Monovalent cation/proton antiporters in membrane vesicles from Bacillus alcalophilus.

Abstract

Alkalophilic bacilli maintain a cytoplasmic pH that is lower than optimal external pH values.The current study, using isolated membrane vesicles from Bacillus alcalophilus, indicates that respiration results in proton extrusion as in other organisms.Cation/proton antiporters then catalyze inward proton movements.Upon energization with ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine, Na'-loaded membrane vesicles from B. alcalophilus exhibited a transmembrane pH gradient (ApH, inside acid) over a range of external pH values from 8 to 10.5; above pH 11, ApH was zero.A transmembrane electrical potential (A$, inside negative), increasing from -125 to -135 mV from pH 8 to 11.5, was observed.Over the same pH range, the internal and external pH values of K'-loaded vesicles were equal (ApH = 0), and the A$ declined from -125 to -98 mV.Vesicles that were prepared without Na' or K' exhibited a small ApH, inside alkaline, and a A$, inside negative, of -64 to -100 mV.These observations are compatible with the presence of: a K+/H+ antiporter which exchanges the protons that are extruded during respiration for K' ; and a Na+/H+ antiporter which acidifies the intravesicular space relative to the external medium.Indeed, everted vesicles catalyzed net proton extrusion and Na' accumulation that were dependent upon a A$ and were half-maximal at a Na' concentration of about 0.7 m ~.Whole cells of a nonalkalophilic strain of B. alcalophilus had been found to lack both Na+ efflux activity and the ability to grow above pH 9.0.Vesicles from this mutant strain lacked Na+/H+ antiporter activity, while still exhibiting apparent K'/H' antiporter activity.Thus, studies of vesicles from both mutant and wild type strains indicate that the ability of B. alcalophilus to maintain an internal pH that is lower than the external pH is related to the activity of the Na'/H' antiporter.Alkalophilic bacteria present special problems with respect to transmembrane concentration gradients of protons.Specifically, at optimal pH values for growth, alkalophiles such as Bacillus alcalophilus (1) and Bacillus firmus RAB ( 2 ) maintain a cytoplasmic pH that is considerably lower than the external pH.Yet, in accord with the chemiosmotic hypothesis, respiring bacteria have been found to extrude protons(3).This proton extrusion establishes an electrochemical gradient