Genomic reconstruction of Shewanella oneidensis MR-1 metabolism reveals a previously uncharacterized machinery for lactate utilization

Abstract

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...

Shewanella oneidensis MR-1 Sensory Box Protein Involved in Aerobic and Anoxic Growth

“…In our study, these (40) study reported that these genes functioned under both aerobic and anaerobic conditions, and the decreased expression observed in our study might be a result of a decline in lactate consumption under anoxic conditions compared to that under aerobic conditions. The results suggested that SO3389 plays a potential role in down-expressing genes involved in lactate consumption when oxygen becomes limited.…”

“…Recent work demonstrated that S. oneidensis MR-1 can utilize both L-and D-lactate, and it was hypothesized that SO1518 to SO1520 encodes an L-lactate dehydrogenase and SO1521 encodes a D-lactate dehydrogenase (40). In our study, these (40) study reported that these genes functioned under both aerobic and anaerobic conditions, and the decreased expression observed in our study might be a result of a decline in lactate consumption under anoxic conditions compared to that under aerobic conditions.…”

Substrate-Level Phosphorylation Is the Primary Source of Energy Conservation during Anaerobic Respiration of Shewanella oneidensis Strain MR-1

“…Two carbon sources entering central metabolism at different locations are N-acetylglucosamine (NAG) and lactate, which enter before and after glycolysis, respectively ( Fig. 1) (24,36). Both are oxidized to acetate and carbon dioxide anaerobically, though lactate yields one ATP and two reducing equivalents per molecule, while NAG yields three ATPs and four reducing equivalents per molecule.…”

Towards patterned bioelectronics: facilitated immobilization of exoelectrogenic Escherichia coli with heterologous pili

“…In S. oneidensis , EET is initiated at the inner cell membrane where electrons, originating from the oxidation of primary fermentation products (e.g. lactate, pyruvate, formate and hydrogen), are transferred to membrane‐embedded menaquinols by formate and NADH dehydrogenases (Meshulam‐Simon et al ., 2007; Pinchuk et al ., 2009; Cordova et al ., 2011; Brutinel and Gralnick, 2012a; Kane et al ., 2016). Menaquinol is in turn oxidized by the inner membrane‐bound quinol dehydrogenase CymA (Marritt et al ., 2012), or in its absence, the enzyme complex SirCD (Cordova et al ., 2011).…”