Evaluating the Involvement of Alternative Sigma Factors SigF and SigG in Clostridium perfringens Sporulation and Enterotoxin Synthesis

Clostridium perfringens type A food poisoning is the second most commonly identified bacterial food-borne illness. Sporulation contributes to this disease in two ways: (i) most food-poisoning strains form exceptionally resistant spores to facilitate their survival of food-associated stresses, and (ii) the enterotoxin (CPE) responsible for the symptoms of this food poisoning is synthesized only during sporulation. In Bacillus subtilis, four alternative sigma factors mediate sporulation. The same four sigma factors are encoded by C. perfringens genomes, and two (SigE and SigK) have previously been shown to be necessary for sporulation and CPE production by SM101, a transformable derivative of a C. perfringens food-poisoning strain (K. H. Harry, R. Zhou, L. Kroos, and S. B. Melville, J. Bacteriol. 2009, 191:2728-2742). However, the importance of SigF and SigG for C. perfringens sporulation or CPE production had not yet been assessed. In the current study, after confirming that sporulating wild-type SM101 cultures produce SigF (from a tricistronic operon) and SigG, we prepared isogenic sigF- or sigG-null mutants. Whereas SM101 formed heat-resistant, phase-refractile spores, spore formation was blocked in the sigF- and sigG-null mutants. Complementation fully restored sporulation by both mutants. By use of these mutants and complementing strains, CPE production was shown to be SigF dependent but SigG independent. This finding apparently involved regulation of the production of SigE and SigK, which Harry et al. showed to be necessary for CPE synthesis, by SigF. By combining these findings with those previous results, it is now apparent that all four alternative sigma factors are necessary for C. perfringens sporulation, but only SigE, SigF, and SigK are needed for CPE synthesis.