The ability to use lactate as a sole source of carbon and energy is one of the key metabolic signatures of Shewanellae, a diverse group of dissimilatory metal-reducing bacteria commonly found in aquatic and sedimentary environments. Nonetheless, homology searches failed to recognize orthologs of previously described bacterial D-or L-lactate oxidizing enzymes (Escherichia coli genes dld and lldD) in any of the 13 analyzed genomes of Shewanella spp. By using comparative genomic techniques, we identified a conserved chromosomal gene cluster in Shewanella oneidensis MR-1 (locus tag: SO1522-SO1518) containing lactate permease and candidate genes for both D-and L-lactate dehydrogenase enzymes. The predicted D-LDH gene (dld-II, SO1521) is a distant homolog of FAD-dependent lactate dehydrogenase from yeast, whereas the predicted L-LDH is encoded by 3 genes with previously unknown functions (lldEGF, SO1520 -SO1518). Through a combination of genetic and biochemical techniques, we experimentally confirmed the predicted physiological role of these novel genes in S. oneidensis MR-1 and carried out successful functional validation studies in Escherichia coli and Bacillus subtilis. We conclusively showed that dld-II and lldEFG encode fully functional D-and L-LDH enzymes, which catalyze the oxidation of the respective lactate stereoisomers to pyruvate. Notably, the S. oneidensis MR-1 LldEFG enzyme is a previously uncharacterized example of a multisubunit lactate oxidase. Comparative analysis of >400 bacterial species revealed the presence of LldEFG and Dld-II in a broad range of diverse species accentuating the potential importance of these previously unknown proteins in microbial metabolism.central carbon metabolism ͉ genome context analysis ͉ lactate dehydrogenase M any aerobic and anaerobic bacteria are able to grow by using D-and/or L-lactate as a sole source of carbon and energy (1-4). Although lactate is a common product of carbohydrate fermentation (5, 6), it is rarely detected in environmental samples (7,8), suggesting that it is either a minor metabolic product or that its conversion rates are very high. In support of the latter possibility, Finke et al. (9) recently reported constant production and consumption of lactate in marine sediments, linking its high turnover rates with microbiological reduction of sulfate and metals.Among microorganisms actively coupling lactate oxidation to the reduction of multiple electron acceptors is a diverse and ubiquitous group of dissimilatory metal-reducing bacteria, which belong to the genus Shewanella (10). Shewanellae are commonly found in complex microbial communities within aquatic and sedimentary systems, many of which are subject to spatial and temporal variations in the type and concentration of organic and inorganic substrates that reflect redox gradients (10). The versatile flexibility of energygenerating pathways, which enables respiration of various electron acceptors including O 2 , Fe(III), Mn(IV), thiosulfate, elemental sulfur, and nitrate, contributes to the ability...