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Characterization of energy-conserving hydrogenase B in Methanococcus maripaludis.

ABSTRACT: The Methanococcus maripaludis energy-conserving hydrogenase B (Ehb) generates low potential electrons required for autotrophic CO(2) assimilation. To analyze the importance of individual subunits in Ehb structure and function, markerless in-frame deletions were constructed in a number of M. maripaludis ehb genes. These genes encode the large and small hydrogenase subunits (ehbN and ehbM, respectively), a polyferredoxin and ferredoxin (ehbK and ehbL, respectively), and an ion translocator (ehbF). In addition, a gene replacement mutation was constructed for a gene encoding a putative membrane-spanning subunit (ehbO). When grown in minimal medium plus acetate (McA), all ehb mutants had severe growth deficiencies except the DeltaehbO::pac strain. The membrane-spanning ion translocator (DeltaehbF) and the large hydrogenase subunit (DeltaehbN) deletion strains displayed the severest growth defects. Deletion of the ehbN gene was of particular interest because this gene was not contiguous to the ehb operon. In-gel activity assays and Western blots confirmed that EhbN was part of the membrane-bound Ehb hydrogenase complex. The DeltaehbN strain was also sensitive to growth inhibition by aryl acids, indicating that Ehb was coupled to the indolepyruvate oxidoreductase (Ior), further supporting the hypothesis that Ehb provides low potential reductants for the anabolic oxidoreductases in M. maripaludis.

SUBMITTER: Major TA

PROVIDER: S-EPMC2916364 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Characterization of energy-conserving hydrogenase B in Methanococcus maripaludis.

Major Tiffany A TA Liu Yuchen Y Whitman William B WB

Journal of bacteriology 20100528 15

The Methanococcus maripaludis energy-conserving hydrogenase B (Ehb) generates low potential electrons required for autotrophic CO(2) assimilation. To analyze the importance of individual subunits in Ehb structure and function, markerless in-frame deletions were constructed in a number of M. maripaludis ehb genes. These genes encode the large and small hydrogenase subunits (ehbN and ehbM, respectively), a polyferredoxin and ferredoxin (ehbK and ehbL, respectively), and an ion translocator (ehbF). ...[more]

PMID: 20511510

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Project description:The rapidly growing mesophilic methanogen Methanococcus maripaludis S2 has a unique ability to consume both CO2 and N2, the main components of a flue gas, and produce methane with H2 as the electron donor. The existing literature lacks experimental measurements of CO2 and H2 uptake rates and CH4 production rates on M. maripaludis. Furthermore, it lacks estimates of maintenance energies for use with genome-scale models. In this paper, we performed batch culture experiments on M. maripaludis S2 using CO2 as the sole carbon substrate to quantify three key extracellular fluxes (CO2, H2, and CH4) along with specific growth rates. For precise computation of these fluxes from experimental measurements, we developed a systematic process simulation approach. Then, using an existing genome-scale model, we proposed an optimization procedure to estimate maintenance energy parameters: growth associated maintenance (GAM) and non-growth associated maintenance (NGAM).The measured extracellular fluxes for M. maripaludis showed excellent agreement with in silico predictions from a validated genome-scale model (iMM518) for NGAM = 7.836 mmol/gDCW/h and GAM = 27.14 mmol/gDCW. M. maripaludis achieved a CO2 to CH4 conversion yield of 70-95 % and a growth yield of 3.549 ± 0.149 g DCW/mol CH4 during the exponential phase. The ATP gain of 0.35 molATP/molCH4 for M. maripaludis, computed using NGAM, is in the acceptable range of 0.3-0.7 mol ATP/molCH4 reported for methanogens. Interestingly, the uptake distribution of amino acids, quantified using iMM518, confirmed alanine to be the most preferred amino acids for growth and methanogenesis.This is the first study to report experimental gas consumption and production rates for the growth of M. maripaludis on CO2 and H2 in minimal media. A systematic process simulation and optimization procedure was successfully developed to precisely quantify extracellular fluxes along with cell growth and maintenance energy parameters. Our growth yields, ATP gain, and energy parameters fall within acceptable ranges known in the literature for hydrogenotrophic methanogens.

Project description:Methanococcus maripaludis is a rapidly growing, fully sequenced, genetically tractable model organism among hydrogenotrophic methanogens. It has the ability to convert CO2 and H2 into a useful cleaner energy fuel (CH4). In fact, this conversion enhances in the presence of free nitrogen as the sole nitrogen source due to prolonged cell growth. Given the global importance of GHG emissions and climate change, diazotrophy can be attractive for carbon capture and utilization applications from appropriately treated flue gases, where surplus hydrogen is available from renewable electricity sources. In addition, M. maripaludis can be engineered to produce other useful products such as terpenoids, hydrogen, methanol, etc. M. maripaludis with its unique abilities has the potential to be a workhorse like Escherichia coli and S. cerevisiae for fundamental and experimental biotechnology studies. More than 100 experimental studies have explored different specific aspects of the biochemistry and genetics of CO2 and N2 fixation by M. maripaludis. Its genome-scale metabolic model (iMM518) also exists to study genetic perturbations and complex biological interactions. However, a comprehensive review describing its cell structure, metabolic processes, and methanogenesis is still lacking in the literature. This review fills this crucial gap. Specifically, it integrates distributed information from the literature to provide a complete and detailed view for metabolic processes such as acetyl-CoA synthesis, pyruvate synthesis, glycolysis/gluconeogenesis, reductive tricarboxylic acid (RTCA) cycle, non-oxidative pentose phosphate pathway (NOPPP), nitrogen metabolism, amino acid metabolism, and nucleotide biosynthesis. It discusses energy production via methanogenesis and its relation to metabolism. Furthermore, it reviews taxonomy, cell structure, culture/storage conditions, molecular biology tools, genome-scale models, and potential industrial and environmental applications. Through the discussion, it develops new insights and hypotheses from experimental and modeling observations, and identifies opportunities for further research and applications.

Dataset Information

Characterization of energy-conserving hydrogenase B in Methanococcus maripaludis.

Publications

Characterization of energy-conserving hydrogenase B in Methanococcus maripaludis.

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