The proton electrochemical gradient in Escherichia coli cells

Abstract

The internal pH of Escherichia coli cells was estimated from the distribution of either 5,5-[14C]dimethyl-2,4-oxazolidinedione or [14C]methylamine. EDTA/valinomycin treatment of cells was employed to estimate delta psi from 86Rb+ distribution concomitant with the delta pH for calculation of delta muH. Respiring intact cells maintained an internal pH more alkaline by 0.63-0.75 unit than that of the milieu at extracellular pH 7, both in growth medium and KCl solutions. The delta pH decreased when respiration was inhibited by anaerobiosis or in the presence of KCN. The delta muH, established by EDTA/valinomycin-treated cells, was constant (122-129 mV) over extracellular potassium concentration of 0.01 mM-1 mM. At the lower potassium concentration delta psi (110-120 mV) was the predominant component, and at the higher concentration delta pH increased to 0.7 units (42 mV). At 150 mM potassium delta muH was reduced to 70 mV mostly due to a delta pH component of 0.89 (53 mV). The interchangeability of the delta muH components is consistent with an electronic proton pump and with potassium serving as a counter ion in the presence of valinomycin. Indeed both parameters of delta muH decreased in the presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone. The highest delta pH of 2 units was observed in the intact cells at pH 6; increasing the extracellular pH decreased the delta pH to 0 at pH 7.65 and to -0.51 at pH 9. A similar pattern of dependence of delta pH on extracellular pH was observed in EDTA/valinomycin-treated cells but the delta psi was almost constant over the whole range of extracellular pH values (6-8) implying electroneutral proton movement. Potassium is specifically required for respiration of EDTA-treated E. coli K12 cells since other monovalent or divalent cations could not replace potassium and valinomycin was not required.