title: 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 micromol of NH3/min/mg of protein in the mutant versus 240 +/- 100 micromol 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 mutant (9.3 +/- 3.7 pmol of Ni2+/min/10(8) bacteria) of H. pylori as compared with the parent strain (30.2 +/- 8.1 pmol of Ni2+/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.
authors.affiliation: Division of Infectious Diseases, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: P
authors.initials: P
authors.lastname: Bauerfeind
authors.forename: R M
authors.initials: RM
authors.lastname: Garner
authors.forename: L T
authors.initials: LT
authors.lastname: Mobley
mesh_terms: D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D027682:Cation Transport Proteins; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000235:genetics* / Q000378:metabolism* / Q000472:pathogenicity; D006801:Humans; D066298:In Vitro Techniques; D017136:Ion Transport / Q000235:genetics; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009532:Nickel / Q000378:metabolism*; D014510:Urease / Q000235:genetics / Q000378:metabolism*; D014774:Virulence / Q000235:genetics / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D008565:Membrane Proteins; C095257:NixA protein, Helicobacter pylori; D009532:Nickel; D014510:Urease