Urease is a virulence determinant, a taxonomic and diagnostic marker, and immunogen for Helicobacter pylori, an aetiologic agent of gastritis and peptic ulceration. This enzyme requires Ni2+ ions in the active site for successful hydrolysis of urea. When expressed in Escherichia coli, recombinant urease is only weakly active unless urease structural subunits are overexpressed, exogenous NiCl2 is added, and the host strain is grown in medium that does not chelate free Ni2+. As wild-type H. pylori does not require such conditions for very high levels of urease expression, we reasoned that additional genes were required to accumulate the metal ion. To isolate such genes, E. coli SE5000 (pHP808), which carries the H. pylori urease gene cluster, was complemented with a lambda ZAP-derived plasmid library of the H. pylori chromosome. One of 1000 ampicillin-resistant clones, plated onto urea segregation agar, produced detectable urease. Urease activity of this co-transformant, grown in Luria broth containing 1 microM NiCl2, was 36 mumol NH3 min-1 mg-1 protein. Urease-enhancing activity, which is not directly linked to the urease gene cluster, was localized by subcloning and nucleotide sequencing. The largest open reading frame, designated nixA, predicted a polypeptide of 34,317 Da that displayed characteristics of an integral membrane protein. In vitro transcription-translation of nixA sequences yielded a polypeptide estimated to be 32 kDa in size. An in-frame Bal31 deletion within nixA abolished urease-enhancing activity. At 50 nM NiCl2, E. coli containing the nixA clone transported 1250 +/- 460 pmol Ni2+ min-1 10(-8) cells, whereas the vector control transported only 140 +/- 85 pmol Ni2+ min-1 10(8) cells, i.e. significantly less (P = 0.01). We conclude that NixA confers upon E. coli a high-affinity nickel-transport system (KT = 11.3 +/- 2.4 nM; Vmax = 1750 +/- 220 pmol Ni2+ min-1 10(-8) cells) and is necessary for expression of catalytically active urease, regardless of growth conditions.

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NixA, the high affinity nickel transport protein of Helicobacter pylori, imports Ni 2؉ ions across the cytoplasmic membrane for insertion into the active site of the urease metalloenzyme, which is essential for colonization of the gastric mucosa.

We concluded that bacteria lacking Hpn, cultured in vitro, are more susceptible than is the wild type to bismuth and Ni2+.

Inactivation of Helicobacter pylori cadA, encoding a putative transition metal ATPase, was only possible in one of four natural competent H. pylori strains, designated 69A. All tested cadA mutants showed increased growth sensitivity to Cd(II) and Zn(II). In addition, some of them showed both reduced 63Ni accumulation during growth and no or impaired urease activity, which was not due to lack of urease enzyme subunits. Gene complementation experiments with plasmid (pY178)-derived H. pylori cadA failed to correct the deficiencies, whereas resistance to Cd(II) and Zn(II) was restored. Moreover, pY178 conferred increased Co(II) resistance to both the cadA mutants and the wild-type strain 69A. Heterologous expression of H. pylori cadA in an Escherichia coli zntA mutant resulted in an elevated resistance to Cd(II) and Zn(II). Expression of cadA in E. coli SE5000 harbouring H. pylori nixA, which encodes a divalent cation importer along with the H. pylori urease gene cluster, led to about a threefold increase in urease activity compared with E. coli control cells lacking the H. pylori cadA gene. These results suggest that H. pylori CadA is an essential resistance pump with ion specificity towards Cd(II), Zn(II) and Co(II). They also point to a possible role of H. pylori CadA in high-level activity of H. pylori urease, an enzyme sensitive to a variety of metal ions.