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H2-independent growth of the hydrogenotrophic methanogen Methanococcus maripaludis.

Costa KC, Lie TJ, Jacobs MA, Leigh JA - MBio (2013)

Bottom Line: In either case, the reduced ferredoxin generated was inefficient at stimulating methanogenesis, resulting in a slow growth phenotype.As methanogenesis is limited by the availability of reduced ferredoxin under these conditions, other electron donors, such as reduced coenzyme F(420), should be abundant.Indeed, when F(420)-reducing hydrogenase was reintroduced into the hydrogenase-free mutant, the equilibrium of H(2) production via an F(420)-dependent formate:H(2) lyase activity shifted markedly toward H(2) compared to the wild type.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Washington, Seattle, Washington, USA.

ABSTRACT

Unlabelled: Hydrogenotrophic methanogenic Archaea require reduced ferredoxin as an anaplerotic source of electrons for methanogenesis. H(2) oxidation by the hydrogenase Eha provides these electrons, consistent with an H(2) requirement for growth. Here we report the identification of alternative pathways of ferredoxin reduction in Methanococcus maripaludis that operate independently of Eha to stimulate methanogenesis. A suppressor mutation that increased expression of the glycolytic enzyme glyceraldehyde-3-phosphate:ferredoxin oxidoreductase resulted in a strain capable of H(2)-independent ferredoxin reduction and growth with formate as the sole electron donor. In this background, it was possible to eliminate all seven hydrogenases of M. maripaludis. Alternatively, carbon monoxide oxidation by carbon monoxide dehydrogenase could also generate reduced ferredoxin that feeds into methanogenesis. In either case, the reduced ferredoxin generated was inefficient at stimulating methanogenesis, resulting in a slow growth phenotype. As methanogenesis is limited by the availability of reduced ferredoxin under these conditions, other electron donors, such as reduced coenzyme F(420), should be abundant. Indeed, when F(420)-reducing hydrogenase was reintroduced into the hydrogenase-free mutant, the equilibrium of H(2) production via an F(420)-dependent formate:H(2) lyase activity shifted markedly toward H(2) compared to the wild type.

Importance: Hydrogenotrophic methanogens are thought to require H(2) as a substrate for growth and methanogenesis. Here we show alternative pathways in methanogenic metabolism that alleviate this H(2) requirement and demonstrate, for the first time, a hydrogenotrophic methanogen that is capable of growth in the complete absence of H(2). The demonstration of alternative pathways in methanogenic metabolism suggests that this important group of organisms is metabolically more versatile than previously thought.

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Related in: MedlinePlus

Growth of the ∆6H2ase mutant overexpressing GAPOR in the presence and absence of H2. The ∆6H2ase mutant overexpressing GAPOR grown on formate plus H2 (black symbols), the ∆6H2ase mutant overexpressing GAPOR grown on formate alone (gray symbols), and the ∆6H2ase mutant grown on formate alone (white symbols) were examined.
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fig4: Growth of the ∆6H2ase mutant overexpressing GAPOR in the presence and absence of H2. The ∆6H2ase mutant overexpressing GAPOR grown on formate plus H2 (black symbols), the ∆6H2ase mutant overexpressing GAPOR grown on formate alone (gray symbols), and the ∆6H2ase mutant grown on formate alone (white symbols) were examined.

Mentions: Although the generation of a putative promoter sequence upstream of GAPOR suggests that overexpression of this gene leads to growth, the nature of the mutation could also result in a promoter reading in the opposite direction (see Fig. S1 in the supplemental material). Therefore, instead of engineering the mutation on the chromosome of the ∆6H2ase mutant to test its efficacy at stimulating growth, we chose to overexpress GAPOR on a replicative vector to recapitulate the effect and avoid possible overexpression of a second operon. GAPOR was placed under control of the Methanococcus vanneilii histone promoter on the replicative vector pLW40neo (17) and introduced into the ∆6H2ase background. The resulting strain displayed moderate growth in the absence of H2 and robust growth in the presence of H2, verifying that either GAPOR or Eha could be used to generate the reduced ferredoxin required for growth (Fig. 4).


H2-independent growth of the hydrogenotrophic methanogen Methanococcus maripaludis.

Costa KC, Lie TJ, Jacobs MA, Leigh JA - MBio (2013)

Growth of the ∆6H2ase mutant overexpressing GAPOR in the presence and absence of H2. The ∆6H2ase mutant overexpressing GAPOR grown on formate plus H2 (black symbols), the ∆6H2ase mutant overexpressing GAPOR grown on formate alone (gray symbols), and the ∆6H2ase mutant grown on formate alone (white symbols) were examined.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585446&req=5

fig4: Growth of the ∆6H2ase mutant overexpressing GAPOR in the presence and absence of H2. The ∆6H2ase mutant overexpressing GAPOR grown on formate plus H2 (black symbols), the ∆6H2ase mutant overexpressing GAPOR grown on formate alone (gray symbols), and the ∆6H2ase mutant grown on formate alone (white symbols) were examined.
Mentions: Although the generation of a putative promoter sequence upstream of GAPOR suggests that overexpression of this gene leads to growth, the nature of the mutation could also result in a promoter reading in the opposite direction (see Fig. S1 in the supplemental material). Therefore, instead of engineering the mutation on the chromosome of the ∆6H2ase mutant to test its efficacy at stimulating growth, we chose to overexpress GAPOR on a replicative vector to recapitulate the effect and avoid possible overexpression of a second operon. GAPOR was placed under control of the Methanococcus vanneilii histone promoter on the replicative vector pLW40neo (17) and introduced into the ∆6H2ase background. The resulting strain displayed moderate growth in the absence of H2 and robust growth in the presence of H2, verifying that either GAPOR or Eha could be used to generate the reduced ferredoxin required for growth (Fig. 4).

Bottom Line: In either case, the reduced ferredoxin generated was inefficient at stimulating methanogenesis, resulting in a slow growth phenotype.As methanogenesis is limited by the availability of reduced ferredoxin under these conditions, other electron donors, such as reduced coenzyme F(420), should be abundant.Indeed, when F(420)-reducing hydrogenase was reintroduced into the hydrogenase-free mutant, the equilibrium of H(2) production via an F(420)-dependent formate:H(2) lyase activity shifted markedly toward H(2) compared to the wild type.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Washington, Seattle, Washington, USA.

ABSTRACT

Unlabelled: Hydrogenotrophic methanogenic Archaea require reduced ferredoxin as an anaplerotic source of electrons for methanogenesis. H(2) oxidation by the hydrogenase Eha provides these electrons, consistent with an H(2) requirement for growth. Here we report the identification of alternative pathways of ferredoxin reduction in Methanococcus maripaludis that operate independently of Eha to stimulate methanogenesis. A suppressor mutation that increased expression of the glycolytic enzyme glyceraldehyde-3-phosphate:ferredoxin oxidoreductase resulted in a strain capable of H(2)-independent ferredoxin reduction and growth with formate as the sole electron donor. In this background, it was possible to eliminate all seven hydrogenases of M. maripaludis. Alternatively, carbon monoxide oxidation by carbon monoxide dehydrogenase could also generate reduced ferredoxin that feeds into methanogenesis. In either case, the reduced ferredoxin generated was inefficient at stimulating methanogenesis, resulting in a slow growth phenotype. As methanogenesis is limited by the availability of reduced ferredoxin under these conditions, other electron donors, such as reduced coenzyme F(420), should be abundant. Indeed, when F(420)-reducing hydrogenase was reintroduced into the hydrogenase-free mutant, the equilibrium of H(2) production via an F(420)-dependent formate:H(2) lyase activity shifted markedly toward H(2) compared to the wild type.

Importance: Hydrogenotrophic methanogens are thought to require H(2) as a substrate for growth and methanogenesis. Here we show alternative pathways in methanogenic metabolism that alleviate this H(2) requirement and demonstrate, for the first time, a hydrogenotrophic methanogen that is capable of growth in the complete absence of H(2). The demonstration of alternative pathways in methanogenic metabolism suggests that this important group of organisms is metabolically more versatile than previously thought.

Show MeSH
Related in: MedlinePlus