Membrane topology of CadA homologous P-type ATPase of   as determined by expression of   fusions in   and the positive inside rule

Melchers, Klaus; Schuhmacher, Alexander; Buhmann, Anita; Weitzenegger, Thomas; Belin, Dominique; Grau, Sandra; Ehrmann, Michael

doi:10.1016/s0923-2508(99)00106-0

Cited by 25 publications

(23 citation statements)

References 41 publications

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“…This finding supports the assumption that the zinc export function of the Czn system is partially compensated for by other metal efflux systems. Since cadmium and zinc are also transported by the P-type ATPase CadA of H. pylori (8,13), this export system might act in concert with Czn, as described for the Czc efflux pump and the P-type ATPases ZntA and CadA in Ralstonia metallidurans (10). It was reported that H. pylori double mutants, lacking CznC and HP0605, displayed increased metronidazole sensi- tivity (33), suggesting a more global role of the Czn system in multidrug detoxification.…”

Section: Discussionmentioning

confidence: 97%

The Novel Helicobacter pylori CznABC Metal Efflux Pump Is Required for Cadmium, Zinc, and Nickel Resistance,Urease Modulation, and Gastric Colonization

et al. 2006

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Maintaining metal homeostasis is crucial for the adaptation of Helicobacter pylori to the gastric environment. Iron, copper, and nickel homeostasis has recently been demonstrated to be required for the establishment of H. pylori infection in animal models. Here we demonstrate that the HP0969-0971 gene cluster encoding the Czc-type metal export pump homologs HP0969, HP0970, and the H. pylori-specific protein HP0971 forms part of a novel H. pylori metal resistance determinant, which is required for gastric colonization and for the modulation of urease activity. Insertional mutagenesis of the HP0971, HP0970, or HP0969 genes in H. pylori reference strain 26695 resulted in increased sensitivity to cadmium, zinc, and nickel (czn), suggesting that the encoded proteins constitute a metal-specific export pump. Accordingly, the genes were designated cznC (HP0971), cznB (HP0970), and cznA (HP0969). The CznC and CznA proteins play a predominant role in nickel homeostasis, since only the cznC and cznA mutants but not the cznB mutant displayed an 8-to 10-fold increase in urease activity. Nickel-specific affinity chromatography demonstrated that recombinant versions of CznC and CznB can bind to nickel and that the purified CznB protein interacted with cadmium and zinc, since both metals competitively inhibited nickel binding. Finally, single cznA, cznB, and cznC mutants did not colonize the stomach in a Mongolian gerbil-based animal model. This demonstrates that the metal export functions of H. pylori cznABC are essential for gastric colonization and underlines the extraordinary importance of metal ion homeostasis for the survival of H. pylori in the gastric environment.

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Section: Discussionmentioning

confidence: 97%

The Novel Helicobacter pylori CznABC Metal Efflux Pump Is Required for Cadmium, Zinc, and Nickel Resistance,Urease Modulation, and Gastric Colonization

et al. 2006

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“…The sense primer, which annealed to the start codon of the pvdA coding sequence, was paired with antisense primers to generate fusions at appropriate positions. In both sense and antisense primers, the KpnI restriction site was included to allow cloning of each amplicon into the compatible restriction site of pBADphoA (Table 1), upstream of the phoA gene without a signal sequence, which encodes a PhoA variant lacking the N-terminal signal peptide essential for PhoA export into the periplasm (Melchers et al, 1999). Fusions 12P and 22P were obtained by the oligonucleotide adaptor technique (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

“…Topology of the Pseudomonas PvdA enzyme phosphate hydrolysis in permeabilized cells (Michaelis et al, 1983), and it was reported in arbitrary units (Melchers et al, 1999). Periplasmic, cytoplasmic and membrane fractions from E. coli cells were prepared as described (Neu & Heppel, 1965), using ice-cold 1 mM MgCl 2 instead of water to reduce the amount of shock-released cytoplasmic proteins (Hantash & Earhart, 2000).…”

Section: D30pvdamentioning

confidence: 99%

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Membrane-association determinants of the ω-amino acid monooxygenase PvdA, a pyoverdine biosynthetic enzyme from Pseudomonas aeruginosa

et al. 2008

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The L-ornithine N d -oxygenase PvdA catalyses the N d -hydroxylation of L-ornithine in many Pseudomonas spp., and thus provides an essential enzymic function in the biogenesis of the pyoverdine siderophore. Here, we report a detailed analysis of the membrane topology of the PvdA enzyme from the bacterial pathogen Pseudomonas aeruginosa. Membrane topogenic determinants of PvdA were identified by computational analysis, and verified in Escherichia coli by constructing a series of translational fusions between PvdA and the PhoA (alkaline phosphatase) reporter enzyme. The inferred topological model resembled a eukaryotic reverse signal-anchor (type III) protein, with a single N-terminal domain anchored to the inner membrane, and the bulk of the protein spanning the cytosol. According to this model, the predicted transmembrane region should overlap the putative FAD-binding site. Cell fractionation and proteinase K accessibility experiments in P. aeruginosa confirmed the membrane-bound nature of PvdA, but excluded the transmembrane topology of its N-terminal hydrophobic region. Mutational analysis of PvdA, and complementation assays in a P. aeruginosa DpvdA mutant, demonstrated the dual (structural and functional) role of the PvdA N-terminal domain.

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“…1A). 25) E. coli LMG194 cells carrying pBADphoA and its merC fusion derivatives were induced by adding 0.4% arabinose, and cell extracts were subjected to western analysis probing with anti-PhoA serum. The MerC-PhoA fusion proteins were detected at the expected sizes (Fig.…”

Section: Resultsmentioning

confidence: 99%

Functional Dissection of a Mercuric Ion Transporter, MerC, fromAcidithiobacillus ferrooxidans

Sasaki

Minakawa

Miyazaki

et al. 2005

Bioscience, Biotechnology, and Biochemistry

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Membrane topology of CadA homologous P-type ATPase of as determined by expression of fusions in and the positive inside rule

Cited by 25 publications

References 41 publications

The Novel Helicobacter pylori CznABC Metal Efflux Pump Is Required for Cadmium, Zinc, and Nickel Resistance,Urease Modulation, and Gastric Colonization

The Novel Helicobacter pylori CznABC Metal Efflux Pump Is Required for Cadmium, Zinc, and Nickel Resistance,Urease Modulation, and Gastric Colonization

Membrane-association determinants of the ω-amino acid monooxygenase PvdA, a pyoverdine biosynthetic enzyme from Pseudomonas aeruginosa

Functional Dissection of a Mercuric Ion Transporter, MerC, fromAcidithiobacillus ferrooxidans

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