<i>topA</i> expression is activated aerobically and anaerobically by NrdR.

<p><b>A)</b> GFP fluorescence was measured in <i>P</i>. <i>aeruginosa</i> strains PAO1 (wild-type) and PW7855 (Δ<i>nrdR</i>) harboring plasmid pETS177 (P<i>topA</i>::GFP). The <i>nrdR</i> cloned into plasmid pUCP20T (pETS176) was used to complement <i>nrdR</i> deficiency in strain PW7855. Plasmid pETS178 harbors a <i>topA</i> promoter with a mutation in the NrdR box. The fluorescence was normalized with the absorbance at 550 nm (A₅₅₀) and it is given in relative fluorescent units. The bars represent the mean of three independent experiments ± standard deviation. <b>B)</b> A gel electrophoresis assay, in an agarose gel containing chloroquine, of plasmid DNA isolated from <i>P</i>. <i>aeruginosa</i> wild-type and Δ<i>nrdR</i> strains, at mid-logarithmic and stationary phases. The direction of migration was from top to bottom. *, Significantly different compared with the wild-type strain in an unpaired <i>t</i>-test (<i>P</i><0.05).</p

Summary of the effects of the <i>nrdR</i> mutation on <i>P</i>. <i>aeruginosa</i> gene expression under aerobic and anaerobic conditions.

<p><b>A)</b> Distribution of the different genes (up-regulated, down-regulated and unchanged) in gene expression (>1.5 Log₂ fold change). The number of gens in each category is indicated. <b>B)</b> Distribution of genes whose expression was either increased or decreased in a Δ<i>nrdR</i> mutant strain, grouped according to fold-changes in expression levels.</p

<i>nrdR</i> operon organization and expression.

<p><b>A)</b> Gene organization scheme of the <i>nrdR-rib</i>D operon. <b>B)</b> Sequence alignment (Clustal W) of <i>P</i>. <i>aeruginosa</i> (PAO-NrdR; Uniprot Q9HWX1) and <i>Escherichia coli</i> (Ecoli-NrdR; Uniprot P0A8D0) NrdR proteins. <b>C)</b> Aerobic and anaerobic growth curve of <i>P</i>. <i>aeruginosa</i> strains PAO1 (wild-type) and PW7855 (Δ<i>nrdR</i>). <b>D)</b> Fluorescence (GFP) was measured in both strains harboring pETS161 (P<i>nrdR</i>-GFP) at different points of growth, at 37ºC in LB medium. The fluorescence was normalized dividing by the optical density (A₅₅₀), and it is given in relative fluorescence units. Each experiment was repeated three times, and the results are the mean ± standard deviation. *: Significantly different compared with wild-type strain in an unpaired <i>t</i>-test (<i>P</i><0.05).</p

NarL-dependent expression of <i>nrdR</i>.

<p><b>A)</b> Representation of the <i>P</i>. <i>aeruginosa</i> PAO1 <i>nrdR</i> promoter region sequence, indicating the different mutated NarL binding sites. Black boxes indicate the putative NarL recognition sites, and mutated sequences are shown in upper case and in bold letters. The transcription start site is indicated in bold. The RFU column shows the relative fluorescence intensity presented by the <i>P</i>. <i>aeruginosa</i> wild-type <i>nrdR</i> promoter fusion (pETS161), compared with their mutated NarL boxes (pETS181, pETS182 and pETS183 for NarL box1, pETS184, pETS185 and pETS186 for NarL box2, and pEST187 harboring the double mutation). The expression of wild-type <i>nrdR</i> promoter under a Δ<i>narL</i> mutant background is also stated. The ratio column shows a comparison of all the conditions with the expression of a wild-type promoter under a wild-type background. Strains were grown anaerobically until the mid-logarithmic phase. Values represent the mean of three independent experiments. Transcriptional start codon is shown in bold. Three independent experiments were performed and the mean±standard deviation is shown). *: Significantly different compared with wild-type promoter region (pETS161) in an unpaired <i>t</i>-test (<i>P</i><0.05).</p

The <i>nrdR</i> mutant of <i>P</i>. <i>aeruginosa</i> does not alter the kinetics of <i>D</i>. <i>melanogaster</i> killing.

<p>Control flies were injected with PBS. Fly survival was monitored for 48 h. Approximately 100 flies were used for each experiment.</p

Global transcriptomic analysis of a Δ<i>nrdR</i> mutant strain compared with the <i>P</i>. <i>aeruginosa</i> PAO1 wild-type strain.

<p>Selected differentially regulated genes, under both aerobic and anaerobic conditions. Complete list of all the genes (>1.5-fold) is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123571#pone.0123571.s007" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123571#pone.0123571.s008" target="_blank">S3</a> Table.</p><p>Global transcriptomic analysis of a Δ<i>nrdR</i> mutant strain compared with the <i>P</i>. <i>aeruginosa</i> PAO1 wild-type strain.</p

Model of NrdR-related control on RNR gene expression.

<p>The degree of repression on each RNR class expression, under aerobic or anaerobic conditions, is opposite to the enzymatic activity of these classes under each condition. Considering the presence of an ATP-cone domain in NrdR, dNTPs level alterations could also be affecting the results.</p

Function of the <i>Pseudomonas aeruginosa</i> NrdR Transcription Factor: Global Transcriptomic Analysis and Its Role on Ribonucleotide Reductase Gene Expression

<div><p>Ribonucleotide reductases (RNRs) are a family of sophisticated enzymes responsible for the synthesis of the deoxyribonucleotides (dNTPs), the building blocks for DNA synthesis and repair. Although any living cell must contain one RNR activity to continue living, bacteria have the capacity to encode different RNR classes in the same genome, allowing them to adapt to different environments and growing conditions. <i>Pseudomonas aeruginosa</i> is well known for its adaptability and surprisingly encodes all three known RNR classes (Ia, II and III). There must be a complex transcriptional regulation network behind this RNR activity, dictating which RNR class will be expressed according to specific growing conditions. In this work, we aim to uncover the role of the transcriptional regulator NrdR in <i>P</i>. <i>aeruginosa</i>. We demonstrate that NrdR regulates all three RNR classes, being involved in differential control depending on whether the growth conditions are aerobic or anaerobic. Moreover, we also identify for the first time that NrdR is not only involved in controlling RNR expression but also regulates topoisomerase I (<i>topA</i>) transcription. Finally, to obtain the entire picture of NrdR regulon, we performed a global transcriptomic analysis comparing the transcription profile of wild-type and <i>nrdR</i> mutant strains. The results provide many new data about the regulatory network that controls <i>P</i>. <i>aeruginosa</i> RNR transcription, bringing us a step closer to the understanding of this complex system.</p></div

NrdR differentially regulates ribonucleotide reductase genes in aerobiosis or anaerobiosis.

<p>Aerobic expression studies are shown in <b>A-C</b> and <b>G</b>, and anaerobic expression studies in <b>D-F</b> and <b>H</b>. <i>P</i>. <i>aeruginosa</i> wild-type strain (black bars), Δ<i>nrdR</i> mutant strain (white bars) and the deficiency-complemented <i>nrdR</i> strain (Δ<i>nrdR+</i>pETS176) (gray bars) bearing the promoter fusions P<i>nrdA</i>-<i>gfp</i> (panel <b>A</b> and <b>D</b>), P<i>nrdJ</i>-<i>gfp</i> (panel <b>B</b> and <b>E</b>) and P<i>nrdD</i>-<i>gfp</i> (panel <b>C</b> and <b>F</b>), were grown as indicated in the material and methods. GFP fluorescence is expressed as arbitrary units subtracting the reads of the control plasmid pETS130. <b>G</b>) and <b>H</b>) Quantitative RT-PCR analysis of genes encoding three different classes of RNR. qRT-PCR was conducted on cDNA synthesized from wild-type, compared with Δ<i>nrdR</i> cells, both grown aerobically (A₅₅₀ = 0.6) (<b>G</b>) and anaerobically (A₅₅₀ = 0.6) (<b>H</b>). The means of three independent experiments are displayed, and the error bars represent the positive standard deviation <b>I)</b> dNTPs pool level of aerobic <i>P</i>. <i>aeruginosa</i> wild-type and <i>nrdR</i> mutant cells treated with 10 mM hydroxyurea (HU), measured by DPA assay. DNA contents were normalized with those of wild-type strain. Three independent experiments were performed and the mean ± standard deviation is shown. *, Significantly different compared with the wild-type strain in an unpaired <i>t</i>-test (<i>P</i><0.05).</p

Primers and probes used in this study.

<p>Primers and probes used in this study.</p