerous hydrogen bonds and electrostatic interactions. Mutations in the salt bridge formed by aspartic acid 242 and arginine 265 (D242N, D242C, R265C, and D242C/R265C) resulted in structurally unstable mutant proteins as is shown by their increased protease sensitivity and lack of biological activity.