| The high-(G+C)-content Gram-positive Corynebacterium glutamicum is not only a typical model organism for a variety of pathogens in systems biology,but also a cell factory for fermentation of biobased products.However,in the fermentation process,it is inevitable to be subjected to reactive oxygen species(ROS)-inducing that produce reactive oxygen species(ROS),affecting the physiological status and properties of bacteria,the quality of fermentation products,and even the strain survival.The robustness of industrial microorganisms is manifested in high antioxidant capacity.In order to resist oxidative stress and survive in extreme environment,microorganisms use various antioxidant enzymes to sense and monitor the changes of redox state in cells at any time,including terminal peroxidase,disulfide oxidoreductase,regulatory protein and so on.Among them,Grx,as a glutathione(GSH)dependent disulfide oxidoreductase,plays an important role in catalyzing various mercaptan disulfide bond exchange reactions,maintaining cell redox balance and antioxidant enzyme activity.Although mycoredoxin-1(Mrx1),which is homologous to Grx in C.glutamicum,has been proved to have the de-mycothiolated activity,there are few studies on the physiological phenotypes,biochemical characteristics and regulatory mechanisms of Mrx1.Therefore,in this study,we took Mrx1 in C.glutamicum as the research object to reveal the inherent physiological phenotypes and biochemical characteristics of Mrx1.1.To assess the physiological role of Mrx1,disk assay and growth curve are performed to test the sensitive phenotype of wild type(WT),mrx1-deficient mutants(?mrx1)and corresponding complementary strains(?mrx1~+)to various oxidants,alkylating agents,heavy metals and antibiotics.The results showed that mrx1 gene deletion mutants in C.glutamicum were highly sensitive to oxidants(hydrogen peroxide,cumene hydrogen peroxide,menadione,hydrazine),alkylating agents(iodoacetamide and 1-chloro-2,4-dinitrobenzene),heavy metals(cadmium chloride),and moderately sensitive to antibiotics(neomycin,gentamicin,vancomycin and azithromycin).These results suggest that Mrx1 mediates the resistance of C.glutamicum to the above stresses,and that Mrx1 plays an important role in maintaining the antioxidant stress of C.glutamicum.2.Mrx1 is specififically regenerated with electrons from the mycothiol(MSH)/the flflavoprotein mycothiol disulphide reductase(Mtr)/NADPH pathway.3.Similar to Grx,Mrx1 can not only catalytically reduce the disulfides in the ribonucleotide reductase Ib(Ib RNR),insulin and 5,5?-dithiobis-(2-nitrobenzoic acid)(DTNB),but also reduce S-mycothiolated mixed disulphides,such as HED-SSM and Prx-SSM by exclusively linking linked to the mycothiol electron transfer pathway.The N-terminal nucleophilic cysteine of Mrx1 is essential for the reduction of S-mycothiolated mixed disulphides,only involves the N-terminal cysteine of CXXC,while the reduction of disulfides by Mrx1 requires the N-terminal and C-terminal cysteines of the CXXC catalytic motif in Mrx1.As such,Mrx1 is functioning as a monothiol mixed disulfide reductase via 1-Cys mechanism in the presense of S-mycothiolated mixed disulphides and dithiol reductase via 2-Cys mechanism in the presense of protein disulfides.4.The stress-responsive extracytoplasmic function-sigma(ECF-?)factor Sig H is involved in the response to oxidative stress in C.glutamicum.The Sig H-driven response to oxidative stress generally includes the upregulation of the thioredoxin system and at least one gene(mtr)of the mycothiol system,which are major antioxidant systems in these bacteria.The expression of mrx1 in C.glutamicum is induced by various stress.Electrophoretic Mobility Shift Assay(EMSA)shows that Sig H directly controls mrx1.In conclusion,This study shows Mrx1 is involved in the protection against oxidative stress by acting as a MSH-dependent reductase exclusively linked to the mycothiol electron transfer pathway,thereby giving new insight in how C.glutamicum survived oxidative stressful conditions. |
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