Mosquitocidal protein ofBacillus thuringiensis subsp.israelensis: Identification and partial isolation of the protein

Abstract

Three mutants were isolated from ethyl-methanesulfonate-treatedBacillus thuringiensis subsp.israelensis ONR-60A strain. The mutants were chosen from 200 colonies on the basis of crystal production and differed from the parent strain in plasmid pattern. Under an electron microscope, the parent strain and one of three mutants, il069-3, appeared to produce at least two distinct crystal sizes. The il042-8 mutant showed only small crystals, and no crystals were seen in the il069-9 mutant. Bioassay revealed that the parent strain and il069-3 had high mosquitocidal activity, but il042-8 and il069-9 appeared to be nontoxic to mosquito larvae. When crystal proteins were extracted in NaOH at pH 10 with 2% 2-mercaptoethanol (NaOH-MeEtOH) and analyzed by two-dimensional electrophoresis, the parent strain showed a complex protein composition in a molecular weight range of 10,000–120,000. The il069-3 crystals had a protein profile similar to that of the parent strain, but lost some proteins with high molecular weights. Both the parent strain and the il069-3 mutant produced 28-and 38-kilodalton (kdal) proteins as major components. The il042-8 mutant retained the 38-kdal protein, but was lacking the 28-kdal protein, and no significant amount of protein was seen in the extract of il069-9. The results from protein analysis and bioassay strongly indicate that the toxicity is associated with the 28-kdal protein. To, prove this, the protein was isolated by Sephacryl S-200 column chromatography and tested against mosquito larvae.