Impedance flow cytometry for viability analysis of Corynebacterium glutamicum

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

“…Prior to studying the interplay of oxidative stress and DNA-damage, the cultures viability was measured in regular intervals after applying oxidative stress using impedance flow cytometry (IFC). This allows one to ensure that the cultures can adapt to the harsh conditions rather than losing their cell viability and integrity and by this the capability of producing the reporter protein (E2-Crimson), as recently demonstrated for a non-growing but glutamate producing C. glutamicum culture (Hartmann et al ., 2021).…”

“…In order to characterize the response of the reporter system P recA _e2-crimson to the DNA-damaging agent MMS, the C. glutamicum WT was equipped with the plasmid pJC1_P recA _e2-crimson or the empty vector pJC1 resulting in the reporter strain C. glutamicum WT (pJC1_P recA _e2-crimson ) and the control strain C. glutamicum WT (pJC1), respectively. To analyze the biosensor signal during cultivation at various MMS concentrations, the strains were cultured using a BioLectorII system (Hartmann et al ., 2021). After four hours of cultivation, the cultivation was paused and 5 μL MMS of differently concentrated stock solutions were added to adjust different final concentrations in the cultivation wells.…”

“…All fluorescence measurements were conducted in black flat-bottomed 96-well microtiter plates using a plate reader device (SpectraMaxiD3). Viability (viable cells*mL -1 *OD 600nm -1 ) was determined using impedance flow cytometry as recently described (Hartmann et al ., 2021). Arrows indicate the addition of sodium-hypochlorite (NaOCl; 35 mM) Error bars represent standard deviation from at least three replicates.…”

“…If not otherwise stated, C. glutamicum main cultures were grown in CGXII minimal medium (Graf et al ., 2018). Growth was performed in 500 mL shaker flasks (50 mL cultures) or as 800 μL cultures using a BioLectorII system (m2p-labs, Baesweiler/DE) and 48-well Flowerplates (m2p-labs, Baesweiler/DE) at 1200 rpm and 30 °C as recently described (Hartmann et al ., 2021). E. coli strains were pre-cultured in LB medium at 37 °C as 5 mL or 50 mL cultures.…”

“…Moreover, the cells need to maintain their viability to respond and adapt to the changed conditions and in turn to produce the fluorescent protein. To ensure that the viability is maintained, impedance flow cytometry measurements (IFC) were conducted as previously described (Hartmann et al ., 2021). Based on this, 35 mM NaOCl was added in order to induce oxidative stress.…”