pH of the Cytoplasm and Periplasm of Escherichia coli : Rapid Measurement by Green Fluorescent Protein Fluorimetry

Abstract

Cytoplasmic pH and periplasmic pH of Escherichia coli cells in suspension were observed with 4-s time resolution using fluorimetry of TorA-green fluorescent protein mutant 3* (TorA-GFPmut3*) and TetR-yellow fluorescent protein. Fluorescence intensity was correlated with pH using cell suspensions containing 20 mM benzoate, which equalizes the cytoplasmic pH with the external pH. When the external pH was lowered from pH 7.5 to 5.5, the cytoplasmic pH fell within 10 to 20 s to pH 5.6 to 6.5. Rapid recovery occurred until about 30 s after HCl addition and was followed by slower recovery over the next 5 min. As a control, KCl addition had no effect on fluorescence. In the presence of 5 to 10 mM acetate or benzoate, recovery from external acidification was diminished. Addition of benzoate at pH 7.0 resulted in cytoplasmic acidification with only slow recovery. Periplasmic pH was observed using TorA-GFPmut3* exported to the periplasm through the Tat system. The periplasmic location of the fusion protein was confirmed by the observation that osmotic shock greatly decreased the periplasmic fluorescence signal by loss of the protein but had no effect on the fluorescence of the cytoplasmic protein. Based on GFPmut3* fluorescence, the pH of the periplasm equaled the external pH under all conditions tested, including rapid acid shift. Benzoate addition had no effect on periplasmic pH. The cytoplasmic pH of E. coli was measured with 4-s time resolution using a method that can be applied to any strain construct, and the periplasmic pH was measured directly for the first time.In order to colonize the human gastrointestinal tract, the enteric bacterium Escherichia coli must be able to grow between pH 4.5 and pH 9 (7). Over this wide pH range, E. coli preserves enzyme activity, as well as protein and nucleic acid stability, by maintaining the cytoplasmic pH in the range from pH 7.2 to 7.8 (26,27,32). E. coli responds rapidly to intracellular pH change; after acidification of the external environment, the intracellular pH of E. coli begins to recover within 1 min, and full recovery occurs within 5 min (28). The efficiency with which E. coli maintains pH homeostasis has been attributed to a combination of constitutive and regulated mechanisms, but the essential requirements remain poorly understood (7,9,14,18,28). Some components of pH homeostasis act in the presence of chloramphenicol, whereas others require ongoing protein synthesis (10).Previously, cytoplasmic pH has been measured using 31 P nuclear magnetic resonance (NMR) of titratable phosphate and methylphosphonate (28) and through transmembrane equilibration of radiolabeled permeant acids (32). Both methods have limitations. Radiolabeled permeant acids have low sensitivity, and they measure only the transmembrane pH difference; they do not measure cytoplasmic pH independent of external pH.31 P NMR requires highly concentrated cell suspensions, typically suspensions with optical densities at 600 nm (OD 600 ) of 20 to 200.The advent of highly pH-sensitive fluorescent proteins...

Ratiometric Imaging of pH Probes

“…The pH-sensitive polar fluorescein derivative 2 0 ,7 0 -bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF) remains the most widely used pHi indicator. However, experimental limitations imposed by pH-sensitive dyes have more recently led to the development of genetically encoded pH biosensors derived from the jellyfish Aequorea victoria green fluorescent protein (GFP; Martinez et al, 2012;Miesenböck, De Angelis, & Rothman, 1998;Wilks & Slonczewski, 2007). Together, these pH-sensitive dyes and biosensors can be imaged using most standard fluorescence microscopes to measure steadystate and dynamic changes in pH within cells.…”

“…In general, cytoplasmic pH in most organisms from bacteria (Martinez et al, 2012;Wilks & Slonczewski, 2007) to yeasts (Bagar, Altenbach, Read, & Bencina, 2009;Grynkiewicz et al, 1985) to mammalian cells (Fig. 23.1) is slightly basic (pH is $7.0-7.8), with the exception of some acidophilic or basophilic bacteria (Paradiso et al, 1984;Slonczewski, Fujisawa, Dopson, & Krulwich, 2009).…”

Segregating metabolic processes into different microbial cells accelerates the consumption of inhibitory substrates

“…We exploited this pH dependence to experimentally manipulate the magnitude of nitrite inhibition by adjusting the pH of the culture medium. This approach is effective because the pH of the culture medium is approximately equal to the pH of the periplasm for Gram-negative bacteria (Wilks and Slonczewski, 2007). We then measured how the magnitude of nitrite inhibition affects the speed at which completely consuming cells and co-cultures of nitrite cross-feeding cells consume nitrogen oxides, where we expect that nitrite crossfeeding increasingly accelerates the consumption of nitrogen oxides as the growth-inhibiting effects of nitrite increase.…”

Membrane Potential as a Regulation Mechanism of Periplasmic Nitrite Reductase Activity: A Mathematical Model

“…Величина мембранного потенциала определяет скорость секреции субъединиц периплазматических ферментов из цитоплазмы в периплазму и зависит от разности концентраций протонов в периплазме и цитоплазме. Концентрации протонов были оценены на основании данных [47], согласно которым внутри клетки поддерживается рН, близкий к оптимальному (7.8), тогда как в периплазме он определяется, в основном, рН окружающей среды. В условиях хемостата рН среды поддерживается постоянным и равным 6.5, что и определяет концентрацию протонов в периплазме (kH out ), равную 3·10 -4 мМ.…”