Analyses of the regulatory mechanism and physiological roles of Pseudomonas aeruginosa OhrR, a transcription regulator and a sensor of organic hydroperoxides

Abstract

ohrR encodes an organic hydroperoxide sensor and a transcriptional repressor that regulates organic hydroperoxide-inducible expression of a thiol peroxidase gene, ohr, and itself. OhrR binds directly to the operators and represses transcription of these genes. Exposure to an organic hydroperoxide leads to oxidation of OhrR and to subsequent structural changes that result in the loss of the repressor's ability to bind to the operators that allow expression of the target genes. Differential induction of ohrR and ohr by tert-butyl hydroperoxide suggests that factors such as the repressor's dissociation constants for different operators and the chemical nature of the inducer contribute to OhrR-dependent organic hydroperoxide-inducible gene expression. ohrR and ohr mutants show increased and decreased resistance to organic hydroproxides, respectively, compared to a parental strain. Moreover, the ohrR mutant had a reduced-virulence phenotype in the Pseudomonas aeruginosa-Caenorhabditis elegans pathogenicity model.

FIG. 1.

Expression analysis of ohrR and ohr. (A) Genome organization of the ohr-ohrR operon. (B) RT-PCR of the intergenic region between the ohr and ohrR genes. The primers BT468 and BT484 were used, giving the expected 222-bp PCR products. +, positive control; −, negative control; M, molecular weight marker (GeneRuler 100-bp DNA ladder; Fermentas); WT, PAO1 DNA as the template. (C and D) Phosphor images of Northern blots of total RNA extracted from wild-type P. aeruginosa cultures treated with various oxidants for 15 min. Northern blots were hybridized with radiolabeled probes specific to ohrR (C) or ohr (D). Positively hybridizing bands are indicated by arrows. UN, uninduced; CHP, 250 μM CHP; tBOOH, 250 μM tBOOH; MD, 500 μM menadione; PQ, 500 μM paraquat; H₂O₂, 500 μM H₂O₂. (E) Quantitative analysis of Northern blot analysis of wild-type P. aeruginosa cultures treated with various concentrations of CHP and tBOOH for 30 min.

FIG. 2.

Primer extension analysis of ohrR (A) or ohr (B) using total RNA extracted from P. aeruginosa cultures with and without cumene hydroperoxide treatment. Arrows indicate the transcription start site. The Sanger sequencing ladder to the left of the primer extension lanes was generated using pGEM-3zf(+) as the template and M13 forward as a primer. UN, uninduced; CHP, 250 μM CHP. (C and D) Promoter sequences of ohrR (C) or ohr (D). The putative −10 and −35 promoter regions, the transcription start site (+1), and the putative ohrR and ohr translation start codons are shown in bold. Locations and names of oligonucleotides (BT483, BT467, BT484, and BT514) used in this study are shown. The DNase I-protected regions for OhrR binding are boxed. The putative OhrR binding sites on each promoter are aligned with the predicted conserved sequence of the OhrR box.

FIG. 3.

Effect of an ohrR mutation on ohrR and ohr expression. (A) β-Galactosidase activities of strains containing an ohr-lacZ fusion. (B) β-Galactosidase activities of strains containing an ohrR-lacZ fusion. (C) β-Galactosidase activities of strains containing ohrR-lacZ and ohr-lacZ fusions in the wild type induced with either 1 mM CHP or 1 mM tBOOH. (D) Northern blot analysis of ohr. Overnight cultures of strains were grown to mid-log phase and collected for β-galactosidase analysis or for mRNA extraction for Northern blot analysis. Experiments were performed three times; error bars represent the standard errors of the means. UN, control sample; CHP, 1 mM CHP; tBOOH, 1 mM tBOOH.

FIG. 4.

(A) Binding of OhrR to the ohrR promoter fragment. The percentage of OhrR bound to the ohrR operator fragment was determined using a gel shift assay with the ohrR promoter and purified OhrR. The various concentrations of OhrR added to the binding reactions are indicated above each lane. The unbound promoter fragment is designated UB, and the protein-DNA complex is designated B. (B and C) Mapping of the OhrR binding sites on the P. aeruginosa ohrR (B) and ohr (C) promoter fragments by DNase I footprinting. PCR-generated probe fragments were labeled on one strand by end labeling one of the primers with ³²P prior to amplification. The sequencing ladder used to localize the binding sites on the ohrR promoter (B) was generated using pGEM-3zf(+) as a template and M13 forward as a primer. The sequencing ladder (G, A, T, C) used to localize the binding sites on the ohr promoter (C) was generated using the promoter fragment itself as a template and the same labeled oligonucleotide as was used to generate the probe as a primer. “M” indicates molecular weight marker, φx174/HinfI (Promega). Numbers above each lane indicate amounts of the OhrR protein (μM) used in each reaction.

FIG. 5.

Effect of OhrR cysteine mutations on expression of ohr. Expression of ohr was monitored by RT-PCR. The first panel shows the level of ohr expression. The bottom panel shows the expression of a housekeeping gene, the 16S rRNA gene. CHP, 250 μM CHP.

FIG. 6.

Binding of OhrR, OhrRC19S, and OhrRC121S on the ohrR promoter (A) or the ohr promoter (B). DTT, 1 mM DTT; CHP, 1 mM CHP. Protein: OhrR, OhrRC19S, or OhrRC121S, as indicated on the top of each panel.

FIG. 7.

Sensitivities of wild-type P. aeruginosa and ohrR and ohr mutants to oxidants. Exponential-phase cells grown in LB medium were serially diluted (10-fold dilutions), and 10 μl of each dilution was spotted onto LB agar containing either 500 μM H₂O₂, 1 M CHP, 2 M CHP, or 1 M tBOOH. After incubation at 28°C for approximately 24 h, growth of the bacteria was observed. The experiment was repeated three times with similar results.

FIG. 8.

Caenorhabditis elegans/P. aeruginosa pathogenicity tests. After incubation of P. aeruginosa with C. elegans, the numbers of live and dead/paralyzed worms were recorded over time. Percentage death was then calculated. The data show percentage death at 5 h. These results are representative of at least three independent experiments. Error bars indicate the standard errors for the technical replicates.