pH homeostasis in Escherichia coli: measurement by 31P nuclear magnetic resonance of methylphosphonate and phosphate.

The intracellular pH of Escherichia coli cells, respiring on endogenous energy sources, was monitored continuously by 31P NMR over an extracellular pH range between 5.5 and 9. pH homeostasis was found to be good over the entire range, with the data conforming to the simple relationship intracellular pH = 7.6 + 0.1(external pH - 7.6) so that the extreme values observed for intracellular pH were 7.4 and 7.8 at external pH 5.5 and 9, respectively. As well as inorganic phosphate, we employed the pH-sensitive NMR probe methylphosphonate, which was taken up by glycerol-grown cells and was nontoxic; its pKa of 7.65 made it an ideal probe for measurement of cytoplasmic pH and alkaline external pH.