Transport of peptidoglycan sub-units across the bacterial cytoplasmic membrane

Abstract

Transport of peptidoglycan sub-units across the bacterial cytoplasmic membrane Vincent van Dam The work described in this thesis focuses on the mechanism by which the lipid-linked cell wall precursor molecule Lipid II is transported from the inner leaflet to the outer leaflet of the cytoplasmic membrane. For this purpose, it was first tested in model membranes using fluorescently labeled Lipid II whether the molecule could move spontaneously across the membrane. This was not the case and also the last protein witnessing Lipid II on the cytoplasmic side of the membrane in E. coli, the Lipid II synthesizing protein MurG, was not able to induce transbilayer movement of Lipid II. This showed that Lipid II synthesis and transport are not obligatory coupled. In isolated inner membrane vesicles of E. coli transport of Lipid II was measured and the process appeared to be independent of an energy source. These observations strongly suggested that a protein is needed for translocation of Lipid II across the bilayer and provided the correct parameters that allow Lipid II transport in isolated membrane vesicles. To identify the proteins involved in this process, a photolabeling study was performed under these conditions. For this purpose Lipid II was labeled with the photoactivatable crosslinker NHS-ASA containing 125I to enable specific detection of crosslinked proteins. A combination of two-dimensional gel electrophoresis and mass spectrometry was used to identify proteins that were in contact with Lipid II and resulted in an interesting set of proteins. Strikingly, not many of the identified proteins could directly be linked to cell wall synthesis. Therefore, inner membrane vesicles derived from E. coli strains overexpressing the Lipid II flippase candidate FtsW. This led to a changed pattern of crosslinked proteins, indicating a direct relation between FtsW and Lipid II. Next, several purified candidate proteins were reconstituted in proteoliposomes to test their ability to translocate Lipid II across the membrane. The sec protein translocation machinery, of which several components were identified in the photocrosslink study, was not able to translocate Lipid II. Also the complete Lipid II synthesis machinery MraY and MurG was reconstituted but did not lead to Lipid II transport. When FtsW was reconstituted into proteoliposomes the fluorescent Lipid II was more accessible to the quenching agent dithionite which possibly indicates that FtsW mediates transbilayer transport of Lipid II. In addition, the appearance of protein complexes was studied. MurG plays a central role in this study, because the protein could be in a complex with the Lipid II transporter protein. MurG is found predominantly in monomeric form but approximately 30 % of the protein migrates on SDS-PAGE to a position of approximately 140 kDa. The composition of this protein complex is not completely clear but its formation appeared to be mediated by a membrane associated protein factor.