Phenotypic screening of strain libraries on agar plates has become a versatile tool to understand gene functions and to optimize biotechnological platform organisms. Screening is supported by genetically encoded biosensors that allow to easily measure intracellular processes.

No abstract

No 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 ΔmshC_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.

Genetically encoded fluorescent biosensors have emerged as a powerful tool to support phenotypic screenings of microbes. Optical analyses of fluorescent sensor signals from colonies grown on solid media can be challenging as imaging devices need to be equipped with appropriate filters matching the properties of fluorescent biosensors. Toward versatile fluorescence analyses of different types of biosensor signals derived from arrayed colonies, we investigate here the use of monochromator equipped microplate readers as an alternative to imaging approaches. Indeed, for analyses of the LacI-controlled expression of the reporter mCherry in Corynebacterium glutamicum, or promoter activity using GFP as reporter in Saccharomyces cerevisiae, an improved sensitivity and dynamic range was observed for a microplate reader-based analyses compared to their analyses via imaging. The microplate reader allowed us to capture signals of ratiometric fluorescent reporter proteins (FRPs) with a high sensitivity and thereby to further improve the analysis of internal pH via the pH-sensitive FRP mCherryEA in Escherichia coli colonies. Applicability of this novel technique was further demonstrated by assessing redox states in C. glutamicum colonies using the FRP Mrx1-roGFP2. By the use of a microplate reader, oxidative redox shifts were measured in a mutant strain lacking the non-enzymatic antioxidant mycothiol (MSH), indicating its major role for maintaining a reduced redox state also in colonies on agar plates. Taken together, analyses of biosensor signals from microbial colonies using a microplate reader allows comprehensive phenotypic screenings and thus facilitates further development of new strains for metabolic engineering and systems biology.