The Citrate Metabolic Pathway in Leuconostoc mesenteroides: Expression, Amino Acid Synthesis, and α-Ketocarboxylate Transport

Citrate metabolism in Leuconostoc mesenteroides subspecies mesenteroides is associated with the generation of a proton motive force by a secondary mechanism. 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, α-ketoglutarate, and α-ketoisocaproate to the cells, indicating a broad specificity of the transamination reaction. The α-ketocarboxylates are readily transported across the cytoplasmic membrane. α-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 F0F1 ATPase and by selective dissipation of the membrane potential and the transmembrane pH gradient. It is concluded that in L. mesenteroides α-ketoglutarate is transported via a secondary transporter that may be a general α-ketocarboxylate carrier.

Membrane Potential-generating Transport of Citrate and Malate Catalyzed by CitP of Leuconostoc mesenteroides

Citrate uptake in Leuconostoc mesenteroides subsp. mesenteroides 19D is catalyzed by a secondary citrate carrier (CitP). The kinetics and mechanism of CitP were investigated in membrane vesicles of L. mesenteroides. The transporter is induced by the presence of citrate in the medium and transports both citrate and malate. In spite of sequence homology to the Na+-dependent citrate carrier of Klebsiella pneumoniae, CitP is not Na+-dependent, nor is CitP Mg2+-dependent. The pH gradient (ΔpH) is a driving force for citrate and malate uptake into the membrane vesicles, whereas the membrane potential (Δψ) counteracts transport. An inverted membrane potential (inside positive) generated by thiocyanide diffusion can drive citrate and malate uptake in membrane vesicles. Analysis of the forces involved showed that a single unit of negative charge is translocated during transport. Kinetic analysis of citrate counterflow at different pH values indicated that CitP transports the dianionic form of citrate (Hcit2-) with an affinity constant of ~20 µM. It is concluded that CitP catalyzes Hcit2-/H+ symport. Translocation of negative charge into the cell during citrate metabolism results in the generation of a membrane potential that contributes to the protonmotive force across the cytoplasmic membrane, i.e. citrate metabolism in L. mesenteroides generates metabolic energy. Efficient exchange of citrate and D-lactate, a product of citrate/carbohydrate co-metabolism, is observed, suggesting that under physiological conditions, CitP may function as an electrogenic precursor/product exchanger rather than a symporter. The mechanism and energetic consequences of citrate uptake are similar to malate uptake in lactic acid bacteria.