Thioredoxin and protein-disulfide isomerase selectivity for redox regulation of proteins in Corynebacterium glutamicum.

Thioredoxins (Trxs) and protein-disulfide isomerases (PDIs) are believed to play a pivotal role in ensuring the proper folding of proteins, facilitating appropriate functioning of proteins, and maintaining intracellular redox homeostasis in bacteria. Two thioredoxins (Trxs) and three thiol-disulfide isomerases (PDIs) have been annotated in Corynebacterium glutamicum. However, nothing is known about their functional diversity in the redox regulation of proteins. Thus, we here analyzed the Trx- and PDI-dependent redox shifts of ribonucleotide reductase (RNR), insulin, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and several thiol-dependent peroxidases by measuring enzyme activity and thiol status in vitro. We found that the two Trxs and the three PDIs had activities in the cleavage of the disulfidebond, whereas the PDIs had a lower efficiency than the two Trxs. Trx2 could activate thiol-dependent peroxidases with an efficiency comparable with that of Trx1, but the PDIs were inefficient. The redox-active Cys-X-X-Cys motif harbored in both Trxs and PDIs was essential to supply efficiently the donor of reducing equivalents for protein disulfides. In addition, stress-responsive extracytoplasmic function (ECF)-sigma factor H (SigH)-dependent Trxs and PDIs expressions were observed. These results contributed importantly to our overall understanding of reducing functionality of the Trx and PDI systems, and also highlighted the complexity and plasticity of the intracellular redox network.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance.

MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)-uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882-ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to ß-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR-uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

OsmC in Corynebacterium glutamicum was a thiol-dependent organic hydroperoxide reductase.

Bacterial antioxidants play a vital role in the detoxification of exogenous peroxides. Several antioxidant defenses including low-molecular-weight thiols (LMWTs) and protective enzymes were developed to help the bacterium withstand the adverse stress. Although osmotically induced bacterial protein C (OsmC), classified as the organic hydroperoxide reductase (Ohr)/OsmC superfamily, has been demonstrated in some mycobacterial species, including M. tuberculosis and M. smegmatis, its physiological and biochemical functions in C. glutamicum remained elusive. Here we found the lack of C. glutamicum osmC gene resulted in decreased cell viability and increased intracellular reactive oxygen species accumulation under organic hydroperoxides (OHPs) stress conditions. The osmC expression was induced in the multiple antibiotic resistance regulator MarR-dependent manner by OHPs, and not by other oxidants or osmotic stress. Peroxide reductase activity showed that OsmC had a narrow range of substrates-only degrading OHPs, and detoxified OHPs mainly by linking the alkyl hydroperoxide reductase (AhpD) system (AhpD/dihydrolipoamide dehydrogenase (Lpd)/dihydrolipoamide acyltransferase (SucB)). Site-directed mutagenesis confirmed Cys48 was the peroxidatic cysteine, while Cys114 was the resolving Cys residue that formed an intramolecular disulfide bond with oxidized Cys48. Therefore, C. glutamicum OsmC was a thiol-dependent OHP reductase and played important role of protection against OHPs together with Ohr.