Mapping of the OspR binding sites on the <i>Pseudomonas aeruginosa gpx</i> promoter region.

<p>Gel shift assay of purified OspR protein with various <i>gpx</i> promoter fragments. The concentrations of OspR protein added in the binding reactions are indicated above each lane. The unbound promoter fragment was designated p_gpx, and the protein-DNA complex was designated p_gpx-OspR.</p

Regulation of <i>ohr</i> by OspR, the alteration of <i>ohr</i> expression in the <i>ospR</i> mutant and the alteration of <i>ospR</i> expression in the <i>ohrR</i> mutant.

<p>Quantitative real-time PCR was performed to measure relative expression levels (2^-ΔΔCt) of <i>gpx</i> (A) and <i>ohr</i> (B) in various strains. (C) Expression of <i>gpx</i> in wild-type and <i>ohrR</i> mutant cultures treated with various concentrations of CHP (mM) for 15 min. (D) Expression of <i>ohr</i> in wild-type and <i>ospR</i> mutant cultures treated with various concentrations of CHP (mM) for 15 min. The expression in the wild-type strain was set to 1. Significant differences (<i>P</i> < 0.05) between samples are denoted with asterisks. For (C) and (D), means were compared between wild-type strain and mutant strain at the same condition.</p

Expression analysis of <i>gpx</i> and <i>ospR</i> in the presence of oxidants.

<p>Quantitative real-time PCR was performed to measure relative expression levels (2^-ΔΔCt) of <i>gpx</i> (A) and <i>ospR</i> (B) in wild-type <i>Pseudomonas aeruginosa</i> with various oxidant treatments for 15 min. The uninduced sample was set to 1. UN, uninduced; CHP500, 500 μM CHP; tBOOH500, 500 μM tBOOH; LOOH100, 100 μM linoleic hydroperoxide; H₂O₂500, 500 μM H₂O₂; H₂O₂1000, 1000 μM H₂O₂; H₂O₂4000, 4000 μM H₂O₂; MD50, 50 μM menadione; MD500, 500 μM menadione; PQ50, 50 μM paraquat; PQ100, 100 μM paraquat; PQ500, 500 μM paraquat. Significant differences (<i>P</i> < 0.05) between the treated samples and uninduced sample are denoted with asterisks.</p

Regulation of Organic Hydroperoxide Stress Response by Two OhrR Homologs in <i>Pseudomonas aeruginosa</i>

<div><p><i>Pseudomonas aeruginosa ohrR</i> and <i>ospR</i> are gene homologs encoding oxidant sensing transcription regulators. OspR is known to regulate <i>gpx</i>, encoding a glutathione peroxidase, while OhrR regulates the expression of <i>ohr</i> that encodes an organic peroxide specific peroxiredoxin. Here, we show that <i>ospR</i> mediated <i>gpx</i> expression, like <i>ohrR</i> and <i>ohr</i>, specifically responds to organic hydroperoxides as compared to hydrogen peroxide and superoxide anion. Furthermore, the regulation of these two systems is interconnected. OspR is able to functionally complement an <i>ohrR</i> mutant, i.e. it regulates <i>ohr</i> in an oxidant dependent manner. In an <i>ohrR</i> mutant, in which <i>ohr</i> is derepressed, the induction of <i>gpx</i> expression by organic hydroperoxide is reduced. Likewise, in an <i>ospR</i> mutant, where <i>gpx</i> expression is constitutively high, oxidant dependent induction of <i>ohr</i> expression is reduced. Moreover, <i>in vitro</i> binding assays show that OspR binds the <i>ohr</i> promoter, while OhrR binds the <i>gpx</i> promoter, albeit with lower affinity. The binding of OhrR to the <i>gpx</i> promoter may not be physiologically relevant; however, OspR is shown to mediate oxidant-inducible expression at both promoters. Interestingly, the mechanism of OspR-mediated, oxidant-dependent induction at the two promoters appears to be distinct. OspR required two conserved cysteines (C24 and C134) for oxidant-dependent induction of the <i>gpx</i> promoter, while only C24 is essential at the <i>ohr</i> promoter. Overall, this study illustrates possible connection between two regulatory switches in response to oxidative stress.</p></div

Mapping of the OspR binding sites on the <i>P</i>. <i>aeruginosa gpx</i> (A) and <i>ohr</i> (B) promoter fragments by DNase I footprinting.

<p>PCR-generated probe fragments were labeled on one strand by end-labeling one of the primers with ³²P prior to amplification. The sequencing ladder (G, A, T, C) used to localize the binding sites on the promoters were generated using the promoter fragment itself as a template and the same labeled oligonucleotide as was used to generate the probe as a primer. Numbers above each lane indicate amounts of the OpsR and OhrR protein (μM) used in each reaction. The regions protected by OspR or OhrR are indicated by vertical lines. Hypersensitive site is indicated by asterisk.</p

Gpx confers resistance to organic hydroperoxide and H₂O₂.

<p>(A) Percent survival of exponential phase <i>P</i>. <i>aeruginosa ohr</i> mutant containing pGpx treated with 1 mM tBOOH in comparison to the wild-type and the <i>ohr</i> mutant. (B) Percent survival of exponential phase <i>P</i>. <i>aeruginosa katA</i> mutant containing pGpx treated with 2.5 mM H₂O₂ in comparison to the wild-type and the <i>katA</i> mutant. Significant differences (<i>P</i> < 0.05) between samples are denoted with asterisks.</p

Determination of paraquat resistance levels in <i>P</i>. <i>aeruginosa</i> strains.

<p>(A) Paraquat resistance levels in PAO1 containing the mnin-Tn7 vector control (PAO1::Tn7T, red) and Δ<i>finR</i> mutants containing Tn7T (dotted green), Tn7T-<i>finR</i> (purple), Tn7T-<i>fprA</i> (dotted blue), Tn7T-<i>fprB</i> (yellow), or Tn7T-<i>fdx1</i> (dotted black) were determined using plate sensitivity assays. (B) Paraquat (150 μM) resistance levels of <i>P</i>. <i>aeruginosa</i> strains were determined using LB with and without 1% (w/v) KNO₃ supplementation and incubated under aerobic and anaerobic atmospheres. The survival is expressed as a percentage of the CFU on LB plates containing paraquat over the CFU on plates without paraquat. Data shown are means ± SD from three independent experiments.</p

Expression analysis <i>finR</i> and <i>fprA</i> in response to various stresses.

<p>The expression levels of <i>finR</i> (A) and <i>fprA</i> (B) were determined using real-time RT-PCR. Exponential-phase cultures of <i>P</i>. <i>aeruginosa</i> PAO1 were subjected to various stress conditions, including 1 mM H₂O₂, superoxide anion-generating agents (0.5 mM plumbagin [PB], 0.5 mM menadione [MD] and 0.5 paraquat [PQ]), organic hydroperoxides (1 mM cumene hydroperoxide [CHP] and 1 mM <i>t</i>-butyl hydroperoxide [tBH]), 1 mM 2,2’-dipyridyl (DIPY), high salts (0.5 M NaCl and 0.5 M KCl), or 0.04% NaOCl for 15 minutes prior to RNA preparation for real-time RT-PCR analysis. Relative expression was analyzed using the 16S rRNA gene as the normalizing gene and was expressed as the fold expression relative to the level of uninduced (UN) PAO1. Data shown are means ± SD of three independent experiments.</p

List of plasmids used in this study.

<p>List of plasmids used in this study.</p

List of primers used in this study.

<p>List of primers used in this study.</p