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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

Generation of suppressor strains of the ∆6H2ase mutant capable of H2-independent growth. The ∆6H2ase mutant was grown in formate-containing medium without H2 or CO. The medium contained 1,000 µM (black symbols), 100 µM (gray symbols), or 0 µM (white symbols) CH3-S-CoM (three replicates each); however, CH3-S-CoM had no stimulatory effect on growth. Each curve represents growth in a single tube.
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fig2: Generation of suppressor strains of the ∆6H2ase mutant capable of H2-independent growth. The ∆6H2ase mutant was grown in formate-containing medium without H2 or CO. The medium contained 1,000 µM (black symbols), 100 µM (gray symbols), or 0 µM (white symbols) CH3-S-CoM (three replicates each); however, CH3-S-CoM had no stimulatory effect on growth. Each curve represents growth in a single tube.

Mentions: The growth of the ∆6H2ase mutant with formate and CO suggests that ferredoxin reduction by Eha is not necessary for growth, provided alternative mechanisms to anaplerotically stimulate methanogenesis are present. CH3-S-CoM addition to cell extracts stimulates methanogenesis (8, 9), and methanogenic Archaea are capable of CH3-S-CoM uptake via a poorly characterized and very inefficient activity (10, 11). Therefore, we tried to grow the ∆6H2ase mutant on formate in the absence of H2 in the presence and absence of CH3-S-CoM. Surprisingly, regardless of the presence or absence of CH3-S-CoM, after prolonged incubation of nine independent cultures, all nine grew (Fig. 2). Upon transfer to new medium, each strain tested routinely grew to maximum optical density at 660 nm (OD660) within 24 h. These results suggested that independent suppressor mutations (∆6H2asesup) were generated that allowed for growth on formate alone. Suppressor strains that were generated in the presence of CH3-S-CoM grew well in its absence. Hence, although CH3-S-CoM failed to stimulate growth, mutants developed that had a novel mechanism to anaplerotically stimulate methanogenesis.


H2-independent growth of the hydrogenotrophic methanogen Methanococcus maripaludis.

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

Generation of suppressor strains of the ∆6H2ase mutant capable of H2-independent growth. The ∆6H2ase mutant was grown in formate-containing medium without H2 or CO. The medium contained 1,000 µM (black symbols), 100 µM (gray symbols), or 0 µM (white symbols) CH3-S-CoM (three replicates each); however, CH3-S-CoM had no stimulatory effect on growth. Each curve represents growth in a single tube.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Generation of suppressor strains of the ∆6H2ase mutant capable of H2-independent growth. The ∆6H2ase mutant was grown in formate-containing medium without H2 or CO. The medium contained 1,000 µM (black symbols), 100 µM (gray symbols), or 0 µM (white symbols) CH3-S-CoM (three replicates each); however, CH3-S-CoM had no stimulatory effect on growth. Each curve represents growth in a single tube.
Mentions: The growth of the ∆6H2ase mutant with formate and CO suggests that ferredoxin reduction by Eha is not necessary for growth, provided alternative mechanisms to anaplerotically stimulate methanogenesis are present. CH3-S-CoM addition to cell extracts stimulates methanogenesis (8, 9), and methanogenic Archaea are capable of CH3-S-CoM uptake via a poorly characterized and very inefficient activity (10, 11). Therefore, we tried to grow the ∆6H2ase mutant on formate in the absence of H2 in the presence and absence of CH3-S-CoM. Surprisingly, regardless of the presence or absence of CH3-S-CoM, after prolonged incubation of nine independent cultures, all nine grew (Fig. 2). Upon transfer to new medium, each strain tested routinely grew to maximum optical density at 660 nm (OD660) within 24 h. These results suggested that independent suppressor mutations (∆6H2asesup) were generated that allowed for growth on formate alone. Suppressor strains that were generated in the presence of CH3-S-CoM grew well in its absence. Hence, although CH3-S-CoM failed to stimulate growth, mutants developed that had a novel mechanism to anaplerotically stimulate methanogenesis.

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