Shewanella oneidensis MR-1 Fluxome under Various Oxygen Conditions

Shewanella oneidensis MR-1 (ATCC 70050) is a gram-negative, facultative anaerobe that was isolated from lake sediment (35). S. oneidensis bacteria are capable of utilizing many carbon sources, including lactate, acetate, pyruvate, and some amino acids. Moreover, they are capable of reducing a variety of electron acceptors besides oxygen, including Fe(III), Mn(IV), sulfur, nitrate, and fumarate (26). There have been extensive studies of this strain, focused primarily on its versatile respiration and its potential to engage in cometabolic bioremediation of toxic metals (17, 18, 34, 36, 37). Recently, the complete S. oneidensis genome was sequenced and annotated. Furthermore, key phenotypic and molecular characteristics have been identified (13). The central carbon metabolism of S. oneidensis under both aerobic and anaerobic conditions has been investigated using enzyme assays and genome information (11, 26, 27), and there are several unusual features (26, 42). First, a serine pathway is proposed to be active under anaerobiosis in S. oneidensis due to the detection of high levels of hydroxypyruvate reductase, which is the key enzyme involved in serine metabolism. Second, S. oneidensis shares some metabolic features with nonfermentative pseudomonads, such as utilization of the Entner-Doudoroff (ED) pathway instead of the Embden-Meyerhof-Parnas (EMP) pathway for the oxidation of glucose. Third, because the tricarboxylic acid (TCA) cycle might be truncated under oxygen-limited conditions, the glyoxylate shunt might be present to synthesize TCA cycle intermediates. However, these features have not been rigorously verified using 13C tracer experiments, and very little is known about the actual balances of intracellular metabolic fluxes under different oxygen conditions. Metabolic flux analysis is necessary to provide a detailed physiological characterization of S. oneidensis MR-1 and may be important for improving its metal reduction ability through rational metabolic engineering or by stimulating metal reduction in the environment through the addition of growth supplements and electron donors. 13C isotopomer analysis is a powerful approach for mapping intracellular fluxes. By feeding a 13C-labeled carbon source to the cells, the labeling pattern of the primary metabolites, often the amino acids, can be measured. Based on these isotopomer data and the biochemical network of S. oneidensis MR-1, a metabolic pathway model can quantify the rates of intracellular reactions (16). In our study, labeling patterns of amino acids were analyzed by both nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). The advantage of NMR is that it provides positional information about the labels in the isotopomers even though detection sensitivity is low (2). GC-MS is a more sensitive detection method and determines what fraction of a particular molecule or molecular fragment contains a specific number of labels (38). By combining GC-MS and NMR data, a complete picture of the isotopomer distribution in amino acids can be obtained. The main goal of this study was to determine the fluxes through key metabolic pathways in S. oneidensis MR-1 under aerobic and microaerobic conditions. The determination of fluxes was accomplished in three steps: (i) the cells were grown in defined medium with 13C-labeled lactate as the sole carbon source, (ii) the labeling patterns in key amino acids of the total protein hydrolysate were characterized using both GC-MS and NMR, and (iii) a flux calculation algorithm was used to quantify the central metabolic pathways. Two sets of conditions were probed to determine the flux distribution, carbon limitation (dissolved oxygen [DO] > 70%) and oxygen limitation (DO < 10%). The results not only widen our understanding of Shewanella metabolism but also demonstrate a powerful approach for investigating metabolic flux analysis using both GC-MS and NMR techniques.