H2-independent growth of the hydrogenotrophic methanogen Methanococcus maripaludis

Abstract

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.

FIG 1

Growth of the ∆6H₂ase mutant with CO and formate. The ∆6H₂ase mutant (solid black lines) and the ∆6H₂ase ∆cdh mutant (broken lines) were grown with formate plus H₂ (black symbols), formate plus 5% CO (gray symbols), or formate alone (white symbols). Data points are averages of three cultures, and error bars represent 1 standard deviation around the mean.

FIG 2

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.

FIG 3

Growth of the ∆7H₂ase_sup mutant. (A) Growth of the ∆7H₂ase_sup mutant with formate alone or formate plus H₂. (B) Growth of the ∆7H₂ase_sup mutant with formate + CO. The ∆7H₂ase_sup 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 ∆7H₂ase_sup (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.

FIG 4

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.

FIG 5

Growth and H₂ production by the ∆7H₂ase_sup mutant expressing F₄₂₀-reducing hydrogenase (∆7H ₂ase_sup-frc). (A) Growth on formate (black symbols) and H₂ production (gray symbols) by the wild-type strain MM901 (circles), the ∆7H₂ase_sup mutant (squares), and the ∆7H₂ase_sup-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 ∆7H₂ase_sup-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.

FIG 6

Glyceraldehyde-3-phosphate:ferredoxin oxidoreductase cycle for ATP-dependent ferredoxin reduction. The GAPOR cycle of M. maripaludis as originally described by Park et al. (29) is shown with potential input from F₄₂₀H₂. Fdh, formate dehydrogenase; Fno, F₄₂₀H₂:NADP⁺ oxidoreductase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GAPOR, glyceraldehyde-3-phosphate: ferredoxin oxidoreductase; PGK, phosphoglycerate kinase. G3P, glyceraldehyde-3-phosphate; 1,3-DPG, 1,3-diphosphoglycerate; 3-PG, 3-phosphoglycerate.