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

• Published in: Microbial biotechnology 2019-07, Vol.12 (4), p.799-813
• Main Authors: Arce‐Rodríguez, Alejandro, Volke, Daniel C., Bense, Sarina, Häussler, Susanne, Nikel, Pablo I.
• Format: Article
• Language: English
• Subjects: Antibiotics; Bacteria; Bacteriological Techniques - methods; Binding sites; Coding; Cytosol - chemistry; Deoxyribonucleic acid; DNA; E coli; Environmental changes; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - physiology; Fluorescent indicators; Fluorometry - methods; Genes, Reporter; Genetic code; Glucose; Gram-negative bacteria; Hydrogen-Ion Concentration; Intracellular; Luminescent Proteins - analysis; Luminescent Proteins - genetics; Metabolism; NMR; Nuclear magnetic resonance; Nucleotide sequence; pH effects; Plasmids; Proteins; Pseudomonas; Pseudomonas - chemistry; Pseudomonas - genetics; Pseudomonas - physiology; Recombinant Proteins - analysis; Recombinant Proteins - genetics; Special Issue; Species; Staining and Labeling - methods; Terrestrial environments; Transcription
• ISSN: 1751-7915; 1751-7915
• DOI: 10.1111/1751-7915.13439

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. We present a standard, non‐invasive and easy‐to‐implement method for the determination of pHi in Pseudomonas species, as well as other Gram‐negative bacteria. This procedure is based on the enhanced expression of a pH‐sensitive variant of GFP, and the ratiometric assessment of pHi against cells equilibrated to a range of physiologically‐relevant pH values.