Clostridium difficile: cell surface biogenesis

On the C. difficile cell surface is a proteinaceous paracrystalline array, known as the S-layer. The S-layer of C. difficile is composed of two proteins: the high molecular weight (HMW) and the low molecular weight (LMW) S-layer proteins, derived from the pre-protein SlpA. PS-II, an anionic polymer found in all C. difficile strains examined to date, has been identified as the ligand responsible for the attachment of S-layer and associated cell wall proteins. Early efforts to knock out slpA proved unsuccessful and the genes thought to encode the PS-II synthesis pathway were also thought to be essential. However, by using bacteriocins that specifically target the S-layer, we recently isolated a mutant which had no evident S-layer due to a mutation in the slpA gene. As the S-layer was previously thought to be essential, it now brings into question whether PS-II is also essential. In the strain lacking an S-layer, we have now created a deletion mutant in the putative PS-II polymerase and we are attempting to generate additional mutations in the polysaccharide synthesis pathway. Analysis of these mutants will provide insights into the mechanism of PS-II synthesis and shed light on its function in cell morphogenesis.