The roles of the two ldh genes of Enterococcus faecalis were studied using knockout mutants. Deletion of ldh-1 causes a metabolic shift from homolactic fermentation to ethanol, formate, and acetoin production, with a high level of formate production even under aerobic conditions. Ldh-2 plays only a minor role in lactate production.Carbohydrate metabolism in economically important lactic acid bacterial species, such as Lactococcus lactis, has been extensively studied. However, in lactic acid bacterial species such as Enterococcus faecalis, with less industrial value, research focus has so far been mostly on medical aspects. E. faecalis has two lactate dehydrogenase (ldh) genes (3, 17), with high similarity to ldhA and ldhB of Lactococcus lactis, respectively (2). In L. lactis, ldhA is responsible for all lactate production, while ldhB remains unexpressed. To understand the role of these genes in E. faecalis, and to gain more insight into energy metabolism, we made and characterized three ldh knockout mutants in E. faecalis V583, removing either ldh-1 (EF_0255), ldh-2 (EF_0641) or both.To achieve the construction of deletion mutants, we made a new shuttle vector based on the thermosensitive replicon of pG ϩ host4 (12). Phusion DNA polymerase (Finnzyme, Espoo, Finland) was used for PCR in accordance with the manufacturer's recommendations. A part of pG ϩ host4 was amplified with PCR using primers pgh41 and pgh42 (Table 1) and cloned in the HincII site of pBluescript SKϩ to produce the plasmid pAS221. The tetM gene of Lactobacillus plantarum 5057 (4) was amplified by PCR using primers Dbd43f and Dbd44r (Table 1) and cloned in pCR2.1 (Invitrogen, United Kingdom), subsequently excised from the recombinant plasmid with EcoRI, and cloned in pAS221 cut with the same enzyme to yield pAS222 (Fig. 1). The various parts of the vector were verified by restriction enzyme analysis. Gene replacement was achieved by double-crossover homologous recombination, using pAS222 as cloning vector. Deletion of about one-third of each ldh gene was done by two-step PCR (7) using the outer primer pairs MJ1-MJ2 for ldh-1 and MJ5-MJ6 for ldh-2 (Table 1). The inner primer pairs carrying regions of homology for the fusion step were MJ3-MJ4 for ⌬ldh-1 and MJ7-MJ8 for ⌬ldh-2 (Table 1). The constructs were cloned into the SnabI site of pAS222 and propagated in Escherichia coli cells. To replace the genes on the chromosome with

One of the ABC transporter systems in Enterococcus faecalis V583 is encoded by the ef0176-ef0180 gene cluster, which differs from orthologous operons in related bacteria in that it contains two genes putatively encoding substrate-binding proteins (SBPs). These SBPs, EF0176 and EF0177, have previously been identified on the surface of E. faecalis. By phenotypic studies of single and double knockout mutants, we show here that EF0176 and EF0177 are specific for ribonucleosides and, by inference, that the EF0176-EF0180 ABC transporter plays a role in nucleoside uptake. The specificity of the SBPs was mapped using growth experiments on a medium, RPMI 1640, that only supports growth of E. faecalis when supplemented with purine nucleosides or their corresponding bases. This analysis was complemented by studies with toxic fluorinated pyrimidine ribonucleoside analogues and competition experiments. The data show that EF0176 and EF0177 have broad and overlapping, but not identical, substrate specificities and that they, together, are likely to bind and facilitate the transport of all common ribonucleosides. Comparative sequence analysis and inspection of an available crystal structure of an orthologue, PnrA from Treponema pallidum, showed that the strongest binding interactions between the protein and the ligand involve the ribose moiety and that sequence variation in the binding site primarily affects interactions with the base. This explains both the broad substrate specificity of these binding proteins and the observed variations therein. The presence of two SBPs in this nucleoside ABC transporter system in E. faecalis may improve the bacterium's ability to scavenge nucleosides.