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 alleles encoding changes at the crystallographically identified sodium-binding site still complement the loss of native murJ, 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. IMPORTANCE The biosynthetic pathway of the peptidoglycan cell wall is one of the most favorable targets for antibiotic development. Lipid II, the lipid-linked PG precursor, is made in the inner leaflet of the cytoplasmic membrane and then transported by the MurJ flippase so that it can be used to build the peptidoglycan cell wall. MurJ functions using an alternating-access mechanism thought to depend on a yet-to-be-identified counterion. This study fills a gap in our understanding of MurJ's energy-coupling mechanism by showing that chloride ions are required for MurJ in some, but not all, organisms. Based on our data and prior studies, we propose that, while the general transport mechanism of MurJ may be conserved, its specific mechanistic details may differ across bacteria, as is common in transporters. These findings are important to understand MurJ function and its development as an antibiotic target.