Membrane topology of the NixA nickel transporter of Helicobacter pylori: two nickel transport-specific motifs within transmembrane helices II and III.

Helicobacter pylori is a well-established etiologic agent of gastritis, as well as duodenal and gastric ulceration (6, 35, 36). The development of more serious sequelae, namely, gastric carcinoma and MALT lymphoma, are also strongly associated with chronic H. pylori infection (18, 41). H. pylori produces a battery of virulence factors, including a urease comprising up to 6% of the soluble cell protein (26). The ammonia produced by the hydrolysis of urea by this 550-kDa multimeric enzyme has been postulated to allow the bacterium to survive and colonize the low pH environment of the gastric mucosa. This is supported by the observation that H. pylori is highly sensitive to acid in the absence of urea (34). An additional role of urease in colonization has been implied by the finding that urease-negative mutants are unable to colonize animal models of infection, even when the gastric pH is maintained near neutrality by the administration of the proton pump inhibitor omeprazole (15, 48). The divalent nickel ion is a requisite cofactor in the urease active site (23). The NixA nickel transport protein has an extremely high-affinity (KT = 11.3 nM) import mechanism (39) well suited for scavenging Ni2+ from the low concentrations found in human serum (2 to 11 nM) (45) and presumably in the gastric mucosa. Isogenic NixA-deficient H. pylori cells show predictably lower levels of Ni2+ uptake (31% of that of the wild type) and urease activity (42% of that of the wild type) (3). Alignment of the 331-amino-acid NixA sequence with three homologous single-component Ni2+ transporters, HoxN of Alcaligenes eutrophus (50), HupN of Bradyrhizobium japonicum (19), and UreH of thermophilic Bacillus sp. strain TB-90 (30), as well as hydropathy and charge dispersion (49), suggested a preliminary model in which NixA is an integral cytoplasmic membrane protein composed of seven transmembrane domains with the amino terminus located in the periplasm (39). Further examination of this alignment identified 12 conserved Asp, Glu, and His residues which were postulated to be involved in Ni2+ transport (20). These included the sequence motif GX2HAXDADH, which was conserved among NixA and its homologs, as well as in the NikC component of the nonhomologous Nik ATP-binding cassette transporter of Escherichia coli (20, 50). The motif GX2FX2GHSSVV, which is also shared among the four single-component Ni2+ transporters, is also present as a slight variant in the NhlF Ni2+-sensitive Co2+ transporter of Rhodococcus rhodochrous (20, 27, 50). Both sequence motifs were also predicted to be in the NicT protein, recently identified as encoded in the genome of Mycobacterium tuberculosis (10), as well as in additional putative homologs identified in the genome sequences of Salmonella typhi (4) and Schizosaccharomyces pombe (29). Initial topological predictions for NixA placed these motifs in the first transmembrane domain and across the cytoplasmic border of the first cytoplasmic loop into the second transmembrane domain, respectively (39). Site-directed mutation of two Asp and three His residues located within these motifs each resulted in the complete loss of NixA-mediated Ni2+ uptake and urease activity when each mutation was expressed in E. coli cells along with the H. pylori urease operon (20). Nonconservative mutations of six additional conserved Asp and Glu residues which were postulated to lie within transmembrane domains reduced rates of Ni2+ transport by >90%, with correlating reductions in urease activities (20). While these findings shed considerable light on the function of specific amino acid residues of NixA, a detailed analysis of the membrane topology and localization of the protein is required to assign specific location and structure to amino acid residues which appear to be critical for Ni2+ transport. We here report a new model of the structure and localization of NixA, based on the analysis of reporter fusions, membrane fractionation, and cross-linking studies.