Proton-Dependent Gating and Proton Uptake by Wzx Support O-Antigen-Subunit Antiport Across the Bacterial Inner Membrane

Wzx flippases are crucial for bacterial cell surface polysaccharide assembly as they transport undecaprenyl pyrophosphate-linked sugar repeat units from the cytoplasmic to the periplasmic leaflets of the inner membrane (IM) for final assembly. Our recently reported three-dimensional (3D) model structure of Wzx from Pseudomonas aeruginosa PAO1 (Wzx(Pa)) displayed a cationic internal vestibule and functionally essential acidic amino acids within transmembrane segment bundles. Herein, we examined the intrinsic transport function of Wzx(Pa) following its purification and reconstitution in phospholipid liposomes. Wzx(Pa) was capable of mediating anion flux, consistent with its cationic interior. This flux was electrogenic and modified by extraliposomal pH. Mutation of the above-mentioned acidic residues (E61, D269, and D359) reduced proton (H(+))-modified anion flux, showing the role of these amino acid side chains in H(+)-dependent transport. Wzx also mediated acidification of the proteoliposome interior in the presence of an outward anion gradient. These results indicate H(+)-dependent gating and H(+) uptake by Wzx(Pa) and allow for the first H(+)-dependent antiport mechanism to be proposed for lipid-linked oligosaccharide translocation across the bacterial IM.