Corynebacterium glutamicum is used in the industrial production of amino acids and nucleotides. During the course of fermentation, C. glutamicum cells face various stresses and employ multiple regulatory genes to cope with the oxidative stress. In this study, ORF cg3230 was selected from among the Corynebacterium glutamicum ORFs involved in stress response and designated as osnR for further analysis. The OsnR protein contains an XRE family domain and helix-turn-helix DNA-binding motif located near the C-terminus. Unlike the osnR-deleted (△osnR) strain, the osnR-overexpressing (P180-osnR) strain developed growth defects and increased sensitivity to various oxidant including H₂O₂. Transcription in the P180-osnR strain of genes such as sodA, ahpD, which are involved in the detoxification of reactive oxygen species, was only 40% that of the wild type. In addition, genes such as trxB and mtr that play roles in redox homeostasis and genes involved in the mycothiol metabolism were also found to be affected by overexpression of the osnR gene in C. glutamicum. The phenotypes of the P180-osnR strain suggest that the osnR gene plays a negative regulatory role in redox-dependent oxidative-stress responses. Furthermore, ChIP-seq analysis revealed that OsnR binds to the promoter region of multiple genes, including osnR and cdbC, which seems to function in the membrane-associated redox metabolism. Studies on the role of the osnR gene involving assays employing purified OsnR proteins and in vivo physiological analyses identified that OsnR inhibits the transcription of its own gene. Further, the oxidant diamide stimulated OsnR-binding to the promoter region of the osnR gene, suggesting that osnR may function as a thiol-based redox switch to respond to cellular redox status. The genes affected by the overexpression of osnR have been found to be under the control of σH. In the osnR-overexpressing strain, the transcription of sigH is significantly decreased and the stimulation of sigH transcription by external stress is lost, suggesting that osnR and sigH form an intimate regulatory network. This study suggests that OsnR not only functions as a transcriptional repressor of its own gene and of those involved in redox-dependent stress responses but also participates in the global transcriptional regulation by controlling the transcription of other master regulators, such as sigH. In addition, direct binding of the OsnR protein to the cdbC promoter region indicated that cdbC is involved in the osnR-mediated redox metabolism of C. glutamicum. The predicted amino acid sequence of the cdbC gene contained the signal sequence and the thioredoxin domain, which shows homology to the disulfide bond oxidoreductase family. Herein, CdbC was assessed for the functional properties in cell physiology. Purified CdbC, which contains 2 cysteine residues (Cys 98 and Cys 101) showed disulfide reductase activity as revealed by enzymatic activity assays. Experiments involving site-directed mutagenesis unveiled that the cysteine 98 in the Cys-Pro-Phe-Cys motif is required for the reductase activity. The cdbC-overexpressing (P180-cdbC) strain exposed to DTT showed growth defects and abnormal cell elongation. In addition, the P180-cdbC strain showed sensitivity to heat, lysozyme, and detergent, suggesting that CdbC plays a functional role in cell envelope. Furthermore, the composition of mycolic acid of the P180-cdbC cells which had been treated with DTT was altered, causing hydrophobic properties. Changes in mycolic acid and abnormal cell elongation might have been induced by mycoloyltransferases encoded by mytA and mytC. Mycoloyltransferases have two cysteine residues capable of forming disulfide bond, suggesting that MytA and MytC could be the targets for CdbC in periplasm. This is the first example of reducing pathway in Gram-positive bacteria that consists of membrane-associated redox metabolism.
Taken together, these data indicate that the osnR gene of C. glutamicum as redox switch suppresses its own expression and initiates the expression of other genes important for redox metabolism such as sigH and cdbC. These findings of redox metabolism provide an in-depth understanding of the regulatory mechanisms of stress responses in C. glutamicum.