ALLELIC EXCHANGE MUTAGENESIS OF NIXA IN HELICOBACTER-PYLORI RESULTS IN REDUCED NICKEL TRANSPORT AND UREASE ACTIVITY

Helicobacter pylori, an etiologic agent of gastritis and peptic ulcera tion in humans, synthesizes urease, a nickel metalloenzyme, as its mos t abundant protein. NixA, a high-affinity nickel transport protein, al lows synthesis of catalytically active urease when coexpressed with H. pylori urease in an Escherichia coli host. To determine whether NixA is essential for the production of active urease in H. pylori, nixA wa s insertionally inactivated with a kanamycin resistance cassette (aphA ) and this construct was electroporated into H. pylori ATCC 43504; all elic exchange mutants were selected on kanamycin-containing medium. Th e nixA mutation, confirmed by PCR, reduced urease activity by 42% (140 +/- 70 mu mol of NH3/min/mg of protein in the mutant versus 240 +/- 1 00 mu mol of NH3/min/mg of protein in the parent (P = 0.037). Rates of nickel transport were dramatically reduced (P = 0.0002) in the nixA m utant (9.3 +/- 3.7 pmol of Ni2+/min/10(8) bacteria) of H. pylori as co mpared with the parent strain (30.2 +/- 8.1 pmol of Ni2+/min/10(8) bac teria). We conclude that NixA is an important mediator of nickel trans port in H. pylori. That residual nickel transport and urease activity remain in the nixA mutant, however, provides evidence for the presence of a redundant transport system in this species.