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

Abstract

Summary 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 (pHi) homoeostasis is a particularly important feature, since the pHi 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 pHi changes in Gram‐negative bacteria with minimal manipulations. Here we describe a convenient, non‐invasive protocol for the quantification of the pHi 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 pHi 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 pHi upon external pH shifts. In summary, our work describes a reliable method for measuring pHi in Pseudomonas, allowing for the detailed investigation of bacterial pHi homoeostasis and its regulation.

Arginine Decarboxylase Is Essential for Pneumococcal Stress Responses

“…However, the pH i of WT and Δ speA strains remained within the physiological pH range ( Figure 4 ). These results conform to reports that bacteria maintain their pH i within a narrow pH range despite exposure and growth under varied extracellular pH conditions [ 51 , 52 ]. Although pneumococci are reported to be tolerant of pH 4.4 [ 51 ] in phagosome vesicles, our results show increased sensitivity of Δ speA to acid stress, suggesting that impaired polyamine synthesis renders pneumococci more susceptible to acid stress.…”

PaeA (YtfL) protects from cadaverine and putrescine stress in Salmonella Typhimurium and E. coli

“…To more directly test the effects of cadaverine, we monitored intracellular pH of wild‐type and Δ paeA strains during cadaverine treatment. We used a green fluorescent protein (GFP) that responds to pH change (Arce‐Rodriguez et al., 2019). We confirmed the response to pH by incubating cells in saline solutions buffered at various pH in the presence of cell‐permeable acids and bases, thereby equilibrating intracellular and extracellular pH.…”

The Dynamic Ion Motive Force Powering the Bacterial Flagellar Motor

“…Intracellular pH was first quantified on the single cell level using membrane-permeable pH-dependent fluorescent dyes (Siegumfeldt et al, 2000;Rasmussen et al, 2008;Kurre et al, 2013;Chao et al, 2017). A more recent approach, which obviates the need for a dye loading step and its potential invasive effects (Han and Burgess, 2010), uses genetically encoded fluorescent proteins, such as pH-sensitive derivatives of GFP (Miesenböck et al, 1998;Martinez et al, 2012;Kurre et al, 2013;Morimoto et al, 2016;Rupprecht et al, 2017;Arce-Rodríguez et al, 2019). Aside from excellent temporal (< 1 s) and pH (< 0.1) resolution (Kralj et al, 2011), a fusion between pHluorin and FliG of the BFM has shown local pH measurements with subcellular spatial resolution (Morimoto et al, 2016).…”