The role of respiration-dependent proton translocation in the acid tolerance of Gluconobacter oxydans
Gluconobacter oxydans is characterized by extreme acid tolerance and the ability to carry out rapid, single-step polyol oxidation catalyzed by membrane bound ehydrogenases. Experiments were designed to determine whether acid tolerance is associated with rapid polyol oxidation in this organism. Washed cells were exposed to 0.1 M or 0.5 !vt NaCI at pH 3.20; subsequent alkalinization of the suspending solution suggested a NaCl-dependent flow of protons (H+) into the cells. Cells were then exposed to NaCI at pH 3.20 followed by the addition of glycerol to determine whether polyol oxidation resulted in H + explusion from the cells. Following glycerol addition, immediate acidification of the suspending solution occurred. To verify that H + effiux was a result of respiration, experiments were conducted using sodium azide and 2,4-dinitrophenol; both compounds prevented the acidification that otherwise occurred following glycerol addition. Because glycerol oxidation reversed the NaCl-induced flow of H + into the cell, it appeared that respiration might function to protect acid-labile cell interiors. Cells exposed to NaCl at pH 3.20 in the presence of glycerol maintained cellular viability while loss of viability occurred in the absence of glyceroL To verify the effect of H+ extrusion on pH homeostasis, radioactively labeled organic-acid probes were used to determine intracellular pH in respiring and nonrespiring cells in the presence of 0.1 M NaCI at pH 3.20. No differences in cytoplasmic pH values between respiring and nonrespiring cells were detected. However, because substantial evidence exists for the role of respiration dependent H + extrusion in the acid tolerance of G. oxydans, use of an alternate method for measurement of internal pH, such as 31 P nuclear magnetic resonance spectroscopy, is suggested.