Quantitative measurements of membrane potential in Escherichia coli

By use of giant cells of Escherichia coli induced by growth in the presence of 6-amidinopenicillanic acid, membrane potentials have been measured by two completely independent techniques: directly with intracellular microelectrodes and indirectly from the steady-state distribution of [3H]tetraphenylphosphonium. Under a variety of conditions, the two methods yield values that agree very closely. Thus, with both techniques, the membrane potential approximates -85 mV (interior negative) at pH 5.0 and -142 mV at pH 8.0, with an average slope of -22 mV/pH unit over the range pH 5.0-7.0. A parallel study of membrane vesicles prepared from giant cells was undertaken using tetraphenylphosphonium distribution alone as a measure of membrane potential. The vesicles were found to exhibit a much smaller slope of membrane potential vs. extracellular pH (about -6 mV/pH unit) than intact giant cells. The results indicate that distribution studies with these lipophilic cations provide an excellent measure of membrane potential and are discussed in relation to calculations of H+/substrate stoichiometry for protonsymport systems in E. coli.