PsrA controls the synthesis of the <i>Pseudomonas aeruginosa</i> quinolone signal via repression of the FadE homolog, PA0506

<div><p><i>Pseudomonas aeruginosa</i> is a ubiquitous, Gram-negative opportunistic pathogen that can cause disease in various sites within the human body. This bacterium is a major source of nosocomial infections that are often difficult to treat due to high intrinsic antibiotic resistance and coordinated virulence factor production. <i>P</i>. <i>aeruginosa</i> utilizes three cell-to-cell signaling systems to regulate numerous genes in response to cell density. One of these systems utilizes the small molecule 2-heptyl-3-hydroxy-4-quinolone (<i>Pseudomonas</i> quinolone signal [PQS]) as a signal that acts as a co-inducer for the transcriptional regulator PqsR. Quinolone signaling is required for virulence in multiple infection models, and PQS is produced during human infections, making this system an attractive target for potential drug development. In this study we have examined the role of a TetR-type transcriptional regulator, PsrA, in the regulation of PQS production by <i>P</i>. <i>aeruginosa</i>. Previous studies showed that PsrA regulates genes of the fatty acid β-oxidation pathway, including <i>PA0506</i>, which encodes a FadE homolog. In this report, we show that deletion of <i>psrA</i> resulted in a large decrease in PQS production and that co-deletion of <i>PA0506</i> allowed PQS production to be restored to a wild type level. We also found that PQS production could be restored to the <i>psrA</i> mutant by the addition of oleic or octanoic acid. Taken together, our data suggest that <i>psrA</i> positively affects PQS production by repressing the transcription of <i>PA0506</i>, which leads to a decrease in the conversion of acyl-CoA compounds to enoyl-CoA compounds, thereby allowing some octanoyl-CoA to escape the ß-oxidation pathway and serve as a PQS precursor.</p></div

Increased expression of <i>PA0506</i> alters PQS production.

<p>The indicated strains were grown for 24 h in LB medium harboring pHERD20T (control plasmid) or pGW-21 (<i>PA0506</i> expression plasmid) indicated by a minus or plus symbol, respectively. Cultures were supplemented with 0.5% L-arabinose to induce <i>PA0506</i> expression, and PQS was then extracted and quantified. Data are presented as the average ± SD of three independent experiments.</p

Bacterial strains and plasmids used in this study.

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

PsrA positively controls PQS production in <i>P</i>. <i>aeruginosa</i>.

<p>Strains PAO1 and PGW-Δ<i>psrA</i> were grown for 24 h in LB medium, then (A and C) PQS or (B and D) pyocyanin was extracted and quantified as described in Materials and Methods. The presence of <i>psrA</i> expression plasmid (pGW-2) or pHERD20T (control plasmid) is indicated by a plus or minus, respectively. For C and D, LB media was supplemented with 0.5% L-arabinose to induce <i>psrA</i>. All data are presented as the average ± SD of three independent experiments.</p

PsrA positively controls <i>pqsA</i>.

<p>(A) qRT-PCR was performed on RNA from strains PAO1 and PGW-Δ<i>psrA</i> to analyze the relative expression of <i>lasR</i>, <i>pqsH</i>, <i>pqsR</i> and <i>pqsA</i>. Data are presented as average fold change ± SD in the <i>psrA</i> mutant compared to the wild type strain (set to a value of 1). The gene <i>clpX</i> was used as a reference gene to normalize expression and each experiment was completely repeated three times. (B and C) PQS production by strains PAO1 or PGW-Δ<i>psrA</i> expressing (B) PQS synthetic genes or (C) RpoS. Cultures were grown for 24 h in LB medium supplemented with 0.5% L-arabinose to induce genes. PQS was then extracted and quantified as described in Materials and Methods. Data are presented as the average ± SD of three independent experiments. (B) Plasmids contained by strains are: open bars, control vector; solid bars, <i>pqsABCD</i> expression vector; and hatched bars, <i>pqsH</i> expression vector. (C) Presence of the <i>rpoS</i> expression vector is indicated by a plus (+) symbol.</p

<i>PA0506</i> is required for C18 fatty acid to serve as a PQS precursor.

<p>Cultures of the indicated strains were grown for 24 h in LB medium alone (open bars) or supplemented with either octanoic acid (black bars) or oleic acid (hatched bars). PQS was extracted and quantified as described in Materials and Methods. Data are presented as the average ± SD of three independent experiments.</p

PsrA directly regulates <i>PA0506</i> to control PQS production.

<p>(A) qRT-PCR was performed on RNA from strains PAO1 and PGW-Δ<i>psrA</i>. Data are presented as average fold change ± SD of expression in the <i>psrA</i> mutant as compared to expression in the wild type strain (set to a value of 1). The gene <i>clpX</i> was used as a reference for normalization and experiments were repeated three separate times. Targeted genes are listed below the columns. (B) The indicated single and double mutants were assessed for PQS production after 24 h growth. (C) EMSA for promoter regions of <i>psrA</i> (positive control) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189331#pone.0189331.ref041" target="_blank">41</a>], <i>PA0506</i>, <i>PA0507</i>, <i>PA0508</i>, and <i>kynA</i> (negative control) was performed using 0, 0.1, 1.0, 10, and 50 ng of PsrA-his tag protein. Binding is indicated by an arrow and data are representative of three independent experiments.</p

<i>P. aeruginosa</i> Zur binds the <i>znuA</i> promoter in a zinc dependent-manner.

<p>Radiolabeled <i>znuA</i> promoter fragment was incubated with increasing concentrations of recombinant <i>P. aeruginosa</i> Zur (rZur) as indicated above the figure. The plus (+) or minus (-) symbols below the lanes indicate whether the reaction contained a 100-fold excess of unlabeled <i>znuA</i> promoter fragment (specific competitor), unlabeled internal <i>znuC</i> DNA fragment (nonspecific competitor), or 5 µM TPEN (zinc chelator).</p

Loss of Zur results in intracellular zinc accumulation.

<p>Intracellular zinc content in strains PAO1 and PAO-Zur after growth in high zinc medium as measured by ICP-AES. The data presented are the mean ± SD of at least four replicates. The asterisk indicates a significant difference between intracellular zinc in stain PAO1 versus PAO-Zur.</p

<i>zur</i> is encoded in an operon with <i>znuC</i> and <i>znuB</i>.

<p>(A) RT-PCR was performed on total RNA isolated from <i>P. aeruginosa</i> strain PA01 using the indicated primer pairs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075389#pone.0075389.s003" target="_blank">Table S2</a>). Reverse Transcriptase was added to the reaction in lanes 2, 5, 8, and 11(RT+) but omitted from reactions in lanes 4, 7, 10, and 13 (RT-). Genomic DNA was amplified using the indicated primers as a positive control in lanes 3, 6, 9, and 12 (DNA); (B) Schematic representation of the <i>zur</i> (np20) operon with relative location of the indicated primers used for RT-PCR.</p