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. 2009 Jun 19;284(25):16736-16742.
doi: 10.1074/jbc.M109.009027. Epub 2009 Apr 29.

Regulation of quinone oxidoreductase by the redox-sensing transcriptional regulator QorR in Corynebacterium glutamicum

Shigeki Ehira  1 , Hidetaka Ogino  1 , Haruhiko Teramoto  1 , Masayuki Inui  1 , Hideaki Yukawa  2
Affiliations

Regulation of quinone oxidoreductase by the redox-sensing transcriptional regulator QorR in Corynebacterium glutamicum

Shigeki Ehira et al. J Biol Chem. .

Abstract

Corynebacterium glutamicum cgR_1435 (cg1552) encodes a protein of the DUF24 protein family, which is a novel family of transcriptional regulators. CgR1435 (QorR) is a negative regulator of cgR_1436 (qor2), which is located upstream of cgR_1435 (qorR) in the opposite orientation, and its structural gene. QorR binds to the intergenic region between qor2 and qorR to repress their expression, which is induced by the thiol-specific oxidant diamide. The DNA-binding activity of QorR is impaired by oxidants such as diamide, H(2)O(2), and cumene hydroperoxide in vitro, and its lone cysteine residue (Cys-17) is essential for redox-responsive regulation of QorR activity both in vivo and in vitro. Moreover, a disruptant of qor2, which is a homologue of the ytfG gene of Escherichia coli encoding quinone oxidoreductase, shows increased sensitivity to diamide. It is concluded that the redox-sensing transcriptional regulator QorR is involved in disulfide stress response of C. glutamicum by regulating qor2 expression.

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Figures

FIGURE 1.
FIGURE 1.
Changes in the transcript levels of qor2 and qorR in response to diamide. The relative transcript levels of qorR (A) and qor2 (B) before (0 min) and at 5, 10, 15, 20, and 30 min after addition of 3 mm diamide were determined by qRT-PCR. The transcript levels were determined in triplicate using two independently grown cultures. The transcript level at 0 min was taken as 1.
FIGURE 2.
FIGURE 2.
Gel mobility shift assays with His-QorR and the intergenic region of the qor2 and qorR genes. A, the binding of His-QorR to probe F1, corresponding to the region from +51, with respect to the translation start site of the qor2 gene to +140, with respect to the translation start site of the qorR gene, was examined. Probe F1 (2 nm) was mixed with His-QorR in the amounts indicated above each lane, and then the mixtures were subjected to electrophoresis. Nonlabeled fragments of F1 (lanes 5 and 6) and F2 (lanes 7 and 8), which corresponds to the intergenic region between cgR_2930 and cgR_2931, were added at the indicated amounts. B, mutation and deletion analyses of QBSs are shown. The binding of His-QorR to DNA probes (2 nm) described below were examined. Lanes 1, 2, 7 and 8, probe F3, corresponding to the region from +51 with respect to the translation start site of the qor2 gene to +33 with respect to the translation start site of the qorR gene; lanes 3 and 4, QBS1-deficient probe D1; lanes 5 and 6; probe M1; in which the QBS1 sequence ACTTACT5GATAGT of probe F3 was replaced with the sequence AGGCCGT5CGGCTT; lanes 9 and 10, QBS2-deficient probe D2; lanes 11 and 12, QBS3-deficient probe D3. Open and closed arrowheads indicate the positions of free probes and the complexes of QorR and DNA probes, respectively. C, shown are the DNA probes used for gel mobility shift assays. Numbers adjacent to the fragments represent nucleotide positions relative to the start codons of the qorR or qor2 genes.
FIGURE 3.
FIGURE 3.
The nucleotide sequence of the qor2-qorR intergenic region. A, the coding regions of the qor2 and qorR genes are shaded in gray. The identified transcription initiation sites and the −10 and −35 promoter regions are indicated by bent arrows and boldface letters, respectively. QorR-binding sites (QBS1–3) are enclosed in boxes. B, alignment of QBSs. A putative QorR recognition sequence alignment is given at the bottom.
FIGURE 4.
FIGURE 4.
Redox-sensitive control of the DNA-binding activity of QorR. A, His-QorR (100 nm) and probe F1 (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-QorR was not added. B, purified His-QorR proteins (1 μg) incubated with 1 mm DTT (lane 2) or 1 mm diamide (lane 3) for 30 min were separated by nonreducing SDS-PAGE. Lane M, molecular mass standard marker; lane 1, His-QorR without additives. C, His-QorR (100 nm) and probe F1 (2 nm) were incubated in the presence of 1 mm DTT for 30 min, and then H2O2 (lanes 3–5) or cumene hydroperoxide (CHP) (lanes 6–8) was added in the amounts indicated above each lane. After 30 min, the mixtures were subjected to electrophoresis. Lane 1, His-QorR was not added.
FIGURE 5.
FIGURE 5.
Redox response of the QorRC17S protein. A, gel mobility shift assays with His-QorRC17S and the qor2-qorR intergenic region. Probe F1 (2 nm) was mixed with His-QorRC17S in the amounts indicated above each lane, and then the mixtures were subjected to electrophoresis. B, effects of diamide on the DNA-binding activity of QorRC17S. His-QorRC17S (300 nm) and probe F1 (2 nm) were incubated in the presence of 1 mm DTT for 30 min, and then diamide (lanes 3–5) was added in the amounts indicated above each lane. After 30 min, the mixtures were subjected to electrophoresis. Lane 1, His-QorRC17S was not added. C, purified His-QorRC17S proteins (1 μg) incubated with 1 mm DTT (lane 2) or 1 and 3 mm diamide (lanes 3 and 4) for 30 min were separated by nonreducing SDS-PAGE. Lane M; molecular mass standard marker; lane 1, His-QorRC17S was incubated without additives.
FIGURE 6.
FIGURE 6.
Response to diamide in the QorRC17S-expressing strain. A, immunoblot analysis of QorR proteins. Crude extracts (20 μg of proteins) from Δ1435 cells expressing QorR (left) or QorRC17S (right) were subjected to SDS-PAGE, and QorR derivatives were detected with polyclonal antibodies to His-QorR. Changes in the transcript levels of qor2 (B) and trxB1 (C) by diamide in Δ1435 strains expressing QorR (filled circles) or QorRC17S (open circles). The relative transcript levels before and after addition of 3 mm diamide were determined by qRT-PCR. Means and S.D. of three independent experiments are shown. The transcript level at 0 min in Δ1435 expressing QorR is taken as 1.
FIGURE 7.
FIGURE 7.
Susceptibility of the qor2 disruptant to diamide. A paper disc impregnated with 10 μl of 1 m diamide solution was placed onto the lawns of the wild type (A) and the qor2 disruptant (Δ1436; B). The plates were incubated at 33 °C for 22 h, and then the size of a clear zone was measured. Experiments were repeated three times with independently grown cultures. Ratio of the size of a clear zone in Δ1436 to the wild type was 1.22 ± 0.02.
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