In cells of Leuconostoc oenos, the fermentation of L-malic acid generates both a transmembrane pH gradient, inside alkaline, and an electrical potential gradient, inside negative. In resting cells, the proton motive force ranged from ؊170 mV to ؊88 mV between pH 3.1 and 5.6 in the presence of L-malate. Membrane potentials were calculated by using a model for probe binding that accounted for the different binding constants at the different pH values at the two faces of the membrane. The ⌬ generated by the transport of monovalent malate, H-malate ؊ , controlled the rate of fermentation. The fermentation rate significantly increased under conditions of decreased ⌬, i.e., upon addition of the ionophore valinomycin in the presence of KCl, whereas in a buffer depleted of potassium, the addition of valinomycin resulted in a hyperpolarization of the cell membrane and a reduction of the rate of fermentation. At the steady state, the chemical gradient for H-malate ؊ was of the same magnitude as ⌬. Synthesis of ATP was observed in cells performing malolactic fermentation.Lactic acid bacteria are strictly fermentative and, with the exception of a few streptococci (22), lack electron transfer chains. Therefore, in these bacteria, generation of a proton motive force (expressed as ⌬p) can be achieved only by proton translocation via the membrane-bound F 0 F 1 H ϩ -ATPase driven by the hydrolysis of ATP or by some other chemiosmotic processes. Michels et al. (15) proposed that ⌬p can be formed by carrier-mediated excretion of fermentation end products in symport with protons; indeed, this was demonstrated in cells of Lactococcus lactis subsp. cremoris (18,29) and Enterococcus faecalis (26) and in membrane vesicles of Escherichia coli (27). In addition, two other chemiosmotic mechanisms for proton motive force generation were described in lactic acid bacteria: electrogenic precursor-product exchange (1, 6, 16, 19, 20) and electrogenic uniport (17, 20, 21, 24) in combination with the metabolic breakdown of the substrate inside the cell. Examples of the former are decarboxylation of oxalate in Oxalobacter formigenes (1), L-malate in L. lactis (20), and histidine in Lactobacillus buchneri (16). Examples of the anion uniport mechanism are the decarboxylation of L-malate in Leuconostoc oenos (24) and Lactobacillus plantarum (17) and citrate metabolism in L. oenos (21). In L. lactis, the transporter responsible for the exchange of malate (precursor) and lactate (product) in the malate decarboxylation pathway (malolactic fermentation) was shown to be able, at least in vitro, to catalyze electrogenic monoanionic H-malate Ϫ uniport (or malate 2Ϫ /H ϩ symport) (20). For the same process in L. plantarum, a variable stoichiometry for L-malate/proton symport which depends on the external L-malate concentration was described (17). In this model, the ratio of H-malate Ϫ to proton transported increased with increasing external concentrations of L-malate. Only at concentrations higher than 5 mM, the transport proceeded by a low-affinity unipo...

L-malate transport was studied in membrane vesicles from Leuconostoc oenos MLE( -) (mutant lacking malolactic enzyme) which were fused with liposomes containing beef heart cytochrome c oxidase as a proton-motive-force-generating system. In these hybrid membranes, accumulation of L-malate was observed in response to a pH gradient (ApH), with the inside alkaline, but was strongly inhibited by a membrane potential ( A ! P) of normal polarity (inside negative). Imposition of a A Y, with the inside positive, by means of valinomycin-mediated potassium influx, resulted in a rapid accumulation of L-malate, indicating that L-malate was taken up in an anionic form. The results are consistent with a uniport mechanism facilitating the uptake of monoanionic L-malate, the dominant species at the low pH of the experiments. Kinetic analysis of ApH-driven L-malate uptake in the pH range 3.0 -5.8, yielded apparent affinity constants that varied less than twofold when calculated on the basis of the concentrations of monoanionic L-malate, whereas the values differed 2 -3 orders of magnitude for the other species. At L-malate concentrations above 1 mM, a non-saturable transport component became apparent which may reflect passive influx of L-malic acid. Substrate specificity studies indicated that citrate and L-malate (and possibly D-lactate and L-lactate) compete for a single general carboxylate transport system. The carboxylate transport system catalysed homologous L-malate and heterologous L-malatekitrate exchange with rates similar to the rate of L-malate efflux. Since metabolic energy is conserved during malolactic fermentation in L. oenos, the underlying mechanism most likely involves electrogenic monoanionic L-malate uptake, in combination with H' consumption in the cytoplasm, followed by diffusion outwards of lactic acid plus carbon dioxide.Malolactic fermentation is performed by some lactic acid bacteria and, in the process, metabolic energy is conserved. The pathway involves the uptake of L-malate, which is stoichiometrically converted to L-lactic acid plus COz, after which the reaction products leave the cell. The AGO' of the L-malate decarboxylation reaction at pH 7.0 and pC0, lo5 Pa is approximately -26.5 kJimol [6]. Part of the free energy (AG), taking the AGO' of the reaction and the concentrations of the reactants into account, may be conserved in a chemiosmotic coupling mechanism [14]. Such a mechanism leads to the generation of a proton motive force (Ap) if the overall transport reaction is electronegative and decarboxylation (proton consumption) takes place in the cytoplasm. Enzyme. Malolactic enzyme (EC 1.1.1.38).buchneri [lo]. In the case of decarboxylation of oxalate and histidine, the corresponding transport reactions have been shown to proceed as oxalateiformate exchange and histidine/ histamine exchange or, in more general terms, as electrogenic precursoriproduct exchange [ 131. For malolactic fermentation in L. lactis, electrogenic malateAactate exchange was demonstrated but the same transport system was als...

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