Visualizing the pH inEscherichia colicoloniesviathe sensor protein mCherryEA allows high-throughput screening of mutant libraries

Abstract

Cytoplasmic pH is tightly regulated by diverse active mechanisms and interconnected regulatory processes in bacteria. Many processes and regulators underlying pH-homeostasis have been identified via phenotypic screening of strain libraries towards non-growth 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 including fluorescent dyes or radioactive probes. Genetically encoded biosensors equip single organisms or strain libraries with an internal sensor molecule already during the generation of the strain. In this study, we used the pH-sensitive mCherry variant mCherryEA as ratiometric pH biosensor. We visualized the internal pH of E. coli colonies on agar plates by the use of a Gel-Doc 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 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 the rssB gene (encoding the anti-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 fast identification of genes involved in pH-homeostasis or pH stress adaptation in E. coli.ImportancePhenotypic 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 fast intracellular processes adequately, fluorescent reporter proteins are extensively applied. But these sensor-types are not utilized for phenotypic screenings so far. To take advantage of their properties, we here established an imaging method, which allows to apply a fast ratiometric sensor protein for assessing the internal pH of colonies in a high-thoughput manner.