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

Abstract

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 CO 2 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 Δ ech Δ frh and Δ ech Δ frh Δ vht 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 producedmore » 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.« less

Authors:
 [1];  [1]; ORCiD logo [1]
  1. Univ. of Illinois at Urbana-Champaign, IL (United States)
Publication Date:
Research Org.:
Univ. of Illinois at Urbana-Champaign, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22). Chemical Sciences, Geosciences & Biosciences Division
OSTI Identifier:
1595343
Grant/Contract Number:  
[FG02-02ER15296]
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Bacteriology
Additional Journal Information:
[ Journal Volume: 200; Journal Issue: 20]; Journal ID: ISSN 0021-9193
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Methanosarcina; hydrogenases; methane; methanogenesis

Citation Formats

Mand, Thomas D., Kulkarni, Gargi, and Metcalf, William W. Genetic, Biochemical, and Molecular Characterization of Methanosarcina barkeri Mutants Lacking Three Distinct Classes of Hydrogenase. United States: N. p., 2018. Web. doi:10.1128/JB.00342-18.
Mand, Thomas D., Kulkarni, Gargi, & Metcalf, William W. Genetic, Biochemical, and Molecular Characterization of Methanosarcina barkeri Mutants Lacking Three Distinct Classes of Hydrogenase. United States. doi:10.1128/JB.00342-18.
Mand, Thomas D., Kulkarni, Gargi, and Metcalf, William W. Mon . "Genetic, Biochemical, and Molecular Characterization of Methanosarcina barkeri Mutants Lacking Three Distinct Classes of Hydrogenase". United States. doi:10.1128/JB.00342-18. https://www.osti.gov/servlets/purl/1595343.
@article{osti_1595343,
title = {Genetic, Biochemical, and Molecular Characterization of Methanosarcina barkeri Mutants Lacking Three Distinct Classes of Hydrogenase},
author = {Mand, Thomas D. and Kulkarni, Gargi and Metcalf, William W.},
abstractNote = {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 Δech Δfrh and Δech Δfrh Δvht 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.},
doi = {10.1128/JB.00342-18},
journal = {Journal of Bacteriology},
number = [20],
volume = [200],
place = {United States},
year = {2018},
month = {7}
}

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