Acetobacter aceti Possesses a Proton Motive Force-Dependent Efflux System for Acetic Acid

Abstract

Acetic acid bacteria are obligate aerobes able to oxidize ethanol, sugar alcohols, and sugars into their corresponding acids. Among them, Acetobacter and Gluconacetobacter species have very high ethanol oxidation capacity, leading to accumulation of vast amounts of acetic acid outside the cell. Since these bacteria are able to grow in media with high concentrations of acetic acid, they must possess a specific mechanism such as an efflux pump by which they can resist the toxic effects of acetic acid. In this study, the efflux pump of Acetobacter aceti IFO 3283 was examined using intact cells and membrane vesicles. The accumulation of acetic acid/acetate in intact cells was increased by the addition of a proton uncoupler and/or cyanide, suggesting the presence of an energy-dependent efflux system. To confirm this, right-side-out and inside-out membrane vesicles were prepared from A. aceti IFO 3283, and the accumulation of acetic acid/acetate in the vesicles was examined. Upon the addition of a respiratory substrate, the accumulation of acetic acid/acetate in the right-side-out vesicles was largely decreased, while its accumulation was very much increased in the inside-out vesicles. These respiration-dependent phenomena observed in both types of membrane vesicles were all sensitive to a proton uncoupler. Acetic acid/acetate uptake in the inside-out membrane vesicles was dependent not on ATP but on the proton motive force. Furthermore, uptake was shown to be rather specific for acetic acid and to be pH dependent, because higher uptake was observed at lower pH. Thus, A. aceti IFO 3283 possesses a proton motive force-dependent efflux pump for acetic acid.

Putative ABC Transporter Responsible for Acetic Acid Resistance in Acetobacter aceti

“…Very recently, Matsushita et al (24) reported the presence of the efflux pump for acetic acid in the other strain of A. aceti. They concluded that the efflux pump is proton motive force dependent because transporter activity was dependent on pH, not on ATP, and was sensitive to a proton uncoupler.…”

“…On the other hand, it is likely that there is some other machinery conferring acetic acid resistance that is located in the cell membrane, because part of acetic acid toxicity is caused by serving as an uncoupling agent, which disturbs the proton motive force (2,7,9,36). Recently, the presence of a proton motive efflux system for acetic acid in A. aceti cultured on the glycerol medium has been reported (24); however, it seems unclear if it contributes to acetic acid resistance in acetic acid fermentation.…”

Solute Carrier 11 Cation Symport Requires Distinct Residues in Transmembrane Helices 1 and 6

“…All experiments were performed at least in triplicate, using protein concentrations of 50 (RSOV), 200 (ISOV), and ϳ700 g/ml (whole cells, corresponding to an OD 420 of 10) as determined by protein assay using a modified Lowry procedure (27) (13). Fluorescence Measurements of Membrane Potential (⌬) in RSOV and ISOV-Fluorescent probes, 3,3Ј-dipropylthiadicarbocyanine iodide (1 M) and bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (1 M), were used to monitor ⌬ changes in energized vesicles as described (30). Fluorescence excitation and emission wavelengths were, respectively, 622 and 670 nm for 3,3Ј-dipropylthiadicarbocyanine iodide, and 588 and 614 nm for bis-(1,3-dibutylbarbituric acid)pentamethine oxonol.…”

A Specialized Citric Acid Cycle Requiring Succinyl-Coenzyme A (CoA):Acetate CoA-Transferase (AarC) Confers Acetic Acid Resistance on the Acidophile Acetobacter aceti

“…The molecular mechanisms of acetic acid resistance in Acetobacter aceti include adaptation of the cytoplasmic components (9,14) to internal acidification (32), acetic acid efflux via the AatA acetic acid:proton antiporter (31,39), and production of acid-inducible proteins identified by proteomic screens, many with undefined biochemical roles (28,52).…”