CONSERVED RESIDUES AND MOTIFS IN THE NIXA PROTEIN OF HELICOBACTER-PYLORI ARE CRITICAL FOR THE HIGH-AFFINITY TRANSPORT OF NICKEL IONS

NixA, the high affinity nickel transport protein of Helicobacter pylor i, imports Ni2+ ions across the cytoplasmic membrane for insertion int o the active site of the urease metalloenzyme, which is essential for colonization of the gastric mucosa, Twelve conserved aspartate (aspart ates 47, 49, 55, 194, 231, and 234), glutamate (glutamates 106, 198, a nd 274), and histidine (histidines 44, 50, and 79) residues were ident ified by alignment of NixA with homologous transporters, Polymerase ch ain reaction-generated site-directed mutants of these residues mere ex pressed in E. coli along with the H. pylori urease gene cluster. Mutat ions in residues within the predicted periplasmic domains of NixA main tained near wild type levels of Ni2+ uptake and urease activity, as di d control mutations of conserved positively charged residues (lysines 140 and 268; arginines 162 and 167). Mutations in highly conserved mot ifs in predicted helices II and III of NixA abolished Ni2+ uptake and urease activity, Mutations in helices V and VI and the cytoplasmic dom ains decreased Ni2+ transport rates by greater than or equal to 90%. R eduction in rates of Ni2+ transport correlated with reduction in ureas e activities (r = 0.77), Ni2+ transport was inhibited in the presence of Co2+, Cu2+ and Zn2+ indicating that these ions may also be bound or trans ported by NixA. We conclude that conserved Asp, Glu, and His re sidues in the transmembrane domains of NixA are critical for the trans port of the divalent cations Ni2+, Co2+, Cu2+, and Zn2+ into the cytop lasm of H. pylori.