Acid-dependent response of <i>cag</i> promoters in Δ<i>fur</i>, Δ<i>nikR</i> and Δ<i>arsS</i> mutant strains.

<p>Cultures were grown to exponential phase and exposed to acid-shock (pH = 5.2) for 30 min. Transcript levels at the P<i>cagζ</i>, P<i>cagU</i>, P<i>cagF</i>, P<i>cagA</i>, P<i>cagS</i> and P<i>cagB</i> promoters were assayed by quantitative primer extensions. Asterisks mark the significant differences of n-fold variations deriving from the average band intensity of three independent primer extension experiments. Error bars indicate the standard deviation.</p

Mapping of <i>cag</i> promoters.

<p><b>A</b>. Genomic organization of the <i>cag</i> pathogenicity island in the <i>H. pylori</i> G27 strain. (1) Transcriptional units mapped in this study; black: plus strand; grey: minus strand. (2) Annotated <i>cag</i> genes and ORFs; white block arrow: ORF encoding effector toxin CagA; dark grey block arrows: ORFs encoding structural T4SS components; light grey block arrows: ORFs encoding components with ancillary or unknown function. (3) Alphabetic classification of <i>cag</i> genes. (4) Numeric classification of <i>cag</i> genes. (5) Annotation of <i>cag</i> genes in reference strain 26695. (6) Transcriptional start sites mapped in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098416#pone.0098416-Sharma1" target="_blank">[20]</a>. (7) Transcriptional units identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098416#pone.0098416-Spohn1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098416#pone.0098416-Joyce1" target="_blank">[33]</a>. (8) Transcriptional units identified by unbiased promoter-trapping experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098416#pone.0098416-Ta1" target="_blank">[30] </a><b>B</b>. Summary of relevant features within the nucleotide sequences of the P<i>cag</i> promoters mapped in this study. The TSSs (+1) are boxed in black boxes and showed in boldface. Sequences corresponding to -10 regions, the extended TGn elements and recognizable -35 region are enlightened in grey boxes. <b>C</b>. Comparison of the transcript levels at the P<i>cag</i> promoters fused with <i>lux</i> reporter genes. mRNA levels of the P<i>cag</i>-<i>lux</i> constructs were assayed by primer extension analysis using the oligonucleotide VSluxC1 and quantified with a phospoimager. The mean values from three independent experiments are reported as arbitrary units of ³²P counts.</p

Stress responses of <i>cag</i> promoters.

<p><b>A</b>. Heat-shock response of the P<i>cag</i> promoters. Primer extension analyses were performed on total RNA extracted from bacterial cultures of <i>H. pylori</i> wild type strain grown to exponentially phase and maintained at 37°C or exposed to 42°C for 30 min. <b>B</b>. Iron-dependent regulation of the P<i>cagA</i> promoter. Liquid cultures of wild type and Δ<i>fur</i> strains were grown to OD₆₀₀ = 0.5 or OD₆₀₀ = 1.7 and treated for 30 min with 1 mM (NH₄)₂Fe(SO₄)₂ (Fe+), or 100 µM 2,2-dipyridyl (Fe-). mRNA levels at the P<i>cagA</i> promoter were assayed by quantitative primer extension on the total RNA extracted. <b>C</b>. In vitro binding of Fur protein to the P<i>cagB</i>-P<i>cagA</i> promoter region in a DNaseI footprinting assay. Concentrations of Fur dimer, 0 nM (lane 1), 21 nM (lane 2), 42 nM (lane 3), 84 nM (lane 4), 168 nM (lane 5) and 336 nM (lane 6). The binding reaction was performed in a final volume of 50 µL in presence of divalent iron ions as cofactors (150 µM (NH4)₂Fe(SO₄)₂). The vertical grey and black bars on the right of the panel (I-IV) indicate the areas of partial and complete DNaseI protection, respectively, resulting from binding of Fur on the probe. Fur binds to nucleotide positions +2 to -14 (I), -20 to -63 (II), -108 to -115 (III) and -145 to -188 (IV) with respect to the +1 transcriptional start site (TSS) of P<i>cagA</i>. An hypersensitivity band that appears at high concentrations of Fur is indicated by a black triangle. On the left side of the panel, the TSS downstream P<i>cagB</i> and P<i>cagA</i> are indicated with bent arrows, while the relative position of the -10 and -35 regions of the 2 promoters are indicated as vertical black boxes. G+A: G+A sequencing reaction ladder. <b>D</b>. DNaseI footprinting of Fur protein to the P<i>cagB</i>-P<i>cagA</i> promoter region without the supplement of iron ions and with 150 µM 2,2-dipyridyl used to sequester the Fe²⁺ ions. The experimental conditions used for the footprinting assay are the same as described in panel C.</p

pH-dependent response of <i>cag</i> promoters.

<p>Primer extension analyses were performed on total RNA extracted from bacterial cultures of <i>H. pylori</i> wild type strain, grown to exponential phase and treated for 30 or 90 min with 22 mM HCl to adjust the pH of the medium to 5.2, or maintained at neutral pH (pH 7.0). The intensity of the bands of four independent experiments were quantified and reported in the graphs as n-fold variation of the transcript levels in the acidic-treated samples with respect to the untreated sample. <b>A</b>. P<i>cag</i> promoters with transcript levels increased after an acidic treatment for 30 min; <b>B</b>. Promoters with reduced mRNA levels after the 30 min acidic treatment; <b>C</b>. Promoters with unchanged transcript levels after the treatment. Error bars indicate the standard deviation and significant variations between treated and untreated samples are marked with asterisks (<i>P</i><0.05). <b>D</b>. Response of P<i>cagV</i>, P<i>cagQ</i>, P<i>cagP</i>, P<i>cagM</i> and P<i>cagC</i> promoters after an acidic treatment of 90 min.</p

Reporter assays with the P<i>cag-lux</i> strains in host cell co-cultures.

<p>Liquid cultures of P<i>cagζ-</i>, P<i>cagQ-</i> and P<i>cagB-lux</i> strains were added at a multiplicity of infection of 5 to 24-wells plates containing human gastric adenocarcinoma (AGS) cells or with the medium only. The luminescence emitted by the reporter strains was recorded by a multilabel reader at regular intervals. Signals were normalized on the samples without AGS cells and averaged. Standard errors were calculated from four independent experiments (each in four technical replicates).</p

Oligonucleotides used in this study.

<p>Capital letters indicate <i>N. meningitidis</i> derived sequences, small letters indicate sequences added for cloning purposes, and underlined letters indicate restriction enzyme recognition sites.</p

Regulatory proteins binding to the <i>nadA</i> promoter.

<p>(A) DNase I footprinting of IHF protein to three different phase variant <i>nadA</i> promoters with 9, 6 and 7 repeats corresponding to low, medium and high transcript level in vivo, respectively, and the PΔ mutant <i>nadA</i> variant with a deletion of the TAAA repeated tract. To 20 fmoles of each radioactively labelled probe, 0, 43 and 172 nM (lanes 1–3) of IHF heterodimer were added. Relevant regions are marked and numbers correspond to nucleotide positions with respect to the transcriptional start site of a promoter with 9 repeats. (B) DNase I footprinting of RNAP or the α-subunit of RNAP to the indicated <i>nadA</i> promoter probe. The probe was incubated with 0, 0.25, 0.5, 1, 2, 4, and 5 U of RNAP (lanes 1–7) or 0, 0.17, 0.68, 2.7, 5.5, 11 µM of purified α-subunit (lanes 8–13).</p