| Clostridium difficile is a strictly anaerobic, spore-forming bacterium that is linked to over 250,000 infections annually in the United States. One of the greatest challenges facing C. difficile research has been the lack of genetic tools. This limited repertoire is due, in part, to the anaerobic nature of C. difficile. For example, most fluorescent protein reporters require O2 for chromophore maturation. Here, we demonstrate that O2-dependent fluorescent proteins produced anaerobically can acquire fluorescence after cells are fixed with cross-linkers to preserve native patterns of protein localization. This was shown using the blue and the red codon-optimized fluorescent proteins, CFPopt and mCherryOpt, respectively.;Little is known about cell division in C. difficile. Here we identify and characterize a three-gene operon encoding cell division proteins found only in C. difficile and a small number of closely related bacteria. These proteins were named MldA, MldB, and MldC, for midcell localizing division proteins. MldA is predicted to be a membrane protein with coiled-coil domains and a peptidoglycan-binding SPOR domain. MldB and MldC are predicted to be cytoplasmic proteins; MldB has two predicted coiled-coil domains, while MldC lacks obvious conserved domains or sequence motifs. Mutants of mldA or mldB had morphological defects, including loss of rod shape (a curved cell phenotype) and inefficient separation of daughter cells (a chaining phenotype). Fusions of CFPopt to MldA, MldB, and MldC revealed that all three proteins localize sharply to the division site. Mutants lacking the Mld proteins are severely attenuated for pathogenesis in a hamster model of C. difficile infection. Because all three Mld proteins are essentially unique to C. difficile, they could be exploited as targets for antibiotics that combat C. difficile without disrupting the intestinal microbiome.;C. difficile pathogenesis is mediated primarily by two large exotoxins called Toxin A (TcdA) and Toxin B (TcdB). Transcription of tcdA and tcdB depends on TcdR, an alternative sigma factor for RNA polymerase. Previous studies have shown both toxins are produced upon entry into stationary phase, and that this response is mediated in part by the CodY repressor, which senses GTP and branched chain amino acids. Here we used mCherryOpt as a reporter of gene expression to visualize toxin expression at the level of individual cells. This approach led to the unexpected discovery that only a subset of cells in the population induces expression of tcdA (and tcdB under specific conditions). In other words, toxin production is a "bistable" phenotype. Further experiments indicated TcdR plays a central role in mediating bistability, while CodY makes a minor but still significant contribution to bistability. Why it is advantageous for only a subset of C. difficile cells to produce toxin is not known, but one interesting possibility is related to conflicting requirements for transmission to a new host. Some cells produce toxin to provoke diarrhea while other cells differentiate into spores that can survive exposure to air. |
