High resolution ³¹P nuclear magnetic resonance studies of metabolism in aerobic Escherichia coli cells

³¹P nuclear magnetic resonance spectra at 145.7 MHz were obtained of concentrated suspensions of E. coli cells. The position of the Pi resonance was used to determine the pH, and in most experiments it was possible to distinguish the intracellular (pHin) and extracellular (pHex) values. During respiration pH in approached 7.55, while pHex varied from 6.0 to 8.0. With succinate as a carbon source and in a N₂ environment, pHin=pHex. Upon addition of glucose, pHin > pHex. In the presence of an ATPase (adenosinetriphosphatase; ATP phosphohydrolase; EC 3.6.1.3) inhibitor dicyclohexylcarbodiimide, pHin remained equal to pHex even in the presence of glucose. In other experiments, oxygenation brought pHin above pHex even in the presence of dicyclohexylcarbodiimide. These experiments are consistent with Mitchell's hypothesis that, first, ΔpH can be created by the reversal of the ATPase reaction and, second, that protons are pumped outward during respiration. In addition to Pi, about 10 more resonances were resolved, several of which were assigned to different phosphate metabolites.