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

Abstract

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.

Inactivation of the Organic Hydroperoxide Stress Resistance Regulator OhrR Enhances Resistance to Oxidative Stress and Isoniazid in Mycobacterium smegmatis

“…This is in line with the observations made with OhrR of other bacterial species (34,51,52) and suggests a common mechanism for its function. Usually, the N-terminal Cys of OhrR is the one which gets oxidized by organic peroxides, and this amino acid is conserved at position 13 in M. smegmatis OhrR.…”

“…The observed higher Ohr levels in the ohrR mutant of M. smegmatis does not appear to be due to the stability of this protein because the upregulation is associated with a concomitant increase in mRNA levels. Further, the observations that Ohr of M. smegmatis can be induced only by alkyl or organic hydroperoxides and not by H 2 O 2 are similar to those noticed for other bacterial species, like B. subtilis (59), P. aeruginosa (29), Streptomyces coelicolor (58), A. tumefaciens (50), Sinorhizobium meliloti (51), and Chromobacterium violaceum (34) and suggest that OhrR is a sensor for organic hydroperoxide stress in M. smegmatis. However, the OhrR of M. smegmatis is different from the OhrR of Shewanella oneidensis, a recently described protein that responds to both organic and H 2 O 2 peroxides (61).…”

“…7B), suggesting that the interaction between OhrR and ohrR-IG is highly specific. Further, previous studies have indicated that the ability of OhrR to bind with the ohrR promoter region is affected by oxidants (34,51,52 Fig. 7C show that addition of these oxidants completely abolished the interaction of ohrR-IG with His 10 OhrR and that this effect of oxidants could be completely reversed by adding 100 mM the reducing agent DTT to the binding reactions.…”

Global Transcriptional Response to Organic Hydroperoxide and the Role of OhrR in the Control of Virulence Traits in Chromobacterium violaceum

“…Among these enzymes, OhrA, glutathione amide peroxidase, and glutathione peroxidase were previously described as OHP-scavenging enzymes (12,13,31). Interestingly, besides ohrA and ohrR, which compose a dedicated system for OHP detoxification in C. violaceum (17), we also observed increased expression levels of trxC (19-fold) and trxB (3-fold). Enzymes of the thioredoxin system were shown to be involved in OhrR reduction in vitro (17).…”

“…After OhrR oxidation, an N-terminal cysteine residue undergoes thiolation by lowmolecular-weight thiols (1-Cys subfamily) or reacts with a C-terminal cysteine residue forming an intermolecular disulfide bond (2-Cys subfamily) (14,15). Recently, bacilliredoxins and thioredoxins were shown to be the reducing systems for the 1-Cys OhrR from Bacillus subtilis (16) and for the 2-Cys OhrR from Chromobacterium violaceum (17), respectively. OhrR belongs to the MarR family of transcription factors, which contains many other thiol-based redox-sensing regulators that respond to ROS or to reactive electrophilic species (14,18).…”