Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: A tool for identifying differentially regulated genes

Pseudomonas aeruginosa is a major cause of nosocomial (hospital-derived) infections, is the predominant pathogen in chronic cystic fibrosis lung infections, and remains difficult to treat due to its high intrinsic antibiotic resistance. The completion of the P. aeruginosa PAO1 genome sequence provides the opportunity for genome-wide studies to increase our understanding of the pathogenesis and biology of this important pathogen. In this report, we describe the construction of a mini-Tn5-luxCDABE mutant library and a high-throughput inverse PCR method to amplify DNA flanking the site of insertion for sequencing and insertion site mapping. In addition to producing polar knockout mutations in nonessential genes, the promoterless luxCDABE reporter present in the transposon serves as a real-time reporter of gene expression for the inactivated gene. A total of 2519 transposon insertion sites were mapped, 77% of which were nonredundant insertions. Of the insertions within an ORF, -55% of total and unique insertion sites were transcriptional luxCDABE fusions. A bias toward low insertion-site density in the genome region that surrounds the predicted terminus of replication was observed. To demonstrate the utility of chromosomal lux fusions, we performed extensive regulatory screens to identify genes that were differentially regulated under magnesium or phosphate limitation. This approach led to the discovery of many known and novel genes necessary for these environmental adaptations, including genes involved in resistance to cationic antimicrobial peptides. This dual-purpose mutant library allows for functional and regulation studies and will serve as a resource for the research community to further our understanding of P. aeruginosa biology

Contribution of the PhoP-PhoQ and PmrA-PmrB Two-Component Regulatory Systems to Mg(2+)-Induced Gene Regulation in Pseudomonas aeruginosa

When grown in divalent cation-limited medium, Pseudomonas aeruginosa becomes resistant to cationic antimicrobial peptides and polymyxin B. This resistance is regulated by the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems. To further characterize Mg(2+) regulation in P. aeruginosa, microarray transcriptional profiling was conducted to compare wild-type P. aeruginosa grown under Mg(2+)-limited and Mg(2+)-replete conditions to isogenic phoP and pmrA mutants grown under Mg(2+)-limited conditions. Under Mg(2+)-limited conditions (0.02 mM Mg(2+)), approximately 3% of the P. aeruginosa genes were differentially expressed compared to the expression in bacteria grown under Mg(2+)-replete conditions (2 mM Mg(2+)). Only a modest subset of the Mg(2+)-regulated genes were regulated through either PhoP or PmrA. To determine which genes were directly regulated, a bioinformatic search for conserved binding motifs was combined with confirmatory reverse transcriptase PCR and gel shift promoter binding assays, and the results indicated that very few genes were directly regulated by these response regulators. It was found that in addition to the previously known oprH-phoP-phoQ operon and the pmrHFIJKLM-ugd operon, the PA0921 and PA1343 genes, encoding small basic proteins, were regulated by Mg(2+) in a PhoP-dependent manner. The number of known PmrA-regulated genes was expanded to include the PA1559-PA1560, PA4782-PA4781, and feoAB operons, in addition to the previously known PA4773-PA4775-pmrAB and pmrHFIJKLM-ugd operons

Copyright © 2006, American Society for Microbiology. All Rights Reserved. Contribution of the PhoP-PhoQ and PmrA-PmrB Two-Component Regulatory Systems to Mg 2 �-Induced Gene Regulation

When grown in divalent cation-limited medium, Pseudomonas aeruginosa becomes resistant to cationic antimicrobial peptides and polymyxin B. This resistance is regulated by the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems. To further characterize Mg 2 � regulation in P. aeruginosa, microarray transcriptional profiling was conducted to compare wild-type P. aeruginosa grown under Mg 2 �-limited and Mg 2 �-replete conditions to isogenic phoP and pmrA mutants grown under Mg 2 �-limited conditions. Under Mg 2 �-limited conditions (0.02 mM Mg 2 �), approximately 3 % of the P. aeruginosa genes were differentially expressed compared to the expression in bacteria grown under Mg 2 �-replete conditions (2 mM Mg 2 �). Only a modest subset of the Mg 2 �-regulated genes were regulated through either PhoP or PmrA. To determine which genes were directly regulated, a bioinformatic search for conserved binding motifs was combined with confirmatory reverse transcriptase PCR and gel shift promoter binding assays, and the results indicated that very few genes were directly regulated by these response regulators. It was found that in addition to the previously known oprH-phoP-phoQ operon and the pmrHFIJKLM-ugd operon, the PA0921 and PA1343 genes, encoding small basic proteins, were regulated by Mg 2 � in a PhoP-dependent manner. The number of known PmrAregulated genes was expanded to include the PA1559-PA1560, PA4782-PA4781, and feoAB operons, in addition to the previously known PA4773-PA4775-pmrAB and pmrHFIJKLM-ugd operons

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

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

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

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