Citrate metabolism in the lactic acid bacterium Leuconostoc mesenteroides generates an electrochemical proton gradient across the membrane 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). Reports on the energetics of citrate metabolism in the related organism Lactococcus lactis are contradictory, and this study was performed to clarify this issue. Cloning of the membrane potential-generating citrate transporter (CitP) of Leuconostoc mesenteroides revealed an amino acid sequence that is almost identical to the known sequence of the CitP ofLactococcus lactis. The cloned gene was expressed in aLactococcus 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 embedded in the cytoplasmic membrane of Lactococcus lactis. Furthermore, using the same criteria and experimental approach, we demonstrated that the endogenous CitP ofLactococcus lactis has the same properties, showing that the few differences in the amino acid sequences of the CitPs of members of the two genera do not result in different catalytic mechanisms. The results strongly suggest that the energetics of citrate degradation inLactococcus lactis and Leuconostoc mesenteroides are the same; i.e., citrate metabolism inLactococcus lactis is a proton motive force-generating process.

The conversion from citrate positive (Cit') to citrate negative (Cit-) phenotype of six strains of Leuconostoc mesenteroides was followed during growth in milk and buffered or unbuffered MRS medium at 30 or 37°C. High rate of loss of Cit' phenotype was observed. The Citphenotype was found to be linked to the loss of 22 to 23 kb plasmids. All Cit-mutants isolated from Leuc. mesenteroides subsp. cremoris 195 reverted spontaneously to the Cit' phenotype. Hybridization experiments using a 0.8 kb fragment of the citP gene of Leuc. mesenteroides showed that all the plasmids which were lost in Cit-mutants encoded for a citrate permease. However, neither plasmid nor genomic DNA from Leuc. mesenteroides subsp. cremoris 195 hybridized with the citP probe.