Regulation of Quinone Oxidoreductase by the Redox-sensing Transcriptional Regulator QorR in Corynebacterium glutamicum^*

Bacterial Strains, Culture Media, and Growth Conditions

C. glutamicum strain R (17) and its derivatives were grown at 33 °C in A medium with 4% (w/v) glucose as described previously (18). Disruptants of the qorR (cgR_1435) and qor2 (cgR_1436) genes were constructed by the transposon-mediated mutagenesis method as described previously (19). Disruption of the qorR and qor2 genes was confirmed by DNA sequencing of thermal asymmetric interlaced-PCR products of mutant cells. Transposon was inserted at 160 bases downstream of the 5′ end of the qorR gene in Δ1435 and at 30 bases downstream of the 5′ end of the qor2 gene in Δ1436.

A DNA fragment containing the promoter region of the bglF2 gene of C. glutamicum strain R was amplified by PCR using the primer pair PbglF2-F and PbglF2-R (supplemental Table S1) and was cloned between the EcoRI and SacI sites of a shuttle vector, pCRC500 (20), to construct pCRC531. DNA fragments containing the qorR coding region were excised from pCRD610 and pCRD611 (see below) with NdeI and HindIII and cloned between the NdeI and HindIII sites of pCRC531 to construct pCRD630 and pCRD631, respectively. The qorR gene is expressed from the bglF2 promoter on these plasmids. A cysteine codon of the qorR gene at position 17 is replaced with a codon for serine on pCRD631. pCRD630 and pCRD631 were used separately to transform Δ1435 cells, and transformants were selected for resistance to kanamycin and chloramphenicol.

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, Inc.) as described previously (18). Global gene expression analysis was performed with the C. glutamicum R DNA microarray as described previously (18). Microarray analyses were carried out using two sets of RNA samples isolated from independently grown cultures with different combinations of Cy dyes (a dye swap strategy). Because 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 Student's t test) by at least a factor of two were determined.

Real-time qRT-PCR

A one-step real-time quantitative reverse transcription-PCR (qRT-PCR)² was performed with the Power SYBR® Green PCR Master Mix (Applied BioSystems) and a pair of gene-specific primers (supplemental Table S1) by using the 7500 Fast Real-time PCR system (Applied BioSystems) as described previously (18). Relative ratios were normalized with the value for 16 S rRNA.

Mapping of Transcription Initiation Sites by Rapid Amplification of cDNA Ends (RACE)-PCR

Transcription initiation sites were determined by using the SMART^TM RACE cDNA amplification kit (Clontech). 5′ RACE-PCRs were carried out as recommended by the supplier with 1 μg of total RNA and gene-specific primers (supplemental Table S1). Resulting PCR products were cloned into a pGEM®-T Easy vector (Promega Corporation). At least 10 clones for each 5′ RACE-PCR product were sequenced.

Expression and Purification of His-QorR and His- QorRC17S

For construction of an expression plasmid for the histidine-tagged QorR protein, a DNA fragment containing the qorR gene was amplified by PCR using the primer pair rcgR1435-F and rcgR1435-R (supplemental 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 pCRD610 contains the qorR gene fused to the His tag sequence. A cysteine residue at position 17 of QorR was replaced with serine by performing site-directed mutagenesis using the PrimeSTAR® mutagenesis basal kit (Takara Bio, Inc.). The pCRD610 plasmid was used as a template for PCR with the primer pair 1435C17S-F and 1435C17S-R (supplemental Table S1) to generate a plasmid, pCRD611. E. coli BL21(DE3) cells harboring pCRD610 or pCRD611 were grown at 37 °C in 500 ml of Luria-Bertani medium supplemented with kanamycin (50 μg ml⁻¹). The recombinant gene was expressed in exponentially growing cells (absorbance at 600 nm of 0.6) by adding 1 mm isopropyl β-d-thiogalactopyranoside. After 1 h of incubation, the cells were harvested by centrifugation. Recombinant proteins were purified by using the Ni-NTA Fast Start kit (Qiagen). Elution fractions containing purified proteins were loaded onto a PD-10 column (GE Healthcare) equilibrated with buffer A (20 mm Tris-HCl (pH 8.0), 0.1 m NaCl, 10% glycerol), and the protein was eluted with buffer A.

Gel Mobility Shift Assay

His-QorR was incubated with Cy3-labeled probes (2 nm) in 20 μl of the binding buffer (20 mm Tris-HCl (pH 8.0), 100 mm NaCl, 10 mm MgCl₂, 1 mm dithiothreitol (DTT), 10% glycerol) for 30 min at room temperature. Diamide, H₂O₂, and cumene hydroperoxide were added with the indicated concentrations. The mixtures were subjected to electrophoresis on a native 5% polyacrylamide gel, and Cy3-labeled probes were detected by the Typhoon Trio+^TM variable mode imager (GE Healthcare).

DNA fragments amplified by PCR using the primer pair RT1436-R and RT1435 and the primer pair RT2930 and RT2931 were cloned into the HincII site of the pHSG298 (Takara Bio, Inc.) to construct pCRD620 and pCRD621, respectively. Probes F1 and F2 were prepared by PCR using the primer pair 1436R+51 and RT1435-R and pCRD620 as a template and the primer pair RT2930-R and 2931R+92 and pCRD621 as a template, respectively (supplemental Table S1). QorR-binding sites (QBS)-deficient probes were generated as follows. QBS1, QBS2, and QBS3 sequences were deleted or mutated using PrimeSTAR® mutagenesis basal kit (Takara Bio, Inc.) with the specific primer pairs and pCRD620 as a template (supplemental Table S1). The resultant plasmids were used as a template for PCR with the primer pair 1436R+51 and 1435R+33 (supplemental Table S1). A Cy3-labeled 1436R+51 primer was used for preparation of Cy3-labeled probes.

Immunoblot Analysis

Cells grown to an absorbance at 610 nm of 2.5 was collected by centrifugation, and pellet was washed with phosphate-buffered saline (0.14 m NaCl, 2.7 mm KCl, 8.1 mm Na₂HPO₄, 1.5 mm KH₂PO₄) and mixed with 0.5 g of glass beads and 1 ml of phosphate-buffered saline. The cells were disrupted with a sonicator (Bioruptor UCD-250, Cosmo Bio) in a water bath at 4 °C. SDS-PAGE was carried out in 15% polyacrylamide gels by the method of Laemmli (21) using 20 μg of protein. Separated proteins were blotted to polyvinylidene difluoride membranes (Immobilon-P, Millipore) and probed with polyclonal antibodies to His-QorR proteins. The bound antibodies were detected with goat anti-rabbit IgG secondary antibodies conjugated to alkaline phosphatase (Sigma). Chemiluminescence reactions were done using the CDP-Star detection reagent (GE Healthcare), and the signal was scanned by a luminescent image analyzer (LAS-3000, FUJIFILM). Protein concentration was determined by using a protein assay (Bio-Rad) with bovine serum albumin as the standard.

Disc Diffusion Assays

Approximately 2 × 10⁷ cells of C. glutamicum strains were uniformly spread onto plates of A medium with 4% glucose, and a paper disc impregnated with 10 μl of 1 m diamide solution was placed onto the lawn. The susceptibility of C. glutamicum strains was determined by measuring of the size of a cleared zone of growth inhibition after 22 h of growth at 33 °C using ImageJ software (National Institutes of Health).

CgR1435 (QorR) Negatively Regulates Expression of qorR and qor2

To ascertain the physiological role of the DUF24 family protein encoded by cgR_1435 as a transcriptional regulator in C. glutamicum, gene expression profiles during exponential growth were compared between the wild type and a cgR_1435 disruptant (Δ1435) using a DNA microarray, and differential expression was confirmed by qRT-PCR analysis. Expression of only cgR_1436, which is located upstream of cgR_1435 in the opposite orientation (see Fig. 3), was affected by the disruption of cgR_1435. The transcript level of cgR_1436 was >500-fold higher in Δ1435 (Table 1). Because promoters of cgR_1435 and cgR_1436 are likely to overlap each other, the transcript level of cgR_1435 was determined by qRT-PCR using the primer pair designed upstream of the transposon insertion site of Δ1435. The transcript level of cgR_1435 in Δ1435 was about five times higher than that of the wild type (Table 1). To confirm that the observed changes in the transcript levels were due to the disruption of cgR_1435, the plasmid pCRD630 carrying the coding region of cgR_1435 and the control plasmid pCRC531 were used separately to transform Δ1435 cells. Whereas the transcript level of cgR_1436 in the resultant strain complemented with cgR_1435 was comparable with that of the wild type, the control plasmid did not effect any measurable changes in the transcript level of cgR_1436 (Table 1). These results indicate that CgR1435 negatively controls expression of cgR_1436 and its structural gene.

cgR_1436 is a homologue of the ytfG gene of E. coli with 54% amino acid identity. Given that ytfG codes for a novel type of NADPH-dependent quinone oxidoreductase (QOR2) (22), we designated cgR_1436 as qor2 and cgR_1435 as qorR for quinone oxidoreductase regulator.

Expression of qor2 and qorR Is Induced by Diamide

As quinone oxidoreductase plays a protective role against oxidative stress (23, 24), changes in expression of qor2 and qorR upon oxidative stress were examined. The transcript levels of qor2 and qorR in exponentially growing cells were determined by qRT-PCR before and after treatment of cells with diamide and H₂O₂ (Fig. 1). The results showed that expression of qorR was induced within 5 min after addition of diamide, with the qorR transcript level increasing about 8-fold after 10 min, before gradually decreasing (Fig. 1A). Expression of qor2 was also induced by diamide, with drastic increases in the transcript level of ∼200-fold after 5 min of diamide treatment (Fig. 1B). There was no increase observed in qor2 or qorR transcripts in response to the oxidative stress caused by H₂O₂ (data not shown). Expression of qor2 and qorR was thus induced only by disulfide stress.

QorR Binds to Promoter Regions of qor2 and qorR

Gel mobility shift assays were carried out with purified His-QorR and a DNA probe F1 of the intergenic region between qor2 and qorR (Fig. 2, A and C). His-QorR reduced the electrophoretic mobility of probe F1, and the amount of the QorR-F1 complex formed increased in proportion to the concentration of His-QorR (Fig. 2A, lanes 1–4). Three forms of the QorR-F1 complex with different electrophoretic mobility (C1, C2, and C3) were detected. The band intensity of the QorR-F1 complex was reduced upon addition of a nonlabeled F1 fragment (Fig. 2A, lanes 5 and 6). However, addition of a fragment F2 that contains the oxidative stress-responsive promoters of cgR_2930 and cgR_2931³ did not affect the amount of the QorR-F1 complex formed (Fig. 2A, lanes 7 and 8). These observations lead to the conclusion that His-QorR binds to the qor2-qorR intergenic region in a sequence-specific manner.

The genetic organization of qor2 and qorR is conserved among many bacterial species belonging to actinobacteria, proteobacteria, and cyanobacteria, based on data from the KEGG Sequence Similarity Database. For example, the ytfH gene encoding a transcriptional regulator of the DUF24 protein family, which is the sole gene of E. coli belonging to this family, is located upstream of the ytfG gene in the opposite orientation on the genome of E. coli. This suggests that regulatory sequences may also be conserved among these bacteria. Searches for conserved DNA motifs within the intergenic regions between qorR and qor2 homologues were performed using the BioProspector program (25). The sequence QBS1 was identified as a conserved sequence (Fig. 3A). In addition, similar sequences were found upstream of QBS1 in the opposite orientation (QBS2) and downstream of QBS1 (QBS3) (Fig. 3A). Interactions between His-QorR and the QBS sequences were elucidated by gel mobility shift assays (Fig. 2, B and C). Deletion of QBS1 abolished the interaction between His-QorR and the qor2-qorR intergenic region (Fig. 2B, lanes 1–4). His-QorR bound to QBS2- and QBS3-deficient probes, but the complex C3 was not observed with either probe (Fig. 2B, lanes 7–12). The inferences that QBS1 is indispensable for QorR binding to the qor2-qorR intergenic region and that QorR also binds to QBS2 and QBS3 follow. The putative QorR recognition sequence contains an inverted repeat sequence, ctTacN_4–5Gttag (Fig. 3B). The QBS1 sequence, ACTTACT₅GATAGT, was replaced with AGGCCGT₅CGGCTT to generate probe M1. No interaction between His-QorR and the probe M1 was observed (Fig. 2B, lanes 5 and 6), confirming that the inverted repeat sequence is recognized by QorR.

The transcription initiation sites of qor2 and qorR were determined by RACE-PCR experiments, and putative −10 and −35 promoter regions, which correspond to those of the SigA-dependent promoter (18), were found upstream of the respective transcription initiation sites (Fig. 3A). Within the qor2 promoter region, QBS1 and QBS2 overlap with the −35 region, and QBS3 overlaps with the −10 region. QBS2 also overlaps with the −35 region of the qorR promoter.

DNA-binding Activity of QorR Is Regulated by Cys-17 Oxidation

The QorR protein contains a lone cysteine residue at position 17 from the N terminus (Cys-17). As this cysteine residue is conserved among QorR homologues (supplemental Fig. S1A), Cys-17 is likely to be involved in the redox-sensitive control of QorR activity. The effect of oxidants on DNA-binding activity of QorR was thus examined by gel mobility shift assays (Fig. 4). Binding of QorR to probe F1 was prevented by addition of diamide (Fig. 4A, lanes 2–6). Addition of an excess of the reducing agent DTT restored DNA-binding activity of QorR that was inactivated by diamide (Fig. 4A, lanes 8–12), indicating that the effects of oxidation and reduction on DNA-binding activity of QorR are reversible. When the purified His-QorR protein was subjected to nonreducing SDS-PAGE, two bands of approximate molecular masses of 15 and 30 kDa were observed (Fig. 4B, lane 1). As the theoretical value of the molecular mass of His-QorR is 16.2 kDa, the 15 and 30 kDa bands are likely to correspond to the monomeric and dimeric forms of His-QorR, respectively. Treatment of QorR with DTT resulted in the loss of the 30-kDa band, whereas the intensity of the 30 kDa band was increased by diamide addition (Fig. 4B, lanes 2 and 3). The DNA-binding activity of QorR was also impaired by the other oxidants H₂O₂ or cumene hydroperoxide (Fig. 4C), and they enhanced dimerization of QorR (data not shown). These results support the conclusion that QorR undergoes dimerization and loses the DNA-binding activity under oxidizing conditions.

To ascertain the role of Cys-17 in the redox-sensitive regulation of QorR activity, Cys-17 was replaced with serine to generate the His-QorRC17S protein. His-QorRC17S reduced the electrophoretic mobility of probe F1 (Fig. 5A) and required three times as much protein as His-QorR to bind all probes (see Fig. 2A). Unlike the His-QorR protein, binding of His-QorRC17S to probe F1 was not inhibited by addition of diamide (Fig. 5B). When His-QorRC17S was treated with diamide, it remained in the monomeric form (Fig. 5C). Thus, Cys-17 of QorR is required for dimerization of proteins and the redox-responsive control of DNA-binding activity.

Cys-17 of QorR Is Required for Regulation of qor2 Expression in Response to Diamide

To investigate the role of Cys-17 of QorR in regulation of qor2 expression, QorR and QorRC17S proteins were expressed from plasmids pCRD630 and pCRD631, respectively, in Δ1435. QorR and QorRC17S were produced at similar levels as shown by immunoblot analysis (Fig. 6A). In Δ1435-expressing QorR, expression of the qor2 gene was induced after treatment with diamide in a similar way to the wild type, whereas its induction level was 10 times lower than that of the wild type (Figs. 1B and 6B). The qor2 transcript level of the strain expressing QorRC17S was three times higher than that of the QorR-expressing strain in exponentially growing cells (time 0 in Fig. 6B). However, qor2 was not induced by diamide in the QorRC17S-expressing strain (Fig. 6B). Expression of the trxB1 gene was induced by diamide in the QorRC17S-expressing strain as shown previously by Nakunst et al. (15), indicating that cells were subjected to disulfide stress (Fig. 6C). These results confirm that Cys-17 of QorR is required for induction of qor2 in response to diamide.

Qor2 Plays a Protective Role against Diamide Stress

The role of qor2 in disulfide stress response was investigated by disc diffusion assays using a disruptant of the qor2 gene (Δ1436) (Fig. 7). A paper disc containing diamide was plated onto a lawn of C. glutamicum cells on an agar plate, and the size of a clear zone of growth inhibition surrounding the paper disc was measured. The clear zone of Δ1436 plates was 20% larger than that of the wild type plates, indicating that disruption of the qor2 gene resulted in increased susceptibility to diamide.