The Proton Electrochemical Gradient in Escherichia coli Cells

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 ΔΨ from ⁸⁶Rb⁺ distribution concomitant with the Δ pH for calculation of d ΔH. 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 Δ pH decreased when respiration was inhibited by anaerobiosis or in the presence of KCN. The ΔH, 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 ΔΨ (110‐120 mV) was the predominant component, and at the higher concentration Δ pH increased to 0.7 units (42 mV). At 150 mM potassium ΔH was reduced to 70 mV mostly due to a Δ pH component of 0.89 (53 mV). The interchangeability of the d ΔH components is consistent with an electrogenic proton pump and with potassium serving as a counter ion in the presence of valinomycin. Indeed both parameters of ΔH decreased in the presence of carbonylcyanide p‐trifluoromethoxyphenylhydrazone. The highest Δ pH of 2 units was observed in the intact cells at pH 6; increasing the extracellular pH decreased the Δ pH to 0 at pH 7.65 and to ‐ 0.51 at pH 9. A similar pattern of dependence of Δ pH on extracellular pH was observed in EDTA/valinomycin‐treated cells but the ΔΨ 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