Generation of an Electrochemical Proton Gradient by Lactate Efflux in Membrane Vesicles of Escherichia coli

Abstract

The ‘energy recycling model’ (Michels et al. (1979) FEMS Microbiol. Lett. 5, 357–364] postulates the generation of an electrochemical gradient across the bacterial cytoplasmic membrane by carrier-mediated efflux of metabolic endproducts in symport with protons. Experimental evidence for this model is presented. In membrane vesicles from Escherichia coli ML 308–225 l-lactate translocation (both uptake and efflux) is carrier-mediated. The H⁺/t-lactate stoichiometry varies, depending on the external pH, between 1 and 2. This change in stoichiometry is most likely the result of a protonation of the lactate carrier protein. This process has a pK of 6.75. l-Lactate efflux from membrane vesicles, loaded with 50 mM potassium l-lactate, results at an external pH of 6.6 in an 11-fold accumulation of proline inside the vesicles. This accumulation is completely inhibited by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The uptake of proline is not the result of a potassium or an osmotic gradient. At an external pH of 6.6 efflux of l-lactate from the vesicles leads to the generation of an electrical potential across the membrane of - 55 mV, as is demonstrated from the accumulation of the lipophilic cation tetraphenylphosphonium.