Spore formation by Clostridium difficile is a significant obstacle to overcoming hospital-acquired C. difficile -associated disease. Spores are resistant to heat, radiation, chemicals, and antibiotics, making a contaminated environment difficult to clean. To cause disease, however, spores must germinate and grow out as vegetative cells. The germination of C. difficile spores has not been examined in detail. In an effort to understand the germination of C. difficile spores, we characterized the response of C. difficile spores to bile. We found that cholate derivatives and the amino acid glycine act as cogerminants. Deoxycholate, a metabolite of cholate produced by the normal intestinal flora, also induced germination of C. difficile spores but prevented the growth of vegetative C. difficile . A model of resistance to C. difficile colonization mediated by the normal bacterial flora is proposed.

To cause disease,Clostridium difficilespores must germinate in the host gastrointestinal tract. Germination is initiated upon exposure to glycine and certain bile acids, e.g., taurocholate. Chenodeoxycholate, another bile acid, inhibits taurocholate-mediated germination. By applying Michaelis-Menten kinetic analysis toC. difficilespore germination, we found that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination and appears to interact with the spores with greater apparent affinity than does taurocholate. We also report that several analogs of chenodeoxycholate are even more effective inhibitors. Some of these compounds resist 7α-dehydroxylation byClostridium scindens, a core member of the normal human colonic microbiota, suggesting that they are more stable than chenodeoxycholate in the colonic environment.

Additional targets of CodY, a GTP-activated repressor of early stationary-phase genes inBacillus subtilis, were identified by combining chromatin immunoprecipitation, DNA microarray hybridization, and gel mobility shift assays. The direct targets of CodY newly identified by this approach included regulatory genes for sporulation, genes that are likely to encode transporters for amino acids and sugars, and the genes for biosynthesis of branched-chain amino acids.

Summary: CodY, a GTP‐activated global transcriptional regulator of early stationary phase genes, is conserved in many Gram‐positive bacterial species. Recently, a number of novel targets regulated by CodY have been identified, including three Bacillus subtilis operons involved in branched‐chain amino acid (BCAA) biosynthesis (Molle, V., et al., 2003, J Bacteriol 185: 1911–1922). The mechanism of involvement of CodY in regulating the ilvB operon was investigated here using in vivo transcriptional fusions, in vitro gel mobility shift assays and DNase I footprinting assays. CodY was found to mediate regulation of the ilvB operon by GTP and BCAAs and to bind to the ilvB promoter region. BCAAs increased the affinity of CodY for the ilvB promoter and for all other CodY targets tested. This effect of BCAAs in vitro was additive with the effect of GTP on CodY DNA‐binding activity.