Characterization of Putative Sporulation and Germination Genes in Clostridium perfringens Food-Poisoning Strain SM101

Bacterial sporulation and spore germination are two intriguing processes that involve the expression of many genes coherently. Phylogenetic analyses revealed gene conservation among spore-forming Firmicutes, especially in Bacilli and Clostridia. In this study, by homology search, we found Bacillus subtilis sporulation gene homologs of bkdR, ylmC, ylxY, ylzA, ytaF, ytxC, yyaC1, and yyaC2 in Clostridium perfringenes food-poisoning Type F strain SM101. The beta-glucuronidase reporter assay revealed that promoters of six out of eight tested genes (i.e., bkdR, ylmC, ytaF, ytxC, yyaC1, and yyaC2) were expressed only during sporulation, but not vegetative growth, suggesting that these genes are sporulation-specific. Gene knock-out studies demonstrated that C. perfringens Delta bkdR, Delta ylmC, Delta ytxC, and Delta yyaC1 mutant strains produced a significantly lower number of spores compared to the wild-type strain. When the spores of these six mutant strains were examined for their germination abilities in presence of known germinants, an almost wild-type level germination was observed with spores of Delta ytaF or Delta yyaC1 mutants; and a slightly lower level with spores of Delta bkdR or Delta ylmC mutants. In contrast, almost no germination was observed with spores of Delta ytxC or Delta yyaC2 mutants. Consistent with germination defects, Delta ytxC or Delta yyaC2 spores were also defective in spore outgrowth and colony formation. The germination, outgrowth, and colony formation defects of Delta ytxC or Delta yyaC2 spores were restored when Delta ytxC or Delta yyaC2 mutant was complemented with wild-type ytxC or yyaC2, respectively. Collectively, our current study identified new sporulation and germination genes in C. perfringens.