Genes for Carbon Metabolism and the ToxA Virulence Factor in <em>Pseudomonas aeruginosa</em> Are Regulated through Molecular Interactions of PtxR and PtxS

<div><p>Homologs of the transcriptional regulator PtxS are omnipresent in <em>Pseudomonas</em>, whereas PtxR homologues are exclusively found in human pathogenic <em>Pseudomonas</em> species. In all <em>Pseudomonas</em> sp., PtxS with 2-ketogluconate is the regulator of the gluconate degradation pathway and controls expression from its own promoter and also from the P<em>_gad</em> and P<em>_kgu</em> for the catabolic operons. There is evidence that PtxS and PtxR play a central role in the regulation of exotoxin A expression, a relevant primary virulence factor of <em>Pseudomonas aeruginosa</em>. We show using DNaseI-footprint analysis that in <em>P. aeruginosa</em> PtxR binds to the -35 region of the P<em>_toxA</em> promoter in front of the exotoxin A gene, whereas PtxS does not bind to this promoter. Bioinformatic and DNaseI-footprint analysis identified a PtxR binding site in the P<em>_kgu</em> and P<em>_gad</em> promoters that overlaps the -35 region, while the PtxS operator site is located 50 bp downstream from the PtxR site. <em>In vitro</em>, PtxS recognises PtxR with nanomolar affinity, but this interaction does not occur in the presence of 2-ketogluconate, the specific effector of PtxS. DNAaseI footprint assays of P<em>_kgu</em> and P<em>_gad</em> promoters with PtxS and PtxR showed a strong region of hyper-reactivity between both regulator binding sites, indicative of DNA distortion when both proteins are bound; however in the presence of 2-ketogluconate no protection was observed. We conclude that PtxS modulates PtxR activity in response to 2-ketogluconate by complex formation in solution in the case of the P<em>_toxA</em> promoter, or <em>via</em> the formation of a DNA loop as in the regulation of gluconate catabolic genes. Data suggest two different mechanisms of control exerted by the same regulator.</p> </div

Schematic diagrams of the P<i>_toxA</i> and P<i>_gad</i> regulation models.

<p>Left.- The PtxR dimer binds the -35 region of P<i>_toxA</i> and PtxS in solution inhibits other interactions; with 2-ketogluconate (2 KG) PtxS is released and PtxR can recruit RNA polymerase to promote transcription. Right.- PtxS and PtxR bound to their operator sites interact and induce DNA bending, when 2-ketogluconate is present the PtxS repressor is released and PtxR recruits RNA polymerase to facilitate transcription from the catabolic promoter.</p

DNAseI footprinting assay of P<i>_gad</i> promoter with PtxS, PtxR or both proteins.

<p>Conditions as in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039390#pone-0039390-g002" target="_blank">Figure 2</a>. Lane 1, free DNA; Lane 2 DNA with 10 µM PtxS; Lane 3, DNA with 10 µM PtxR; lane 4 DNA with 10 µM PtxS +10 µM PtxR, lane 5 as lane 4 but with 1 mM 2-ketogluconate.</p

Analysis of the P<i>_kgu</i> and P<i>_gad</i> promoters.

<p>A) Determination of the transcription start point using primer extension analysis of the P<i>_kgu</i> (left), P<i>_gad</i> (central) and P<i>_toxA</i> (right) promoters. The sequencing ladder was used to estimate the size of the transcript. B) Sequences of the three promoters. The transcriptional start sites are indicated by arrows. The palindromic PtxS and PtxR binding sites, and the -10 and -35 binding sites for the RNA polymerase are shown.</p

Genetic organization of the open reading frames which are under the control of PtxS and PtxR.

<p>The physical organization of the region <i>ptxS</i> and <i>ptxR</i> genes was established by Hamood <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039390#pone.0039390-Hamood2" target="_blank">[8]</a>.</p

Interaction of PtxR with promoters P<i>_kgu</i>, P<i>_toxA</i>, P<i>_gad</i> and P<i>_ptxS</i>.

<p><b>A</b>) Electrophoretic mobility shift assays for the binding of PtxR to P<i>_kgu</i>_, P<i>_toxA</i>_, P<i>_gad</i> and P<i>_ptxS</i> and of PtxS to P<i>toxA</i>. The size of the fragments used in EMSA were 289-, 248-, 495-, and 324-bp for the P<i>_kgu</i>, P<i>_gad</i>, P<i>_toxA</i> and P<i>_txS</i> respectively. Experiments were carried out with PtxR or PtxS concentrations in the range between 0.4 and 10 µM as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039390#s4" target="_blank">Materials and Methods</a>. Free DNA and DNA/protein complex are indicated. <b>B</b>) <b>DNAseI footprinting assays of promoter P</b><b><i>_gad</i></b> and P<i>_toxA</i><b>.</b> Experiments were conducted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039390#s4" target="_blank">Materials and Methods</a>. From left to right Lane 1: free DNA, lanes 2 and 3: DNA +10 or 20 µM PtxR, respectively, lanes 4 to 7: DNA sequencing ladder. The region protected by PtxR is indicated by a vertical line and the corresponding sequence is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039390#pone-0039390-g003" target="_blank">Figure 3B</a>.</p

Strains and plasmids used in this study.

<p>Km^r, Str^r, Tc^r and Ap^r stand for resistance to kanamycin, streptomycin, tetracycline and ampicillin, respectively.</p

Binding studies of PtxS to native and mutant PtxR.

<p>(A) In this series of experiments 3 µM PtxR was titrated with 3.2 µl aliquots of 50 µM of PtxS in the absence (a), and in the presence (b) of 2-ketogluconate added at a final concentration of 1 mM. Lower panel: Integrated and dilution corrected raw data for the titration of PtxR with PtxS. Data were fitted with the “Two binding site model” of the MicroCal version of ORIGIN. (B) EMSA with target DNA in the absence (Lane 1) or in the presence of 10 µM PtxS (Lane 2), 10 µM PtxR (Lane 4) or 10 µM PtxS and PtxR (Lane 3).</p

Functional categories in environmental and clinical <i>P</i>. <i>putida</i> isolates.

<p>Functional categories in environmental and clinical <i>P</i>. <i>putida</i> isolates.</p

Phenotypical array characterization of clinical strains.

<p>Graphics show the growth of the studied <i>P</i>. <i>putida</i> clinical strains and KT2440 in the presence of heavy metals <b>(A)</b>; oxidative and other stressors <b>(B)</b>; DNA intermediates as the only nitrogen source <b>(C)</b>; amino acids <b>(D)</b> or fatty acid <b>(E)</b> as the only carbon source; and cysteine (cys) as the only sulfur (S), nitrogen (N), carbon (C) or carbon+nitrogen source (C+N). Blue bars, HB13667; red bars, H8234; green bars, HB3267 and white bars, KT2440. Error bars indicate standard deviation from three experimental repetitions. In parentheses concentration of stressor used, if concentration is not indicated means this was 5 mM. HB4184 was not included in this study because it forms lumps and thick biofilms in these culture conditions.</p