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 β-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 Δ bkdR , Δ ylmC , Δ ytxC , and Δ 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 Δ ytaF or Δ yyaC1 mutants; and a slightly lower level with spores of Δ bkdR or Δ ylmC mutants. In contrast, almost no germination was observed with spores of Δ ytxC or Δ yyaC2 mutants. Consistent with germination defects, Δ ytxC or Δ yyaC2 spores were also defective in spore outgrowth and colony formation. The germination, outgrowth, and colony formation defects of Δ ytxC or Δ yyaC2 spores were restored when Δ ytxC or Δ yyaC2 mutant was complemented with wild-type ytxC or yyaC2 , respectively. Collectively, our current study identified new sporulation and germination genes in C. perfringens .