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The crystalline inclusion of Bacillus thuringiensis, dissolved in 8 m urea containing 10% 2-mercaptoethanol and dialyzed to pH 8.3 to 8.5, was compared with a fraction obtained by the same extraction procedure from spores broken by dry rupture. The two fractions behaved similarly on chromatography with Sephadex G-100 and diethylaminoethyl cellulose. The preparations behaved identically on acrylamide gel electrophoresis at pH 12 and pH 9.5. Further, peptide maps of the two fractions obtained after digestion with trypsin were almost superimposable. Amino acid analyses of the crystal and spore fraction were closely similar; discrepancies are attributed to contamination of the spore extract with small amounts of other proteins. It is concluded that a significant portion of the spore protein is identical with the crystal protein.

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 alkali- or 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 double-diffusion 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.

Thin sections of sporulating cells of Bacillus thuringiensis show a marked association between the crystalline inclusion and the exosporium.

Treatment with urea-mercaptoethanol of purified spores of Bacillus thuringiensis, other Bacillus species, and Clostridium roseum solubilizes a protein fraction between 5 and 12% of the dry weight of the spores. This fraction behaves identically to the crystal protein of B. thuringiensis on acrylamide-gel electrophoresis. The protein from all of the Bacillus species shows partial homology with crystal protein, using the Ouchterlony immunodiffusion technique. A further fraction, similar in amount, can be removed from spores of B. thuringiensis by the addition of sodium lauryl sulfate to the urea-mercaptoethanol. Spores of B. thuringiensis extracted in these ways show no difference when compared to untreated spores with respect to viability or resistance to heat and ultraviolet-irradiation. The extracted spores do show differences in their germination requirements and their susceptibility to phase-darkening by lysozyme. It is concluded that an urea-mercaptoethanol-soluble protein or class of protein is a widespread component of bacterial spores, possibly located in the spore coat, and that this protein may be related to the crystal protein of B. thuringiensis.