NixA and NiuBDE are necessary for resistance to extreme acidity in the presence of urea.

<p>Wild type strains and mutants were exposed during 40 min at pH 2, without or with 6 mM urea. <i>ΔniuB</i> corresponds to a <i>ΔniuB1-ΔniuB2</i> double mutant. Controls were performed at pH 7 without urea. Survival of the bacteria was assessed using an Alamar blue-based test, in which a pink staining reveals metabolically active cells, while a blue staining means that cells are metabolically inactive. The pink staining is proportional to the amount of metabolically active cells. The pH was evaluated for each strain and results were compared to control cells (exposed at pH 7) and to a pH-scale (right side of the figure). A reminder of the results obtained in other experiments for urease activity and ⁶³Ni(II) uptake is given at the bottom of the figure for comparison.</p

NiuD and NiuB are required for mouse colonization by <i>H</i>. <i>pylori</i> strain SS1.

<p>Each point corresponds to the colonization load for one mouse one month after infection with the strain indicated below. Horizontal bars represent the geometric means of the colonization load for the wild type, each mutant and the chromosomally complemented mutants (designated c-). <i>ΔniuB</i> corresponds to a <i>ΔniuB1-ΔniuB2</i> double mutant. The results presented correspond to a representative experiment out of two. The detection limit is shown by a dashed horizontal line. <b>***</b> indicates that the geometric value is significantly different (<i>P</i> ≤ 0.001) from that of the wild type strain.</p

NiuD and NixA mediate <i>H</i>. <i>pylori</i> sensitivity to high nickel concentrations.

<p>Effect of 1.5 and 2 mM NiCl₂ on growth of <i>H</i>. <i>pylori</i> B128-S wild type strain, isogenic mutants and complemented strains. The results are presented as % of growth in the presence of nickel relative to growth without nickel after 24h incubation. The data correspond to the mean value of three independent experiments. Error bars represent the standard deviation. <b>***</b> indicates that the mean value is significantly different from that of the wild type strain (<i>P</i> ≤ 0.001).</p

Role of NixA and NiuBDE in urease activity of <i>H</i>. <i>pylori</i> at pH 5 and 7.

<p>Urease activity was measured on whole cells of the different mutants strains by measuring ammonia production. For each condition, ammonia production is expressed as a percentage of the wild type strain put at 100%. These data indicate that NiuBDE functions at both pH 5 and 7, while NixA is mainly active at pH 5. <i>ΔniuB</i> corresponds to a <i>ΔniuB1-ΔniuB2</i> double mutant. The mutant strains were either complemented by c-<i>niuDE</i> (c- for chromosome) inserted at a neutral site on the chromosome under the control of the P_<i>ureI</i> promoter or by p-<i>niuB1</i> and p-<i>niuB2</i> (p- for plasmid) expressed from derivatives of plasmid pILL2157 under the control of an IPTG-inducible P_<i>ureI</i> promoter in the presence of IPTG. The data correspond to the mean value of three independent experiments and error bars represent the standard deviation. *** and <b>**</b> indicate that the mean value is significantly different from that of the wild type strain (<i>P</i> ≤ 0.001 and <i>P</i> ≤ 0.01, respectively). For the complemented strains, <i>ΔnixA</i> Δ<i>niuD</i> + c-<i>niuDE</i> or <i>ΔnixA</i> Δ<i>niuB</i> p-<i>niuB1/2</i>, the <i>P</i> values correspond to comparison with the corresponding parental <i>ΔnixA ΔniuD</i> and <i>ΔnixA</i> Δ<i>niuB</i> mutants, respectively.</p

RNase J depletion leads to massive changes in mRNA abundance in <i>Helicobacter pylori</i>

<p>Degradation of RNA as an intermediate message between genes and corresponding proteins is important for rapid attenuation of gene expression and maintenance of cellular homeostasis. This process is controlled by ribonucleases that have different target specificities. In the bacterial pathogen <i>Helicobacter pylori</i>, an exo- and endoribonuclease RNase J is essential for growth. To explore the role of RNase J in <i>H. pylori</i>, we identified its putative targets at a global scale using next generation RNA sequencing. We found that strong depletion for RNase J led to a massive increase in the steady-state levels of non-rRNAs. mRNAs and RNAs antisense to open reading frames were most affected with over 80% increased more than 2-fold. Non-coding RNAs expressed in the intergenic regions were much less affected by RNase J depletion. Northern blotting of selected messenger and non-coding RNAs validated these results. Globally, our data suggest that RNase J of <i>H. pylori</i> is a major RNase involved in degradation of most cellular RNAs.</p

NiuD and NixA control the intracellular nickel content and uptake in <i>H</i>. <i>pylori</i>.

<p>Panel A: Nickel amounts measured by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) and expressed as μg of nickel.g^-1 of protein. Strains were grown either without added nickel, with 10 μM or with 100 μM NiCl₂. Panel B: ß-galactosidase activity expressed by the P_<i>fecA3</i>::<i>lacZ</i> reporter fusion in wild type and mutant strains. The expression of the fusion decreases in a NikR-dependent manner with increasing intracellular nickel concentration. ß-galactosidase activities are presented as the ratio of activity measured in strains grown in the presence of 200 μM nickel versus without nickel, expressed in percentages. Panel C: Measurements of radioactive nickel uptake rates in wild type and mutant strains expressed in cpm/min. On the right side scale, the uptake rates were normalized with respect to the value measured for the wild type strain. Complementation with the <i>niuDE</i> operon inserted at a neutral locus on the chromosome is designated c-<i>niuDE</i> (c- stands for “chromosomally inserted”). In these different experiments, the data correspond to the mean value of three independent experiments and error bars represent the standard deviation. <b>***</b> indicates that the mean value is significantly different (<i>P</i> ≤ 0.001) from that of the wild type strain.</p

NiuB1 and NiuB2 control the nickel sensitivity and uptake in <i>H</i>. <i>pylori</i>.

<p>Panel A: Effect of nickel on growth of <i>H</i>. <i>pylori</i> B128-S wild type strain and isogenic mutants. <i>ΔniuB</i> corresponds to a <i>ΔniuB1-ΔniuB2</i> double mutant. Results are presented as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006018#ppat.1006018.g001" target="_blank">Fig 1</a>. Panel B: ß-galactosidase activity of the P_<i>fecA3</i>::<i>lacZ</i> reporter fusion in different backgrounds presented as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006018#ppat.1006018.g002" target="_blank">Fig 2</a>. Panel C: Measurements of radioactive nickel uptake rates in wild type and mutant strains. On the right scale, the uptake rates were normalized with respect to the value measured for the wild type strain. The mutant strains were either complemented by c-<i>niuB1</i> or c-<i>niuB2</i> (c- for chromosomally inserted) inserted at a neutral site on the chromosome under the control of the P_<i>ureI</i> promoter or by p-<i>niuB1</i> and p-<i>niuB2</i> (p- for inserted on a plasmid) expressed from derivatives of plasmid pILL2157 under the control of an IPTG-inducible P_<i>ureI</i> promoter in the presence of IPTG. In these different experiments, the data correspond to the mean value of three independent experiments and error bars represent the standard deviation. <b>***</b> indicates that the mean value is significantly different (<i>P</i> ≤ 0.001) from that of the wild type strain.</p

Model for nickel transport in <i>H</i>. <i>pylori</i>.

<p>In <i>H</i>. <i>pylori</i>, nickel (small blue circles) is transported across the outer membrane by FrpB4 (purple barrel), a TonB-dependent transporter. This uptake activity is most efficient at acidic pH. Once in the periplasm, uptake of nickel through the inner membrane can be performed by the NixA permease (green). Alternatively, nickel can form a Ni(II)-(L-His)₂ complex, that is specifically recognized by the periplasmic solute binding protein NiuB (orange). In <i>H</i>. <i>pylori</i> strains with two NiuB paralogs, NiuB1 (dark orange) seems to be the major contributor for nickel uptake, while NiuB2 (light orange) is less efficient. Then, NiuB docks onto the NiuD permease (orange), and nickel is transferred across the inner membrane upon ATP consumption by NiuE (light blue), and delivered to the cytoplasm. There, it is stored by histidine-rich proteins, such as HspA (GroES, purple), Hpn (blue) or Hpn-2 (green) and/or channeled by the UreEFGH machinery toward urease, or by the HypAB machinery towards hydrogenase. The figure presents urease activation through nickel delivery by UreEFGH (to which HypAB also contributes) in the cytoplasm. Ultimately, this pathway results in nickel-dependent urease activation, this enzyme catalyzing urea breakdown into ammonia and carbon dioxide, both contributing to maintaining the intracellular pH close to neutrality and allowing the bacteria to resist acidity.</p

Sensitivity to cobalt and bismuth of wild type strain and isogenic mutants.

<p>Effect of NiCl_2, CoCl₂ and bismuth subcitrate potassium on growth of <i>H</i>. <i>pylori</i> B128-S wild type strain, isogenic mutants and complemented strains. <i>ΔniuB</i> corresponds to a <i>ΔniuB1-ΔniuB2</i> double mutant. Strains were grown 24 h in the presence or absence of the metal examined. Results are presented as a Growth ratio G_Ratio = 100x[(OD_+metal/OD_-metal)_test−(OD_+metal/OD_-metal)_ref] / (OD_+metal/OD_-metal)_ref allowing to normalize the growth ratio of the test strain compared to the growth ratio of the reference strain. The reference is the wild type strain on panel A and the <i>ΔnixA ΔniuB</i> mutant on panel B. Bars correspond to the G_Ratio values. This representation allows to visualize the results in such a way that bars on the left relative to the vertical axis correspond to G_Ratio values of strains that are more sensitive to the metal than the wild type strain (negative values) and those on the right side to G_Ratio values of strains that are more tolerant (positive values) to the metal. The results are presented together with those with nickel for comparison. The data correspond to the mean value of three independent experiments and error bars represent the standard deviation.</p

Effect of nickel on the growth of <i>H</i>. <i>pylori</i> B128, SS1 and X47-2AL wild type strain and isogenic mutants.

<p>The strains with pureI are controls in which only the pUreI is inserted at the locus at which the <i>hpn</i> or <i>hpn-2</i> genes are introduced in the complemented strains <i>∆hpn + phpn</i> and <i>∆hpn-2</i> + <i>phpn-2</i>. Panel A, nickel disk diffusion assay and sensitivity to 200 μM NiCl₂ in liquid medium. Panel B, nickel sensitivity in liquid medium (range of NiCl₂ concentrations) of mutants and MIC₅₀ for the B128 ∆<i>hpn</i>, ∆<i>hpn</i> +p<i>hpn</i> and ∆<i>hpn-∆hpn-2</i> strains. The data correspond to the mean value of three independent experiments with at least triplicate tests for each strain. Error bars represent the standard deviation.</p