| Peptidoglycan synthesis is a well studied pathway in bacteria both because of its essential role in cell-wall containing bacteria, and also because it serves as a target for antibiotic development. The cytoplasmic steps starting with the synthesis of the membrane-linked peptidoglycan precursor Lipid II from fructose-6-phosphate and the extracytoplasmic steps leading to the synthesis of insoluble cross-linked glycan strands on the outside of the cell are largely well understood. However, the mechanism underlying the membrane translocation of lipid II from its site of synthesis in the cytoplasm to its site of incorporation into mature peptidoglycan on the outside of the cell remains mysterious. In this thesis, I have examined several potential candidates for proteins that mediate this process. These include SEDS (shape, elongation, division, sporulation) proteins, a family of polytopic membrane proteins that are highly conserved among bacteria that make peptidoglycan and most often essential, but have unknown roles in peptidoglycan synthesis. Using B. subtilis as a model organism to study peptidoglycan synthesis, I have used genetic and biochemical approaches to investigate the role of SpoVE, a sporulation-specific SEDS homolog, and SpoVD, its sporulation-specific class B Penicillin Binding Protein. My studies suggest that SEDS proteins are directly coupled to class B PBPs, but that they extend past mere recruitment of class B PBPs. I have also investigated MviN homologs in B. subtilis since MviN was previously hypothesized to be the essential lipid II flippase in E. coli. My studies have shown that a strain containing mutations in four genes encoding putative MviN homologs is viable and had no detectable growth or cell morphology defects. Finally, to test putative flippase candidates I have developed assays for flipping of lipid II in proteoliposomes and flipping in inverted vesicles from B. subtilis . I describe progress in these experiments in detail. |
