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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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Growth and H2 production by the ∆7H2asesup mutant expressing F420-reducing hydrogenase (∆7H 2asesup-frc). (A) Growth on formate (black symbols) and H2 production (gray symbols) by the wild-type strain MM901 (circles), the ∆7H2asesup mutant (squares), and the ∆7H2asesup-frc mutant (triangles) in batch culture. Data are from a single representative experiment, but two replicate experiments gave similar results (see Fig. S2 in the supplemental material). (B) CH4 (black curve) and H2 production (bars) of the ∆7H2asesup-frc mutant in continuous culture. Actively growing cultures (gray bars) and cultures after metronidazole (50 µg ml−1) was added to completely oxidize ferredoxin (white bars) are shown. The medium dilution rate, gas flow rate, and culture optical density are shown on the x axis.
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fig5: Growth and H2 production by the ∆7H2asesup mutant expressing F420-reducing hydrogenase (∆7H 2asesup-frc). (A) Growth on formate (black symbols) and H2 production (gray symbols) by the wild-type strain MM901 (circles), the ∆7H2asesup mutant (squares), and the ∆7H2asesup-frc mutant (triangles) in batch culture. Data are from a single representative experiment, but two replicate experiments gave similar results (see Fig. S2 in the supplemental material). (B) CH4 (black curve) and H2 production (bars) of the ∆7H2asesup-frc mutant in continuous culture. Actively growing cultures (gray bars) and cultures after metronidazole (50 µg ml−1) was added to completely oxidize ferredoxin (white bars) are shown. The medium dilution rate, gas flow rate, and culture optical density are shown on the x axis.

Mentions: The ∆7H2asesup mutant grows more slowly than the wild type on formate, suggesting that reduced ferredoxin is limiting. Reduced ferredoxin limitation of methanogenesis implies that other reduced cofactors that feed into the pathway, such as F420H2, are present in excess. Wild-type M. maripaludis possesses an F420-dependent formate:H2 lyase activity that is catalyzed by Fdh and F420-reducing hydrogenase. The wild-type strain grown on formate can accumulate H2 in the culture headspace to a concentration of 0.16% ± 0.02% of the gas phase at 2 atm pressure (mean ± standard deviation [SD] for three biological replicates) (18, 19). An excess of F420H2 should drive the equilibrium of this activity toward increased H2 production. frc encoding F420-reducing hydrogenase was placed on the replicative vector pLW40 (17) and reintroduced into the ∆7H2asesup mutant to restore formate:H2 lyase activity. When grown with formate as the only electron donor for methanogenesis, the ∆7H2asesup-frc mutant was capable of producing H2 up to a concentration of 2.32% ± 0.79% (Fig. 5A; see Fig. S2 in the supplemental material). When cultures entered stationary phase, H2 reuptake occurred, presumably due to depletion of formate and an equilibrium shift back toward F420H2 production from H2. The ∆7H2asesup mutant, which lacks formate:H2 lyase activity, was incapable of H2 production.


H2-independent growth of the hydrogenotrophic methanogen Methanococcus maripaludis.

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

Growth and H2 production by the ∆7H2asesup mutant expressing F420-reducing hydrogenase (∆7H 2asesup-frc). (A) Growth on formate (black symbols) and H2 production (gray symbols) by the wild-type strain MM901 (circles), the ∆7H2asesup mutant (squares), and the ∆7H2asesup-frc mutant (triangles) in batch culture. Data are from a single representative experiment, but two replicate experiments gave similar results (see Fig. S2 in the supplemental material). (B) CH4 (black curve) and H2 production (bars) of the ∆7H2asesup-frc mutant in continuous culture. Actively growing cultures (gray bars) and cultures after metronidazole (50 µg ml−1) was added to completely oxidize ferredoxin (white bars) are shown. The medium dilution rate, gas flow rate, and culture optical density are shown on the x axis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Growth and H2 production by the ∆7H2asesup mutant expressing F420-reducing hydrogenase (∆7H 2asesup-frc). (A) Growth on formate (black symbols) and H2 production (gray symbols) by the wild-type strain MM901 (circles), the ∆7H2asesup mutant (squares), and the ∆7H2asesup-frc mutant (triangles) in batch culture. Data are from a single representative experiment, but two replicate experiments gave similar results (see Fig. S2 in the supplemental material). (B) CH4 (black curve) and H2 production (bars) of the ∆7H2asesup-frc mutant in continuous culture. Actively growing cultures (gray bars) and cultures after metronidazole (50 µg ml−1) was added to completely oxidize ferredoxin (white bars) are shown. The medium dilution rate, gas flow rate, and culture optical density are shown on the x axis.
Mentions: The ∆7H2asesup mutant grows more slowly than the wild type on formate, suggesting that reduced ferredoxin is limiting. Reduced ferredoxin limitation of methanogenesis implies that other reduced cofactors that feed into the pathway, such as F420H2, are present in excess. Wild-type M. maripaludis possesses an F420-dependent formate:H2 lyase activity that is catalyzed by Fdh and F420-reducing hydrogenase. The wild-type strain grown on formate can accumulate H2 in the culture headspace to a concentration of 0.16% ± 0.02% of the gas phase at 2 atm pressure (mean ± standard deviation [SD] for three biological replicates) (18, 19). An excess of F420H2 should drive the equilibrium of this activity toward increased H2 production. frc encoding F420-reducing hydrogenase was placed on the replicative vector pLW40 (17) and reintroduced into the ∆7H2asesup mutant to restore formate:H2 lyase activity. When grown with formate as the only electron donor for methanogenesis, the ∆7H2asesup-frc mutant was capable of producing H2 up to a concentration of 2.32% ± 0.79% (Fig. 5A; see Fig. S2 in the supplemental material). When cultures entered stationary phase, H2 reuptake occurred, presumably due to depletion of formate and an equilibrium shift back toward F420H2 production from H2. The ∆7H2asesup mutant, which lacks formate:H2 lyase activity, was incapable of H2 production.

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