Immunological Homology Between Crystal and Spore Protein of Bacillus thuringiensis

Abstract

Spore suspensions containing about 0.3% crystals and crystal suspensions containing about 0.1% spores were obtained from cultures of Bacillus thuringiensis by extraction with a two-phase system. Both preparations were tested for the presence of contaminating material from vegetative cells and were judged to be clean. Solutions of spore protein were obtained by extracting broken spores with phosphate buffer followed by extraction with either alkalior urea-mercaptoethanol. The alkali spore or urea spore extracts had the same isoelectric point as crystal protein solubilized with these reagents. An antiserum prepared against alkali crystal solution precipitated alkali or urea spore extracts and crystal solutions but not phosphate spore extracts or extracts of whole cells. Lines of identity between spore and crystal precipitates were observed by using the Ouchterlony doublediffusion technique. Absorption of the antiserum with an excess of urea spore extract caused a disappearance of the precipitin bands originating from the spore protein and the homologous bands from the crystal protein. The results suggest that the crystal and endospore contain one or more common proteins.

Biogenesis of the Crystalline Inclusion of Bacillus thuringiensis during Sporulation

“…These observations point to a possible relationship, both in nature and origin, between the crystal protein and one of the spore-coat proteins; such homology has also been suggested by Delafield et al [7,8]. In both instances the substance is an "insoluble" protein whose synthesis appears to be controlled by the same factors.…”

“…The most recent work of Delafield et al [7] and Somerville et al [8] demonstrates that the crystal proteins have certain immunological and biochemical characteristics in common with one or more of the spore proteins (presumed spore coats).…”

Neither barium nor calcium prevents the inhibition by Bacillus thuringiensis δ‐endotoxin of sodium‐ or potassium gradient‐dependent amino acid accumulation by tobacco hornworm midgut brush border membrane vesicles

“…Parasporal crystals were separated from the other components of sporulated cultures using a two-phase extraction system with dextran sulfate and polyethylene glycol [20]. This procedure has been shown routinely to yield crystals contaminated by less than 1% spores [21].…”

Immunocytochemical Localization of Spore Specific Antigens in Ultrathin Sections

“…Samples were removed and processed for electron microscopy using the fixation and embedding procedure described by Walker (1969). Mature spores were prepared in the medium described by Delafield et al (1968) at 37 "C with continuous shaking for 3 d. A further 3 d at room temperature allowed for autolysis of the vegetative cells. After washing at least four times with distilled water the spores were cleaned further by separation in the two-phase system of Sacks & Alderton (1961).…”