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CELL AND MOLECULAR BIOLOGY OF MICROBES

A novel redox-sensing transcriptional regulator CyeR controls expression of an Old Yellow Enzyme family protein in Corynebacterium glutamicum

Shigeki Ehira, Haruhiko Teramoto, Masayuki Inui, Hideaki Yukawa

  • Research Institute of Innovative Technology for the Earth (RITE), 9-2 Kizugawadai, Kizugawa, Kyoto 619-0292, Japan
  • Correspondence
    Hideaki Yukawa
    mmg-lab{at}rite.or.jp

    • Microbiology 2010; 156(5):1335–1341 · https://doi.org/10.1099/mic.0.036913-0

    View at publisher PubMed

    Abstract

    Corynebacterium glutamicum cgR_2930 (cyeR) encodes a transcriptional regulator of the ArsR family. Its gene product, CyeR, was shown here to repress the expression of cyeR and the cgR_2931 (cye1)–cgR_2932 operon, which is located upstream of cyeR in the opposite orientation. The cye1 gene encodes an Old Yellow Enzyme family protein, members of which have been implicated in the oxidative stress response. CyeR binds to the intergenic region between cyeR and cye1. Expression of cyeR and cye1 is induced by oxidative stress, and the DNA-binding activity of CyeR is impaired by oxidants such as diamide and H2O2. CyeR contains two cysteine residues, Cys-36 and Cys-43. Whereas mutation of the former (C36A) has no effect on the redox regulation of CyeR activity, mutating the latter (C43A, C43S) abolishes the DNA-binding activity of CyeR. Cys-43 of CyeR and its C36A derivative are modified upon treatment with diamide, suggesting an important role for Cys-43 in the redox regulation of CyeR activity. It is concluded that CyeR is a redox-sensing transcriptional regulator that controls cye1 expression.

    • ↵†Present address: Department of Biological Science, Chuo University, 1-13-27 Kasuga, Bunkyo, Tokyo 112-8551, Japan.

    • A supplementary figure, showing an amino acid sequence alignment of CyeR homologues, and a supplementary table, showing the primers used in this study, are available with the online version of this paper.

    Edited by: J.-H. Roe

    INTRODUCTION

    Corynebacterium glutamicum is a non-pathogenic, high-G+C, Gram-positive bacterium that belongs to the actinobacteria, which include the genera Mycobacterium and Streptomyces. C. glutamicum is widely used for the industrial production of various amino acids and nucleic acids (Hermann, 2003; Terasawa & Yukawa, 1993). We have previously demonstrated a high productivity of ethanol and organic acids using C. glutamicum (Inui et al., 2004a, b; Okino et al., 2008a, b). Meanwhile, this species is of increasing interest as a model organism for closely related pathogenic species such as Corynebacterium diphtheriae and Mycobacterium tuberculosis (Brune et al., 2005; Mishra et al., 2007). Since the C. glutamicum genomic sequence was determined (Ikeda & Nakagawa, 2003; Kalinowski et al., 2003; Yukawa et al., 2007), knowledge of transcriptional regulation in C. glutamicum has drastically increased as a result of combining DNA microarray analysis with targeted mutagenesis (Baumbach et al., 2009). However, of more than 100 genes encoding transcriptional regulators in its genome (Brune et al., 2005), many remain uncharacterized.

    Oxidative stress is an inescapable consequence of aerobic metabolism, which produces reactive oxygen species that can damage cellular components, including nucleic acids, proteins and lipids. Redox-sensing transcriptional regulators play central roles in the cellular response to oxidative stress by regulating expression of resistance genes against oxidative stress (Paget & Buttner, 2003). The reduction-oxidation of the thiol groups of cysteine residues is the basis of various mechanisms that sense changes in cellular redox conditions in response to oxidative stress. For example, a LysR family transcriptional regulator, OxyR, of Escherichia coli is activated by oxidation through the formation of an intramolecular disulfide bond between Cys-199 and Cys-208 (Zheng et al., 1998). OxyR regulates the expression of genes involved in peroxide metabolism and protection (katG, ahpCF, dps), and redox balance (gor, grxA, trxC) (Zheng et al., 2001). A MarR family transcriptional regulator, OhrR, of Bacillus subtilis is an organic peroxide sensor and represses expression of ohrA, which encodes a peroxiredoxin (Fuangthong et al., 2001). The lone cysteine residue (Cys-15) of OhrR is essential for redox sensing (Fuangthong & Helmann, 2002). Oxidation of Cys-15 leads to S-thiolation by cysteine, coenzyme A or an unknown thiol with a molecular mass of 398 Da (Lee et al., 2007).

    It has recently been shown that a DUF24 family protein, QorR, of C. glutamicum is a redox-sensing transcriptional regulator, and represses expression of both qor2, which encodes a quinone oxidoreductase, and its own structural gene (Ehira et al., 2009a). QorR contains a lone cysteine residue (Cys-17). Under oxidizing conditions, QorR undergoes dimerization and loses DNA-binding activity through the formation of an intermolecular disulfide bond between Cys-17 of each subunit.

    In this study, we characterized an ArsR family transcriptional regulator, CgR2930 (CyeR), encoded by the gene which is located immediately upstream of the cgR_2931 (cye1) operon in the opposite orientation. The cye1 gene encodes an Old Yellow Enzyme (OYE) family protein (Williams & Bruce, 2002). The OYE family members are proposed to be involved in the oxidative stress response in bacteria, e.g. in B. subtilis and Shewanella oneidensis (Brigé et al., 2006; Fitzpatrick et al., 2003), although the gene regulation mechanism remains unknown. Our present results show that CyeR directly controls expression of the cye1 operon and its own structural gene as a transcriptional repressor. Expression of cyeR and cye1 is induced by thiol oxidative stress, and the DNA-binding activity of CyeR is impaired by oxidants. Furthermore, site-directed mutagenesis studies show that one of two cysteine residues of CyeR plays a key role in its DNA-binding activity. These findings suggest that CyeR is a redox-sensing transcriptional regulator involved in the oxidative stress response of C. glutamicum by regulating cye1 expression.

    METHODS

    Bacterial strains, culture media and growth conditions.

    C. glutamicum strain R (Yukawa et al., 2007) and its derivative were grown at 33 °C in A medium (Inui et al., 2007) with 4 % (w/v) glucose on a rotary shaker at 180 r.p.m. A disruptant of cyeR (Δ2930) was constructed by the transposon-mediated mutagenesis method, as described previously (Suzuki et al., 2006). Disruption of cyeR was confirmed by DNA sequencing of thermal asymmetrical interlaced-PCR products of mutant cells.

    RNA isolation and DNA microarray analysis.

    Total RNA was extracted from C. glutamicum cells by using the RNeasy Mini kit (Qiagen) and was treated with DNase I (Takara Bio), as described previously (Ehira et al., 2008). Global gene expression analysis was performed with the C. glutamicum R DNA microarray, as described previously (Ehira et al., 2008). Microarray analysis was carried out using two sets of RNA samples isolated from independently grown cultures with different combinations of Cy dyes (a dye swap strategy). Since the C. glutamicum R DNA microarray contains two replicates per gene, a total of four replicates per gene were available to determine changes in gene expression. Genes with significantly differential transcript levels (P<0.05 in a Student's t test) by at least a factor of two were determined.

    Real-time quantitative RT-PCR (qRT-PCR) analysis.

    A one-step real-time qRT-PCR was performed with the Power SYBR Green PCR Master Mix (Applied BioSystems) and a pair of gene-specific primers (Supplementary Table S1) by using the 7500 Fast Real-Time PCR system (Applied Biosystems), as described previously (Ehira et al., 2008). Relative ratios were normalized with the value for 16S rRNA.

    RT-PCR analysis.

    cDNA was synthesized from total RNA with a reverse primer as follows. An aliquot (1 μg) of total RNA and 1 pmol primer RT2932-R were denatured at 90 °C for 5 min and then gradually cooled to 55 °C. The reverse transcription reaction was performed in 20 μl First-Strand Buffer [50 mM Tris/HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2] containing total RNA, the primer RT2932-R, 5 mM DTT, 0.5 mM each of dATP, dGTP, dCTP and dTTP, and 200 U SuperScript III RNase H− reverse transcriptase (Invitrogen). The reaction mixture was incubated at 55 °C for 1 h, and then the reaction was stopped by addition of 80 μl TE buffer [10 mM Tris/HCl (pH 8.0), 1 mM EDTA]. An aliquot (1 μl) of the cDNA solution was taken for PCR analysis using the primer pair RT2931-F and RT2932-R.

    Mapping of transcription initiation sites (TISs) by rapid amplification of cDNA ends (RACE)-PCR.

    TISs were determined by using the SMART RACE cDNA Amplification kit (Clontech). 5′ RACE-PCRs were carried out as described previously (Ehira et al., 2009b), with 1 μg total RNA and gene-specific primers (Supplementary Table S1). The resulting PCR products were cloned into a pGEM-T Easy vector (Promega). At least 20 clones for each 5′ RACE-PCR product were sequenced.

    Expression and purification of His–CyeR, His–CyeRC36A and His–CyeRC43A.

    For construction of an expression plasmid for the histidine-tagged CyeR (His–CyeR) protein, a DNA fragment containing the ORF of cyeR was amplified by PCR using the primer pair rcgR2930-F and rcgR2930-R (Supplementary Table S1). The amplified DNA fragment was cloned between the NdeI and EcoRI sites of the pET-28a expression vector (Merck KGaA). The resulting plasmid, pCRD612, contained cyeR fused to the His-tag sequence. A cysteine residue at position 36 or position 43 of CyeR was replaced with alanine by performing site-directed mutagenesis using a PrimeSTAR Mutagenesis Basal kit (Takara Bio). pCRD612 was used as a template for PCR with the primer pair 2930C36A-F and 2930C36A-R or the primer pair 2930C43A-F and 2930C43A-R (Supplementary Table S1) to generate plasmid pCRD613 or plasmid pCRD614, respectively. Expression and purification of recombinant proteins were performed as described previously (Ehira et al., 2009a) using E. coli BL21 (DE3) cells harbouring pCRD612, pCRD613 or pCRD614.

    Gel mobility shift assay.

    The gel mobility shift assay was performed with purified recombinant proteins and a Cy3-labelled probe, as described previously (Ehira et al., 2009a). Probes 2930-1 and 1435-6 were prepared by PCR using the primer pair RT2930-R and 2931R+92 and pCRD621 (Ehira et al., 2009a) as a template, and the primer pair 1436R+51 and RT1435-R and pCRD620 (Ehira et al., 2009a) as a template, respectively. A Cy3-labelled 2931R+92 primer was used for preparation of the Cy3-labelled probe.

    Thiol redox state analyses.

    After incubation with 1 mM DTT or diamide for 30 min, His–CyeR and His–CyeRC36A (1 μg) were incubated at room temperature for 2 h in 200 mM Tris/HCl (pH 8.0) containing 1 % SDS and 15 mM 4-acetamido-4′-maleimidylstibene-2,2′-disulfonic acid (AMS) (Molecular Probes). The mixtures were then separated by non-reducing SDS-PAGE.

    RESULTS

    CgR2930 (CyeR) negatively regulates expression of the cye1 operon and cyeR

    C. glutamicum cgR_2930 encodes a transcriptional regulator of the ArsR family. To ascertain the physiological role of CgR2930 as a transcriptional regulator in C. glutamicum, gene expression profiles during exponential growth were compared between the wild-type strain and a cgR_2930 disruptant (Δ2930) using a DNA microarray. Expression of only cgR_2931 and cgR_2932, which are located upstream of cgR_2930 in the opposite orientation, was shown to be upregulated in Δ2930. Differential expression of cgR_2931 and cgR_2932 was confirmed by qRT-PCR. In Δ2930, the transcript levels of cgR_2931 and cgR_2932 were more than 20-fold higher (25.9±3.5 and 23.7±5.4, respectively) than the wild-type levels. As the distance between cgR_2931 and cgR_2932 is 12 bp, the two genes are likely to be co-transcribed. The dicistronic transcript of cgR_2931 and cgR_2932 was indeed detected by RT-PCR analysis (data not shown). It was not possible to determine the TIS of cgR_2931 by RACE-PCR and primer extension analysis because of the detection of multiple 5′ ends of transcripts. However, putative −10 and −35 promoter sequences were found upstream of cgR_2931 (Fig. 1⇓). The TIS of cgR_2930 was determined by RACE-PCR and the −10 and −35 promoter regions were identified (Fig. 1⇓). Since the promoters of cgR_2930 and cgR_2931 are supposed to overlap, the transcript level of cgR_2930 in Δ2930 was determined by qRT-PCR using a primer pair designed based on a sequence upstream of the transposon insertion site of Δ2930. The transcript level of cgR_2930 in Δ2930 was about 10 times higher (10.9±2.0) than that of the wild-type. These results indicate that CgR_2930 negatively controls expression of the cgR_2931–cgR_2932 operon and its structural gene.

    Figure image not available in archive
    Fig. 1.

    Nucleotide sequence of the cgR_2930–cgR_2931 intergenic region. The coding regions of cgR_2930 and cgR_2931 are shaded in grey. The identified TIS and the putative −10 and −35 promoter regions are indicated by the bent arrow and bold type, respectively.

    The cgR_2931 gene encodes a protein with 42 % amino acid sequence identity to YqjM of B. subtilis, which is a member of the OYE family (Fitzpatrick et al., 2003). The entire amino acid sequence encoded by the cgR_2932 gene does not show high similarity to that of any other protein characterized functionally so far. We designated cgR_2931 as cye1 (Corynebacterium yellow enzyme 1) and cgR_2930 as cyeR (Corynebacterium yellow enzyme regulator).

    Expression of cyeR and the cye1 operon is induced by oxidative stress

    The OYE family members are implicated in the oxidative stress response in bacteria (Brigé et al., 2006; Fitzpatrick et al., 2003). As expression of B. subtilis YqjM and S. oneidensis SYE4 is induced in response to oxidative stress, changes in expression of cyeR and cye1 upon oxidative stress were examined. The transcript levels of cyeR and cye1 in exponentially growing cells were determined by qRT-PCR before and after treatment of cells with the thiol-specific oxidant diamide (Fig. 2⇓). cyeR and cye1 were upregulated within 5 min of the addition of diamide and the transcript levels remained high for another 5 min, before gradually decreasing. The cye1 transcript level also increased upon addition of H2O2 (data not shown).

    Figure image not available in archive
    Fig. 2.

    Changes in the transcript levels of cyeR and cye1 in response to diamide. The relative transcript levels of cyeR (squares) and cye1 (circles) before (0 min) and at 5, 10, 15 and 20 min after addition of 3 mM diamide were determined by qRT-PCR. The transcript level at 0 min was taken as 1. Experiments were repeated twice and representative data are shown.

    CyeR binds to the intergenic region between cyeR and cye1

    Gel mobility shift assays were carried out with purified His–CyeR and a DNA probe, 2930-1, for the intergenic region between cyeR and cye1 (Fig. 3⇓). His–CyeR reduced the electrophoretic mobility of probe 2930-1 (Fig. 3⇓, lanes 1–4). The band intensity of the protein–DNA complex was reduced upon addition of a non-labelled 2930-1 fragment (Fig. 3⇓, lanes 5 and 6). However, addition of a probe, 1435-6, that contains the oxidative stress-responsive promoters of qorR and qor2 (Ehira et al., 2009a) did not affect the amount of complex formed (Fig. 3⇓, lanes 7 and 8). It was concluded that His–CyeR binds to the cyeR and cye1 intergenic region in a sequence-specific manner.

    Figure image not available in archive
    Fig. 3.

    Gel mobility shift assays with His–CyeR and the cyeR–cye1 intergenic region. The binding of His–CyeR to probe 2930-1 was examined. Probe 2930-1 (2 nM) was mixed with His–CyeR in the amounts indicated above each lane, and then the mixtures were subjected to electrophoresis. Non-labelled fragments of 2930-1 (lanes 5 and 6) and 1435-6 (lanes 7 and 8) were added at the indicated amounts. Filled arrow, complex of His–CyeR and probe 2930-1; open arrow, probe 2930-1 alone.

    CyeR is a redox-responsive transcriptional regulator

    The predicted amino acid sequence of CyeR contains two cysteine residues at position 36 (Cys-36) and 43 (Cys-43). Cys-43 is conserved among uncharacterized CyeR homologues not only in actinobacteria but also in proteobacteria, firmicutes and cyanobacteria (Supplementary Fig. S1). Reduction-oxidation of these cysteine residues is presumed to be involved in the control of CyeR activity. The effect of oxidants on the DNA-binding activity of CyeR was examined by gel mobility shift assays (Fig. 4a⇓). Binding of His–CyeR to probe 2930-1 was prevented by addition of diamide (Fig. 4a⇓, lanes 2–6). The addition of an excess of the reducing agent DTT restored the DNA-binding activity of His–CyeR that was inactivated by diamide (Fig. 4a⇓, lanes 8–12), indicating that the effects of oxidation and reduction on the DNA-binding activity of CyeR are reversible. The DNA-binding activity of CyeR was also impaired by H2O2 (Fig. 4b⇓).

    Figure image not available in archive
    Fig. 4.

    Redox-sensitive control of DNA-binding activity of CyeR. (a) His–CyeR (300 nM) and probe 2930-1 (2 nM) were incubated in the presence of 1 mM DTT (lanes 2–6) or 1 mM diamide (lanes 8–12) for 30 min, and then diamide (lanes 2–6) or DTT (lanes 8–12) was added in the amounts indicated above each lane. After 30 min, the mixtures were subjected to electrophoresis. Lanes 1 and 7, His–CyeR not added. (b) His–CyeR (300 nM) and probe 2930-1 (2 nM) were incubated in the presence of 1 mM DTT for 30 min, and then H2O2 (lanes 2–5) was added in the amounts indicated above each lane. After 30 min, the mixtures were subjected to electrophoresis. Lane 1, His–CyeR not added.

    Cys-43 plays a pivotal role in the DNA-binding activity of CyeR

    To examine the role of cysteine residues in the redox-responsive regulation of CyeR activity, Cys-36 and Cys-43 were replaced with alanine or serine. His–CyeRC36A reduced the electrophoretic mobility of probe 2930-1 (Fig. 5a⇓, lanes 1–4) and binding of His–CyeRC36A to probe 2930-1 was prevented by addition of diamide (Fig. 5b⇓). However, no interaction between His–CyeRC43A (Fig. 5a⇓, lanes 5–8) or His–CyeRC43S (data not shown) and probe 2930-1 was observed. It was concluded that Cys-36 is dispensable for redox regulation of CyeR activity and that substitutions of Cys-43 inactivate CyeR.

    Figure image not available in archive
    Fig. 5.

    DNA-binding activity of His–CyeRC36A and His–CyeRC43A. (a) Gel mobility shift assays were performed with His–CyeRC36A (lanes 1–4) or His–CyeRC43A (lanes 5–8) and probe 2930-1, as described in Fig. 3. (b) His–CyeRC36A (300 nM) and probe 2930-1 (2 nM) were incubated in the presence of 1 mM DTT for 30 min, and then 1 mM (lane 3) or 3 mM diamide (lane 4) was added. After 30 min, the mixtures were subjected to electrophoresis. Lane 1, His–CyeRC36A not added.

    We examined whether Cys-36 and Cys-43 form an inter- or intramolecular disulfide bond under oxidizing conditions. When the His–CyeR protein oxidized with diamide was subjected to non-reducing SDS-PAGE, only one band of a molecular mass of approximately 14 kDa, corresponding to the monomeric form of His–CyeR, was detected (Fig. 6⇓, lane 2), indicating that diamide-inactivated His–CyeR does not undergo dimerization. We next examined whether the thiol groups of cysteine residues of CyeR are modified by treatment with diamide using AMS. AMS covalently modifies free thiol groups, which retards electrophoretic mobility. The electrophoretic mobility of His–CyeR treated with DTT was retarded by AMS modification (Fig. 6⇓, lanes 1 and 3), while AMS did not affect the mobility of diamide-treated His–CyeR (Fig. 6⇓, lanes 2 and 4). When diamide-inactivated His–CyeRC36A was subjected to non-reducing SDS-PAGE, an additional band of molecular mass 28 kDa, which is likely to correspond to the dimeric form of His–CyeRC36A, appeared (Fig. 6⇓, lane 6). AMS modification was not observed for either the monomeric or the dimeric form of diamide-treated His–CyeRC36A (Fig. 6⇓, lane 8). His–CyeRC36A treated with DTT was modified with AMS, although the electrophoretic mobility of the AMS-modified His–CyeRC36A was faster than that of the AMS-modified His–CyeR (Fig. 6⇓, lanes 3 and 7). These results indicate that the diamide treatment prevents AMS modification of two cysteine residues, Cys-36 and Cys-43, of His–CyeR, and a lone cysteine residue, Cys-43, of His–CyeRC36A. Therefore, it is likely that modification of Cys-43 under oxidizing conditions inactivates the CyeR activity.

    Figure image not available in archive
    Fig. 6.

    Oxidation of cysteine residues of CyeR. Purified His–CyeR (lanes 1–4) and His–CyeRC36A (lanes 5–8) were treated as follows. Proteins (1 μg) treated with 1 mM DTT (lanes 1, 3, 5 and 7) or 1 mM diamide (lanes 2, 4, 6 and 8) were further incubated with (lanes 3, 4, 7 and 8) or without (lanes 1, 2, 5 and 6) AMS. The modified samples were separated by non-reducing SDS-PAGE. Lanes M, molecular mass standard marker.

    DISCUSSION

    In the present study, we demonstrated that CyeR is a redox-sensing transcriptional regulator that represses expression of the cye1 operon and its structural gene. Cye1 belongs to the OYE family, members of which are found in yeasts, bacteria, plants and nematodes, and share characteristic biochemical properties: the protein binds flavin and catalyses the reduction of broad substrates, such as α,β-unsaturated aldehydes and ketones, using NADPH as a cofactor (Williams & Bruce, 2002). Expression of bacterial OYE family members such as B. subtilis YqjM and S. oneidensis SYE4 is induced by oxidative stress. Based on their biochemical and expression properties, the OYE family members are implicated in the control of the cellular redox state in response to oxidative stress, although exact substrates in vivo remain elusive (Brigé et al., 2006; Fitzpatrick et al., 2003). We observed that expression of the cye1 gene is induced by treatment with diamide (Fig. 2⇑) or H2O2. The redox-responsive DNA-binding activity of CyeR suggests that expression of cye1 is derepressed by inactivation of the transcriptional repressor CyeR, in which thiol groups of cysteine residues are modified under oxidizing conditions. The effects of site-directed mutagenesis on the DNA-binding activity imply that Cys-43 plays a pivotal role in the redox regulation of CyeR activity. To our knowledge, this is the first report to characterize a transcriptional regulator that controls expression of OYE family proteins in bacteria. It is noteworthy that a CyeR homologue in Pseudomonas putida is located upstream of xenA, which encodes a xenobiotic reductase A (Blehert et al., 1999). XenA and Cye1 belong to the YqjM family, a new bacterial subfamily of OYE homologues (Kitzing et al., 2005).

    Unlike C. glutamicum QorR, which becomes dimeric and inactive under oxidizing conditions (Ehira et al., 2009a), CyeR loses the DNA-binding activity but remains monomeric (Fig. 6⇑, lane 2). Since both Cys-36 and Cys-43 are protected from AMS modification by treatment with diamide (Fig. 6⇑, lane 4), an intramolecular disulfide bond between these two cysteine residues may be formed. However, the DNA-binding activity of CyeRC36A remains redox-responsive (Fig. 5⇑). Most of the diamide-inactivated CyeRC36A protein remains monomeric, although some undergoes dimerization (Fig. 6⇑, lane 6). As Cys-43 of the monomeric form of CyeRC36A is modified upon diamide treatment (Fig. 6⇑, lane 8), CyeR is likely to be inactivated by Cys-43 modification, although the mechanism of CyeRC36A inactivation upon diamide treatment is currently unknown. As reported for B. subtilis YodB, a DUF24 family redox-sensing transcriptional regulator, modification of cysteine residues with diamide might occur in vitro, although detection of such molecular species has not yet been realized (Leelakriangsak et al., 2008). Replacement of Cys-43 with alanine or serine inactivates CyeR, suggesting that the thiol group of Cys-43, which is predicted to be positioned in the helix–turn–helix domain, is essential for maintaining the correct structure for DNA binding of CyeR (Fig. 5a⇑). Further study is needed to fully understand the role of Cys-43 in the redox-sensing mechanism of CyeR.

    B. subtilis displays a complex adaptive response to oxidative stress that is coordinated by a sigma factor, SigB, along with transcriptional regulators PerR and OhrR (Helmann et al., 2003). In C. glutamicum, two extracytoplasmic function sigma factors, SigH and SigM, are involved in the response to oxidative stress (Kim et al., 2005; Nakunst et al., 2007). SigH controls transcription of sigM, and SigM regulates expression of trxB1 and trxC, encoding thioredoxins, and trxB, encoding a thioredoxin reductase. Although there are no C. glutamicum homologues of PerR and OhrR, two transcriptional regulators, QorR (Ehira et al., 2009a) and CyeR, contribute to the oxidative stress response. Moreover, Streptomyces coelicolor OxyR has been shown to regulate the expression of the ahpCD operon, which encodes an alkyl hydroperoxide reductase system, and its structural gene (Hahn et al., 2002). An OxyR homologue is also encoded in the C. glutamicum genome. Therefore, the oxidative stress response of C. glutamicum as well as other actinobacteria appears to be controlled by a wide variety of regulatory mechanisms, more than is the case for other bacteria that have been examined to date (den Hengst & Buttner, 2008).

    Acknowledgments

    We thank Crispinus A. Omumasaba (RITE) for critical reading of the manuscript. This study was partially supported by a grant from the New Energy and Industrial Technology Development Organization (NEDO), Japan.

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