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 (Delta pH) is a driving force for citrate and malate uptake into the membrane vesicles, whereas the membrane potential (Delta psi) 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 (Hcit(2-)) with an affinity constant of similar to 20 mu M. It is concluded that CitP catalyzes Hcit(2-)/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.