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. 2008 Feb 13;3(2):e1602.
doi: 10.1371/journal.pone.0001602.

A novel OxyR sensor and regulator of hydrogen peroxide stress with one cysteine residue in Deinococcus radiodurans

Huan Chen  1 , Guangzhi Xu, Ye Zhao, Bing Tian, Huiming Lu, Xiaomin Yu, Zhenjian Xu, Nanjiao Ying, Songnian Hu, Yuejin Hua
Affiliations

A novel OxyR sensor and regulator of hydrogen peroxide stress with one cysteine residue in Deinococcus radiodurans

Huan Chen et al. PLoS One. .

Abstract

In bacteria, OxyR is a peroxide sensor and transcription regulator, which can sense the presence of reactive oxygen species and induce antioxidant system. When the cells are exposed to H(2)O(2), OxyR protein is activated via the formation of a disulfide bond between the two conserved cysteine residues (C199 and C208). In Deinococcus radiodurans, a previously unreported special characteristic of DrOxyR (DR0615) is found with only one conserved cysteine. dr0615 gene mutant is hypersensitive to H(2)O(2), but only a little to ionizing radiation. Site-directed mutagenesis and subsequent in vivo functional analyses revealed that the conserved cysteine (C210) is necessary for sensing H(2)O(2), but its mutation did not alter the binding characteristics of OxyR on DNA. Under oxidant stress, DrOxyR is oxidized to sulfenic acid form, which can be reduced by reducing reagents. In addition, quantitative real-time PCR and global transcription profile results showed that OxyR is not only a transcriptional activator (e.g., katE, drb0125), but also a transcriptional repressor (e.g., dps, mntH). Because OxyR regulates Mn and Fe ion transporter genes, Mn/Fe ion ratio is changed in dr0615 mutant, suggesting that the genes involved in Mn/Fe ion homeostasis, and the genes involved in antioxidant mechanism are highly cooperative under extremely oxidant stress. In conclusion, these findings expand the OxyR family, which could be divided into two classes: typical 2-Cys OxyR and 1-Cys OxyR.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Alignment of OxyR homologs from different organisms.
Using CLUSTAL W software aligned amino acid sequences of the Streptomyces coelicolor A3(2), Neisseria gonorrhoeae, E. coli, and D. radiodurans. Identical amino acids are highlighted in black, and conserved residues are highlighted with grey. The DrOxyR helix-turn-helix region has four conserved residues (R4, L32, S33 and R50) . At its LysR-substrate binding domain, D142 and R273 possibly define an activating region on OxyR (contact with RNA polymerase), A233 residue is involved in tetramerization, V110, L124, and A233 form a hydrophobic core. Numbering is based on the E. coli OxyR sequence.
Figure 2
Figure 2. Survival curves of D. radiodurans strains exposed to (A) H2O2 and (B) ionizing irradiation.
(A) Wild-type D. radiodurans R1 (○) compared to MOxyR (▪), MOxyR_wtC (•), and MOxyR_sdC (□) under 20 mM H2O2 treatment at the five recovery time points (0, 20, 40, 60, and 80 min). (B) Wild-type D. radiodurans R1 (○) compared to MOxyR (▪) after ionizing irradiation. Error bars represent standard deviations from four replicate experiments.
Figure 3
Figure 3. Effect of oxyR disruption on the antioxidant ability of D. radiodurans.
(A) Catalase activities of R1, MOxyR, MOxyR_wtC, and MOxyR_sdC after H2O2 treatment (grey bar) or not (white bar). (B) ROS accumulate in four strains after H2O2 treatment (grey bar) or not (white bar). Data reported represent the average and standard deviations of three independent experiments.
Figure 4
Figure 4. Absorbance of NBD chloride-treated purified OxyR. Air-oxidized OxyR (▪), along with the CHP (10 mM) treated OxyR (□), shows maximal absorption at 347 nm.
The shoulder at 420 nm is reduced OxyR (▴) reacted with NBD chloride. The absorbance spectrum is from 285 to 600 nm.
Figure 5
Figure 5. Q-RT PCR of the expression of potential OxyR-dependent genes in D. radiodurans R1 wild-type compared to wild type after H2O2 treatment (white bar), MOxyR (grey bar), and MOxyR H2O2 treatment (black bar).
(A) genes positively regulated by DrOxyR; (B) genes negatively regulated by DrOxyR; (C) the expression patterns of DR1709 and DR2263 were measured in MOxyR_sdC; (D) genes not regulated by DrOxyR.
Figure 6
Figure 6. Binding of reduced and oxidized OxyR to the upstream region of (A) negative control (dr0207 and coding sequence for dr1709); (B) positivly regulated genes (dr1998 and drB0125); (C) negativly regulated genes (dr1709, dr2263, and drB0036).
To generate reduced protein, 200 mM DTT was added to the binding reactions. Column 1, 2, and 3 indicate non-added protein, reduced OxyR added, and oxidized OxyR added, respectively.
Figure 7
Figure 7. Model for reduced and oxidized OxyR binding to and activation at the two classes genes.
For Class I (katE): OxyR activates gene in the presence of H2O2, whereas under non-stressed conditions, reduced OxyR is bound to two pairs of adjacent major grooves separated by one helical turn of the DNA duplex and acts to repress its own synthesis. For Class II (mntH): mutation of OxyR can greatly enhance gene expression, reduced OxyR binds to DNA and minimally induced genes, whereas oxidized OxyR significantly decreases the gene expression levels.
See this image and copyright information in PMC

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