CdbC: a disulfide bond isomerase involved in the refolding of mycoloyltransferases in Corynebacterium glutamicum cells exposed to oxidative conditions.

In Corynebacterium glutamicum cells, cdbC, which encodes a protein containing the CysXXCys motif, is regulated by the global redox-responsive regulator OsnR. In this study, we assessed the role of the periplasmic protein CdbC in disulfide bond formation and its involvement in mycomembrane biosynthesis. Purified CdbC efficiently refolded scrambled RNaseA, exhibiting prominent disulfide bond isomerase activity. The transcription of cdbC was decreased in cells grown in the presence of the reductant dithiothreitol (DTT). Moreover, unlike wild-type and cdbC-deleted cells, cdbC-overexpressing (P180-cdbC) cells grown in the presence of DTT exhibited retarded growth, abnormal cell morphology, increased cell surface hydrophobicity and altered mycolic acid composition. P180-cdbC cells cultured in a reducing environment accumulated trehalose monocorynomycolate, indicating mycomembrane deformation. Similarly, a two-hybrid analysis demonstrated the interaction of CdbC with the mycoloyltransferases MytA and MytB. Collectively, our findings suggest that CdbC, a periplasmic disulfide bond isomerase, refolds misfolded MytA and MytB and thereby assists in mycomembrane biosynthesis in cells exposed to oxidative conditions.

Gene nceA encodes a Ni/Co-sensing transcription factor to regulate metal efflux in Corynebacterium glutamicum.

The function of Corynebacterium glutamicum open reading frame (ORF) NCgl2684 (named nceA in this study), which was annotated to encode a metalloregulator, was assessed using physiological, genetic, and biochemical approaches. Cells with deleted-nceA (ΔnceA) showed a resistant phenotype to NiSO4 and CoSO4 and showed faster growth in minimal medium containing 20 µM NiSO4 or 10 µM CoSO4 than both the wild-type and nceA-overexpressing (P180-nceA) cells. In the ΔnceA strain, the transcription of the downstream-located ORF NCgl2685 (nceB), annotated to encode efflux protein, was increased approximately 4-fold, whereas gene transcription decreased down to 30% level in the P180-nceA strain. The transcriptions of the nceA and nceB genes were stimulated, even when as little as 5 nM NiSO4 was added to the growth medium. Protein NceA was able to bind DNA comprising the promoter region (from -14 to + 18) of the nceA--nceB operon. The protein-DNA interaction was abolished in the presence of 20 µM NiSO4, 50 µM CoSO4, or 50 µM CdSO4. Although manganese induced the transcription of the nceA and nceB genes, it failed to interrupt protein-DNA interaction. Simultaneously, the P180-nceA cells showed increased sensitivity to oxidants such as menadione, hydrogen peroxide, and cumene hydroperoxide, but not diamide. Collectively, our data show that NceA is a nickel- and cobalt-sensing transcriptional regulator that controls the transcription of the probable efflux protein-encoding nceB. The genes are able to suppress intracellular levels of nickel to prevent reactions, which can cause oxidative damage to cellular components.

The cysS gene (ncgl0127) of Corynebacterium glutamicum is required for sulfur assimilation and affects oxidative stress-responsive cysteine import.

The OsnR protein functions as a transcriptional repressor of genes involved in redox-dependent stress responses. Here, we studied Corynebacterium glutamicum ORF ncgl0127 (referred to as cysS in this study), one of the target genes of OsnR, to reveal its role in osnR-mediated stress responses. The ΔcysS strain was found to be a cysteine auxotroph, and the transcription levels of the sulfur assimilatory genes and cysR, the master regulatory gene for sulfur assimilation, were low in this strain. Complementation of the strain with cysR transformed the strain into a cysteine prototroph. Cells challenged with oxidants or cysteine showed transcriptional stimulation of the cysS gene and decreased transcription of the ncgl2463 gene, which encodes a cysteine/cystine importer. The transcription of the ncgl2463 gene was increased in the ΔcysS strain and further stimulated by cysteine. Unlike the wild-type strain, ΔcysS cells grown with an excess amount of cysteine showed an oxidant- and alkylating agent-resistant phenotype, suggesting deregulated cysteine import. Collectively, our data suggest that the cysS gene plays a positive role in sulfur assimilation and a negative role in cysteine import, in particular in cells under oxidative stress.

Trehalose biosynthetic gene otsB of Corynebacterium glutamicum is regulated by whcE in response to oxidative stress.

The gene whcE of Corynebacterium glutamicum plays a positive role in oxidative stress responses and the WhcE protein interacts with SpiE. By utilizing 2D-PAGE analysis, we identified the otsB gene to be under the control of whcE. The transcription of otsB, encoding trehalose 6-phosphatase, was stimulated by oxidative stress, and whcE and spiE were involved in diamide-mediated transcriptional stimulation. The ΔotsB strain was created and found to be sensitive to the thiol-specific oxidant diamide, suggesting a role of the gene in stress responses. Genes located upstream of otsB, such as NCgl2534 and otsA, formed an operon and purified WhcE was able to bind to the promoter region of the operon (PNCgl2534), but the binding was only possible in the presence of the oxidant diamide. In addition, the transcriptional activation of PNCgl2534 by WhcE was demonstrated in in vivo assays and the transcription was stimulated in cells exposed to the oxidant diamide. These findings indicate that WhcE is a transcriptional activator, and otsB, which is involved in trehalose biosynthesis, has a role in oxidative stress responses in C. glutamicum.

OsnR is an autoregulatory negative transcription factor controlling redox-dependent stress responses in Corynebacterium glutamicum.

BACKGROUND: 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. The osnR gene plays a negative regulatory role in redox-dependent oxidative-stress responses, but the underlying mechanism is not known yet. RESULTS: Overexpression of the osnR gene in C. glutamicum affected the expression of genes involved in the mycothiol metabolism. ChIP-seq analysis revealed that OsnR binds to the promoter region of multiple genes, including osnR and cg0026, which seems to function in the membrane-associated redox metabolism. Studies on the role of the osnR gene involving in vitro assays employing purified OsnR proteins and in vivo physiological analyses have identified that OsnR inhibits the transcription of its own gene. Further, oxidant diamide stimulates OsnR-binding to the promoter region of the osnR gene. 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. CONCLUSIONS: Our 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.

Thiol-specific oxidant diamide downregulates whiA gene of Corynebacterium glutamicum, thereby suppressing cell division and metabolism.

The whiA (NCgl1527) gene from Corynebacterium glutamicum plays a crucial role during cell growth, and WhiA is recognized as the transcription factor for genes involved in cell division. In this study, we assessed the regulatory role of the gene in cell physiology. Transcription of the gene was specifically downregulated by the thiol-specific oxidant, diamide, and by heat stress. Cells exposed to diamide showed decreased transcription of genes involved in cell division and these effects were more profound in ΔwhiA cells. In addition, the ΔwhiA cells showed sensitivity to thiol-specific oxidants, DNA-damaging agents, and high temperature. Further, downregulation of sigH (NCgl0733), the central regulator in stress responses, along with master regulatory genes in cell metabolism, was observed in the ΔwhiA strain. Moreover, the amount of cAMP in the ΔwhiA cells in the early stationary phase was only at 30% level of that for the wild-type strain. Collectively, our data indicate that the role of whiA is to downregulate genes associated with cell division in response to heat or thiol-specific oxidative stress, and may suggest a role for the gene in downshifting cell metabolism by downregulating global regulatory genes when growth condition is not optimal for cells.

Corynebacterium glutamicum whiA plays roles in cell division, cell envelope formation, and general cell physiology.

The whiA gene is widely distributed among Gram-positive bacteria. Although the encoded protein has conserved N-terminal homing endonuclease scaffold and C-terminal helix-turn-helix DNA-binding domains, whiA plays a unique physiological role in its host organisms, reflecting a long history of evolution. Here, we used genetic approaches to unveil the physiological function of whiA in Corynebacterium glutamicum. We found that cells lacking whiA (ΔwhiA) were unable to grow in minimal medium containing glucose, although reduced growth was observed in complex medium. The ΔwhiA strain showed altered transcription of the cell division genes ftsZ, sepF, ftsK, crgA, divIVA, and amiC genes. Accordingly, ΔwhiA cells exhibited large, elongated, branched, and bud-shaped morphologies. In addition, some genes, including fas-IA, fas-IB, accD1, and cmrA, which help synthesize the fatty acid and cell envelope component mycolic acid, showed altered transcription in the ΔwhiA strain. Further, treS, treY, otsA, and otsB, which are involved in the biosynthesis of the outer envelope component trehalose, were down-regulated in the ΔwhiA strain. 2D-PAGE analysis of the ΔwhiA mutant showed that proteins involved in other cellular activities were also affected by the loss of whiA. These findings suggest that C. glutamicum whiA plays a critical role in cell division, envelope formation, and general cell physiology.

The osnR gene of Corynebacterium glutamicum plays a negative regulatory role in oxidative stress responses.

Among the Corynebacterium glutamicum ORFs that have been implicated in stress responses, we chose ORF cg3230, designated osnR, and analyzed it further. Unlike the osnR-deleted strain (ΔosnR), the osnR-overexpressing strain (P180-osnR) developed growth defects and increased sensitivity to various oxidants including H2O2. Transcription in the P180-osnR strain of genes such as sodA (superoxide dismutase), ftn (ferritin biosynthesis), and ahpD (alkyl hydroperoxide reductase; cg2674), which are involved in the detoxification of reactive oxygen species, was only 40% that of the wild type. However, transcription of katA, encoding H2O2-detoxifying catalase, was unchanged in this strain. Genes such as trxB (thioredoxin reductase) and mtr (mycothiol disulfide reductase), which play roles in redox homeostasis, also showed decreased transcription in the strain. 2D-PAGE analysis indicated that genes involved in redox reactions were considerably affected by osnR overexpression. The NADPH/NADP+ ratio of the P180-osnR strain (1.35) was higher than that of the wild-type stain (0.78). Collectively, the phenotypes of the ΔosnR and P180-osnR strains suggest a global regulatory role as well as a negative role for the gene in stress responses, particularly in katA-independent oxidative stress responses.

The ahpD gene of Corynebacterium glutamicum plays an important role in hydrogen peroxide-induced oxidative stress response.

In this study, we analysed the ahpD gene from Corynebacterium glutamicum, which may function in a H2O2-mediated stress responses. Cells overexpressing C. glutamicum ahpD (P180-ahpD) showed increased sensitivity to H2O2 when exposed to the latter in concentrations of 8 mM or greater while showing reduced expression of katA, which encodes catalase. On the other hand, cells that lack ahpD (ΔahpD) displayed increased sensitivity when exposed to low levels of H2O2 while showing katA transcription that was comparable to the level in the wild-type strain. Accordingly, transcription of ahpD and katA was stimulated by low and high concentration of H2O2, respectively. Further, the NAD+/NADH ratio was severely reduced in the ΔahpD (3.03) and P180-ahpD (0.47) strains as compared with that in the wild-type (4.55) strain. Transcriptional analysis indicated that ahpD and upstream genes such as cg2675, cg2676, cg2677 and cg2678, which were annotated as ABC-type transporter, were organized into an operon. Collectively, these findings indicate that C. glutamicum possesses bi-level defence pathways against hydrogen peroxide, involving katA and ahpD. Further, ahpD, along with cg2675-cg2678 genes, may play a novel role in cellular activities against oxidative stress.