Abstract
Mycothiol (MSH) functions as major low molecular weight (LMW) thiol in the industrially important Corynebacterium glutamicum. In this study, we genomically integrated an Mrx1-roGFP2 biosensor in C. glutamicum to measure dynamic changes of the MSH redox potential (EMSH) during the growth and under oxidative stress. C. glutamicum maintains a highly reducing intrabacterial EMSH throughout the growth curve with basal EMSH levels of ~− 296 mV. Consistent with its H2O2 resistant phenotype, C. glutamicum responds only weakly to 40 mM H2O2, but is rapidly oxidized by low doses of NaOCl. We further monitored basal EMSH changes and the H2O2 response in various mutants which are compromised in redox-signaling of ROS (OxyR, SigH) and in the antioxidant defense (MSH, Mtr, KatA, Mpx, Tpx). While the probe was constitutively oxidized in the mshC and mtr mutants, a smaller oxidative shift in basal EMSH was observed in the sigH mutant. The catalase KatA was confirmed as major H2O2 detoxification enzyme required for fast biosensor re-equilibration upon return to non-stress conditions. In contrast, the peroxiredoxins Mpx and Tpx had only little impact on EMSH and H2O2 detoxification. Further live imaging experiments using confocal laser scanning microscopy revealed the stable biosensor expression and fluorescence at the single cell level. In conclusion, the stably expressed Mrx1-roGFP2 biosensor was successfully applied to monitor dynamic EMSH changes in C. glutamicum during the growth, under oxidative stress and in different mutants revealing the impact of Mtr and SigH for the basal level EMSH and the role of OxyR and KatA for efficient H2O2 detoxification under oxidative stress.
Highlights
The Gram-positive soil bacterium Corynebacterium glutamicum is the most important industrial platform bacterium that produces millions of tons of L-glutamate and L-lysine every year as well as other value-added products [1,2,3,4]
Previous studies have revealed a specific response of the Mrx1roGFP2 biosensor to mycothiol disulfide (MSSM) in vitro, which was based on a fusion of mycobacterial Mrx1 to redox-sensitive green fluorescent protein (roGFP2) [36]
We have successfully designed the first genome-integrated Mrx1-roGFP2 biosensor that was applied in the industrial platform bacterium C. glutamicum which is of high biotechnological importance
Summary
The Gram-positive soil bacterium Corynebacterium glutamicum is the most important industrial platform bacterium that produces millions of tons of L-glutamate and L-lysine every year as well as other value-added products [1,2,3,4]. In its natural soil habitat and during industrial production, C. glutamicum is exposed to reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) which is generated as consequence of the aerobic lifestyle [6,7,8]. The low molecular weight (LMW) thiol mycothiol (MSH) functions as glutathione surrogate in detoxification of ROS and other thiol-reactive compounds in all actinomycetes, including C. glutamicum and mycobacteria to maintain the reduced state of the cytoplasm [9,10,11]. MSH-deficient mutants are sensitive to various thiol-reactive compounds, the secreted histidine-derivative ergothioneine (EGT) functions as alternative LMW thiol [12,13,14,15,16]
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