Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori

Essential trace metals play key roles in the survival, replication, and virulence of bacterial pathogens. Helicobacter pylori (H. pylori), the main bacterial cause of gastric ulcers, requires Ni(II) to colonize and persist in the acidic environment inside the stomach, exploiting the nickel-containing enzyme urease to catalyze the hydrolysis of urea to ammonia and bicarbonate and create a pH-buffered microenvironment. Urease utilizes Ni(II) as a catalytic cofactor for its activity. In ureolytic bacteria, unique transmembrane (TM) transporters evolved to guarantee the selective uptake and efflux of Ni(II) across cellular membranes to meet the cellular requirements. NixA is an essential Ni(II) transporter expressed by H. pylori when the extracellular environment experiences a drop in pH. This Class I Nickel-Cobalt transporter of the NiCoT family catalyzes the uptake of Ni(II) across the inner membrane from the periplasm. In this study, we characterized NixA using a platform whereby, for the first time on a NiCoT transporter, recombinantly expressed and purified NixA, and key mutants in the translocation pathway, have been reconstituted in artificial lipid bilayer vesicles (proteoliposomes). Fluorescent sensors responsive to Ni(II) transport (Fluozin-3-Zn(II)), luminal pH changes (pyranine), and membrane potential (oxonol VI) were encapsulated in proteoliposome lumen to monitor, in real-time, NixA transport properties and translocation mechanism. Kinetic transport analysis revealed that NixA is highly selective for Ni(II) with no substrate promiscuity towards Co(II), the other putative metal substrate of the NiCoT family, nor Zn(II). NixA-mediated Ni(II) transport exhibited a Michaelis-Menten-type saturable substrate concentration dependence, with an experimental KM, Ni(II) = 30.97 +/- 1.20 uM. Moreover, Ni(II) transport by NixA was demonstrated to be electrogenic, and metal translocation did not require a proton motive force, resulting in the generation of a positive-inside transmembrane potential in the proteoliposome lumen. Mutation analysis characterized key transmembrane residues for substrate recognition, binding, and/or transport, suggesting the presence of a three-step transmembrane translocation conduit. Taken together, these investigations reveal that NixA is a Ni(II)-selective Class I NiCoT electrogenic uniporter. The work also provides an in vitro approach to characterize the transport properties of metal transporters responsible for Ni(II) acquisition and extrusion in prokaryotes.