Electrogenic malate uptake and improved growth energetics of the malolactic bacterium Leuconostoc oenos grown on glucose-malate mixtures

Abstract

Growth of the malolactic bacterium Leuconostoc oenos was improved with respect to both the specific growth rate and the biomass yield during the fermentation ofglucose-malate mixtures as compared with those in media lacking malate. Such a finding indicates that the malolactic reaction contributed to the energy budget of the bacterium, suggesting that growth is energy limited in the absence of malate. An energetic yield (YATP) of 9.5 g of biomass mol ATP'1 was found during growth on glucose with an ATP production by substrate-level phosphorylation of 1.2 mol of ATP. mol of glucose-'. During the period of mixed-substrate catabolism, an apparent YATP of 17.7 was observed, indicating a mixotrophy-associated ATP production of 2.2 mol of ATP-mol of glucose 1, or more correctly an energy gain of 0.28 mol of ATP-mol of malate-1, representing proton translocation flux from the cytoplasm to the exterior of 0.56 or 0.84 H+ mol of malate'1 (depending on the H+/ATP stoichiometry). The growth-stimulating effect of malate was attributed to chemiosmotic transport mechanisms rather than proton consumption by the malolactic enzyme. Lactate efflux was by electroneutral lactatef/H+ symport having a constant stoichiometry, while malate uptake was predominantly by a malate-/H+ symport, though a low-affinity malate-uniport was also implicated. The measured electrical component (A*) of the proton motive force was altered, passing from -30 to -60 mV because of this translocation of dissociated organic acids when malolactic fermentation occurred.Certain species of lactic acid bacteria (belonging to the genera Lactobacillus, Pediococcus, and Leuconostoc) possess the capacity to convert malate to lactate via a direct decarboxylation reaction referred to as malolactic fermentation. This fermentation is of use in the enological industry since it enables the removal of excess acidity, enhances organoleptic properties, and increases the bacteriological stability of wine (7,33).Those bacteria able to metabolize malate via malolactic fermentation show improved growth characteristics when presented with glucose-malate mixtures as compared with the growth characteristics during fermentation of glucose alone. This response to an auxiliary substrate is not, at first glance, surprising: increased growth yields attributable to a second catabolic substrate have been previously reported (2, 13). In these reports the maximum specific growth rates remained unaltered, being fixed by carbon flux limitations. More recently, improved growth yields and specific growth rates have been achieved with substrate mixtures which overcome both carbon flux and energetic limitations simultaneously (15). In the case of the malolactic bacteria, the situation is somewhat different in that the malate cannot contribute directly to the anabolic carbon flux and, furthermore, there is no energy conservation at the enzyme level (either directly by substrate-level phosphorylation [SLP] or indirectly via reducing equivalent generation). The reaction involved in the decarboxylatio...

An organic acid blend can modulate swine intestinal fermentation and reduce microbial proteolysis

“…This finding could be explained by the fact that citric acid (Lutgens and Gottschalk 1980;Marty-Teysset et al 1996), malic acid (Renault et al 1988;Loubiere et al 1992) and fumaric acid (Tran et al 1997;Tielens and Van Hellemond 1998) may positively modulate the energy metabolism of some bacterial strains usually residing in the hindgut. Lopez et al (1999) observed that sodium fumarate at 5 and 10 mmol L -1 was able to stimulate ruminal proliferation of cellulolytic bacteria and digestion of fiber.…”

Article

“…The transmembrane pH and electrical gradients, e.g., ∆pH and ∆ψ, were measured by determining the internal to external gradient of 14 C-benzoic acid and 3 H-tetraphenylphosphonium bromide (TPP + ), respectively, after centrifugation of the cells through silicon oil, as previously described [13]. The ATPase activity was determined as previously described [5].…”

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

“…Only at concentrations higher than 5 mM, the transport proceeded by a low-affinity uniport which was able to generate ⌬p. For L. oenos, three different models for L-malate uptake and ⌬p generation, all based on L-malate uniport, have been proposed (12,24,30 Other authors proposed simultaneous low-affinity H-malate Ϫ uniport and passive diffusion of nondissociated L-malate, the relative contribution being dependent on the external pH (30). In a previous report on studies in membrane vesicles (24), we proposed a model showing H-malate Ϫ uniport in the pH range 3 to 5.6 and at low concentrations of L-malate.…”

“…Only at concentrations higher than 5 mM, the transport proceeded by a low-affinity uniport which was able to generate ⌬p. For L. oenos, three different models for L-malate uptake and ⌬p generation, all based on L-malate uniport, have been proposed (12,24,30). Loubiere et al suggested a system similar to that of L. plantarum, in which the combined action of H-malate Ϫ /H ϩ symport and H-malate Ϫ uniport depends on the external malate concentration (12).…”

“…Intracellular pH was determined from the distribution of [ 14 C]benzoic acid (23), using the silicone oil centrifugation technique (28). Cells (0.5 mg [dry weight]/ml) were incubated in K-MES-glycylglycine buffer at pH 3.1 to 5.6 for 12 min at 25ЊC in the presence of 50 mM L-malate and 1 Ci of [7][8][9][10][11][12][13][14] C] benzoic acid per ml. Samples of 500 l were centrifuged through 300 l of silicone oil (density ϭ 1.02 g/cm 3 ) at 16,000 ϫ g for 10 min at room temperature.…”