The citrate metabolic pathway in Leuconostoc mesenteroides: expression, amino acid synthesis, and alpha-ketocarboxylate transport

Abstract

Citrate metabolism in Leuconostoc mesenteroides subspecies mesenteroides is associated with the generation of a proton motive force by a secondary mechanism (C. Marty-Teysset, C. Posthuma, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Bacteriol. 178:2178-2185, 1996). The pathway consists of four steps: (i) uptake of citrate, (ii) splitting of citrate into acetate and oxaloacetate, (iii) pyruvate formation by decarboxylation of oxaloacetate, and (iv) reduction of pyruvate to lactate. Studies of citrate uptake and metabolism in resting cells of L. mesenteroides grown in the presence or absence of citrate show that the citrate transporter CitP and citrate lyase are constitutively expressed. On the other hand, oxaloacetate decarboxylase is under stringent control of the citrate in the medium and is not expressed in its absence, thereby blocking the pathway at the level of oxaloacetate. Under those conditions, the pathway is completely directed towards the formation of aspartate, which is formed from oxaloacetate by transaminase activity. The data indicate a role for citrate metabolism in amino acid biosynthesis. Internalized radiolabeled aspartate produced from citrate metabolism could be chased from the cells by addition of the amino acid precursors oxaloacetate, pyruvate, ␣-ketoglutarate, and ␣-ketoisocaproate to the cells, indicating a broad specificity of the transamination reaction. The ␣-ketocarboxylates are readily transported across the cytoplasmic membrane. ␣-Ketoglutarate uptake in resting cells of L. mesenteroides was dependent upon the presence of an energy source and was inhibited by inhibition of the proton motive force generating F 0 F 1 ATPase and by selective dissipation of the membrane potential and the transmembrane pH gradient. It is concluded that in L. mesenteroides ␣-ketoglutarate is transported via a secondary transporter that may be a general ␣-ketocarboxylate carrier.Cometabolism of glucose with citrate results in growth stimulation of Leuconostoc species that has been explained by a shift in glucose metabolism from ethanol to acetate production via acetate kinase, yielding additional ATP (2, 30). However, in recent studies, citrate metabolism in Leuconostoc mesenteroides was shown to be a secondary proton motive force (PMF)-generating pathway (14,18,21,22). The mechanism of PMF generation is similar to that observed during malolactic fermentation in Lactococcus lactis (25), and by analogy the pathway was termed citrolactic fermentation. Studies of the mechanism of the citrate transporter CitP performed in membrane vesicles of L. mesenteroides showed that CitP catalyzes two modes of transport, symport of dianionic citrate with one proton (CitH 2Ϫ /H ϩ ) and exchange of citrate and D-lactate. DLactate is a product of citrate-carbohydrate cometabolism, which suggested that CitP might function as a precursor/product exchanger (21). Subsequent studies of the energetics of citrate metabolism in resting cells of L. mesenteroides showed that citrate/lactate exchange was indeed the ph...

Membrane Potential-generating Malate (MleP) and Citrate (CitP) Transporters of Lactic Acid Bacteria Are Homologous Proteins

“…Possibly, hydrogen bonding between a residue on the transporter and the hydroxyl or keto group on the substrates is essential for translocation. The transport activity of CitP with oxaloacetate is remarkable since oxaloacetate is the first metabolic intermediate in the citrate degradation pathway in lactic acid bacteria (39). Since the 2-hydroxycarboxylates are the physiological and preferred substrates of these carriers we have termed the family the 2-hydroxycarboxylate transporter family.…”

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

“…The answer is provided by the different induction profiles of the citrate metabolic pathways in Leuconostoc and Lactococcus species. In Leuconostoc mesenteroides, the enzymes of the pathway are induced by the presence of citrate in the medium, while in L. lactis, the induction additionally requires a low pH in the medium, suggesting a role in acid stress (40,85,89,92,93,95). The conversion of carbohydrate to lactic acid results in acidification of the medium, which eventually inhibits growth.…”

Substrate Specificity of the Citrate Transporter CitP of Lactococcus lactis

“…CitP catalyzes uptake of divalent citrate (Hcit 2Ϫ ) in exchange with monovalent L-lactate (lac Ϫ ) (precursor-product exchange), which results in generation of a membrane potential (⌬) (13,14,15). Together with proton consumption in decarboxylation reactions in the citrate metabolic pathway, which results in a transmembrane pH gradient (⌬pH), the pathway generates proton motive force (PMF) (10,11,16). Recognition by CitP of two structurally related but different substrates, i.e., the tricarboxylate citrate and the monocarboxylate L-lactate, suggests an inherent broad substrate specificity of the transporter.…”

“…he citrate transporter CitP functions in citrate fermentation by lactic acid bacteria (LAB) such as Lactococcus lactis and Leuconostoc mesenteroides (16,18). Internalized citrate is split into acetate and oxaloacetate, after which the latter is decarboxylated, yielding pyruvate.…”

“…Many of the substrates of CitP are intermediates or products of amino acid metabolism, suggesting that CitP may have a broader physiological function than its role in citrate fermentation alone. (16,18). Internalized citrate is split into acetate and oxaloacetate, after which the latter is decarboxylated, yielding pyruvate.…”