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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 of the ∆7H2asesup mutant. (A) Growth of the ∆7H2asesup mutant with formate alone or formate plus H2. (B) Growth of the ∆7H2asesup mutant with formate + CO. The ∆7H2asesup mutant grown on formate (from Fig. 3A) and the ∆6H2ase mutant grown on formate plus CO (from Fig. 1) are shown for comparison. Wild-type strain MM901 (black symbols) and the ∆7H2asesup (gray symbols) and ∆6H2ase (white symbols) mutants were studied. Broken lines indicate that the cultures were grown with H2 or 5% CO in the culture headspace. Data points are averages of three cultures, and error bars represent 1 standard deviation around the mean.
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fig3: Growth of the ∆7H2asesup mutant. (A) Growth of the ∆7H2asesup mutant with formate alone or formate plus H2. (B) Growth of the ∆7H2asesup mutant with formate + CO. The ∆7H2asesup mutant grown on formate (from Fig. 3A) and the ∆6H2ase mutant grown on formate plus CO (from Fig. 1) are shown for comparison. Wild-type strain MM901 (black symbols) and the ∆7H2asesup (gray symbols) and ∆6H2ase (white symbols) mutants were studied. Broken lines indicate that the cultures were grown with H2 or 5% CO in the culture headspace. Data points are averages of three cultures, and error bars represent 1 standard deviation around the mean.

Mentions: A suppressor mutation that allows growth of ∆6H2asesup on formate alone could have produced a novel H2 production activity, or it could have generated a new ferredoxin reducing activity that is independent of H2. If a novel H2 production pathway were responsible, eha would still be essential (4). We attempted deletion of the genes encoding the active site subunits of Eha (ehaNO) in one of the ∆6H2asesup strains (12, 13). Deletion of ehaNO was indeed possible, suggesting that another ferredoxin reduction activity is present in the suppressor background. The new strain (∆7H2asesup) lacks the genes encoding the active sites for all genomically encoded hydrogenases: ∆vhuAU ∆vhcA ∆fruA ∆frcA ∆hmd ∆ehbN ∆ehaNO. The ∆7H2asesup mutant grew in the absence of H2, and H2 did not stimulate growth (Fig. 3A).


H2-independent growth of the hydrogenotrophic methanogen Methanococcus maripaludis.

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

Growth of the ∆7H2asesup mutant. (A) Growth of the ∆7H2asesup mutant with formate alone or formate plus H2. (B) Growth of the ∆7H2asesup mutant with formate + CO. The ∆7H2asesup mutant grown on formate (from Fig. 3A) and the ∆6H2ase mutant grown on formate plus CO (from Fig. 1) are shown for comparison. Wild-type strain MM901 (black symbols) and the ∆7H2asesup (gray symbols) and ∆6H2ase (white symbols) mutants were studied. Broken lines indicate that the cultures were grown with H2 or 5% CO in the culture headspace. Data points are averages of three cultures, and error bars represent 1 standard deviation around the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Growth of the ∆7H2asesup mutant. (A) Growth of the ∆7H2asesup mutant with formate alone or formate plus H2. (B) Growth of the ∆7H2asesup mutant with formate + CO. The ∆7H2asesup mutant grown on formate (from Fig. 3A) and the ∆6H2ase mutant grown on formate plus CO (from Fig. 1) are shown for comparison. Wild-type strain MM901 (black symbols) and the ∆7H2asesup (gray symbols) and ∆6H2ase (white symbols) mutants were studied. Broken lines indicate that the cultures were grown with H2 or 5% CO in the culture headspace. Data points are averages of three cultures, and error bars represent 1 standard deviation around the mean.
Mentions: A suppressor mutation that allows growth of ∆6H2asesup on formate alone could have produced a novel H2 production activity, or it could have generated a new ferredoxin reducing activity that is independent of H2. If a novel H2 production pathway were responsible, eha would still be essential (4). We attempted deletion of the genes encoding the active site subunits of Eha (ehaNO) in one of the ∆6H2asesup strains (12, 13). Deletion of ehaNO was indeed possible, suggesting that another ferredoxin reduction activity is present in the suppressor background. The new strain (∆7H2asesup) lacks the genes encoding the active sites for all genomically encoded hydrogenases: ∆vhuAU ∆vhcA ∆fruA ∆frcA ∆hmd ∆ehbN ∆ehaNO. The ∆7H2asesup mutant grew in the absence of H2, and H2 did not stimulate growth (Fig. 3A).

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