H₂-Independent Growth of the Hydrogenotrophic Methanogen Methanococcus maripaludis

ABSTRACT

Hydrogenotrophic methanogenic Archaea require reduced ferredoxin as an anaplerotic source of electrons for methanogenesis. H₂ oxidation by the hydrogenase Eha provides these electrons, consistent with an H₂ 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 H₂-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 F₄₂₀, should be abundant. Indeed, when F₄₂₀-reducing hydrogenase was reintroduced into the hydrogenase-free mutant, the equilibrium of H₂ production via an F₄₂₀-dependent formate:H₂ lyase activity shifted markedly toward H₂ compared to the wild type.

IMPORTANCE

Hydrogenotrophic methanogens are thought to require H₂ as a substrate for growth and methanogenesis. Here we show alternative pathways in methanogenic metabolism that alleviate this H₂ requirement and demonstrate, for the first time, a hydrogenotrophic methanogen that is capable of growth in the complete absence of H₂. The demonstration of alternative pathways in methanogenic metabolism suggests that this important group of organisms is metabolically more versatile than previously thought.

Introduction

Methanogenic Archaea can be grouped into two physiologically distinct groups, the methylotrophs and the hydrogenotrophs (1). Methylotrophic methanogens are relatively versatile, as their substrate repertoire for methanogenesis includes H₂ and CO₂, acetate, methyl compounds, such as methanol and methylamines, and CO. Hydrogenotrophic methanogens are more restricted, using H₂, formate, or for a few species, certain alcohols as electron donors for CO₂ reduction to CH₄.

Hydrogenotrophic methanogens are distinct from methylotrophic methanogens in their use of electron bifurcation as an energy-conserving step in methanogenesis from CO₂ (1–4). Two pairs of electrons enter the methanogenic pathway at a protein complex that contains two key enzymes, heterodisulfide reductase (Hdr) and formylmethanofuran dehydrogenase (Fwd). One pair of electrons is used by Hdr in an exergonic reaction to reduce the heterodisulfide CoM-S-S–CoB to the sulfhydryl coenzymes HS–CoB and HS-CoM, which in turn serve as reductants for reduction of the methyl group on coenzyme M (CH₃-S-CoM) to CH₄. The other pair of electrons is used in an endergonic reaction by Fwd to reduce CO₂ to the formyl group of formyl-methanofuran (formyl-MFR) (for a diagram of the methanogenic pathway, see reference 4).

By coupling the final, methane-producing step to the initial, CO₂-reducing step, electron bifurcation renders the pathway cyclic. The cyclic model of methanogenesis was recently named the Wolfe cycle in honor of the contributions of Ralph S. Wolfe (5). The cyclic nature of the pathway can explain in part why hydrogenotrophs have not evolved the metabolic versatility of the methylotrophs. The latter organisms use acetate by oxidizing the carbonyl to provide electrons for reduction of the methyl to methane, and they use methyl compounds by disproportionation where some of the substrate is oxidized and some is reduced to methane. However, in hydrogenotrophs, the stoichiometric coupling of the methane-producing step to the recruitment of CO₂ into the pathway prohibits the input of additional intermediates. Furthermore, in the case of acetate, only one pair of electrons is available from the oxidation of the carbonyl, yet electron bifurcation requires that two pairs of electrons must feed into the heterodisulfide reductase complex. Although many hydrogenotrophic methanogens contain acetyl coenzyme A (acetyl-CoA) synthase/CO dehydrogenase (ACS/CODH) (the essential enzyme for aceticlastic methanogenesis), they appear to use it only anabolically for CO₂ fixation and not catabolically for acetate utilization.

Either H₂ or formate donates electrons to all four reduction steps in methanogenesis from CO₂. Both substrates supply electrons directly to the Hdr complex via an Hdr-associated hydrogenase or an Hdr-associated formate dehydrogenase (Fdh) (2), thus providing for the first and last reduction steps in the pathway. The two intermediate reduction steps use the coenzyme F₄₂₀ as the direct electron donor, and H₂ or formate directly reduces F₄₂₀ via an F₄₂₀-reducing hydrogenase or Fdh. Hence, H₂ or formate alone can provide the stoichiometric reducing requirements for CO₂ reduction to methane. However, as another consequence of the Wolfe cycle, the levels of intermediates in methanogenesis must be anaplerotically maintained in order to avoid decaying flux in the pathway (5). In the model species Methanococcus maripaludis we showed how this occurs by genetically eliminating six of the seven hydrogenases encoded in the genome (4). The resulting mutant, designated ∆6H₂ase, was unable to use H₂ as the stoichiometric electron donor for methanogenesis. The mutant also lacked a formate-H₂ lyase activity found in the wild-type strain. As a result of these two deficiencies, the ∆6H₂ase mutant required both formate and H₂ for growth and methanogenesis. While formate was needed stoichiometrically for the four reduction steps of methanogenesis, H₂ was required only in small amounts sufficient to support the anaplerotic needs of methanogenesis. In the ∆6H₂ase mutant, only one hydrogenase remained: the membrane-bound energy-converting hydrogenase Eha, which harvests chemiosmotic energy to drive the reduction of a low-potential ferredoxin which in turn is thought to reduce CO₂ to formyl-MFR. Hence, during growth on H₂ or formate, Eha is essential in wild-type M. maripaludis to anaplerotically replenish methanogenesis at the first reduction step. Indeed, a knockout of the genes encoding Eha was not successful. Consistent with these results, H₂ stimulated methanogenesis from formate in cell suspensions (4).

Here we show that the essentiality of H₂ and Eha does not always hold, and that in M. maripaludis, there are at least two additional pathways by which the anaplerotic requirements of methanogenesis can be satisfied. We delete genes encoding Eha in a strain derived from the ∆6H₂ase mutant that expresses one of these pathways, thus eliminating all H₂ metabolism in a hydrogenotrophic methanogen. By reintroducing a single hydrogenase—the F₄₂₀-reducing hydrogenase—into the hydrogenase-free strain and observing its effects in isolation from other H₂-metabolizing pathways, we find that substantial electron flux is diverted to H₂ production. These findings show that hydrogenotrophic methanogens are metabolically more versatile than previously thought.

RESULTS

CO stimulates growth of the ∆6H₂ase mutant in the absence of H₂.

Eha provides anaplerotic input to the methanogenic pathway by acting as a supplement to electron bifurcation for the reduction of a ferredoxin that in turn is used to reduce CO₂ to formyl-MFR (4). However, other pathways of ferredoxin reduction/oxidation exist in methanogenic Archaea. The ACS/CODH enzyme complex converts CO₂ to CO, which ultimately becomes the carbonyl carbon of acetyl-CoA (6, 7). This reduction is dependent on reduced ferredoxin as an electron donor.

To test whether the reverse reaction of ACS/CODH—CO oxidation to CO₂ with the production of reduced ferredoxin—can stimulate methanogenesis in an H₂-independent manner, the ∆6H₂ase mutant was grown on formate medium with or without the addition of H₂ or CO to the culture headspace. As expected, H₂ promoted robust growth. In addition, CO promoted growth in the absence of H₂ (Fig. 1). Growth rates were much lower with CO in place of H₂, suggesting that ferredoxin reduction by Eha is preferred. Growth promoted by CO was directly attributable to ACS/CODH, since when the CO₂-reducing subunits were genetically eliminated (∆6H₂ase-∆cdh), growth no longer occurred with CO and formate as the only electron donors (Fig. 1).

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.

Isolation of a suppressor mutation that allows growth of the ∆6H₂ase mutant on formate alone.

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 (∆6H₂ase_sup) 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.

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.

Deletion of eha is possible due to a suppressor mutation.

A suppressor mutation that allows growth of ∆6H₂ase_sup 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 ∆6H₂ase_sup strains (12, 13). Deletion of ehaNO was indeed possible, suggesting that another ferredoxin reduction activity is present in the suppressor background. The new strain (∆7H₂ase_sup) lacks the genes encoding the active sites for all genomically encoded hydrogenases: ∆vhuAU ∆vhcA ∆fruA ∆frcA ∆hmd ∆ehbN ∆ehaNO. The ∆7H₂ase_sup mutant grew in the absence of H₂, and H₂ did not stimulate growth (Fig. 3A).

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.

CO is a potent inhibitor of nickel-containing hydrogenases (14, 15). Five percent CO inhibited wild-type M. maripaludis grown on formate but had no inhibitory effect on the ∆7H₂ase_sup mutant. In fact, the ∆7H₂ase_sup mutant grew better on formate in the presence of 5% CO than it did on formate alone (Fig. 3B), suggesting that in the ∆7H₂ase_sup mutant, like the ∆6H₂ase mutant, CO was oxidized to CO₂, leading to the production of reduced ferredoxin. The growth of the ∆7H₂ase_sup mutant is apparently limited by the availability of reduced ferredoxin, and CO oxidation partially relieves its slow growth phenotype. The reduced ferredoxins generated by the suppressor activity and ACS/CODH appear additive in stimulating methanogenesis. The ∆7H₂ase_sup mutant grew faster with formate and CO than either the ∆7H₂ase_sup mutant grown with formate alone or the ∆6H₂ase mutant grown with formate and CO.

Genome sequencing reveals a suppressor mutation allowing for growth of the ∆6H₂ase_sup and ∆7H₂ase_sup mutants on formate alone.

To determine the genetic background that allowed growth of the ∆6H₂ase_sup and ∆7H₂ase_sup mutants on formate in the absence of H₂ or CO, we performed Illumina sequencing on the ∆7H₂ase_sup mutant and six of the isolated suppressors and compared the sequence to the ∆6H₂ase parent (see Table S1 in the supplemental material). Four of six of the ∆6H₂ase_sup mutants, as well as the ∆7H₂ase_sup mutant, shared a common insertion (AT at position 931341) in an intergenic region directly upstream of the gene for glyceraldehyde-3-phosphate (G3P):ferredoxin oxidoreductase (GAPOR) (see Fig. S1 in the supplemental material). The insertion generated the sequence AATATATA upstream of GAPOR which is very similar to the consensus methanogen promoter TTTA(T/A)ATA (16). Therefore, it appears that generation of a promoter upstream of GAPOR increases expression of this ferredoxin-reducing enzyme to allow for H₂-independent growth. Sanger sequencing confirmed the presence of this mutation in the ∆7H₂ase_sup mutant and its absence in the ∆6H₂ase mutant. The nature of the suppressor mutations allowing growth in the other two ∆6H₂ase_sup strains was not readily apparent from the genome sequencing data.

Overexpression of GAPOR in the ∆6H₂ase mutant allows growth without H₂.

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).

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.

The ∆7H₂ase_sup mutant expressing F₄₂₀-reducing hydrogenase can produce substantial amounts of H₂.

The ∆7H₂ase_sup 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 ∆7H₂ase_sup mutant to restore formate:H₂ lyase activity. When grown with formate as the only electron donor for methanogenesis, the ∆7H₂ase_sup-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 ∆7H₂ase_sup mutant, which lacks formate:H₂ lyase activity, was incapable of H₂ production.

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.

We also attempted continuous culture of the ∆7H₂ase_sup-frc mutant to assess how additional factors affect the equilibrium of formate:H₂ lyase activity. The ∆7H₂ase_sup-frc mutant was maintained at a low OD₆₆₀ under conditions where the medium dilution rate (0.125 liters h⁻¹) slightly exceeded the growth rate to ensure that the culture was continuously growing. Under these conditions, the ∆7H₂ase_sup-frc mutant produced between 100 and 500 µmol H₂ gdw⁻¹ (gdw stands for grams [dry weight]) (Fig. 5B). The higher production rates were observed when the gas flow rate was increased from 25 to 230 ml min⁻¹, demonstrating that increased removal of H₂ results in an equilibrium shift toward even greater H₂ production. When the growth rate was allowed to exceed the medium dilution rate (0.031 liters h⁻¹), H₂ production was not greatly affected.

As reduced ferredoxin limitation of growth appears to lead to increased H₂ production, we sought to take this to the extreme case of the absence of reduced ferredoxin. Metronidazole, an antibiotic capable of oxidizing ferredoxin (20), was added to the chemostat, and after 1 h, H₂ production was found to have increased 5-fold to ~2.5 mmol gdw⁻¹ h⁻¹. With increased gas flow, this rate approached 5 mmol gdw⁻¹ h⁻¹. Robust H₂ production upon metronidazole addition verifies that limiting reduced ferredoxin leads to an excess of F₄₂₀H₂.

DISCUSSION

Hydrogenotrophic methanogens have unappreciated metabolic versatility.

Although they do not share pathways of methylotrophic methanogens, hydrogenotrophic methanogens have a different kind of metabolic versatility. We have shown previously that this is due in part to the ability of hydrogenotrophs to use either formate or H₂ as the electron donor to all four reduction steps of methanogenesis (2, 4). In addition, we have shown here that hydrogenotrophs also have versatility in their pathways of ferredoxin reduction for the anaplerotic electron input to methanogenesis. First, our results show clearly that H₂ is not the only possible reductant for this purpose. The ∆6H₂ase mutant grows in the complete absence of H₂ as long as CO is present along with formate. In the case of the ∆7H₂ase_sup strain, even CO was not needed, and formate was the sole electron donor. H₂ addition to cultures of the ∆7H₂ase_sup mutant grown on formate had no stimulatory effect, confirming that H₂ uptake did not occur (Fig. 3B). CO, a hydrogenase inhibitor, also had no inhibitory effect in the ∆7H₂ase_sup background but did in the wild type (14, 15). Additionally, the ∆7H₂ase_sup mutant lacked all formate:H₂ lyase activity (Fig. 5A). Taken together, these data confirm that all hydrogenase activity had been eliminated. The ∆6H₂ase, ∆6H₂ase_sup and ∆7H₂ase_sup strains represent, to our knowledge, the first examples of hydrogenotrophic methanogens capable of growth in the complete absence of H₂.

H₂-independent growth occurs via novel electron flow pathways.

Here we have demonstrated two pathways by which H₂ may be replaced as the anaplerotic electron donor for methanogenesis. Like Eha with H₂, both pathways reduce ferredoxin which then presumably reduces CO₂ to formyl-MFR. First, CO served this purpose in the ∆6H₂ase and ∆7H₂ase_sup strains. To our knowledge, only one other study reported the use of CO for methanogenesis in a hydrogenotrophic methanogen. In that report, CO as the sole substrate supported slow growth of Methanothermobacter thermautotrophicus by disproportionation to CO₂ and CH₄, and an F₄₂₀-reducing carbon monoxide dehydrogenase activity was found (21). This differs from our results in which formate as well as CO was present. In addition, our ∆6H₂ase and ∆7H₂ase_sup mutants are capable of producing F₄₂₀H₂ directly from formate, so an F₄₂₀-reducing carbon monoxide dehydrogenase activity is likely of little importance. Instead, the oxidation of CO leads to reduced ferredoxin. CO formation from H₂ and CO₂ with reduced ferredoxin as an intermediate has been observed in cell suspensions of hydrogenotrophic methanogens possessing ACS/CODH (7, 22), and the process we observed here, which depended on ACS/CODH, appears to be the reverse.

The CO utilization demonstrated here for M. maripaludis contrasts with methylotrophic methanogens. First, in methylotrophic methanogens, CO can be converted to methane and CO₂ or to formate and acetate with the concomitant generation of ATP (23, 24). In contrast, 5% CO, as was used in our growth experiments, is insufficient as a stoichiometric electron donor for the amount of growth observed and functioned only anaplerotically. In both kinds of methanogens, CO oxidation results in reduced ferredoxin, but only the methylotrophs are known to use reduced ferredoxin as a stoichiometric source of electrons for methanogenesis. Second, methylotrophic methanogens can carry out methanogenesis solely from acetate by using ACS/CODH to cleave acetyl-CoA. M. maripaludis can utilize acetate anabolically (25) and can also use ACS/CODH anabolically for CO₂ fixation to acetyl-CoA (6). Nevertheless, although CO utilization as an anaplerotic stimulant of methanogenesis in M. maripaludis occurred via ACS/CODH, M. maripaludis is apparently unable to use acetate for methanogenesis, even anaplerotically. Thus, our ∆6H₂ase mutant, which was always grown in the presence of acetate and Casamino Acids, would not grow on formate in the absence of H₂ without a suppressor mutation occurring.

As an additional novel pathway, overexpression of GAPOR could substitute for Eha and H₂. GAPOR is found throughout the Archaea (26–29) and functions in glycolysis, catalyzing the oxidation of G3P to 3-phosphoglycerate (3-PG) with the concomitant reduction of ferredoxin (26, 29). The corresponding gluconeogenic reactions are catalyzed by G3P dehydrogenase (GAPDH), a NADPH-dependent enzyme, and phosphoglycerate kinase (PGK), an ATP-dependent enzyme (Fig. 6). Running both pathways simultaneously would result in:

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.

NADPH + ATP + Fd_ox → NADP⁺ + ADP + P_i + Fd_red where Fd is ferredoxin, Fd_ox is oxidized Fd, and Fd_red is reduced Fd.

M. maripaludis also encodes an F₄₂₀H₂:NADP⁺ oxidoreductase (Fno) and the F₄₂₀-dependent Fdh (12, 30). When these activities are taken into account, an ATP-dependent F₄₂₀H₂:ferredoxin oxidoreductase activity is possible (Fig. 6):

F₄₂₀H₂ + ATP + Fd_ox → F₄₂₀ + ADP + P_i + Fd_red

This pathway evidently operates in the ∆6H₂ase_sup strains and the ∆7H₂ase_sup strain, as well as in the ∆6H₂ase strain with GAPOR overexpressed on a plasmid.

Reduced ferredoxin abundance versus inefficient electron transfer.

The CO-dependent and GAPOR-dependent pathways of ferredoxin reduction appear less efficient than Eha in supplying anaplerotic electrons to methanogenesis, since the ∆6H₂ase strain with formate and CO and the ∆7H₂ase_sup strain with formate alone grew more slowly than Eha⁺ strains with formate and H₂. Indeed, combining the two pathways by including CO with formate for growth of the ∆7H₂ase_sup strain increased growth in an additive manner. The low efficiency of the alternative pathways may be due to low concentrations of reduced ferredoxin produced compared to what can be produced by Eha, or the reduced ferredoxin could be abundant but inefficient at transferring electrons to Fwd. The latter interpretation is consistent with the proposal that the ferredoxin pools for anabolism and catabolism are normally separated in M. maripaludis (4), since the anabolic ferredoxin-reducing hydrogenase Ehb and the anaplerotic ferredoxin-reducing Eha reduce ferredoxins that substitute inefficiently for each other (4, 25).

Robust H₂ production by the ∆7H₂ase_sup-frc mutant suggests abundant F₄₂₀H₂.

A low availability of reduced ferredoxin, limiting methanogenesis and growth, should result in a buildup of F₄₂₀H₂. The equilibrium between F₄₂₀H₂ and H₂ should then result in significant H₂ production upon reintroducing F₄₂₀-reducing hydrogenase into the ∆7H₂ase_sup strain. This proved to be the case. Furthermore, the enhancement of H₂ production by the addition of metronidazole supported the notion that an abundance of F₄₂₀H₂ resulted from a depletion of reduced ferredoxin.

MATERIALS AND METHODS

Growth conditions.

All strains were grown as described previously in McCas medium containing 200 mM sodium formate and 200 mM morpholinepropanesulfonic acid (MOPS) (pH 7) buffer (19, 31). H₂-CO₂ (20:80; 40 lb/in²) or N₂-CO₂ (20:80; 15 lb/in²) was added to the culture headspace unless otherwise indicated. Antibiotics (neomycin sulfate [5 mg ml⁻¹], puromycin [2.5 µg ml⁻¹], or metronidazole [50 µg ml⁻¹]) were used where appropriate. For growth with CO in the culture headspace, 50% CO was injected to a final concentration of 5% (vol/vol). For growth curves, triplicate cultures were inoculated with a 10% inoculum, and the optical density at 660 nm (OD₆₆₀) was monitored.

For growth under continuous culture, a modified version of a previously established chemostat system was employed (32–34). The ∆7H₂ase_sup-frc mutant was grown under steady-state conditions with 380 mM sodium formate. NaCl was removed to maintain osmotic balance. Casamino Acids (0.2% [wt/vol]) and ampicillin (100 µg ml⁻¹) were included in the growth medium. A gas mixture containing Ar−CO₂-1% H₂S (29:8:3) was used. Medium and gas flow rates are indicated in the text. The pH of steady-state samples was monitored and maintained at 6.95 by the automated addition of 10% H₂SO₄. Agitation in the vessel was maintained at 50 rpm throughout steady-state growth.

For continuous culture, the ∆7H₂ase-frc mutant was grown to a maximum OD₆₆₀ of 0.7 with a medium dilution rate of 0.083 liters h⁻¹ and a gas flow rate of 26 ml ⋅ min⁻¹. Medium dilution was changed to 0.125 liters h⁻¹ until the culture OD₆₆₀ dropped to 0.28 at which point the first sample was taken. The low OD₆₆₀ ensured the culture was constantly growing with excess formate. When the medium dilution rate was changed throughout the course of sampling, culture was given at least 24 h to equilibrate before samples were collected. For changes in the gas flow rate, the gas phase was allowed at least 1 h to equilibrate before sample collection.

Generation of mutants and plasmids.

Strains used in this study are listed in Table 1. Plasmids and primers can be found in Table S2 in the supplemental material. Strain MM901, a derivative of M. maripaludis S2 that lacks uracil phosphoribosyl transferase (upt), was used as the wild-type strain (2, 35). Mutants were constructed using methods described in reference 31 and modified in reference 2. Briefly, to generate deletion mutants, a PCR product containing an in-frame deletion of the gene of interest was ligated into the vector pCRuptneo (neo stands for neomycin) (2). This was then transformed into strain MM901. Neomycin sulfate (5 mg ml⁻¹) was used to select for a merodiploid, and 6-azauracil (250 µg ml⁻¹) was used as negative selection to isolate the mutant of interest. To place genes onto a replicative vector, PCR products were cloned directly into pLW40 or pLW40neo and transformed into the strain of interest. Either neomycin sulfate (5 mg ml⁻¹) or puromycin (2.5 µg ml⁻¹) was included as appropriate. Suppressor strains of the ∆6H₂ase mutant capable of growth in the absence of H₂ were grown in formate medium either with or without the addition of 100 µM or 1 mM ammonium 2-(methylthio)ethanesulfonate (CH₃-S-CoM); however, CH₃-S-CoM was found to have no stimulatory effect on growth and was excluded from all subsequent experiments. All mutants were confirmed by PCR screen, and deletion of ehaNO was also confirmed by Southern blotting and Illumina sequencing (see below).

TABLE 1 .

Methanococcus maripaludis strains used in this study

Strain	Description	Relevant locus	Reference
MM901	Wild-type M. maripaludis with an in-frame deletion of upt	See reference	2
MM1284	Δ6H2ase, MM901 ΔvhuAU ΔvhcA ΔfruA ΔfrcA Δhmd ΔehbN	See reference	4
MM1310	Δ7H2asesup, MM1316 ΔehaNO	Mmp1461, Mmp1462	This study
MM1315	Δ6H2ase mutant with a suppressor allowing for growth without H2		This study
MM1316	Δ6H2ase mutant with a suppressor allowing for growth without H2		This study
MM1317	Δ6H2ase mutant with a suppressor allowing for growth without H2		This study
MM1318	Δ6H2ase mutant with a suppressor allowing for growth without H2		This study
MM1319	Δ6H2ase mutant with a suppressor allowing for growth without H2		This study
MM1320	Δ6H2ase mutant with a suppressor allowing for growth without H2		This study
MM1327	MM1284 with a deletion of CO dehydrogenase (cdh)	Mmp0983-0985	This study
MM1338	MM1284 with GAPOR overexpressed on pLW40neo	Mmp0945	This study
MM1339	MM1310 with F420-reducing hydrogenase (frc) on pLW40	Mmp0817-0820	This study

Genome sequencing.

Genome sequencing was carried out for the ∆6H₂ase mutant (MM1284), the ∆7H₂ase_sup mutant (MM1310), and six of the ∆6H₂ase strains that had developed suppressor mutations (MM1315, MM1316, MM1317, MM1318, MM1319, and MM1320). ∆6H₂ase strains were isolated by dilution to extinction before processing. High-molecular-weight DNA for sequencing was extracted using the Qiagen PureGene kit according to the manufacturer’s instructions. Genomes were sequenced as described in reference 36, using unpaired 36-bp reads with the Illumina Genome Analyzer GA IIX according to the manufacturer’s instructions (Illumina, San Diego, CA). For each genome, a random-fragment library was constructed using a custom protocol. Briefly, genomic DNA (gDNA) samples were sheared using a Bioruptor UCD-200 (Diagenode Inc., Denville, NJ), and end repaired using an End-It DNA end repair kit (Epicentre). Repaired fragments were subjected to A tailing using Taq DNA polymerase (Roche Inc., USA, Chicago, IL), and custom adaptors ligated to A-tailed fragments using T4 DNA ligase (New England Biolabs, Beverly, MA). Libraries were size selected using automated electrophoresis on a Pippen Prep (Sage Science, Beverly, MA) and assessed for size range and concentration using a Qubit (Invitrogen Inc., Carlsbad, CA) and a Bioanalyzer (Agilent Inc., San Diego, CA).

Raw sequence data were compared directly to the M. maripaludis strain S2 reference genome (NCBI reference NC_005791.1) to identify possible suppressor mutations allowing for H₂-independent growth (see Table S1 in the supplemental material). Putative mutations upstream of the gene encoding glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAPOR) were identified and verified in ∆6H₂ase and ∆7H₂ase_sup mutants with Sanger sequencing (GeneWiz, South Plainfield, NJ).

CH₄ and H₂ measurements.

To measure H₂ from batch culture, 2.5 ml of culture headspace was collected and stored for no longer than 24 h in a 5-ml serum vial preflushed with 100% N₂. To measure gas concentration from chemostat-grown cells, gas was collected directly into 5-ml serum vials after flushing with at least 350 ml of chemostat gas outflow. For batch culture measurements, the headspace contained a pressure of 2 atm. H₂ and CH₄ concentrations were measured by gas chromatography as described previously (4). To completely oxidize ferredoxin, metronidazole (50 µg ml⁻¹) was used. Upon the addition of metronidazole, gas flow through the chemostat vessel was allowed to equilibrate for 1 h prior to collection and analysis. Rates of H₂ production were calculated assuming a liter of cell material at an OD₆₆₀ of 1.0 yields 0.34 gram (dry weight) (18).

SUPPLEMENTAL MATERIAL

Figure S1

Genomic context of a suppressor mutation allowing for H₂-independent growth of the Δ7H₂ase_sup mutant Download

Figure S2

Growth and H₂ production by the Δ7H₂ase_sup-frc mutant. Growth on formate (black symbols) and H₂ production (gray symbols) by the wild-type strains(circles), the Δ7H₂ase_sup mutant (squares), and the Δ7H₂ase_sup-frc mutant (triangles) in batch culture. Download

TABLE S1 

Genome sequencing results for Δ6H₂ase, Δ7H₂ase_sup, and Δ6H₂ase_sup mutants. Mutations due to the upt and hydrogenase deletions are not shown.

TABLE S2 

Primers and plasmids used in this study