MECHANISM OF THE CITRATE TRANSPORTERS IN CARBOHYDRATE AND CITRATE COMETABOLISM IN LACTOCOCCUS AND LEUCONOSTOC SPECIES

Citrate metabolism in the lactic acid bacterium Leuconostoc mesenteroi des generates an electrochemical proton gradient across the membrane b y 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). Reports on the energetics of citrate metabolism in the relate d organism Lactococcus lactis are contradictory, and this study was pe rformed to clarify this issue. Cloning of the membrane potential-gener ating citrate transporter (CitP) of Leuconostoc mesenteroides revealed an amino acid sequence that is almost identical to the known sequence of the CitP of Lactococcus lactis. The cloned gene was expressed in a Lactococcus lactis Cit-strain, and the gene product was functionally characterized in membrane vesicles. Uptake of citrate was counteracted by the membrane potential, and the transporter efficiently catalyzed heterologous citrate-lactate exchange. These properties are essential for generation of a membrane potential under physiological conditions and show that the Leuconostoc CitP retains its properties when it is e mbedded in the cytoplasmic membrane of Lactococcus lactis. Furthermore , using the same criteria and experimental approach, we demonstrated t hat the endogenous CitP of Lactococcus lactis has the same properties, showing that the few differences in the amino acid sequences of the C itPs of members of the two genera do not result in different catalytic mechanisms. The results strongly suggest that the energetics of citra te degradation in Lactococcus lactis and Leuconostoc mesenteroides are the same; i.e., citrate metabolism in Lactococcus lactis is a proton motive force-generating process.