Alkyl hydroperoxide reductase (Ahp), comprised of four different subunits AhpC, AhpD, AhpE, and AhpF, is a thiol-based antioxidative enzyme with the ability to protect bacteria against oxidative stress. Functionally, AhpC and AhpE considered as peroxidases directly detoxify peroxides, while AhpD and AhpF as oxidoreductases restore oxidized peroxidases to their reduced form. Corynebacterium glutamicum ncgl0877 encodes a putative Ahp with a unique Cys-Pro-Phe-Cys (C-P-G-C) active-site motif, similar with those of the thiol-disulfide oxidoreductases such as thioredoxin (Trx), mycoredoxin-1 (Mrx1) and AhpD. However, its physiological and biochemical functions remain unknown in C. glutamicum. Here, we report that NCgl0877, designated CgAhp, is involved in the protection against organic peroxide (OP) stress. The cgahp-deleted strain is notably more sensitive to OP stress. The cgahp expression is controlled by a MarR-type transcriptional repressor OasR (organic peroxide- and antibiotic-sensing regulator). The physiological role of CgAhp in resistance to OP stresses is corroborated by its induced expression under stresses. Although CgAhp has a weak peroxidase activity toward OP, it mainly supports the OP-scavenging activity of the thiol-dependent peroxidase preferentially linked to the dihydrolipoamide dehydrogenase (Lpd)/dihydrolipoamide succinyltransferase (SucB)/NADH system. The C-P-G-C motif of CgAhp is essential to maintain the reductase activity. In conclusion, our study identifies CgAhp, behaving like AhpD, as a key disulfide oxidoreductase involved in the oxidative stress tolerance and the functional electron donor for peroxidase. © 2023, The Author(s).

以C.glutamicum中的NCgl0877(命名为CgAhp)为研究对象,揭示了CgAhp内在生理表型及其发挥这些功能的生化特性和调控机制.结果表明:C.glutamicum中cgahp基因缺失突变体仅对有机过氧化物异丙苯过氧化氢(CHP)和叔丁基过氧化氢(t-BHP)高度敏

Background The TetR (tetracycline repressor) family is one of the major transcription factor families that regulate expression of genes involved in bacterial antimicrobial resistance systems. NCgl0886 protein, designated as AtsR, is a member of the TetR family identified in Corynebacterium glutamicum, which is conserved in several species of the genera Corynebacterium, also including the well-known pathogen C. diphtheriae. AtsR is located at no far upstream of the identically oriented ncgl0884 gene, encoding a putative multidrug efflux pump protein, and in the same operon with ncgl0887, encoding a resistance, nodulation and cell division (RND) superfamily drug exporter. However, the role of AtsR is not clearly understood. Results Here we showed that dimeric AtsR directly repressed the expression of the ncgl0887-atsR operon, as well as indirectly controlled the ncgl0884 transcription. Antibiotics and toxic compounds induced the expression of ncgl0887-atsR operon. A perfect palindromic motif (5GREEK TONOS-TGCAA-N-2-TTGCA-3GREEK TONOS; 12 bp) was identified in the upstream region of ncgl0887-atsR operon. Electrophoretic mobility shift assays (EMSAs) demonstrated specific binding of AtsR to this motif, and hydrogen peroxide (H2O2) blocked binding. H2O2 oxidized cysteine residues to form Cys123-Cys187 intermolecular disulfide bonds between two subunits in AtsR dimer, which altered its DNA-binding characteristics and caused its dissociation, thereby leading to derepression of the drug efflux protein. Deletion of ncgl0884 and ncgl0887 increased the susceptibilities of C. glutamicum for several toxic compounds, but overexpression of atsR decreased the drug tolerance of C. glutamicum. Conclusions Our study revealed that AtsR was a redox regulator that sensed oxidative stress via thiol modification. The results obtained here will contribute to our understanding of the drug response mechanism not only in C. glutamicum but also in the related bacteria C. diphtheriae.

Background CssR, the product of the Corynebacterium glutamicum ncgl1578 gene cotranscribed with ncgl1579, is a TetR (tetracycline regulator) family repressor. Although many TetR-type regulators in C. glutamicum have been extensively described, members of the TetR family involved in the stress response remain unidentified. Results In this study, we found that CssR regulated the transcription of its own gene and the ncgl1576-ncgl1577 operon. The ncgl1576-ncgl1577 operon, which is located upstream of cssR in the orientation opposite that of the cssR operon, encodes an ATP-binding cassette (ABC), some of which are involved in the export of a wide range of antimicrobial compounds. The cssR-deletion (Delta cssR) mutant displayed increased resistance to various stresses. An imperfect palindromic motif (5 '-TAA(G)TGN(13)CA(G)TTA-3 '; 25 bp) located at the intergenic region between cssR and ncgl1577 was identified as the sole binding site for CssR. Expression of cssR and ncgl1577 was induced by antibiotics and heavy metals but not H2O2 or diamide, and the DNA-binding activity of CssR was impaired by antibiotics and heavy metals but not H2O2. Antibiotics and heavy metals caused CssR dissociation from target gene promoters, thus derepressing their transcription. Oxidant treatment neither altered the conformation of CssR nor modified its cysteine residues, indicating that the cysteine residues in CssR have no redox activity. In the Delta cssR mutant strain, genes involved in redox homeostasis also showed increased transcription levels, and the NADPH/NADP(+) ratio was higher than that of the parental strain. Conclusion The stress response mechanism of CssR in C. glutamicum is realized via ligand-induced conformational changes of the protein, not via cysteine oxidation-based thiol modification. Moreover, the crucial role of CssR in the stress response was demonstrated by negatively controlling the expression of the ncgl1576-ncgl1577 operon, its structural gene, and/or redox homeostasis-related genes.

ncgl2478 gene from Corynebacterium glutamicum encodes a thiol-disulfide oxidoreductase enzyme annotated as dithiol-disulfide isomerase DsbA. It preserves a Cys-Pro-Phe-Cys active-site motif, which is presumed to be an exclusive characteristic of the novel DsbA-mycoredoxin 1 (Mrx1) cluster. However, the real mode of action, the nature of the electron donor pathway and biological functions of NCgl2478 in C. glutamicum have remained enigmatic so far. Herein, we report that NCgl2478 plays an important role in stress resistance. Deletion of the ncgl2478 gene increases the size of growth inhibition zones. The ncgl2478 expression is induced in the stress-responsive extra-cytoplasmic function-sigma (ECF-sigma) factor SigH-dependent manner by stress. It receives electrons preferentially from the mycothiol (MSH)/mycothione reductase (Mtr)/NADPH pathway. Further, NCgl2478 reduces S-mycothiolated mixed disulfides and intramolecular disulfides via a monothiol-disulfide and a dithiol-disulfide exchange mechanism, respectively. NCgl2478 lacks oxidase activity; kinetic properties of its demycothiolation are different from those of Mrx1. Site-directed mutagenesis confirms Cys24 is the resolving Cys residue, while Cys21 is the nucleophilic cysteine that is oxidized to a sulfenic acid and then forms an intramolecular disulfide bond with Cys24 or a mixed disulfide with MSH under oxidative stress. In conclusion, our study presents the first evidence that NCgl2478 protects against various stresses by acting as an MSH-dependent thiol-disulfide reductase, belonging to a novel DsbA-Mrx1 cluster.

Glutaredoxins (Grxs) with Cys-Pro-Phe (Tyr)-Cys motif and a thioredoxin fold structure play an important role in the anti-oxidant system of bacteria by catalyzing a variety of thiol-disulfide exchange reactions with a 2-Cys mechanism or a 1-Cys mechanism. However, the catalytic and physiological mechanism of Corynebacterium glutamicum Mycoredoxin 1 (Mrx1) that shares a high amino acid sequence similarity to Grxs has not been fully elucidated. Here, we report that Mrx1 has a protective function against various adverse conditions, and the decrease of cell viability to various stress conditions by deletion of the Mrx1 in C. glutamicum was confirmed in the mrx1 mutant. The physiological roles of Mrx1 in defence to oxidative stress were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic function-sigma (ECF-s) factor SigH. As well as reducing mycothiol (MSH) mixed disulfide bonds via a 1-Cys mechanism, C. glutamicum Mrx1 catalytically reduced the disulfides in the Ib RNR, insulin and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) by exclusively linking the MSH/Mtr (mycothiol disulfide reductase)/ NADPH electron pathway via a 2-Cys mechanism. Thus, we present the first evidence that the Mrx1 is able to protect against the damaging effects of various exogenous stresses by acting as a disulfide oxidoreductase, thereby giving a new insight in how C. glutamicum survives oxidative stressful conditions.

Corynebacterium glutamicum is an important industrial strain for amino acids and a key model organism for human pathogens. The study of C. glutamicum oxidoreductases, such as mycoredoxin 1 ( Mrx1), dithiol-disulfide isomerase DsbA, and DsbA-like Mrx1, is helpful for understanding the survival, pathogenic infection, and stress resistance of its homologous species. However, the action mode and enzymatic function of C. glutamicum NCgl0018 preserving the Cys-Pro-Phe- Cys motif, annotated as a putative DsbA, have remained enigmatic. Here, we report that the NCgl0018-deleted strain increased sensitivity to various oxidative stresses. The ncgl0018 expression was induced in the stress-responsive extracytoplasmic function-sigma (ECF-sigma) factor SigH- and organic peroxide- and antibioticsensing regulator (OasR)-dependent manner by stress. NCgl0018 reduced S- mycothiolated mixed disulfides and intramolecular disulfides via a monothiol-disulfide mechanism preferentially linking the mycothiol/mycothione reductase/NADPH electron pathway. Site-directed mutagenesis confirmed Cys107 was the resolving Cys residue, while Cys104 was the nucleophilic cysteine that was oxidized to a sulfenic acid and then could form an intramolecular disulfide bond with Cys107 or a mixed disulfide with mycothiol under stress. Biochemical analyses indicated that NCgl0018 lacked oxidase properties like the classical DsbA. Further, enzymatic rates and substrate preferences of NCgl0018 were highly similar to those of DsbA-like Mrx1. Collectively, our study presented the first evidence that NCgl0018 protected against stresses by functioning as a novel DsbA- like Mrx1 but not DsbA and Mrx1.

Background Corynebacterium glutamicumthrives under oxidative stress caused by the inevitably extreme environment during fermentation as it harbors antioxidative stress genes. Antioxidant genes are controlled by pathway-specific sensors that act in response to growth conditions. Although many families of oxidation-sensing regulators inC. glutamicumhave been well described, members of the xenobiotic-response element (XRE) family, involved in oxidative stress, remain elusive. Results In this study, we report a novel redox-sensitive member of the XER family, MsrR (multiple stress resistance regulator). MsrR is encoded as part of themsrR-3-mst(3-mercaptopyruvate sulfurtransferase) operon;msrR-3-mstis divergent from multidrug efflux proteinMFS. MsrR was demonstrated to bind to the intergenic region betweenmsrR-3-mstandmfs. This binding was prevented by an MsrR oxidation-mediated increase in MsrR dimerization. MsrR was shown to use Cys62 oxidation to sense oxidative stress, resulting in its dissociation from the promoter. Elevated expression ofmsrR-3-mstandmfswas observed under stress. Furthermore, a Delta msrRmutant strain displayed significantly enhanced growth, while the growth of strains lacking either3-mstormfswas significantly inhibited under stress. Conclusion This report is the first to demonstrate the critical role of MsrR-3-MST-MFS in bacterial stress resistance.

Corynebacterium glutamicum, an important industrial and model microorganism, inevitably encountered stress environment during fermentative process. Therefore, the ability of C. glutamicum to withstand stress and maintain the cellular redox balance was vital for cell survival and enhancing fermentation efficiency. To robustly survive, C. glutamicum has been equipped with many types of redox sensors. Although cysteine oxidation-based peroxide-sensing regulators have been well described in C. glutamicum, redox sensors involving in multiple environmental stress response remained elusive. Here, we reported an organic peroxide- and antibiotic-sensing MarR (multiple antibiotics resistance regulators)-type regulator, called OasR (organic peroxide- and antibiotic-sensing regulator). The OasR regulator used Cys95 oxidation to sense oxidative stress to form S-mycothiolated monomer or inter-molecular disulfide-containing dimer, resulting in its dissociation from the target DNA promoter. Transcriptomics uncovered the strong up-regulation of many multidrug efflux pump genes and organic peroxide stress-involving genes in oasR mutant, consistent with the phenomenon that oasR mutant showed a reduction in sensitivity to antibiotic and organic peroxide. Importantly, the addition of stress-associated ligands such as cumene hydroperoxide and streptomycin induced oasR and multidrug efflux pump protein NCgl1020 expression in vivo. We speculated that cell resistance to antibiotics and organic peroxide correlated with stress response-induced up-regulation of genes expression. Together, the results revealed that OasR was a key MarR-type redox stress-responsive transcriptional repressor, and sensed oxidative stress generated through hydroxyl radical formation to mediate antibiotic resistance in C. glutamicum.