A constructed lactate dehydrogenase (LDH)-negative mutant of Enterococcus faecalis V583 grows at the same rate as the wild type but ferments glucose to ethanol, formate, and acetoin. Microarray analysis showed that LDH deficiency had profound transcriptional effects: 43 genes in the mutant were found to be upregulated, and 45 were found to be downregulated. Most of the upregulated genes encode enzymes of energy metabolism or transport. By two-dimensional (2D) gel analysis, 45 differentially expressed proteins were identified. A comparison of transcriptomic and proteomic data suggested that for several proteins the level of expression is regulated beyond the level of transcription. Pyruvate catabolic genes, including the truncated ldh gene, showed highly increased transcription in the mutant. These genes, along with a number of other differentially expressed genes, are preceded by sequences with homology to binding sites for the global redox-sensing repressor, Rex, of Staphylococcus aureus. The data indicate that the genes are transcriptionally regulated by the NADH/ NAD ratio and that this ratio plays an important role in the regulatory network controlling energy metabolism in E. faecalis.Lactic acid bacteria (LAB) are widely used for production of lactic acid in fermented food. During the fermentation process, pyruvate is converted to lactate in addition to a number of minor metabolites, such as acetic acid, acetaldehyde, ethanol, acetoin, and acetate. However, under certain conditions, these bacteria shift from homolactic to heterolactic (or mixed-acid) fermentation, with formate, acetate, acetoin, ethanol, and CO 2 as end products. In Lactococcus lactis, mixed-acid fermentation has been shown to take place at low grow rates under microaerobic conditions (11), under true carbon-limited conditions, and while growing at low pH on carbon sources other than glucose (15,20).Mixed-acid fermentation was also seen after removing the lactate dehydrogenase (LDH) activity in Enterococcus faecalis V583 (12). This bacterium has two ldh genes, but ldh-1 is the main contributor to lactate production. A mutant with deletions in both ldh genes (the ⌬ldh1.2 mutant) was constructed and shown to direct its carbon flow from pyruvate away from lactate toward formate, acetoin, and alcohol production (12). Alternative carbon fluxes in different knockout mutants have also been reported for Lactococcus lactis (22).The mechanism of the shift from homolactic to mixed-acid fermentation is still not fully understood. During transformation of pyruvate to lactate, LDH regenerates NAD ϩ from NADH formed during glycolysis. When pyruvate is converted to acetyl-coenzyme A (acetyl-CoA) by either pyruvate formate lyase (PFL) or pyruvate dehydrogenase (PDH), reduction of acetyl-CoA to ethanol regenerates NAD ϩ from NADH and is an alternative to lactate formation in redox balancing. The carbon flux is biochemically regulated (4, 5). Fructose-1,6-bisphosphate is an allosteric activator of lactate production, and dihydroxyacetone phosphate and D-glyce...