Time course of <i>t</i>-BOOH degradation by various <i>PA</i> strains.

<p>The rate of PA <i>t</i>-BOOH degradation, an organic hydroperoxide, was investigated using xylenol orange–iron reaction system as described in the materials and methods section. The exponential phase of PAO1 (white bar), <i>oxyR</i> (light gray bar), <i>oxyR</i>/p<i>oxyR</i> (dotted bar), <i>phnW</i>::Gm/p<i>phnW</i> (black bar) and <i>phnW</i>::Gm (dark gray bar) cells were used in this study in the present of 200 μM of <i>t</i>-BOOH and measured the rate of degradation of <i>t</i>-BOOH in each cell at difference time points. The percentage of <i>t</i>-BOOH remaining was reported after 12 min of incubation. The experiments were independently repeated at least three times and typical results are shown.</p

A model of <i>phnW</i> expression under difference conditions.

<p><b>(A).</b> In wild-type bacteria, <i>phnW</i> is continuously expressed in both untreated bacteria and during exposure to <i>t</i>-BOOH, while oxidized OxyR triggers activation of <i>ahpC</i> expression as previously shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.ref006" target="_blank">6</a>]. Wild-type bacteria used both of these proteins and Ohr to assist in either the direct or indirect degradation of <i>t</i>-BOOH. This results in efficient <i>t</i>-BOOH degradation to a by-product that is not toxic to the bacteria, thereby preventing damage to proteins, DNA and lipid. (<b>B).</b> Expression of the <i>phnW</i> gene is significant lowered in <i>oxyR</i> mutant bacteria (80%) when compared to wild-type bacteria with no induction of <i>ahpC</i> expression when exposed to <i>t</i>-BOOH. This results in greater susceptibility to <i>t</i>-BOOH of this mutant relative to wild-type bacteria since this strain likely only uses Ohr to help detoxify <i>t</i>-BOOH. (<b>C).</b> Lower expression of <i>phnW</i> was also detected in a PAO1 <i>ahpC</i> mutant compared to wild type bacteria (30%). A lack of this major AHP in response to <i>t</i>-BOOH coupled with lower expression of <i>phnW</i> with only Ohr detoxifying power remaining results in less protection of this mutant from <i>t</i>-BOOH toxicity. (<b>D).</b> An <i>ahpC ohr</i> mutant showed a slightly reduced expression of <i>phnW</i> when bacteria were exposed to <i>t</i>-BOOH, but no difference were observed under control conditions when compared to wild-type expression. Thus, a lack of both of the major <i>t</i>-BOOH detoxification proteins and a lower expression of <i>phnW</i> results in this mutant being the most susceptible to <i>t</i>-BOOH. This may aid in bacterial protection from endogenous free radicals that are continuously generated under aerobic conditions and/or at the earliest time period, when exposed to <i>t</i>-BOOH while the responding gene is not yet expressed. When the bacteria are exposed to <i>t</i>-BOOH, oxidized OxyR governs over-expression of AhpCF to help in its detoxification and together with Ohr, an OxyR independent <i>t</i>-BOOH detoxification protein (<b>Fig 7A</b>). A significantly lower expression level of <i>phnW</i> gene (~80%) was revealed in the <i>oxyR</i> mutant under both reduced and oxidized conditions, indicative of OxyR-mediated regulation of the <i>phnW</i> gene (<b>Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.g001" target="_blank">1</a></b>). When the <i>oxyR</i> mutant that had significantly reduced expression of AhpC and PhnW was exposed to <i>t</i>-BOOH, this event triggered an increased susceptibility to this oxidant when compared to wild-type bacteria (<b>Fig 7B</b>). Interestingly, <i>phnW</i> expression levels were also ~30% lower when compared to wild-type levels but are complemented when exposed to <i>t</i>-BOOH in the <i>ahpC</i> but not in the <i>ohr</i> mutant (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.g002" target="_blank">Fig 2</a></b>). This could be yet another mechanism by which bacteria used to protect themselves from <i>t</i>-BOOH toxicity when they lack the major <i>t</i>-BOOH detoxifying protein, AhpC (<b>Fig 7C</b>). Both <i>PA oxyR</i> and <i>PA ahpC</i> mutants still have an Ohr (organic hydroperoxide resistance), one of the major proteins that can contribute to <i>t</i>-BOOH detoxification. A genome search for “peroxidase” and “hydroperoxide” revealed 6 and 5 hits, respectively. This indicates that there are likely multiple redundant mechanisms to dispose of hyperoxides such as <i>t</i>-BOOH. Therefore, we expected that <i>phnW</i> expression should be higher in the <i>ohr ahpC</i> double mutant to protect bacteria cell from <i>t</i>-BOOH. Surprisingly, expression was ~20% lower in this strain after exposure to <i>t</i>-BOOH. The lower expression of PhnW after exposure to <i>t</i>-BOOH in the <i>ohr ahpC</i> double mutant may also contribute to the sensitivity of this double mutant to <i>t</i>-BOOH (<b>Fig 7D</b>). AhpC is OxyR-dependenct but Ohr is not. Therefore, it appears that <i>phnW</i> expression levels depend on the present of OxyR in the cell and also the level of AhpC in <i>ahpC</i> or <i>ahpC ohr</i> mutants. Therefore, it is likely that OxyR directly regulated <i>phnW</i> expression level through binding on an upstream sequence of this gene in certain condition to help protect cell from <i>t</i>-BOOH toxicity (<b>Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.g004" target="_blank">4</a></b>). Frustratingly, though the mechanism of PhnW in responding to <i>t</i>-BOOH or regulated by OxyR is still unclear, this study clearly indicates that this protein has the ability to assist in the protection of <i>PA</i> cell from <i>t</i>-BOOH toxicity.</p

Semi-quantitative expression of PA <i>phnW</i>.

<p>Total RNA was isolated from exponential phase <i>PA</i> PAO1 or its isogenic <i>oxyR</i> mutant. Then, 1 μl of cDNA was used to amplify the <i>phnW</i> promoter region with specific primers. The <i>omlA</i> gene was used as an internal constitutive control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.ref014" target="_blank">14</a>] and <i>ahpC</i> was used as a positive gene under OxyR control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.ref006" target="_blank">6</a>].</p

Localization of the OxyR binding domain of the <i>phnW</i> promoter by DNase I footprinting analysis.

<p><b>(</b><u><b>A).</b></u> The 199-bp upstream sequence of <i>phnW</i> containing a putative OxyR domain was used to treat with DNase I in the presence of OxyR at 0, 500 and 1000 nM. The digested DNA was analyzed on a 5% denaturing polyacrylamide gel, followed by autoradiography. The sequence to which OxyR binds on this fragment was identified. (<u><b>B).</b></u> The upstream sequences of <i>phnW</i> that are underlined are the primers used in this study. The bold letters indicate the OxyR binding domain within the <i>phnW</i> upstream sequence while the lower case bold letters indicate the putative OxyR binding site based upon a match to the consensus ATAG-N₇-CTAT-N₇-ATAG-N₇-CTAT sequence used to search for OxyR-dependent gene candidates. The underlined and bold ATG indicates the translational initiation codon of the <i>phnW</i> gene.</p

Determination of <i>phnW</i> expression levels in wild-type and mutant strains when exposed to <i>t</i>-BOOH.

<p>All bacteria which contained a <i>phnW</i>-<i>lacZ</i> transcriptional fusion plasmid (pQF-<i>phnW</i>) were grown to exponential phase and exposed to 250 μM <i>t</i>-BOOH. ß-galactosidase activity assays were reported as the mean +/- standard error compared to untreated bacteria. The assays were performed using three independent experiments. The white bars represent untreated bacteria while the gray bars represent <i>t</i>-BOOH treated organisms, respectively.</p

Sensitivity of bacteria to <i>t</i>-BOOH.

<p>Bacteria from the aerobic exponential phase (<u><b>A,B).</b></u> PAO1, <i>phnW</i>::Gm and <i>phnW</i>::Gm/p<i>phnW</i>) or (<u><b>E,F).</b></u> <i>ohr ahpC</i> and <i>ohr ahpC</i>/p<i>phnW</i>) or stationary phase (<u><b>C,D</b></u>). PAO1, <i>oxyR</i>/p<i>oxyR</i>, <i>phnW</i>::Gm, <i>oxyR phnW</i>::Gm, <i>oxyR</i> and <i>oxyR</i>/p<i>phnW</i>) were used to determine sensitivity to <i>t</i>-BOOH at 0.3 M (exponential) or 0.5 M (stationary), respectively. The experiments were independent and repeated at least three times. The values shown are means +/- standard error.</p

The OxyR-regulated <i>phnW</i> gene encoding 2-aminoethylphosphonate:pyruvate aminotransferase helps protect <i>Pseudomonas aeruginosa</i> from <i>tert</i>-butyl hydroperoxide

<div><p>The LysR member of bacterial transactivators, OxyR, governs transcription of genes involved in the response to H₂O₂ and organic (alkyl) hydroperoxides (AHP) in the Gram-negative pathogen, <i>Pseudomonas aeruginosa</i>. We have previously shown that organisms lacking OxyR are rapidly killed by <2 or 500 mM H₂O₂ in planktonic and biofilm bacteria, respectively. In this study, we first employed a bioinformatic approach to elucidate the potential regulatory breadth of OxyR by scanning the entire <i>P</i>. <i>aeruginosa</i> PAO1 genome for canonical OxyR promoter recognition sequences (ATAG-N₇-CTAT-N₇-ATAG-N₇-CTAT). Of >100 potential OxyR-controlled genes, 40 were strategically selected that were <u><b><i>not</i></b></u> predicted to be involved in the direct response to oxidative stress (e.g., catalase, peroxidase, etc.) and screened such genes by RT-PCR analysis for potentially positive or negative control by OxyR. Differences were found in 7 of 40 genes when comparing an <i>oxyR</i> mutant vs. PAO1 expression that was confirmed by ß-galactosidase reporter assays. Among these, <i>phnW</i>, encoding 2-aminoethylphosphonate:pyruvate aminotransferase, exhibited reduced expression in the <i>oxyR</i> mutant compared to wild-type bacteria. Electrophoretic mobility shift assays indicated binding of OxyR to the <i>phnW</i> promoter and DNase I footprinting analysis also revealed the sequences to which OxyR bound. Interestingly, a <i>phnW</i> mutant was more susceptible to <i>t</i>-butyl-hydroperoxide (<i>t</i>-BOOH) treatment than wild-type bacteria. Although we were unable to define the direct mechanism underlying this phenomenon, we believe that this may be due to a reduced efficiency for this strain to degrade <i>t</i>-BOOH relative to wild-type organisms because of modulation of AHP gene transcription in the <i>phnW</i> mutant.</p></div

Electrophoretic mobility shift assay (EMSA) to indicate OxyR binding to the promoter region upstream of <i>phnW</i>.

<p>Purified OxyR was added to 0.8 ng of DIG-nonradioactive labeled 199-bp DNA fragment of <i>phnW</i> containing the putative <i>phnW</i> OxyR binding domain in the binding buffer and separated on a polyacrylamide gel as described in the materials methods section. The binding reaction consisted of a labeled <i>phnW</i> fragment and various quantities of OxyR protein. UP is the addition of 2 μg of <u><b>u</b></u>nrelated <u><b>p</b></u>rotein (BSA) to the binding reaction; F is <u><b>f</b></u>ree probe; the addition of increasing concentrations of OxyR (100, 250, 500 nM) to labeled <i>phnW</i> probe are listed; CP is the addition of 125-fold excess of unlabeled <i>phnW</i> DNA to the binding reaction; UD, the addition of 125-fold excess of <u><b>u</b></u>nrelated <u><b>D</b></u>NA (pUCP20 plasmid) to the binding reaction. The positions of free and bound <i>phnW</i> probe are shown on the left (see arrows).</p