The proton motive force generated in Leuconostoc oenos by L-malate fermentation

Abstract

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...

Biochemical basis for glucose-induced inhibition of malolactic fermentation in Leuconostoc oenos

“…Although L. oenos was recently reclassified as Oenococcus oeni (13), the old designation will be used throughout this work. The malate fermentation pathway in L. oenos generates a proton motive force that drives ATP synthesis (9,29), thus explaining the early report of a pH-independent stimulation of growth by malate (24). Glucose and fructose, the major sugars present in wine, can be utilized by L. oenos as energy sources for growth (41), and another important component in wine, citric acid, also plays an important role in the bioenergetics of this bacterium (26).…”

Surviving the Acid Test: Responses of Gram-Positive Bacteria to Low pH

“…Malolactic fermentation (MLF) is the conversion of the dicarboxylic malic acid to the monocarboxylic lactic acid. MLF plays a major role in the activity of the wine bacteria Oenococcus oeni (199) and has also been identified in Lactobacillus sakei (31), Lactobacillus plantarum (174), and Lactococcus lactis (192). The L-lactate produced is excreted via either a lactate-malate antiporter (L. lactis [182]) or an electrogenic uniport (O. oeni and L. planatum), both of which allow the synthesis of ATP at low pH (Fig.…”

The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism

“…Lactic acid leaves the cell by passive diffusion. The energetic consequences of the pathway are the same, but the pathway is better adapted to the acidic conditions of wine fermentation (119). The transporter that catalyzes the electrogenic uniport reaction is a member of the auxin efflux carrier (AEC) family (TC 2.A.69 in the transporter classification system [17,117]), which contains many members from bacteria, archaea, and plants (70).…”