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  • 1
    Electronic Resource
    Electronic Resource
    Role of Hpn and NixA of Helicobacter pylori in Susceptibility and Resistance to Bismuth and Other Metal Ions (1999)
    Boston, MA, USA : Blackwell Science Inc
    Helicobacter 4 (1999), S. 0 
    Details
    ISSN: 1523-5378
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background. Helicobacter pylori produces Hpn, a 60-amino acid, histidine-rich protein that avidly binds nickel and zinc ions, and NixA, a high-affinity nickel transporter in the cytoplasmic membrane. We tested the hypothesis that Hpn and NixA govern susceptibility to metal ions in H. pylori.Materials and Methods. Hpn-negative mutants of four H. pylori strains were constructed by standard allelic exchange techniques to yield isogenic Hpn+/Hpn-deficient pairs. A metal concentration that inhibited growth by 50% (IC50) was calculated for Ni2+, Zn2+, Cu2+, and Co2+ by comparing OD600 of cultures in metal-supplemented and control media.Results. Among all four pairs of isogenic strains, the tolerance for Ni2+ was reduced significantly (p 〈 .001) in the Hpn mutants; the mean IC50 value for wild-type strains was 1.9 mM; for the mutant, it was 0.8 mM. In   contrast, growth inhibition by Zn2+ was identical within the fours pairs, as was Cu2+ and Co2+ tolerance in one pair tested. We also found that deletion of the hpn gene increases susceptibility to therapeutic forms of bismuth by testing a mutant and wild-type pair with ranitidine bismuth citrate, bismuth citrate, and four antibiotics. Minimal inhibitory concentrations of ranitidine bismuth citrate dropped from 9.2 to 2.3 μg/ml, and those of bismuth citrate dropped from 7.4 to 3.2 μg/ml (p 〈 .05 for both comparisons), while susceptibility to the antibiotics was unaffected. Disruption of the nixA gene encoding the specific Ni2+ transport protein of H. pylori did not change susceptibility to bismuth.Conclusion. We concluded that bacteria lacking Hpn, cultured in vitro, are more susceptible than is the wild type to bismuth and Ni2+.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1523-5378.1999.99286.x
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  • 2
    Electronic Resource
    Electronic Resource
    Helicobacter pylori nickel-transport gene nixA: synthesis of catalytically active urease in Escherichia coli independent of growth conditions (1995)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 16 (1995), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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. coll SE5000 (pHP808), which carries the H. pylori urease gene cluster, was complemented with a λ 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 μM NiCl2, was 36μmol NH3min−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 34317 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−110−8 cells, whereas the vector control transported only 140±85pmol Ni+2 min−1 108 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±2.4nM; Vmax=1750±220 pmol Ni2+ min−110−8 cells) and is necessary for expression of catalytically active urease, regardless of growth conditions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1995.tb02395.x
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  • 3
    Electronic Resource
    Electronic Resource
    Helicobacter pylori cadA encodes an essential Cd(II)–Zn(II)–Co(II) resistance factor influencing urease activity (1999)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 33 (1999), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1999.01496.x
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