Nickel and cobalt are essential for survival, replication, and virulence of bacterial pathogens. Helicobacter pylori depends on Ni(II) to colonize the acidic stomach environment. H. pylori utilizes the nickel-dependent enzyme urease to hydrolyze urea, producing ammonia and bicarbonate to create a pH-buffered niche for survival. Maintaining Ni(II) homeostasis is crucial for H. pylori’s pathogenicity, which has driven the evolution of specialized transmembrane transporter proteins that regulate Ni(II) uptake and export. Among these, NixA, a key Ni(II) transporter from the NiCoT family, facilitates Ni(II) import from the periplasm into the cytosol.
Despite the key role of NixA in Ni(II) acquisition and virulence, molecular level information about its structure, cargo selectivity, and mechanism of transport remains limited. We successfully expressed and purified NixA and several key mutants, and reconstituted them into artificial lipid bilayer vesicles (proteoliposomes). Upon encapsulation in the proteoliposome lumen of fluorescent probes (Fluozin-3-Zn(II), pyranine, and oxonol VI) repsonsive to various stimuli (metal substrates, pH, transmembrane potential), we achieved, for the first time in a NiCoT transporter, real-time monitoring of NixA’s transport properties and mechanism. Translocation kinetic studies showed that NixA is highly selective for Ni(II). Ni(II) transport by NixA follows saturable Michaelis-Menten kinetics and is electrogenic, not relying on a proton motive force. Mutation analysis pinpointed crucial transmembrane residues involved in substrate recognition and transport, indicating a three-step transmembrane translocation process. This methodology has been extended to other Ni(II) and Co(II) NiCoT transporters belonging to other pathogenic bacteria featuring different Ni(II) and Co(II) requirements for Ni(II)- and non-corrinoid Co(II)-enzymes. By applying our developed proteoliposome platform we reveal the molecular determinants of NiCoT cargo selectivity and promiscuity. Overall, this research introduces new methods to characterize transmembrane transporters responsible for Ni(II) and Co(II) acquisition in prokaryotes at an unprecedented level of molecular detail.