Structure of the subunit protein of bipyramidal .DELTA.-endotoxin produced by Bacillus thuringiensis kurstaki strain HD-1.

Abstract

The structure of the subunit protein of the bipyramidal <S-endotoxin of Bacillus thuringiensis kurstaki strain HD-1 was analyzed by limited hydrolysis with a gut juice protease (P-III) of the silkworm, Bombyxmori, and by neutralization tests using subunit protein IgG. The molecular weight of the subunit protein was 1 30,000 by SDS-polyacrylamide gel electrophoresis. Hydrolysis of the subunit protein by P-III produced a toxic protein with 60,000 dalton (P-59) and nontoxic peptides. A preparation of subunit IgG was put through P-59-agarose affinity chromatography, and the adsorbed fraction (TR-IgG) was separated. The unadsorbed fraction was reapplied to a subunit protein-agarose affinity column, and the adsorbed fraction (FR-IgG) was obtained. TR-IgG was capable of reacting to both subunit protein and P-59, and neutralized the toxicity of subunit protein. FR-IgG reacted to subunit protein but not to P-59. The toxicity ofsubunit protein was not neutralized by FR-IgG. These results indicate that the subunit protein is composed of proteaseresistant region with a molecular weight of about 60,000 and a residual protease-sensitive nontoxic region, which was surmised to be involved in the construction of the crystalline inclusion. Purified P-59 appeared to be a mixture of two protein species with similar molecular weights but with different charges.

An analysis of Bacillus thuringiensisδ‐endotoxin action on insect‐midgut‐membrane permeability using a light‐scattering assay

“…A similar effect was observed in vitro where lepidopteran midgut-membrane vesicles challenged with activated toxin exhibited morphological changes and swelling (Tojo, 1986). Subsequent investigations have shown that CryI toxins alter the permeability of lepidopteran midgut apical membranes for monovalent cations (Sacchi et al, 1986;Crawford and Harvey, 1988;Wolfersberger, 1989;Hendrickx et al, 1990).…”

Facile autoplast generation and transformation in Bacillus thuringiensis subsp. kurstaki

“…The organism produces parasporal crystals, which are comprised of two polypeptides, P1 and P2, having endotoxic activity against lepidopteran and dipteran larvae (7,21). In recent years much research has been devoted to the isolation of the specific toxin-encoding genes and determination of their nucleotide sequences (14,19,20). However, with the advent of gene fusion and site-specific mutagenesis techniques, it has become important to transport genetically constructed molecules into B. thuringiensis.…”

Mode of Action of Bipyramidal δ-Endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1

“…A total of 42 fifth-instar larvae of the silkworm (mean body weight, 580 mg) were used. Each larva was force fed with 10 p.g of inclusions containing approximately 2 ,ug of P-59 (20). At specified intervals between 5 and 240 min, 50 p.l of hemolymph was collected from each larva, and the larvae were discarded after this bleeding.…”

“…At specified intervals between 5 and 240 min, 50 p.l of hemolymph was collected from each larva, and the larvae were discarded after this bleeding. The hemolymph was diluted with 150 p. of PBS containing 1% bovine serum albumin, and the presence of P-59 antigen was examined by an enzyme-linked immunosorbent assay (ELISA) as described previously (20). The sensitivity limit of the assay for determination of P-59 in the hemolymph was 1.6 ng/ml.…”