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Genetic, Biochemical, and Molecular Characterization of Methanosarcina barkeri Mutants Lacking Three Distinct Classes of Hydrogenase

Mand, Thomas D. and Kulkarni, Gargi and Metcalf, William W. (2018) Genetic, Biochemical, and Molecular Characterization of Methanosarcina barkeri Mutants Lacking Three Distinct Classes of Hydrogenase. Journal of Bacteriology, 200 (20). Art. No. e00342-18. ISSN 0021-9193. PMCID PMC6153667. doi:10.1128/JB.00342-18.

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The methanogenic archaeon Methanosarcina barkeri encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F_(420)-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 H_2-CO_2, 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-H_2 unless they were supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the Δech Δfrh and Δech Δfrh Δvht mutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H_2, which prevents production of the reducing equivalents needed for biosynthesis. Quantification of the methane and CO_2 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 F_(420) dehydrogenase operon were significantly higher in all strains lacking frh, suggesting a mechanism to sense the redox state of F_(420). The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed.

Item Type:Article
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URLURL TypeDescription Paper CentralArticle
Metcalf, William W.0000-0002-0182-0671
Additional Information:© 2018 American Society for Microbiology. Received 5 June 2018; Accepted 11 July 2018; Accepted manuscript posted online 16 July 2018. We acknowledge the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences, of the U.S. Department of Energy for funding this work through grant DE-FG02-02ER15296. Accession number(s).Raw and processed data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE98441.
Funding AgencyGrant Number
Department of Energy (DOE)DE-FG02-02ER15296
Subject Keywords:Methanosarcina, hydrogenases, methane, methanogenesis
Issue or Number:20
PubMed Central ID:PMC6153667
Record Number:CaltechAUTHORS:20181011-121629877
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Official Citation:Genetic, Biochemical, and Molecular Characterization of Methanosarcina barkeri Mutants Lacking Three Distinct Classes of Hydrogenase. Thomas D. Mand, Gargi Kulkarni, William W. Metcalf. Journal of Bacteriology Sep 2018, 200 (20) e00342-18; DOI: 10.1128/JB.00342-18
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:90245
Deposited By: Tony Diaz
Deposited On:11 Oct 2018 20:18
Last Modified:01 Jun 2023 23:32

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