Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Regulation of the Expression of the <i>Vibrio parahaemolyticus peuA</i> Gene Encoding an Alternative Ferric Enterobactin Receptor

<div><p>A <i>pvsB</i>-<i>vctA</i>-<i>irgA</i> triple deletion mutant of <i>Vibrio parahaemolyticus</i> can utilize enterobactin under iron-limiting conditions by inducing a previously undescribed receptor, PeuA (VPA0150), in response to extracellular alkaline pH and enterobactin. <i>In silico</i> analyses revealed the existence of a two-component regulatory system operon, <i>peuRS</i>, immediately upstream of <i>peuA</i>, which constitutes an operon with the TonB2 system genes. Both the <i>peuRS</i> and <i>peuA-tonB2</i> operons were found to be upregulated under iron-limiting conditions in a ferric uptake regulator (Fur)-dependent manner. The involvement of <i>peuA</i> and <i>peuRS</i> in enterobactin utilization was analyzed by complementation experiments using deletion mutants. Primer extension analysis indicated that, under iron-limiting conditions, the transcription of <i>peuA</i> was initiated from the +1 site at pH 7.0 and from both the +1 and +39 sites at pH 8.0 in the presence of enterobactin. The +39 transcript was absent from the <i>peuRS</i> deletion mutant. Secondary structure prediction of their 5′-untranslated regions suggested that translation initiation is blocked in the +1 transcript, but not in the +39 transcript. Consistent with this, <i>in vitro</i> translation analysis demonstrated that production of PeuA was determined only by the +39 transcript. These studies establish a novel gene regulation mechanism in which the two-component regulatory system PeuRS enhances expression of the alternative +39 transcript that possesses non-inhibitory structure, allowing the <i>peuA</i> expression to be regulated at the translation stage.</p></div

Relative levels of <i>peuA</i> mRNA, assessed by RT-qPCR.

<p>Total RNA samples were prepared from VPD54 (<i>vctA</i>- and <i>irgA</i>-deficient mutant derived from VPD5) and VPD102 (<i>peuRS</i>-deficient mutant derived from VPD54) grown at pH 7.0 and 8.0 in LB-Tris, LB-Tris/+EDDA, and LB-Tris/+EDDA/+Ent media. Data are shown as means ± SD from 3 separate experiments. An asterisk indicates <i>P</i><0.05 compared to other samples.</p

Genetic map and operon structure of <i>VPA0148</i>–<i>VPA0156</i> locus.

<p>(A) Genetic map of the <i>peuA</i> gene and the flanking genes. Thick arrows indicate genes and their orientations. The –35 and –10 promoter elements and putative Fur box sequences in the promoter regions of <i>peuR</i> (<i>VPA0148</i>) and <i>peuA</i> (<i>VPA0150</i>) are indicated. The transcription start sites for <i>peuR</i> (+1) and <i>peuA</i> (+1 and +39) are indicated by right-angled arrows. The putative terminator signal between the <i>peuS</i> and <i>peuA</i> genes, the predicted RBS for the <i>peuA</i> gene, the start codons for <i>peuR</i> and <i>peuA</i> genes, and the stop codon for <i>peuS</i> are also indicated. The amino acid sequence consistent with the N-terminal sequence determined for the iron-repressible OMR induced in LB-Tris/+EDDA and LB-Tris/+EDDA/+Ent media at pH 8.0 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105749#pone-0105749-g003" target="_blank">Figure 3</a>) is indicated by a double underline. (B) Schematic representation of mRNAs transcribed from the <i>VPA0148</i>-<i>VPA0156</i> genes and the primer pairs used for RT-PCR. For preparation of cDNAs by RT, VPpeuS-R and VP0156-R were used. (C) RT-PCR analysis of RT-PCR products. +RT and –RT, RT-PCR was performed with and without reverse transcriptase, respectively. M, 100-bp DNA ladder.</p

Schematic representation of the +1-<i>peuA</i>′-<i>flag</i> (A) and +39-<i>peuA</i>′-<i>flag</i> (B) DNA fragments.

<p>Each of these DNA fragments includes a nucleotide sequence corresponding to the <i>peuA</i> 5′-UTR from the +1 or +39 sites and the nucleotide sequence for the N-terminal 99 amino acid residues (in gray), in addition to a T7 promoter and a FLAG tag preceding the stop codon (TAA). The secondary structures of the 5′-UTRs of the +1 transcript (A) and the +39 transcript (B) of <i>peuA</i> are shown, both of which were predicted by the CentroidFold software (<a href="http://www.ncrna.org/centroidfold/" target="_blank">http://www.ncrna.org/centroidfold/</a>). The RBS and start codon of <i>peuA</i> mRNA are boxed in the secondary structures.</p

<i>In vitro</i> translation of <i>peuA</i> mRNA.

<p>(A) <i>In vitro</i> translation analysis of the +1 and +39 <i>peuA</i> transcripts labeled with the FLAG tag. The +1-<i>peuA</i>′-<i>flag</i> RNA and +39-<i>peuA</i>′-<i>flag</i> RNA were first synthesized by <i>in vitro</i> transcription, as described in the MATERIALS AND METHODS, and a mixture containing either the +1-<i>peuA</i>′-<i>flag</i> RNA (30 pmol)/<i>fur</i>-<i>flag</i> RNA (3 pmol) or the +39-<i>peuA</i>′-<i>flag</i> RNA (30 pmol)/<i>fur</i>-<i>flag</i> RNA (3 pmol) as the template was subjected to <i>in vitro</i> translation. The FLAG-fused proteins translated were separated on 15% SDS-polyacrylamide gels, and were detected by western blotting using anti-FLAG IgG. (B) Confirmation of the presence of +1-<i>peuA</i>′-<i>flag</i> RNA and +39-<i>peuA</i>′-<i>flag</i> RNA in the reaction mixture for <i>in vitro</i> translation. These RNA fragments were detected in the reaction mixture by northern blotting using a DIG-labeled <i>peuA</i> probe.</p

SDS-PAGE analysis of Sarkosyl-insoluble OMPs of <i>V. parahaemolyticus</i>.

<p>SDS-PAGE analysis was performed with VPD5, VPD107 (seven iron-repressible OMRs-deficient mutant derived from VPD5), VPD108 (<i>peuA</i>-deficient mutant derived from VPD107), VPD108/pRK415-peuA, VPD109 (<i>peuRS</i>-deficient mutant derived from VPD107), and VPD109/pRK415-peuRS. The OMP fractions were prepared from cells grown in LB-Tris medium at pH 7.0, LB-Tris/+EDDA media at pH 7.0 and 8.0, or LB-Tris/+EDDA/+Ent media at pH 7.0 and 8.0. Lanes 1–7 and 9–10 were loaded with 20 µg OMPs, and lane 8 was loaded with 3 µg OMPs. Electrophoresis was performed on 7.5% SDS-polyacrylamide gels (130 mm long) at a constant current of 15 mA at 4°C. The gel was stained with Coomassie Brilliant Blue. The figure shows only the relevant portions of the gel. The iron-repressible OMPs expressed by VPD5 at pH 7.0 under iron-limiting conditions are boxed in lane 2. Lane M, molecular weight marker proteins; closed arrowheads, PeuA.</p

Involvement of <i>peuA</i> and <i>peuRS</i> in Ent utilization.

<p>The growth assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105749#pone-0105749-g001" target="_blank">Figure 1</a>. Data are shown as means ± SD from 3 separate experiments.</p

Alkaline pH-dependent utilization of Ent in VPD54.

<p>VPD54, which is a <i>vctA</i> and <i>irgA</i> deletion mutant generated from the VPD5 vibrioferrin-deficient mutant, was grown in LB-Tris/+EDDA medium (at indicated pH) at 37°C for 18 h with shaking at 70 rpm. When required, Ent was added at 5 µM. Cultures were monitored by measuring the OD₆₀₀ every 3 h. Data are shown as means ± SD from 3 separate experiments.</p

Primer extension analyses of total RNA from VPD54 or VPD102 to determine the transcription start sites of <i>peuR</i> (A) and <i>peuA</i> (B and C).

<p>Total RNAs were isolated from VPD54 (<i>vctA</i>- and <i>irgA</i>-deficient mutant derived from VPD5) and VPD102 (<i>peuRS</i>-deficient mutant derived from VPD54) grown at pH 7.0 and 8.0 in LB-Tris, LB-Tris/+EDDA, and LB-Tris/+EDDA/+Ent media. The amounts of total RNA and primers used for reverse transcription were as follows: (A) 10 µg VppeuR-PE, (B) 10 µg VppeuA-PE, and (C) 150 µg VppeuA-PE. The same primers used for primer extension analysis were used to generate the sequence ladders (A, C, G, T). The transcription start sites and putative Fur boxes are indicated at the top of panels A and B (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105749#pone-0105749-g003" target="_blank">Figure 3A</a>).</p