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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+.

SUBMITTER: Duhl KL

PROVIDER: S-EPMC7561040 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Shewanella oneidensis NADH dehydrogenase mutants exhibit an amino acid synthesis defect.

Duhl Kody L KL TerAvest Michaela A MA

Frontiers in energy research 20191024

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 amin ...[more]

PMID: 33072733

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Project description:Shewanella oneidensis MR-1 is a metal-reducing bacterium with the ability to utilize many different terminal electron acceptors, including oxygen and solid-metal oxides. Both metal oxide reduction and aerobic respiration have been studied extensively in this organism. However, electron transport chain processes upstream of the terminal oxidoreductases have been relatively understudied in this organism, especially electron transfer from NADH to respiratory quinones. Genome annotation indicates that S. oneidensis MR-1 encodes four NADH dehydrogenases, a proton-translocating dehydrogenase (Nuo), two sodium ion-translocating dehydrogenases (Nqr1 and Nqr2), and an "uncoupling" dehydrogenase (Ndh), but none of these complexes have been studied. Therefore, we conducted a study specifically focused on the effects of individual NADH dehydrogenase knockouts in S. oneidensis MR-1. We observed that two of the single-mutant strains, the ?nuoN and ?nqrF1 mutants, exhibited significant growth defects compared with the wild type. However, the defects were minor and only apparent under certain growth conditions. Further testing of the ?nuoN ?nqrF1 double-mutant strain yielded no growth in minimal medium under oxic conditions, indicating that Nuo and Nqr1 have overlapping functions, but at least one is necessary for aerobic growth. Coutilization of proton- and sodium ion-dependent energetics has important implications for the growth of this organism in environments with varied pH and salinity, including microbial electrochemical systems.IMPORTANCE Bacteria utilize a wide variety of metabolic pathways that allow them to take advantage of different energy sources, and to do so with varied efficiency. The efficiency of a metabolic process determines the growth yield of an organism, or the amount of biomass it produces per amount of substrate consumed. This parameter has important implications in biotechnology and wastewater treatment, where low growth yields are often preferred to minimize the production of microbial biomass. In this study, we investigated respiratory pathways containing NADH dehydrogenases with varied efficiency (i.e., the number of ions translocated per NADH oxidized) in the metal-reducing bacterium Shewanella oneidensis MR-1. We observed that two different respiratory pathways are used concurrently, and at least one pathway must be functional for growth under oxic conditions.

Project description:Shewanella oneidensis MR-1 is a facultative anaerobe that grows by respiration using a variety of electron acceptors. This organism serves as a model to study how bacteria thrive in redox-stratified environments. A glucose-utilizing engineered derivative of MR-1 has been reported to be unable to grow in glucose minimal medium (GMM) in the absence of electron acceptors, despite this strain having a complete set of genes for reconstructing glucose to lactate fermentative pathways. To gain insights into why MR-1 is incapable of fermentative growth, this study examined a hypothesis that this strain is programmed to repress the expression of some carbon metabolic genes in the absence of electron acceptors. Comparative transcriptomic analyses of the MR-1 derivative were conducted in the presence and absence of fumarate as an electron acceptor, and these found that the expression of many genes involved in carbon metabolism required for cell growth, including several tricarboxylic acid (TCA) cycle genes, was significantly downregulated in the absence of fumarate. This finding suggests a possibility that MR-1 is unable to grow fermentatively on glucose in minimal media owing to the shortage of nutrients essential for cell growth, such as amino acids. This idea was demonstrated in subsequent experiments that showed that the MR-1 derivative fermentatively grows in GMM containing tryptone or a defined mixture of amino acids. We suggest that gene regulatory circuits in MR-1 are tuned to minimize energy consumption under electron acceptor-depleted conditions, and that this results in defective fermentative growth in minimal media. IMPORTANCE It is an enigma why S. oneidensis MR-1 is incapable of fermentative growth despite having complete sets of genes for reconstructing fermentative pathways. Understanding the molecular mechanisms behind this defect will facilitate the development of novel fermentation technologies for the production of value-added chemicals from biomass feedstocks, such as electro-fermentation. The information provided in this study will also improve our understanding of the ecological strategies of bacteria living in redox-stratified environments.

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Shewanella oneidensis NADH dehydrogenase mutants exhibit an amino acid synthesis defect.

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<i>Shewanella oneidensis</i> NADH dehydrogenase mutants exhibit an amino acid synthesis defect.

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