Stable integration of the Mrx1-roGFP2 biosensor to monitor dynamic changes of the mycothiol redox potential in Corynebacterium glutamicum.

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 (E) during the growth and under oxidative stress. C. glutamicum maintains a highly reducing intrabacterial E throughout the growth curve with basal E levels of ~- 296 mV. Consistent with its HO resistant phenotype, C. glutamicum responds only weakly to 40 mM HO, but is rapidly oxidized by low doses of NaOCl. We further monitored basal E changes and the HO 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 E was observed in the sigH mutant. The catalase KatA was confirmed as major HO 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 E and HO 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 E 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 E and the role of OxyR and KatA for efficient HO detoxification under oxidative stress.