Abstract
Shewanella oneidensis MR-1 is a dissimilatory metal reducing bacterium with a highly branched respiratory electron transport chain. The S. oneidensis MR-1 genome encodes four NADH dehydrogenases, any of which may be used during respiration. We previously determined that a double-knockout of two NADH dehydrogenases, Nuo and Nqr1, eliminated aerobic growth in minimal medium. However, the double-knockout strain was able to grow aerobically in rich medium. Here, we determined that amino acid supplementation rescued growth of the mutant strain in oxic minimal medium. To determine the mechanism of the growth defect, we monitored growth, metabolism, and total NAD(H) pools in S. oneidensis MR-1 and the NADH dehydrogenase knockout strain. We also used a genetically encoded redox sensing system and determined that NADH/NAD+ was higher in the mutant strain than in the wild-type. We observed that the double-knockout strain was able to metabolize d,l-lactate and N-acetylglucosamine when supplemented with tryptone, but excreted high concentrations of pyruvate and acetate. The requirement for amino acid supplementation, combined with an apparent inability of the mutant strain to oxidize pyruvate or acetate suggests that TCA cycle activity was inhibited in the mutant strain by a high NADH/NAD+.
Highlights
Shewanella oneidensis MR-1 is a model organism studied for its ability to respire with a variety of terminal electron acceptors, including oxygen, nitrate, TMAO, metal oxides, and electrodes (Beliaev et al, 2005; Gralnick et al, 2006; Meshulam-Simon et al, 2007; Pinchuk et al, 2011; Coursolle and Gralnick, 2012; TerAvest and Angenent, 2014; Duhl et al, 2018)
We previously developed NADH dehydrogenase knockout strains of S. oneidensis MR-1 and found a severe growth defect for a strain lacking Nuo and Nqr1
Previous work indicated that S. oneidensis MR-1 is incapable of using individual amino acids as carbon sources but is capable a wide variety of defined dipeptides (Serres and Riley, 2006)
Summary
Shewanella oneidensis MR-1 is a model organism studied for its ability to respire with a variety of terminal electron acceptors, including oxygen, nitrate, TMAO, metal oxides, and electrodes (Beliaev et al, 2005; Gralnick et al, 2006; Meshulam-Simon et al, 2007; Pinchuk et al, 2011; Coursolle and Gralnick, 2012; TerAvest and Angenent, 2014; Duhl et al, 2018). Underlying the respiratory versatility of S. oneidensis MR-1 is a highly branched electron transport chain, including four NADH dehydrogenases and 3 aerobic terminal oxidases, along with specialized terminal oxidases for a variety of electron acceptors (Heidelberg et al, 2002; Pinchuk et al, 2010; Deutschbauer et al, 2011). Even within use of a single electron acceptor, S. oneidensis MR-1 can remodel its electron transport chain depending on environmental conditions. A third aerobic terminal oxidase, the caa cytochrome oxidase, is rarely expressed in S. oneidensis MR-1 and has only been observed under high oxygen, low organic carbon conditions (Le Laz et al, 2016). The variety of respiratory complexes and their differential regulation may allow S. oneidensis MR-1 to optimize metabolic flux and energy conservation under the wide range of redox conditions it experiences in the environment
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