Allelic exchange mutagenesis of nixA in Helicobacter pylori results in reduced nickel transport and urease activity

Abstract

Helicobacter pylori, an etiologic agent of gastritis and peptic ulceration in humans, synthesizes urease, a nickel metalloenzyme, as its most abundant protein. NixA, a high-affinity nickel transport protein, allows 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 was insertionally inactivated with a kanamycin resistance cassette (aphA) and this construct was electroporated into H. pylori ATCC 43504; allelic exchange mutants were selected on kanamycin-containing medium. The nixA mutation, confirmed by PCR, reduced urease activity by 42% (140 ؎ 70 mol of NH 3 /min/mg of protein in the mutant versus 240 ؎ 100 mol of NH 3 /min/mg of protein in the parent (P ‫؍‬ 0.037). Rates of nickel transport were dramatically reduced (P ‫؍‬ 0.0002) in the nixA mutant (9.3 ؎ 3.7 pmol of Ni 2؉ /min/10 8 bacteria) of H. pylori as compared with the parent strain (30.2 ؎ 8.1 pmol of Ni 2؉ /min/10 8 bacteria). We conclude that NixA is an important mediator of nickel transport 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.

Contribution of dppA to Urease Activity in Helicobacter pylori 26695

“…Amplification using primers DPP1-DPP2 resulted in a band of approximately two kb in both parental strains and approximately 2.7 kb in the dppA mutant strains, resulting from insertion of the 624 bp cat cassette. PCR analysis of nixA mutants was carried out with primers NIXA1-NIXA2 as previously described [14]. All mutants remained motile and catalase-positive.…”

“…© 2005 Blackwell Publishing Ltd, Helicobacter, 10, 416-423 as recipient cells. Construction of the H. pylori nixA mutant has been previously described [14,15].…”

“…Allelic exchange mutagenesis of nixA significantly reduced nickel uptake and production of catalytically active urease in H. pylori (14). However, urease activity is not completely abolished in these mutants suggesting the presence of alternative nickel transporters.…”

The role of Helicobacter pylori urease in the pathogenesis of gastritis and peptic ulceration.

“…In activation of rrixA in H. pylori results in decreased urease activity, presumably beca u se of' low ni ckel ion content in th e enzyme. 27 However, Signifi cant activity remains, suggestin g that alternate nickel ion tra nsport systems are also present in H. pylori that allow the accumulation of these ions in the absence of a functiona l NixA ( Figure 2).…”

“…tO Additional backup nickel transport proteins arc also present. 27 The net result of the interaction of these genes and proteins is a ata lytically active urease.…”