Shewanella oneidensis NADH Dehydrogenase Mutants Exhibit an Amino Acid Synthesis Defect

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 + .

NADH dehydrogenases drive inward electron transfer in Shewanella oneidensisMR‐1

“…We observed that both the control and NADH dehydrogenase double mutant strain produced ~1 mM 2,3‐butanediol from 10 mM acetoin under this condition (Figure 4A ). In this condition, the NADH dehydrogenase mutant strain produced slightly higher levels of 2,3‐butanediol, possibly due to a build‐up of NADH in the cytoplasm, as previously observed (Duhl & Ter Avest, 2019 ). The 2,3‐butanediol concentration decreased during stationary phase for both strains, possibly due to back conversion to acetoin after major electron donors in the medium were consumed.…”

“…We speculate that this was due to a greater amount of stored organic carbon accumulated in the cells during aerobic growth in flasks. The ∆ nuoN ∆ nqrF1 NADH dehydrogenase mutant has a reduced respiration rate which reduces its ability to oxidize residual organic carbon during the aerobic phases of the experiment (Duhl & Ter Avest, 2019 ). It is not likely that cell death was responsible for the lack of acetoin reduction as the strain was capable of elevated electron transfer to fumarate (Figure 2C ), albeit at a reduced rate compared to the hydrogenase mutant.…”

“…To test whether NADH dehydrogenases participate in inward electron transfer to acetoin, we measured the ability of strains lacking NADH dehydrogenases to carry out this process. We previously studied the NADH dehydrogenases of S. oneidensis under a range of growth conditions and found that only Nuo and Nqr1 have a significant impact on growth (Duhl et al, 2018 ; Duhl & Ter Avest, 2019 ; Madsen & TerAvest, 2019 ). Therefore, we focused only on those two complexes in this work.…”

Engineering extracellular electron transfer pathways of electroactive microorganisms by synthetic biology for energy and chemicals production

“…However, with the increase in the NAD(H) pool size, the activities of citrate synthase and pyruvate dehydrogenase (PDH) could be gradually inhibited, thus reducing the energy conversion efficiency. 54,55 In fact, increasing the NAD(H) pool capacity without strengthening the electron flux of the electron transport chain inevitably resulted in the excessive accumulation of intracellular electrons, which may lead to an imbalance in intracellular redox homeostasis and interfere with cellular activities. 150,151 Fig.…”

“…However, EET currently proceeds at a low rate due to (i) inefficient energy conversion capacity, [50][51][52] (ii) inadequate intracellular electron generation, [53][54][55][56][57] and (iii) high transmembrane or cell-electrode interfacial electron transfer resistance, [58][59][60][61][62][63] which seriously restrict practical and costefficient application of BESs. Over the last decade, synthetic biology and materials engineering approaches have been adopted to rationalize the design and construction of EET conduits to overcome these deficiencies.…”