Dataset Information

Genetic, Genomic, and Transcriptomic Studies of Pyruvate Metabolism in Methanosarcina barkeri Fusaro.

ABSTRACT: Pyruvate, a central intermediate in the carbon fixation pathway of methanogenic archaea, is rarely used as an energy source by these organisms. The sole exception to this rule is a genetically uncharacterized Methanosarcina barkeri mutant capable of using pyruvate as a sole energy and carbon source (the Pyr(+) phenotype). Here, we provide evidence that suggests that the Pyr(+) mutant is able to metabolize pyruvate by overexpressing pyruvate ferredoxin oxidoreductase (por) and mutating genes involved in central carbon metabolism. Genomic analysis showed that the Pyr(+) strain has two mutations localized to Mbar_A1588, the biotin protein ligase subunit of the pyruvate carboxylase (pyc) operon, and Mbar_A2165, a putative transcriptional regulator. Mutants expressing the Mbar_A1588 mutation showed no growth defect compared to the wild type (WT), yet the strains lacked pyc activity. Recreation of the Mbar_A2165 mutation resulted in a 2-fold increase of Por activity and gene expression, suggesting a role in por transcriptional regulation. Further transcriptomic analysis revealed that Pyr(+) strains also overexpress the gene encoding phosphoenolpyruvate carboxylase, indicating the presence of a previously uncharacterized route for synthesizing oxaloacetate in M. barkeri and explaining the unimpaired growth in the absence of Pyc. Surprisingly, stringent repression of the por operon was lethal, even when the media were supplemented with pyruvate and/or Casamino Acids, suggesting that por plays an unidentified essential function in M. barkeri.The work presented here reveals a complex interaction between anabolic and catabolic pathways involving pyruvate metabolism in Methanosarcina barkeri Fusaro. Among the unexpected findings were an essential role for the enzyme pyruvate-ferredoxin oxidoreductase and an alternate pathway for synthesis of oxaloacetate. These results clarify the mechanism of methanogenic catabolism of pyruvate and expand our understanding of carbon assimilation in methanogens.

SUBMITTER: Lopez Munoz MM

PROVIDER: S-EPMC4621090 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Genetic, Genomic, and Transcriptomic Studies of Pyruvate Metabolism in Methanosarcina barkeri Fusaro.

López Muñoz Madeline M MM Schönheit Peter P Metcalf William W WW

Journal of bacteriology 20150908 22

<h4>Unlabelled</h4>Pyruvate, a central intermediate in the carbon fixation pathway of methanogenic archaea, is rarely used as an energy source by these organisms. The sole exception to this rule is a genetically uncharacterized Methanosarcina barkeri mutant capable of using pyruvate as a sole energy and carbon source (the Pyr(+) phenotype). Here, we provide evidence that suggests that the Pyr(+) mutant is able to metabolize pyruvate by overexpressing pyruvate ferredoxin oxidoreductase (por) and ...[more]

PMID: 26350133

Similar Datasets

Project description:The methanogenic archaeon Methanosarcina barkeri encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F420-dependent (Frh), methanophenazine-dependent (Vht), and ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, we characterized a series of mutants lacking each hydrogenase in various combinations. Mutants lacking Frh, Vht, or Ech in any combination failed to grow on H2-CO2, whereas only Vht and Ech were essential for growth on acetate. In contrast, a mutant lacking all three grew on methanol with a final growth yield similar to that of the wild type and produced methane and CO2 in the expected 3:1 ratio but had a ca. 33% lower growth rate. Thus, hydrogenases play a significant, but nonessential, role during growth on this substrate. As previously observed, mutants lacking Ech failed to grow on methanol-H2 unless they were supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the ?ech ?frh and ?ech ?frh ?vht mutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H2, which prevents production of the reducing equivalents needed for biosynthesis. Quantification of the methane and CO2 produced from methanol by resting cell suspensions of various mutants supported this conclusion. On the basis of the global transcriptional profiles, none of the hydrogenases were upregulated to compensate for the loss of the others. However, the transcript levels of the F420 dehydrogenase operon were significantly higher in all strains lacking frh, suggesting a mechanism to sense the redox state of F420 The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed.IMPORTANCE Methanogenic archaea are key players in the global carbon cycle due to their ability to facilitate the remineralization of organic substrates in many anaerobic environments. The consequences of biological methanogenesis are far-reaching, with impacts on atmospheric methane and CO2 concentrations, agriculture, energy production, waste treatment, and human health. The data presented here clarify the in vivo function of hydrogenases during methanogenesis, which in turn deepens our understanding of this unique form of metabolism. This knowledge is critical for a variety of important issues ranging from atmospheric composition to human health.

Project description:BackgroundAnaerobic digestion of easily degradable biowaste can lead to the accumulation of volatile fatty acids, which will cause environmental stress to the sensitive methanogens consequently. The metabolic characteristics of methanogens under acetate stress can affect the overall performance of mixed consortia. Nevertheless, there exist huge gaps in understanding the responses of the dominant methanogens to the stress, e.g., Methanosarcinaceae. Such methanogens are resistant to environmental deterioration and able to utilize multiple carbon sources. In this study, transcriptomic and proteomic analyses were conducted to explore the responses of Methanosarcina barkeri strain MS at different acetate concentrations of 10, 25, and 50 mM.ResultsThe trend of OD600 and the regulation of the specific genes in 50 mM acetate, indicated that high concentration of acetate promoted the acclimation of M. barkeri to acetate stress. Acetate stress hindered the regulation of quorum sensing and thereby eliminated the advantages of cell aggregation, which was beneficial to resist stress. Under acetate stress, M. barkeri allocated more resources to enhance the uptake of iron to maintain the integrities of electron-transport chains and other essential biological processes. Comparing with the initial stages of different acetate concentrations, most of the genes participating in acetoclastic methanogenesis did not show significantly different expressions except hdrB1C1, an electron-bifurcating heterodisulfide reductase participating in energy conversion and improving thermodynamic efficiency. Meanwhile, vnfDGHK and nifDHK participating in nitrogen fixation pathway were upregulated.ConclusionIn this work, transcriptomic and proteomic analyses are combined to reveal the responses of M. barkeri to acetate stress in terms of central metabolic pathways, which provides basic clues for exploring the responses of other specific methanogens under high organics load. Moreover, the results can also be used to gain insights into the complex interactions and geochemical cycles among natural or engineered populations. Furthermore, these findings also provide the potential for designing effective and robust anaerobic digesters with high organic loads.

Dataset Information

Genetic, Genomic, and Transcriptomic Studies of Pyruvate Metabolism in Methanosarcina barkeri Fusaro.

Publications

Genetic, Genomic, and Transcriptomic Studies of Pyruvate Metabolism in Methanosarcina barkeri Fusaro.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets