Isolation of a relatively nontoxic 65-kilodalton protein inclusion from the parasporal body of Bacillus thuringiensis subsp. israelensis

Abstract

Ultrastructural studies of the mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis revealed that the parasporal body contained three major inclusion types, designated types 1, 2, and 3, which could be differentiated on the basis of electron opacity and size and, to some extent, shape. The type-2 inclusion, which was of moderate electron density and often appeared as a bar-shaped polyhedral body, was isolated on NaBr gradients from purified parasporal bodies and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transmission electron microscopy, and bioassays against neonate larvae of Aedes aegypti. Purified inclusions averaged 150 to 200 nm by 500 to 700 nm in transverse sections and consisted almost exclusively of a 65-kilodalton (kDa) protein contaminated with minor quantities of 38- and 28-kDa proteins. Lethal concentration values at the 50% level for preparations of the purified parasporal body and the type-2 inclusion were, respectively, 0.66 and 43 ng/ml, indicating that the 65-kDa protein is only slightly toxic to mosquitoes in comparison to the intact parasporal body. Analysis of the type-2 polyhedral inclusion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bioassays during different stages of purification demonstrated a positive correlation between the toxicity of the preparation and the degree of contamination with the 28-kDa protein. These results indicate that the 65-kDa protein is not the primary larvicidal toxin, although it may act in conjunction with other parasporal body proteins to produce the high mosquitocidal toxicity characteristic of this bacterium.