A transmembrane pH gradient in Streptococcus faecalis: Origin, and dissipation by proton conductors and N, N′-dicyclohexylcarbodiimide

Abstract

The distribution of [ 14C]dimethyloxazolidinedione was employed to measure the internal pH of Streptococcus faecalis. Glycolyzing cells maintained an internal pH more alkaline than that of the medium by 0.5–1 unit. The pH difference disappeared upon exhaustion of glucose. Arginine metabolism did not establish a pH gradient. The pH gradient was abolished by metabolic inhibitors of two kinds: inhibitors of the membrane-bound ATPase including N, N′-dicyclohexylcarbodiimide and chlorhexidine; and proton conductors including tetrachlorosalicylanilide, carbonylcyanide m-chlorophenylhydrazone and the antibiotics nigericin and monensin. The antibiotics valinomycin and monactin which facilitate K + diffusion did not affect the pH gradient so long as the external K + concentration was relatively high; at low K + concentrations they lowered the internal pH. To study the relationship of internal pH to K + accumulation we employed cells in which K + was completely replaced by Na +. Such Na +-loaded cells also maintained a pH gradient during glycolysis, albeit a small one (0.3 unit), which was again abolished by dicyclohexylcarbodiimide and by proton conductors. Addition of K + raised the internal pH to the level characteristic of K +-loaded cells. The results suggest that the pH gradient is formed by energy-dependent extrusion of protons from the cell; this renders the interion alkaline and generates a membrane potential (interior negative). K + accumulation apparently results from movement of K + down its electrochemical gradient.