Chloride Ions Are Required for Thermosipho africanus MurJ Function

Most bacteria have a peptidoglycan cell wall that determines their cell shape and helps them resist osmotic lysis. Peptidoglycan synthesis depends on the translocation of the lipid-linked precursor lipid II across the cytoplasmic membrane by the MurJ flippase. Structure-function analyses of MurJ from Thermosipho africanus (MurJ(Ta)) and Escherichia coli (MurJ(Ec)) have revealed that MurJ adopts multiple conformations and utilizes an alternating-access mechanism to flip lipid II. MurJ(Ec) activity relies on membrane potential, but the specific counterion has not been identified. Crystal structures of MurJ(Ta) revealed a chloride ion bound to the N-lobe of the flippase and a sodium ion in its C-lobe, but the role of these ions in transport is unknown. Here, we investigated the effect of various ions on the function of MurJ(Ta) and MurJ(Ec) in vivo. We found that chloride, and not sodium, ions are necessary for MurJ(Ta) function, but neither ion is required for MurJ(Ec) function. We also showed that murJ(Ta) alleles encoding changes at the crystallographically identified sodium-binding site still complement the loss of native murJ(Ec), although they decreased protein stability and/or function. Based on our data and previous work, we propose that chloride ions are necessary for the conformational change that resets MurJ(Ta) after lipid II translocation and suggest that MurJ orthologs may function similarly but differ in their requirements for counterions.