Analysis of the Organic Hydroperoxide Response of Chromobacterium violaceum Reveals That OhrR Is a Cys-Based Redox Sensor Regulated by Thioredoxin

Organic hydroperoxides are oxidants generated during bacterial-host interactions. Here, we demonstrate that the peroxidase OhrA and its negative regulator OhrR comprise a major pathway for sensing and detoxifying organic hydroperoxides in the opportunistic pathogen Chromobacterium violaceum. Initially, we found that an ohrA mutant was hypersensitive to organic hydroperoxides and that it displayed a low efficiency for decomposing these molecules. Expression of ohrA and ohrR was specifically induced by organic hydroperoxides. These genes were expressed as monocistronic transcripts and also as a bicistronic ohrR-ohrA mRNA, generating the abundantly detected ohrA mRNA and the barely detected ohrR transcript. The bicistronic transcript appears to be processed. OhrR repressed both the ohrA and ohrR genes by binding directly to inverted repeat sequences within their promoters in a redox-dependent manner. Site-directed mutagenesis of each of the four OhrR cysteine residues indicated that the conserved Cys21 is critical to organic hydroperoxide sensing, whereas Cys126 is required for disulfide bond formation. Taken together, these phenotypic, genetic and biochemical data indicate that the response of C. violaceum to organic hydroperoxides is mediated by OhrA and OhrR. Finally, we demonstrated that oxidized OhrR, inactivated by intermolecular disulfide bond formation, is specifically regenerated via thiol-disulfide exchange by thioredoxin (but not other thiol reducing agents such as glutaredoxin, glutathione and lipoamide), providing a physiological reducing system for this thiol-based redox switch.INCT de Processos Redox em Biomedicina-Redoxoma (FAPESP/CNPq/CAPES) [2008/57721-3, 2008/573530]INCT de Processos Redox em BiomedicinaRedoxoma (FAPESP/CNPq/CAPES)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [07/58147-6]CNPqCNP

<i>Chromobacterium violaceum</i> possesses four proteins belonging to the OsmC/Ohr family.

<p>(<b>A</b>) Phylogenetic tree of OsmC/Ohr proteins (PF02566) from six selected bacterial genomes. Protein sequences from <i>Chromobacterium violaceum</i> (CHRVO), <i>Pseudomonas aeruginosa</i> (PSEAE), <i>Xylella fastidiosa</i> (XYLFA), <i>Xanthomonas campestris</i> pv. <i>phaseoli</i> (XANCH), <i>Bacillus subtilis</i> (BACSU) and <i>Escherichia coli</i> (ECOLI) were aligned using the CLUSTAL W 2.1 program. The tree was obtained by using the neighbor-joining method and Blosum62 from the CLUSTAL W alignment. (<b>B</b>) The schematic depicts the chromosomal region of <i>C. violaceum</i> around the <i>ohrA</i> and <i>ohrR</i> genes.</p

Investigation of intermolecular disulfide bond formation on OhrR by nonreducing SDS-PAGE.

<p>Purified OhrR variants were reduced by DTT treatment. Samples of reduced proteins were either untreated (−) or treated (+) with 100 µM cumene hydroperoxide (CuOOH) for 30 min before alkylation. The NEM-alkylated proteins were separated by nonreducing SDS-PAGE, stained with Coomassie Brilliant Blue and subsequently destained. The monomeric (M) and dimeric (D) forms of OhrR are indicated by arrows. M represents the protein molecular mass standards.</p

Primers used in this study.

a<p>Underlined letters indicate the restriction enzyme recognition sites, used for cloning purposes.</p

Degradation of peroxides by the <i>C. violaceum</i> ATCC 12472 and Δ<i>ohrA</i> and Δ<i>ohrR</i> mutant strains.

<p>Cultures grown in LB medium to an OD at 600 nm of 0.7 were divided and either treated with 300 µM tBOOH (<b>A</b>), 200 µM CuOOH (<b>B</b>) or 500 µM H₂O₂ (<b>C</b>). The level of residual peroxides remaining in the culture medium was determined at 5-min intervals using a xylenol orange assay.</p

Effect of thiol systems on OhrR reduction <i>in vitro</i>, as monitored by EMSA.

<p>Samples of purified <i>C. violaceum</i> OhrR (5 µM air-oxidized protein) were treated with the indicated reductants for 30 min at 30°C. All lanes contain the <i>ohrA</i> promoter region as a probe and 250 nM OhrR except lanes 1 (−), which contain the DNA probe only, or lanes 6 and 10 (<b>*</b>), where OhrR was omitted in the reaction mixtures. (<b>A</b>) Oxidized OhrR (ox) treated with increasing concentrations of the low molecular weight thiols dithiothreitol (DTT), β-mercaptoethanol (βME), reduced glutathione (GSH) and dyhydrolipoic acid (DHLA). (<b>B</b>) Oxidized OhrR incubated with 0.5 mM NADPH, 10 µg/ml glutathione reductase (GR), 10 mM glutathione (GSH) plus 1, 10 and 20 µM glutaredoxin 3 (GrxC) (lanes 3–5). (<b>C</b>) Oxidized OhrR incubated with 0.5 mM NADH plus 1, 10 and 50 µM LpdA (lanes 3–5). (<b>D</b>) Oxidized OhrR incubated with 0.5 mM NADH, 10 µM lipoamide plus 1, 5 and 10 µM dihydrolipoamide dehydrogenase (Lpd) (lanes 3–5). Lanes 2 in panels B, C and D contain OhrR treated with 10 mM DTT (positive controls). (<b>E</b>) Oxidized OhrR (lane 2) was incubated with 10 mM DTT (lane 3), 0.5 mM DTT (lane 4), and 0.5 mM DTT plus 10 µM thioredoxin (TrxA) (lane 5). (<b>F</b>) Samples of oxidized OhrR (lane 2) were incubated with 1 mM DTT (lane 3); 0.5 mM NADPH plus 5 µM thioredoxin reductase (TrxB) (lane 4); 0.5 mM NADPH, 1 µM TrxB plus 10, 20 and 40 µM TrxA (lanes 5–7); 0.5 mM NADPH, 5 µM TrxB plus 20 and 40 µM TrxA (lanes 8–9). The control reactions without OhrR to confirm the absence of unspecific DNA binding by the enzymes (lanes 6 and 10, indicated by an asterisk) contain the same mixtures of the samples in lanes 5 and 9 of each respective panel.</p

Mapping transcription start sites for <i>orhR</i> and <i>ohrA</i>.

<p>(<b>A and B</b>) Primer extension analysis of <i>ohrR</i> (<b>A</b>) and <i>ohrA</i> (<b>B</b>) using RNA extracted from <i>C. violaceum</i> cultures either untreated (UN) or treated with 200 µM linoleic acid hydroperoxide (LaOOH) for 15 min. The Sanger sequencing ladders (G, C, A, T) were generated using the same primers as those used for the primer extension. (<b>C and D</b>) Sequences of the <i>ohrR</i> (<b>C</b>) and <i>ohrA</i> (<b>D</b>) promoters. The putative −10 and −35 promoter elements, the transcription start sites (+1, bent arrows), the stop codons, and the putative <i>ohrR</i> and <i>ohrA</i> translation initiation codons (ATG) are shown in bold. The locations of the multiple primer extension products observed for <i>ohrA</i> are indicated in the <i>ohrA</i> promoter sequence (underlined nucleotides). The DNase I-protected regions for OhrR binding are shaded, and the inverted repeat motifs are indicated by arrows. The primers used in primer extension assays are indicated.</p

The essential role of OhrR Cys21 in cumene hydroperoxide sensing <i>in vivo</i>.

<p>(<b>A</b>) Cultures of wild type (ATCC 12472) and Δ<i>ohrR</i> mutant strains containing empty vector (pMR20) or the Δ<i>ohrR</i> mutant strain complemented with OhrR, OhrR C7S, OhrR C21S, OhrR C126S and OhrR C143S were either untreated or treated with 200 µM cumene hydroperoxide (CuOOH). The expression of <i>ohrA</i> was monitored by Northern blot. Ethidium bromide staining of rRNA was used as a loading control. (<b>B</b>) Effect of OhrR cysteine mutations on the degradation of cumene hydroperoxide. Cultures of the indicated strains were growth in LB medium with 10 µg/ml tetracycline. CuOOH (200 µM) was then added, and the level of residual peroxide was determined using the xylenol orange assay.</p

Northern blot analysis of the <i>C. violaceum ohr</i> genes.

<p>(<b>A</b>) Total RNA was extracted from exponential-phase cultures of wild type ATCC 12472 strain under uninduced conditions (UN) and after exposure to 200 µM tBOOH, 200 µM CuOOH, 200 µM H₂O₂, 3% NaCl and 4% ethanol (EtOH) for 15 min. RNA was separated on 1.5% agarose gel, transferred to a nylon membrane and hybridized with radiolabeled probes specific to <i>ohrA</i>, <i>ohrR</i> or <i>ohrB</i>. (<b>B</b>) <i>ohrA</i> induction by lipid hydroperoxides. Wild type cells were exposed to 50 µM and 200 µM linoleic acid hydroperoxide (LaOOH) or to 50 µM oleic acid hydroperoxide (OaOOH) for 15 min prior to Northern blot. (<b>C</b>) Time course of <i>ohrA</i> and <i>ohrR</i> induction. RNA samples from wild type cells untreated (0) or treated with 100 µM tBOOH for the indicated times (0 to 90 min) were hybridized with probes specific to <i>ohrA</i> or <i>ohrR</i>. Ethidium bromide staining of rRNA was used as a loading control and is shown below each Northern blot experiment. The sizes of the bands were estimated by ethidium bromide staining of the molecular weight marker Low Range RNA Ladder Riboruler (Fermentas).</p

Interaction of OhrR with the promoter of <i>ohrA</i> and <i>ohrR</i>, as determined by EMSA.

<p>Labeled probes containing the promoter region of <i>ohrA</i> (<b>A</b>) and <i>ohrR</i> (<b>B</b>) were incubated with increasing concentrations of purified OhrR protein, as indicated, and the mixture was separated in a polyacrylamide non-denaturing gel. Competition assays <b>(right panels)</b> using 250 nM OhrR were performed in the presence of 30-fold excess of unlabeled fragments of the same region (S) or the <i>ohrR</i> coding region (N) as competitors. (<b>C and D</b>) Effects of oxidants and DTT on OhrR binding. EMSA assays using OhrR with the <i>ohrA</i> promoter (<b>C</b>) or <i>ohrR</i> promoter (<b>D</b>). ³²P-Labeled probes were incubated with 250 nM purified OhrR. Either 0.3 mM <i>tert</i>-butyl hydroperoxide (tBOOH), 0.3 mM cumene hydroperoxide (CuOOH) or 1 mM H₂O₂ was added to the binding reaction and incubated for 10 min at 25°C. When indicated, DTT was then added into the reactions, and incubation continued at 25°C for 15 min.</p