Non‐invasive, ratiometric determination of intracellular pH in Pseudomonas species using a novel genetically encoded indicator

The ability of Pseudomonas species to thrive in all major natural environments (i.e. terrestrial, freshwater and marine) is based on its exceptional capability to adapt to physicochemical changes. Thus, environmental bacteria have to tightly control the maintenance of numerous physiological traits across different conditions. The intracellular pH (pH (i)) homoeostasis is a particularly important feature, since the pH (i) influences a large portion of the biochemical processes in the cell. Despite its importance, relatively few reliable, easy‐to‐implement tools have been designed for quantifying in vivo pH (i) changes in Gram‐negative bacteria with minimal manipulations. Here we describe a convenient, non‐invasive protocol for the quantification of the pH (i) in bacteria, which is based on the ratiometric fluorescent indicator protein PHP (pH indicator for Pseudomonas). The DNA sequence encoding PHP was thoroughly adapted to guarantee optimal transcription and translation of the indicator in Pseudomonas species. Our PHP‐based quantification method demonstrated that pH (i) is tightly regulated over a narrow range of pH values not only in Pseudomonas, but also in other Gram‐negative bacterial species such as Escherichia coli. The maintenance of the cytoplasmic pH homoeostasis in vivo could also be observed upon internal (e.g. redirection of glucose consumption pathways in P. putida) and external (e.g. antibiotic exposure in P. aeruginosa) perturbations, and the PHP indicator was also used to follow dynamic changes in the pH (i) upon external pH shifts. In summary, our work describes a reliable method for measuring pH (i) in Pseudomonas, allowing for the detailed investigation of bacterial pH (i) homoeostasis and its regulation.