Two types of entomocidal toxins in the parasporal crystals of Bacillus thuringiensis kurstaki

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Abstract

Two types of entomocidal proteins of Bacillus thuringiensis kurstaki were isolated from the parasporal bodies (crystals), and their structures were compared with each other in relation to the toxic activity. When the crystals were dissociated in 2% 2-mercaptoethanol at pH 10, a protein of Mr = 135,000, called δ-endotoxin, was liberated. The crystals of a strain of B. thuringiensis kurstaki, the HD-1 strain, also released another protein in small quantities. This minor component of HD-1, which had been discovered and named mosquito factor by Yamamoto and McLaughlin (T. Yamamoto and R. E. McLaughlin (1981)Biochem. Biophys. Res. Commun. 103, 414–421) because of its toxicity to mosquito larvae, could be liberated selectively from the crystals by alkali treatment without any thiol reagent at pH 11. Electron microscopic observation suggested that the bipyramidal crystal is composed of a homogeneous component, presumably the δ-endotoxin, and the mosquito factor is not within the crystal matrix. The liberated toxins, including the mosquito factor, were purified by Sephacryl S-300 column chromatography and activated by proteinases obtained from gut juice of the cabbage looper (Trichoplusia ni). The activated toxins were characterized by peptide mapping using techniques of HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide mapping revealed that the mosquito factor is a protein distinctly different from the δ-endotoxin. Furthermore, a comparison between two strains of B. thuringiensis kurstaki indicated that minor differences in the structure of the δ-endotoxins, in particular the differences in their proteinase-resistant region, caused significant variations in their toxicity to susceptible insects.

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A part of this study was done in cooperation with the Texas Agricultural Experiment Station, Texas A&M University, College Station, Texas 77843. Mention of a commercial product does not constitute recommendation for use by the U. S. Department of Agriculture.

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