Mechanisms Of Mycothiol In Resistace To Multiple Environmental Stresses In Corynebacterium Glutamicum

Besides being a very important industrial microorganism (mainly used for the productionof amino acids), Corynebacterium glutamicum is also used as a typical model organism inmicroorganism. In the fermentation process of biological products, however, it will inevitablymeet a variety of environmental stimulus. These adverse environmental stimuluses led to acommon effect-oxidative stress, which inactivates biomacromolecules by forming ROS.Therefore, its robustness against adversity is critical to the quality and quantity offermentation products. And how to improve robustness has became the focus of lots ofresearch on Corynebacterium glutamicum. Organisms have evolved a powerful antioxidantdefense mechanism for reducing the damage effects of ROS, which consisting of antioxidantenzyme systems and low molecular mass antioxidants (LMMA). The latter refered mainly tolow molecular mass thiol, including cysteine (Cys), coenzyme A (CoA), vitamin C, vitamin E,glutathione (glutathione, GSH) and Mycothiol (Mycothiol, MSH). Among them, GSH hasbeen well-studied on its metabolic pathway, physiological function and roles in detoxificationand response to antioxidant stresses. However, Actinomycetales, such as Corynebacteriumglutamicum, did not produce GSH, instead they produce MSH. Studies showed that MSH hadphysiological functions similar to GSH, being essential in detoxification of oxidants, toxins,antibiotics and the degradation of aromatic compounds. Our previous studies have shown thatoverexpression of MSH can obviously improve robustness and survival of Corynebacteriumglutamicum under various environmental stresses. Therefore, expression of MSH became animportant means of improving resistance to environmental stress. The study of MSH in recentyears mainly focused on its metabolic pathways, physiological function and new function asdrug target, while molecular mechanism of resistance to environmental stresses which MSHtake part in was neglected. Thus, by taking Corynebacterium glutamicums as a modelorganism, we aim to uncover the underlying molecular mechanism of MSH in resistance tomultiple environmental stresses. The main results are as follows:1. Protein S-glutathionylation, the reversible formation of a mixed-disulfide betweenglutathione and thiols of protein, is involved not only in protection of protein cysteines fromirreversible oxidation, but also in protein redox regulation. Since MSH can helpCorynebacterium glutamicum enhancing its tolerance to environmental stress, the existing of similar S-mycothiolated protein modification was proposed but well-studied. Considering thepotential importance of mycothiolation, a novel method for identification and analysis of S-mycothiolated proteins was designed in this study. The method was developed based on thecombination of the biotin-streptavidin technique, Mrx1:C15S/MSH/Mtr system and Trx/TrxRsystem. Among them, specific reduction of MSH-protein mixed disulphides can be performedby Mrx1:C15S/MSH/Mtr system, and intramolecular or intermolecular disulfide bond wasexclusively reduced by Trx/TrxR system. By using ΔmshC (the mutant strains completely lostthe ability to synthetic MSH) as a reference,12S-mycothiolated proteins were identified fromthe wild type Corynebacterium glutamicum, including the methionine synthase (MetE), themyoinositol-1-phosphate synthase (Ino1), sugar (pentulose and hexulose) kinase (XylB) andadenylate kinase (Adk); enzymes for the biosynthesis of nucleotides (GuaB1and GuaB2);translation proteins (PheT, RplM and RpsC); antioxidant enzymes (Prx, Mpx and MsrA). Wefurther confirmed S-mycothiolation of Ino1, MsrA, Mpx, MetE and Prx by Western Blottingand LC-MS/MS analysis.2. We revealed the inherent mechanisms of MSH in resistance to multiple environmentalstress:(1) The redox buffer MSH could be directly involved in ROS detoxification leading toMSSM formation;(2) S-mycothiolation of the active site Cys residues of MetE and Ino1ledto metabolite auxotrophy during the detoxification process, then swith off the metabolicpathways to prevent further destroying of the cells;(3) MSH could protect cells from peroxideor ROS by acting as a cofactor for antioxidant enzymes such as Mpx and MsrA;(4) MSH wasconjugated to alkylating agents, oxidants (menadione), antibiotics (rifamycin), heavy metalsand other environmental stressors through its thiol moiety, forming the MSH S-conjugates.MSH S-conjugates were then cleaved by an amidase (Mca) to accomplish detoxification;(5)Reversible S-mycothiolation modification of Prx acted as a major regulatory mechanism inregulating its oligomer state and activity in response to certain concentrations of peroxide.3. We revealed the mechanism of SigH in regulation of antioxidant enzymes as well asenzymes related to the synthesis of MSH under different environmental stimulus. Theexpression of mca, mtr, msrA as well as mpx was induced by various environmental stresses(including oxidant, alkylating agents, antibiotic and heavy metal) and directly mediated bySigH. On the contrary, the expression of prx which coding for the thiol peroxidase wasdirectly inhibited by SigH. In addition, SigH plays no role in regulation of Inol, whichexpressed in a constitutive manner.