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 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.more »
- Authors:
-
- 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). Chemical Sciences, Geosciences, and 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. https://doi.org/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. https://doi.org/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 = {Mon Jul 16 00:00:00 EDT 2018},
month = {Mon Jul 16 00:00:00 EDT 2018}
}
Web of Science
Works referenced in this record:
Development of a Markerless Genetic Exchange Method for Methanosarcina acetivorans C2A and Its Use in Construction of New Genetic Tools for Methanogenic Archaea
journal, March 2004
- Pritchett, M. A.; Zhang, J. K.; Metcalf, W. W.
- Applied and Environmental Microbiology, Vol. 70, Issue 3
Genetic Basis for Metabolism of Methylated Sulfur Compounds in Methanosarcina Species
journal, February 2015
- Fu, He; Metcalf, William W.
- Journal of Bacteriology, Vol. 197, Issue 8
Hydrogen is a preferred intermediate in the energy-conserving electron transport chain of Methanosarcina barkeri
journal, September 2009
- Kulkarni, Gargi; Kridelbaugh, Donna M.; Guss, Adam M.
- Proceedings of the National Academy of Sciences, Vol. 106, Issue 37
Molecular, genetic, and biochemical characterization of the serC gene of Methanosarcina barkeri Fusaro.
journal, October 1996
- Metcalf, W. W.; Zhang, J. K.; Shi, X.
- Journal of Bacteriology, Vol. 178, Issue 19
Genetic analysis of mch mutants in two Methanosarcina species demonstrates multiple roles for the methanopterin-dependent C-1 oxidation/reduction pathway and differences in H 2 metabolism between closely related species : Methyl reduction in
journal, January 2005
- Guss, Adam M.; Mukhopadhyay, Biswarup; Zhang, Jun Kai
- Molecular Microbiology, Vol. 55, Issue 6
Methanogenesis by Methanosarcina acetivorans involves two structurally and functionally distinct classes of heterodisulfide reductase
journal, February 2010
- Buan, Nicole R.; Metcalf, William W.
- Molecular Microbiology, Vol. 75, Issue 4
Energy Conservation via Hydrogen Cycling in the Methanogenic Archaeon Methanosarcina barkeri
journal, July 2018
- Kulkarni, Gargi; Mand, Thomas D.; Metcalf, William W.
- mBio, Vol. 9, Issue 4
Development and quantitative analyses of a universal rRNA-subtraction protocol for microbial metatranscriptomics
journal, March 2010
- Stewart, Frank J.; Ottesen, Elizabeth A.; DeLong, Edward F.
- The ISME Journal, Vol. 4, Issue 7
Small-sample estimation of negative binomial dispersion, with applications to SAGE data
journal, July 2007
- Robinson, M. D.; Smyth, G. K.
- Biostatistics, Vol. 9, Issue 2
Differences in Hydrogenase Gene Expression between Methanosarcina acetivorans and Methanosarcina barkeri
journal, February 2009
- Guss, A. M.; Kulkarni, G.; Metcalf, W. W.
- Journal of Bacteriology, Vol. 191, Issue 8
Generation of Dominant Selectable Markers for Resistance to Pseudomonic Acid by Cloning and Mutagenesis of the ileS Gene from the Archaeon Methanosarcina barkeri Fusaro
journal, May 2000
- Boccazzi, P.; Zhang, J. K.; Metcalf, W. W.
- Journal of Bacteriology, Vol. 182, Issue 9
Protein content and enzyme activities in methanol- and acetate-grown Methanosarcina thermophila.
journal, January 1990
- Jablonski, P. E.; DiMarco, A. A.; Bobik, T. A.
- Journal of Bacteriology, Vol. 172, Issue 3
Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri
journal, October 1999
- Meuer, Jorn; Bartoschek, Stefan; Koch, Jurgen
- European Journal of Biochemistry, Vol. 265, Issue 1
Metabolic, Phylogenetic, and Ecological Diversity of the Methanogenic Archaea
journal, March 2008
- Liu, Yuchen; Whitman, William B.
- Annals of the New York Academy of Sciences, Vol. 1125, Issue 1
Comparison of Unitrophic and Mixotrophic Substrate Metabolism by an Acetate-Adapted Strain of Methanosarcina barkeri
journal, January 1982
- Krzycki, J. A.; Wolkin, R. H.; Zeikus, J. G.
- Journal of Bacteriology, Vol. 149, Issue 1
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
journal, May 1976
- Bradford, Marion M.
- Analytical Biochemistry, Vol. 72, Issue 1-2
[FeFe]- and [NiFe]-hydrogenase diversity, mechanism, and maturation
journal, June 2015
- Peters, John W.; Schut, Gerrit J.; Boyd, Eric S.
- Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1853, Issue 6
Bioenergetics and anaerobic respiratory chains of aceticlastic methanogens
journal, July 2014
- Welte, Cornelia; Deppenmeier, Uwe
- Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1837, Issue 7
Purification of the F420-reducing hydrogenase from Methanosarcina barkeri (strain Fusaro)
journal, September 1989
- Fiebig, Klaus; Friedrich, Barbel
- European Journal of Biochemistry, Vol. 184, Issue 1
The Membrane-Bound Electron Transport System of Methanosarcina Species
journal, February 2004
- Deppenmeier, Uwe
- Journal of Bioenergetics and Biomembranes, Vol. 36, Issue 1
Methanogenic archaea: ecologically relevant differences in energy conservation
journal, June 2008
- Thauer, Rudolf K.; Kaster, Anne-Kristin; Seedorf, Henning
- Nature Reviews Microbiology, Vol. 6, Issue 8
A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR.
journal, June 1988
- Miller, V. L.; Mekalanos, J. J.
- Journal of Bacteriology, Vol. 170, Issue 6
A Ferredoxin- and F 420 H 2 -Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea
journal, February 2017
- Yan, Zhen; Wang, Mingyu; Ferry, James G.
- mBio, Vol. 8, Issue 1
Genetic analysis of the archaeon Methanosarcina barkeri Fusaro reveals a central role for Ech hydrogenase and ferredoxin in methanogenesis and carbon fixation
journal, April 2002
- Meuer, J.; Kuettner, H. C.; Zhang, J. K.
- Proceedings of the National Academy of Sciences, Vol. 99, Issue 8
Biochemical Characterization of the 8-hydroxy-5-deazaflavin-reactive Hydrogenase from Methanosarcina barkeri Fusaro
journal, November 1995
- Michel, Regina; Massanz, Christian; Kostka, Susanne
- European Journal of Biochemistry, Vol. 233, Issue 3
Electron Transport in the Pathway of Acetate Conversion to Methane in the Marine Archaeon Methanosarcina acetivorans
journal, December 2005
- Li, Q.; Li, L.; Rejtar, T.
- Journal of Bacteriology, Vol. 188, Issue 2
Two F 420 -reducing hydrogenases in Methanosarcina barkeri
journal, February 1998
- Vaupel, Martin; Thauer, R. K.
- Archives of Microbiology, Vol. 169, Issue 3
Disaggregation of Methanosarcina spp. and Growth as Single Cells at Elevated Osmolarity
journal, January 1993
- Sowers, Kevin R.; Boone, Jane E.; Gunsalus, Robert P.
- Applied and Environmental Microbiology, Vol. 59, Issue 11
Novel regulatory mutants of the phosphate regulon in Escherichia coli K-12
journal, September 1986
- Wanner, Barry L.
- Journal of Molecular Biology, Vol. 191, Issue 1
Classification and phylogeny of hydrogenases
journal, August 2001
- Vignais, Paulette M.; Billoud, Bernard; Meyer, Jacques
- FEMS Microbiology Reviews, Vol. 25, Issue 4, p. 455-501
Directed Mutagenesis and Plasmid-Based Complementation in the Methanogenic Archaeon Methanosarcina acetivorans C2A Demonstrated by Genetic Analysis of Proline Biosynthesis
journal, March 2002
- Zhang, J. K.; White, A. K.; Kuettner, H. C.
- Journal of Bacteriology, Vol. 184, Issue 5
Hydrogenases from Methanogenic Archaea, Nickel, a Novel Cofactor, and H 2 Storage
journal, June 2010
- Thauer, Rudolf K.; Kaster, Anne-Kristin; Goenrich, Meike
- Annual Review of Biochemistry, Vol. 79, Issue 1
Essential anaplerotic role for the energy-converting hydrogenase Eha in hydrogenotrophic methanogenesis
journal, August 2012
- Lie, T. J.; Costa, K. C.; Lupa, B.
- Proceedings of the National Academy of Sciences, Vol. 109, Issue 38
A genetic system for Archaea of the genus Methanosarcina: Liposome-mediated transformation and construction of shuttle vectors
journal, March 1997
- Metcalf, W. W.; Zhang, J. K.; Apolinario, E.
- Proceedings of the National Academy of Sciences, Vol. 94, Issue 6
New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species
journal, January 2008
- Guss, Adam M.; Rother, Michael; Zhang, Jun Kai
- Archaea, Vol. 2, Issue 3
Classification and phylogeny of hydrogenases
journal, August 2001
- Vignais, P.
- FEMS Microbiology Reviews, Vol. 25, Issue 4
Works referencing / citing this record:
Methanogens: pushing the boundaries of biology
journal, December 2018
- Buan, Nicole R.
- Emerging Topics in Life Sciences, Vol. 2, Issue 4
Extracellular Electron Uptake by Two Methanosarcina Species
journal, April 2019
- Yee, Mon Oo; Snoeyenbos-West, Oona L.; Thamdrup, Bo
- Frontiers in Energy Research, Vol. 7
Responses of Methanosarcina barkeri to acetate stress
journal, December 2019
- He, Pinjing; Duan, Haowen; Han, Wenhao
- Biotechnology for Biofuels, Vol. 12, Issue 1
Methane-Linked Mechanisms of Electron Uptake from Cathodes by Methanosarcina barkeri
journal, March 2019
- Rowe, Annette R.; Xu, Shuai; Gardel, Emily
- mBio, Vol. 10, Issue 2
Redox cycling of Fe(II) and Fe(III) in magnetite accelerates aceticlastic methanogenesis by Methanosarcina mazei
journal, October 2019
- Wang, Hui; Byrne, James M.; Liu, Pengfei
- Environmental Microbiology Reports, Vol. 12, Issue 1
Energy Conservation and Hydrogenase Function in Methanogenic Archaea, in Particular the Genus Methanosarcina
journal, September 2019
- Mand, Thomas D.; Metcalf, William W.
- Microbiology and Molecular Biology Reviews, Vol. 83, Issue 4
NanoFe3O4 as Solid Electron Shuttles to Accelerate Acetotrophic Methanogenesis by Methanosarcina barkeri
journal, March 2019
- Fu, Li; Zhou, Ting; Wang, Jingyuan
- Frontiers in Microbiology, Vol. 10
Electron and Proton Flux for Carbon Dioxide Reduction in Methanosarcina barkeri During Direct Interspecies Electron Transfer
journal, December 2018
- Holmes, Dawn E.; Rotaru, Amelia-Elena; Ueki, Toshiyuki
- Frontiers in Microbiology, Vol. 9