title: Proton-dependent gating and proton uptake by Wzx support O-antigen-subunit antiport across the bacterial inner membrane.
abstract: 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 (WzxPa) displayed a cationic internal vestibule and functionally essential acidic amino acids within transmembrane segment bundles. Herein, we examined the intrinsic transport function of WzxPa following its purification and reconstitution in phospholipid liposomes. WzxPa 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 WzxPa and allow for the first H(+)-dependent antiport mechanism to be proposed for lipid-linked oligosaccharide translocation across the bacterial IM. IMPORTANCE Many bacterial cell surface polysaccharides that are important for survival and virulence are synthesized at the periplasmic leaflet of the inner membrane (IM) using precursors produced in the cytoplasm. Wzx flippases are responsible for translocation of lipid-linked sugar repeat units across the IM and had been previously suggested to simply facilitate passive substrate diffusion. Through our characterization of purified Wzx in a reconstitution system described herein, we have observed protein-dependent intrinsic transport producing a change in the electrical potential of the system, with H(+) identified as the coupling ion. These results provide the first evidence for coupled (i.e., secondary active) transport by these proteins and, in conjunction with structural data, allow for an antiport mechanism to be proposed for the directed transport of lipid-linked sugar substrates across the IM. These findings bring our understanding of lipid-linked polysaccharide transporter proteins more in line with the efflux pumps to which they are evolutionarily related.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
authors.forename: Salim T
authors.initials: ST
authors.lastname: Islam
authors.forename: Paul D W
authors.initials: PD
authors.lastname: Eckford
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Jones
authors.forename: Timothy
authors.initials: T
authors.lastname: Nugent
authors.forename: Christine E
authors.initials: CE
authors.lastname: Bear
authors.forename: Christian
authors.initials: C
authors.lastname: Vogel
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Lam
mesh_terms: D020816:Amino Acid Motifs; D000838:Anions / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019081:O Antigens / Q000378:metabolism; D011522:Protons; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000838:Anions; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D019081:O Antigens; D011522:Protons
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