Solubilization and characterization of spore coat proteins of Clostridium perfringens

Coat proteins from mature spores of two enterotoxin-positive (Ent$\sp+$) and two enterotoxin-negative (Ent$\sp-$) strains of Clostridium perfringens were solubilized using 50 mM dithiothreitol and 1% sodium dodecyl sulphate at pH 9.7, and alkylated using 110 mM iodoacetamide to prevent aggregation. SDS-PAGE patterns of solubilized spore coat proteins from different strains of C. perfringens show that the Ent$\sp+$ strains have similar major polypeptides (34- and 19-kDa). The Ent$\sp-$ strains also have similar coat protein composition (28- and 19-kDa major protein) distinct from that of the Ent$\sp+$ strains. The spore coat proteins seem to be synthesized under similar genetic control among strains of C. perfringens regardless of their ability to produce enterotoxin. The aggregation of enterotoxin in the presence of SDS did not occur at concentrations below 15 $\mu$g/ml. Enterotoxin was not a major structural component of the coat fraction of C. perfringens spores. But two enterotoxin-related proteins (34- and 48-kDa) were found in the solubilized spore coat protein of Ent$\sp+$ strains while only the 48 kDa enterotoxin-related protein was found in the spore coat fraction of Ent$\sp-$ strains. The possible precursor of enterotoxin (48-kDa) was detected in sporulating cell extracts of all seven strains of C. perfringens tested (NCTC 8239, NCTC 10240, ATCC 3624, ATCC 3626, FD1, 2498, 748) and in the vegetative cell extracts of four of these (NCTC 8239, FD1, 2498, 748). However enterotoxin itself was present in only Ent$\sp+$ strains (NCTC 8239, NCTC 10240). There was no clear relationship between the enterotoxin synthesis and the spore coat protein production even though netropsin and distamycin study showed that the syntheses of both were closely related to sporulation process of C. perfringens. In the absence of caffeine, netropsin and distamycin unexpectedly stimulated sporulation of C. perfringens NCTC 8679 grown in D sporulation medium although the majority of sporulating cells were defective