Combined sensor-based monitoring of mycothiol redox potential and DNA-damage response in Corynebacterium glutamicum

Abstract

Excessive amounts of reactive oxygen species (ROS) can cause irreversible damages to essential cellular components such as DNA. Genetically encoded biosensors targeting oxidative stress and DNA-stress have emerged to a powerful analytical tool to assess physiological states in a non-invasive manner. In this study, we aimed to combine the redox biosensor protein Mrx1-roGFP2 with a transcriptional biosensor for DNA-damage based on the PrecA promoter fused to a reporter gene (e2-crimson) in Corynebacterium glutamicum. Therefore, the redox biosensor strains C. glutamicum WT_Mrx1-roGFP2 and the mycothiol (MSH)-deficient mutant strain C. glutamicum {Delta}mshC_Mrx1-roGFP2 were equipped with the DNA-stress reporter plasmid pJC1_PrecA_e2-crimson. Exposure of the double-sensor equipped C. glutamicum WT strain to hypochlorite resulted in an oxidative redox shift, accompanied by an induction of the DNA-stress reporter system. In absence of the major non-enzymatic antioxidant MSH, the induction of the DNA-stress response was even more pronounced. This confirms the linkage of oxidative stress and DNA-damage response, and therefore making antioxidants a crucial player to protect DNA. Furthermore, exposure of the double biosensor strains to a DNA-damage inducing agent resulted in an oxidative redox shift. These results suggest a direct link of DNA-damage and oxidative stress response in C. glutamicum. Finally, we observed that inhibition of cell wall biosynthesis by penicillin caused both an oxidative redox shift and a DNA-damage response in C. glutamicum. The excellent compatibility of Mrx1-roGFP2 with E2-Crimson shown here provides a powerful combinatorial biosensor concept for in-depth studies of redox-related physiology in future studies.