Visualizing the pH in Escherichia coli Colonies via the Sensor Protein mCherryEA Allows High-Throughput Screening of Mutant Libraries.

Cytoplasmic pH in bacteria is tightly regulated by diverse active mechanisms and interconnected regulatory processes. Many processes and regulators underlying pH homeostasis have been identified via phenotypic screening of strain libraries for nongrowth at low or high pH values. Direct screens with respect to changes of the internal pH in mutant strain collections are limited by laborious methods, which include fluorescent dyes and radioactive probes. Genetically encoded biosensors equip single organisms or strain libraries with an internal sensor molecule during the generation of the strain. Here, we used the pH-sensitive mCherry variant mCherryEA as a ratiometric pH biosensor. We visualized the internal pH of Escherichia coli colonies on agar plates by the use of a GelDoc imaging system. Combining this imaging technology with robot-assisted colony picking and spotting allowed us to screen and select mutants with altered internal pH values from a small transposon mutagenesis-derived E. coli library. Identification of the transposon (Tn) insertion sites in strains with altered internal pH levels revealed that the transposon was inserted into trkH (encoding a transmembrane protein of the potassium uptake system) or rssB (encoding the adaptor protein RssB, which mediates the proteolytic degradation of the general stress response regulator RpoS), two genes known to be associated with pH homeostasis and pH stress adaptation. This successful screening approach demonstrates that the pH sensor-based analysis of arrayed colonies on agar plates is a sensitive approach for the rapid identification of genes involved in pH homeostasis or pH stress adaptation in E. coli. IMPORTANCE 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. For this purpose, transcription factor-based biosensors have emerged as the sensor type of choice. Here, the target stimulus initiates the activation of a response gene (e.g., a fluorescent protein), followed by transcription, translation, and maturation. Due to this mechanistic principle, biosensor readouts are delayed and cannot report the actual intracellular state of the cell in real time. To capture rapid intracellular processes adequately, fluorescent reporter proteins are extensively applied. However, these sensor types have not previously been used for phenotypic screenings. To take advantage of their properties, we established here an imaging method that allows application of a rapid ratiometric sensor protein for assessing the internal pH of colonies in a high-throughput manner.

Impedance flow cytometry for viability analysis of Corynebacterium glutamicum.

Corynebacterium glutamicum efficiently produces glutamate when growth is inhibited. Analyses of viability in this non-growing state requires time consuming plating and determination of colony forming units. We here establish impedance flow cytometry measurements to assess the viability of non-growing, glutamate producing C. glutamicum cultures within minutes.

The Industrial Organism Corynebacterium glutamicum Requires Mycothiol as Antioxidant to Resist Against Oxidative Stress in Bioreactor Cultivations.

In aerobic environments, bacteria are exposed to reactive oxygen species (ROS). To avoid an excess of ROS, microorganisms are equipped with powerful enzymatic and non-enzymatic antioxidants. Corynebacterium glutamicum, a widely used industrial platform organism, uses mycothiol (MSH) as major low molecular weight (LMW) thiol and non-enzymatic antioxidant. In aerobic bioreactor cultivations, C. glutamicum becomes exposed to oxygen concentrations surpassing the air saturation, which are supposed to constitute a challenge for the intracellular MSH redox balance. In this study, the role of MSH was investigated at different oxygen levels (pO2) in bioreactor cultivations in C. glutamicum. Despite the presence of other highly efficient antioxidant systems, such as catalase, the MSH deficient ΔmshC mutant was impaired in growth in bioreactor experiments performed at pO2 values of 30%. At a pO2 level of 20%, this growth defect was abolished, indicating a high susceptibility of the MSH-deficient mutant towards elevated oxygen concentrations. Bioreactor experiments with C. glutamicum expressing the Mrx1-roGFP2 redox biosensor revealed a strong oxidative shift in the MSH redox potential (EMSH) at pO2 values above 20%. This indicates that the LMW thiol MSH is an essential antioxidant to maintain the robustness and industrial performance of C. glutamicum during aerobic fermentation processes.