Production of multiple δ-endotoxins by Bacillus thuringiensis: δ-endotoxins produced by strains of the subspecies galleriae and wuhanensis

Abstract

A method was developed to assess the number of δ-endotoxins contained in Bacillus thuringiensis entomocidal crystals. It utilized proteolytic conversion of 130-kDa protoxin into 60- to 65-kDa "true" toxin via limited proteolysis with trypsin and separation of stable N-terminal domains by fast-performance liquid chromatography. Immunodiffusion experiments and N-terminal sequence determination (applied to the major component isolated by SDS-PAGE) completed the analysis of the crystal protein composition. The application of this approach to crystals produced by cells of B. thuringiensis subsp. galleriae and wuhanensis allowed us to identify at least seven and eight different δ-endotoxins, respectively. Among those δ-endotoxins assigned to previously described families, CryIA, CryID, Cry IF, and CryIG were found, as well as crystal proteins, which possess N-terminal amino acid sequences very different from those of all known δ-endotoxins. Possible functional consequences of δ-endotoxin multiplicity are discussed.Key words: Bacillus thuringiensis, δ-endotoxins, multiplicity of crystal proteins, separation of true toxins.