Proton Electrochemical Gradient in Escherichia coli Cells and Its Relation to Active Transport of Lactose

The electrochemical potential gradients of protons (Δ+) and lactose (Δlac) maintained by respiring Escherichia coli ML 308-225 (i-z-y+a+) cells have been measured as a function of the external pH. The proton gradient (ΔpH) was determined from the distribution across the cell membrane of the weak acid 5, 5-dimethyloxazolidine-2, 4-dione (DMO) after a rapid centrifugation step. The accumulation of tetraphenylphosphonium (TPP+; a lipophylic cation) and 86Rb+ in the presence of valinomycin by EDTA-treated cells incubated in a flow dialysis system has been used to calculate the membrane potential (Δѱ. Simultaneous measurements of ΔH+and lactose gradient were conducted using [14C]lactose and [3H]TPP+. Respiring EDTA-treated cells maintain, at neutrality, a ΔH+ of 170 mV (ΔpH=35 mV, Δѱ=135 mV, interior negative and alkaline). When the external pH was changed from 6.0 to 8.0, Δѱ increased from 95 to 150 mV, while ΔpH decreased from 1.8 to-0.2 units (at pH 6 interior alkaline and at pH 8 interior acid). Thus, Δѱ significantly compensates for the decrease in ΔpH, yielding a A£H+ of 200 mV at pH 6 and 140 mV at pH 8. In contrast to intact cells, inverted membrane vesicles from E. coli maintain a large ApH at external pH 8.0, while Δѱ was undetectable. Lactose steady-state gradients were determined in cells maintaining different ΔH+ levels in the presence of various concentrations of the uncoupler FCCP at pH 8. The results indicate that one proton is translocated with each molecule of lactose at pH 8 similar to the case at pH 6. This conclusion is supported by direct measurements of H+ and lactose fluxes induced by a lactose pulse in nonmetabolizing cells.