pH of the cytoplasm and periplasm of Escherichia coli: rapid measurement by green fluorescent protein fluorimetry

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.