Essential anaplerotic role for the energy-converting hydrogenase Eha in hydrogenotrophic methanogenesis

Abstract

Despite decades of study, electron flow and energy conservation in methanogenic Archaea are still not thoroughly understood. For methanogens without cytochromes, flavin-based electron bifurcation has been proposed as an essential energy-conserving mechanism that couples exergonic and endergonic reactions of methanogenesis. However, an alternative hypothesis posits that the energy-converting hydrogenase Eha provides a chemiosmosis-driven electron input to the endergonic reaction. In vivo evidence for both hypotheses is incomplete. By genetically eliminating all nonessential pathways of H₂ metabolism in the model methanogen Methanococcus maripaludis and using formate as an additional electron donor, we isolate electron flow for methanogenesis from flux through Eha. We find that Eha does not function stoichiometrically for methanogenesis, implying that electron bifurcation must operate in vivo. We show that Eha is nevertheless essential, and a substoichiometric requirement for H₂ suggests that its role is anaplerotic. Indeed, H₂ via Eha stimulates methanogenesis from formate when intermediates are not otherwise replenished. These results fit the model for electron bifurcation, which renders the methanogenic pathway cyclic, and as such requires the replenishment of intermediates. Defining a role for Eha and verifying electron bifurcation provide a complete model of methanogenesis where all necessary electron inputs are accounted for.

Methanogenesis is an anaerobic respiration carried out by a phylogenetically related group of Archaea within the phylum Euryarchaeota. Methanogens are divided into two metabolic types, those without and those with cytochromes (1). Methanogens without cytochromes use H₂ as an electron donor and are termed hydrogenotrophic. Some species can substitute H₂ with formate, and a few can use secondary alcohols. CO₂ is the electron acceptor and is reduced to methane. Methanogens with cytochromes reduce certain methyl compounds or the methyl carbon of acetate to methane and are called methylotrophic. Many can also use H₂ and CO₂, as can hydrogenotrophic methanogens.

Although the pathways of methanogenesis have long been known, an understanding of energy conservation has been slower to emerge. Methanogens with and without cytochromes both export Na⁺ when a methyl group is transferred from the carrier tetrahydromethanopterin (H₄MPT) to coenzyme M (CoM) (Fig. 1). The Na⁺ gradient across the membrane is used directly for ATP synthesis or is converted by an antiporter to a proton gradient. However, for methanogenesis from CO₂, the initial reduction of CO₂ to a formyl group attached to methanofuran (MFR) is endergonic. How energy is provided to drive this reaction is not well understood. Methanogens with and without cytochromes have membrane-associated energy-converting hydrogenases that couple the reduction of low-potential ferredoxins (Fd) to a chemiosmotic membrane gradient (2). If such a Fd donates electrons for CO₂ reduction, an energy-converting hydrogenase is the conduit of energy for this reaction. Indeed, for methanogens with cytochromes, an energy-converting hydrogenase is required for CO₂ reduction (3). However, the energy requirement for the first step in the pathway results in a need for additional energy conservation. This could be provided by the final step of methanogenesis, which involves an exergonic reduction of a heterodisulfide of two methanogenic cofactors (CoM-S-S-CoB) by heterodisulfide reductase (Hdr). Methanogens with cytochromes harvest the energy yielded in heterodisulfide reduction with a proton-exporting electron transport chain. However, methanogens without cytochromes lack this electron transport chain and an alternative explanation is required.

Fig. 1.

The methanogenic pathway. Eha, energy-converting hydrogenase A; Fdh, formate dehydrogenase; Fru and Frc, F₄₂₀-reducing hydrogenases; Ftr, formyl-MFR:H₄MPT formyltransferase; Fwd, formyl-MFR dehydrogenase; Hdr, heterodisulfide reductase; Hmd, H₂-dependent methylene-H₄MPT dehydrogenase; Mch, methenyl-H₄MPT cyclohydrolase; Mcr, methyl-CoM reductase; Mer, methylene-H₄MPT reductase; Mtd, F₄₂₀-dependent methylene-H₄MPT dehydrogenase; Mtr, methyl-H₄MPT-CoM methyltransferase; Vhu and Vhc, F₄₂₀-nonreducing (Hdr-associated) hydrogenases.

Here we present results supporting an emerging view of methanogenesis without cytochromes. The emerging model diverges from the conventional picture of a linear pathway of CO₂ reduction to methane. Instead, a cyclical pathway involving electron bifurcation has been proposed (1) (Fig. 1). The reductions of the heterodisulfide and CO₂ are coupled in the flavin-containing enzyme complex centered around Hdr. For each pair of electrons accepted, one electron is used for the exergonic reduction of CoM-S-S-CoB, and one is used to reduce a low-potential ferredoxin that in turn donates electrons for the reduction of CO₂ to formyl-MFR. Hence, electron bifurcation, a nonchemiosmotic form of energy conservation, couples the exergonic and endergonic steps of methanogenesis and allows for the net availability of chemiosmotic energy for ATP synthesis. The electron bifurcation model renders methanogenesis a cyclic process, in which late steps are coupled by electron flow to the initial step, and explains why in cell extracts, CH₄ production from CO₂ requires an input of C-1 intermediates (4). Electron bifurcation is supported by experiments with whole cells (5), with purified enzymes (6), and by the characterization of an enzyme complex in which it could take place (7). However, these studies do not explain the presence in most methanogens without cytochromes of the energy-converting hydrogenase Eha that is apparently linked to the first step (2). Electron flux from this hydrogenase would appear to compete with flux from electron bifurcation as well as to consume chemiosmotic energy, leaving a deficit for ATP synthesis.

Whatever the correct model for energy conservation, it likely centers around reactions that reduce low-potential ferredoxins. Three such reactions are proposed to occur in methanogens without cytochromes. Two of these reactions are those mentioned above, the concomitant reduction of Fd and CoM-S-S-CoB that occurs in electron bifurcation, and the H₂-dependent reduction of Fd by the energy-converting hydrogenase Eha, both of which are proposed to lead to the endergonic reduction of CO₂ to formyl-MFR. A third such reaction, which reduces a Fd with another energy-converting hydrogenase, Ehb, functions in anabolic CO₂ fixation reactions and does not appear to be involved in methanogenesis (8, 9).

Here we present an analysis of electron flow in methanogens without cytochromes, focusing on the role of H₂ when formate is the electron donor for methanogenesis. We show that there are two pools of electrons that are distinguished by their substrate origins, their carriers, and their functions. One pool of electrons feeds into methanogenesis via coenzyme F₄₂₀ as well as directly to Hdr from electron-donating growth substrates. Surprisingly, these electrons need not come from H₂, even in hydrogenotrophic methanogens, but instead can come directly from formate. Another pool of electrons supports critical biosynthetic or anaplerotic steps, are carried by low-potential ferredoxins, and come only from H₂. We show that only one hydrogenase, Eha, is the essential conduit of electrons from H₂ and that Eha supports methanogenesis, but it does so in an anaplerotic and not a stoichiometric manner. Eha is needed only to replenish intermediates that are removed from the methanogenesis cycle by diversion to biosynthetic pathways, dilution of intermediates due to growth, or imperfect coupling in electron bifurcation as proposed previously (1). Electron bifurcation still accounts for the stoichiometric flow of electrons for methanogenesis. Our results therefore support the electron bifurcation model in vivo as well as demonstrating the function of Eha.

Results

Identification of an Additional H₂:F₄₂₀ Oxidoreductase Activity and Demonstration of a H₂ Requirement for Growth.

Our initial question was whether H₂ is a necessary substrate or intermediate for growth of hydrogenotrophic methanogens. Methanococcus maripaludis was an ideal species for addressing this question because it can substitute formate for H₂ and mutations are easily generated (10). In the conventional view, during growth on formate, H₂ generated from formate serves as the electron donor. Indeed, H₂ is generated from formate and recycled in a poorly understood manner (5, 11). However, if there is no direct requirement for H₂ in methanogenesis (5, 7), most of the hydrogenases encoded in the genome ought to be dispensable during growth on formate. The Hdr-associated hydrogenases (Vhu and Vhc) (Fig. 1), which provide electrons to the last reductive step of methanogenesis and potentially the first step via electron bifurcation (1, 6, 7), can be substituted by formate dehydrogenase (Fdh) during growth on formate (7). The F₄₂₀-reducing hydrogenases (Fru and Frc) generate F₄₂₀H₂ for the second and third reductive steps of methanogenesis, but the Fdh is also F₄₂₀ reducing (12, 13). The hydrogenase Hmd catalyzes the second reductive step directly with H₂, but its function is redundant with Mtd, which uses reduced F₄₂₀ for the same purpose (11). Finally, the anabolic energy-converting hydrogenase Ehb is nonessential in the presence of fixed carbon and is not required for methanogenesis (8, 9). Only Eha remains as possibly essential.

Based on the above considerations, we expected that H₂ would not be needed as an intermediate for methanogenesis from formate. Indeed, experiments with cell suspensions have already shown that rates of methanogenesis can substantially exceed rates of H₂ production from formate (5). As a further test, our approach here was to genetically remove formate-hydrogen lyase activity, so that H₂ would not be produced from formate. If growth still occurred on formate without added H₂, then H₂ was not a required intermediate. Because Fdh is F₄₂₀ reducing, removal of formate-hydrogen lyase activity amounts to the removal of F₄₂₀:H₂ oxidoreductase activity. Two such activities are known, the direct Fru or Frc activity and the Hmd-Mtd cycle (Fig. 1) (11). Therefore, deletion of fru, frc, and hmd should eliminate both modes of formate-hydrogen lyase activities. However, cell suspensions of a ∆fru∆frc∆hmd mutant (MM1290, henceforth designated ∆3H₂ase) still produced substantial H₂ from formate (Fig. 2). Furthermore, the mutant grew not only on formate as predicted, but also on H₂, albeit poorly (Fig. 3A). Because F₄₂₀H₂ is essential for methanogenesis, a third pathway must exist for F₄₂₀ reduction by H₂.

Fig. 2.

H₂ production by cell suspensions in the absence or presence of formate. Values in x axis are in minutes.

Fig. 3.

Requirements of the ∆3H₂ase (A) and ∆5H₂ase (B) mutants for H₂ and formate for growth. For growth of the ∆5H₂ase mutant on H₂ and formate, 14.3 μmoles of H₂ was added.

In a further attempt to remove F₄₂₀:H₂ oxidoreductase activity, vhu and vhc were deleted in the ∆3H₂ase background, resulting in strain MM1289 containing deletions in five hydrogenases (∆fru∆frc∆hmd∆vhu∆vhc, ∆5H₂ase). This strain required both formate and H₂ for growth (Fig. 3B). This result suggested that F₄₂₀:H₂ oxidoreductase activity had been reduced to below the level needed to support growth on H₂ alone. The low level of F₄₂₀:H₂ oxidoreductase activity in ∆5H₂ase was verified by low H₂ production from formate compared with wild type and ∆3H₂ase in cell suspensions (Fig. 2). The third F₄₂₀:H₂ oxidoreductase activity is evidently Vhu/Vhc dependent and represents a previously uncharacterized electron flow pathway in methanogenic Archaea. Although further experiments are needed to characterize this pathway, it could involve Vhu/Vhc, Hdr, and Fdh, which is F₄₂₀ reducing and like Vhu/Vhc is Hdr associated. The H₂ requirement of the ∆5H₂ase mutant demonstrates that, contrary to our initial expectation, H₂ is a required intermediate during growth on formate and H₂ is indeed required for growth of hydrogenotrophic methanogens.

H₂ Requirement Is Quantitatively Low.

It was unclear whether the H₂ that is required for growth of the ∆5H₂ase mutant supports the catabolic process of methanogenesis or the anabolic process of CO₂ fixation. Cultures of M. maripaludis grown on our formate medium use 1 mmole of formate (11). Therefore, if H₂ were required for just one reductive step of methanogenesis, the amount of H₂ needed would be ∼0.33 mmoles. However, we observed maximum growth with H₂ as low as 10–15 μmoles (Fig. 4). At this level, H₂ cannot be a substantial electron donor for methanogenesis. Instead, the H₂ requirement may be anabolic and/or anaplerotic (see below). In fact, during autotrophic growth, about 35 μmol of H₂ was required for each mg of cell dry weight formed in the ∆5H₂ase mutant (Fig. 4) (for M. maripaludis, milligrams of dry weight/OD₆₆₀/milliliter = 0.34) (5). Under these conditions, cells require 10.7 μmol of pyruvate and 3.9 μmol of acetate per milligram of cell dry weight for autotrophic growth (14). In methanogens, acetate biosynthesis requires two pairs of low potential electrons, one for formation of formyl-MFR and one for the acetyl-CoA synthase step. Pyruvate is formed from acetyl-CoA and requires one additional pair of low potential electrons. Thus, about 40 μmol of low potential electron pairs per milligram of cell dry weight are required for autotrophic growth, close to the value observed. If the H₂ was required for generation of low potential electrons for anabolism, the addition of carbon sources to the medium should decrease the amount of H₂ needed for growth. In fact, in the presence of acetate and acetate plus casamino acids, the amount of H₂ required decreased to 17 and 8 μmol of H₂ per milligram of cell dry weight, respectively (Fig. 4). Therefore, the H₂ requirement appears at least partially anabolic.

Fig. 4.

H₂ dose–response of ∆5H₂ase mutant. Clear bars, mineral medium; checkered bars, 10 mM acetate added; solid bars, 10 mM acetate and casamino acids (0.2% wt/vol) added. All cultures contained 200 mM formate. Five-milliliter cultures were incubated until stationary phase and OD₆₆₀ was measured.

H₂ Is Not Required for Methanogenesis in Vitro but Stimulates Methanogenesis in Cell Suspensions.

We performed two additional experiments to further examine the nature of the H₂ requirement. For both experiments, first Ehb was genetically eliminated from the ∆5H₂ase mutant background to generate strain MM1284, which contained deletions in six hydrogenases (∆fruA∆frcA∆hmd∆vhuAU∆vhcA∆ehbN, ∆6H₂ase). Eha was the sole remaining hydrogenase. Similar to the ∆5H₂ase mutant, ∆6H₂ase required H₂ as well as formate for growth (Fig. S1). In the first experiment, in vitro CH₄ production assays were performed (Fig. 5). These assays followed published reports (4, 15), which show that CH₄ production from CO₂ in vitro requires stimulation by the intermediate CH₃-S-CoM and that the yield of CH₄ is limited by the CH₃-S-CoM added. In our assays, CH₃-S-CoM was added and CH₄ production continued presumably until CH₃-S-CoM was depleted. CH₄ production was measured in extracts of ∆6H₂ase mutant or wild-type cells with either H₂ or formate as electron donor. Extract of wild-type M. maripaludis produced substantial CH₄ from CO₂ with either electron donor. In contrast, the mutant extract produced substantial CH₄ only from formate. H₂ had no stimulatory effect on CH₄ production from formate (Fig. S2).

Fig. 5.

Methanogenesis in cell extracts from CH₃-S-CoM and CO₂ using H₂ (solid line) or formate (dashed line) as the electron donor in cell extracts from (A) wild-type or (B) ∆6H₂ase mutant. Each reaction contained 300 nmols of CH₃-S-CoM and 200–350 μg of protein. Extracts with no CH₃-S-CoM added are represented by open diamonds.

Next, we assayed CH₄ production by cell suspensions of the ∆6H₂ase mutant. With cell suspensions, stimulation by an intermediate in the pathway was not needed and CH₃-S-CoM was not added. Methanogenesis occurred with formate but not with H₂. Significantly, methanogenesis was greatly enhanced by H₂, either present initially or added during the course of the assay (Fig. 6). Hence, H₂ did not contribute to methanogenesis in vitro where the pathway intermediate CH₃-S-CoM was added, but H₂, presumably acting through Eha, stimulated methanogenesis in cell suspensions.

Fig. 6.

CH₄ production by cell suspensions of the ∆6H₂ase mutant. Black diamonds, H₂ alone; open triangles, formate alone with (+) or without H₂ addition [8% (vol/vol) final concentration] at 60 min; black triangles, H₂ and formate.

Eha Is Essential for Growth of M. maripaludis.

To test whether Eha is essential, mutagenesis of ehaHIJ was attempted. The genes ehaH, I, and J encode the presumed cation translocator of the enzyme complex. Each is homologous to a portion of ehbF, for which a null allele has a strong phenotype (9). In preliminary experiments, the construction of an ehaHIJ allelic replacement with a puromycin resistance cassette was unsuccessful. An additional test of essentiality was sought. As before (11), our strategy was to determine whether negative selection to resolve a merodiploid would result in a deletion allele. All other things equal, if there is no growth disadvantage for a null allele, deletion mutants should arise with roughly the same frequency as wild-type alleles, and this occurred in a control experiment where a ehbF⁺-ΔehbF merodiploid was resolved (Table 1). However, resolution of the ehaHIJ⁺-ΔehaHIJ merodiploid in standard medium with H₂ resulted in only wild-type clones. Similar results were obtained when formate rather than H₂ was used. Eha could be involved in 2-ketoglutarate biosynthesis, because 2-ketoglutarate oxidoreductase depends on Fd (9), and high levels of glutamate in the medium (10 mM) could provide sufficient 2-ketoglutarate and remove the requirement for Eha. Alternatively, Eha might be involved in NAD⁺ reduction, and alanine dehydrogenase in methanococci generates NADH (16). However, when glutamate or alanine was added, still no mutations were obtained. In contrast, when the ehaHIJ mutagenesis experiment was performed in the presence of trans-complementation (Pnif-ehaHIJ), the majority of clones contained the deletion. These results strongly suggest that Eha is essential, consistent with the H₂ requirement for growth of the ∆6H₂ase mutant.

Table 1.

Resolution of merodiploids of ehb or eha

	No. of clones
Merodiploid	WT	Mutant
ΔehbF	26	34
ΔehaHIJ	108	0
ΔehaHIJ, formate	24	0
ΔehaHIJ, glu	12	0
ΔehaHIJ, ala	38	0
ΔehaHIJ + Pnif-ehaHIJ	5	52

Number of clones with the wild-type (WT) or mutant allele following resolution of the merodiploids is given. Resolution was performed in complex medium with the indicated additions or trans-complementation by the plasmid expressing ehaHIJ from the P_nif promoter.

Discussion

Distinct Electron Pools Function in Hydrogenotrophic Methanogens.

Until recently, it was not known whether any of the hydrogenase activities in hydrogenotrophic methanogens could be eliminated. However, in past work we reported that some of these hydrogenases were unnecessary under some conditions. Thus, in separate strains we deleted genes encoding the F₄₂₀-reducing hydrogenases (11), the Hdr-associated hydrogenases (7), and the hydrogen-using methylene-H₄MPT dehydrogenase (11). Here we eliminated all three of these hydrogenase activities in a single strain (∆5H₂ase) and found that both formate and H₂ were required for growth, the former in quantities stoichiometrically sufficient for methanogenesis, and the latter in much smaller quantities. The mutant effectively separates two pools of electrons that ordinarily exchange via H₂. One pool of electrons provides a stoichiometric supply of electrons for methanogenesis and flows through F₄₂₀ and Hdr (Fig. 1). In the wild-type strain, either formate or H₂ functions as electron donor for this pool. The ∆5H₂ase and ∆6H₂ase mutants, by eliminating H₂:F₄₂₀ oxidoreductase activities, disrupt electron flow from H₂ and, as a result, formate is required as the stoichiometric electron donor for methanogenesis. The other pool of electrons supports biosynthesis, and as demonstrated here, anaplerotically replenishes methanogenesis (see below). This pool is carried by ferredoxins, and only H₂ functions as electron donor. In the wild-type strain, H₂ produced from formate allows the latter to function as sole electron donor. However, in the ∆5H₂ase and ∆6H₂ase mutants where electron flow between the two pools is blocked, H₂ must be provided.

Function of Eha Is Essential, Anaplerotic, and Ancillary to Electron Bifurcation.

In the ∆5H₂ase mutant, two hydrogenases remain, Eha and Ehb. Previous work has suggested that the role of Ehb is the reduction of Fd for anabolic CO₂ fixation via acetyl-CoA synthase, pyruvate oxidoreductase, 2-ketoglutarate oxidoreductase, indole-pyruvate oxidoreductase, and 2-oxoisovalerate oxidoreductase (8, 9). Eha may play a role analogous to a related energy converting hydrogenase, Ech, which in methanogens with cytochromes generates reduced Fd for CO₂ reduction to formyl-MFR (2). Because Eha and Ehb have different functions, they may reduce different ferredoxins. Although not yet proven biochemically, Eha could have specificity for a polyferredoxin associated with formylmethanofuran dehydrogenase (Fwd), the enzyme that catalyzes CO₂ reduction to formyl-MFR (17), whereas Ehb could reduce a Fd associated with anabolic CO₂ fixation reactions.

In methanogens without cytochromes, if the role of Eha is to reduce CO₂ to formyl-MFR, how is this reconciled with electron bifurcation as the main pathway for delivery of electrons for the same step? Our data show that Eha needs to provide only a small portion of the electrons for this reaction. In the ∆5H₂ase mutant, formate provided nearly all electrons for methanogenesis and the H₂ requirement accounted for only up to 4% of the electrons for growth, much of this apparently for anabolic purposes. We propose that Eha functions in the reduction of CO₂ to formyl-MFR, but does so anaplerotically. In the electron bifurcation model, hydrogenotrophic methanogenesis is a cyclic pathway where the first step is dependent on the last step (1). However, this model presents a dilemma. A constant pool of CoM-S-S-CoB is required, yet intermediates in methanogenesis will inevitably be diluted by growth and cell division, or lost due to a leaky electron bifurcating Hdr complex (1, 6). In addition, intermediates will diminish when methyl-H₄MPT is diverted from methanogenesis to generate acetyl-CoA for autotrophic CO₂ fixation (18). Our results show that Eha solves this dilemma by priming or recharging the cycle: it anaplerotically restores intermediates to the methanogenic pathway at the level of formyl-MFR. This model accounts for all of the following observations. First, only small amounts of H₂ are required for growth. Second, Eha is essential even though electron bifurcation can account for a stoichiometric supply of electrons for methanogenesis. Third, even though H₂ and Eha are essential, there is no need for H₂ for methanogenesis in an in vitro assay where the intermediate CH₃-S-CoM is added. Finally, for methanogenesis in cell suspensions where no intermediate is provided, H₂ is stimulatory.

Why do most hydrogenotrophic methanogens, including M. maripaludis, maintain two ion-translocating energy-converting hydrogenases that reduce Fd and provide electrons to fix CO₂? One such hydrogenase could suffice, and indeed, Eha can apparently recognize the Ehb-type ferredoxin and substitute for Ehb, albeit inefficiently (8, 9). Having both hydrogenases separates the recharging of methanogenesis from other anabolic activities and may optimize control over the separate processes. When conditions limit growth, anabolic CO₂ fixation is unimportant but CO₂ reduction to formyl-MFR for methanogenesis and ATP synthesis are still necessary for survival. Under these conditions, functional Eha is essential, and a functional Ehb could be detrimental.

Electron Flow and Energy Conservation in Hydrogenotrophic Methanogens.

Electron bifurcation at Hdr explains a decades-old dilemma regarding methanogenesis: How is net energy conservation achieved in hydrogenotrophic methanogens (1)? The results presented here verify that electron bifurcation must function in vivo and elaborate on the mechanisms that allow this to be the case, filling in the known gaps of a pathway that has been incomplete since methanogens were first discovered and grown in culture (19). A complete model for electron flow in methanogens without cytochromes can now be described: Methanogenesis is dependent upon F₄₂₀-reducing enzymes and enzymes that feed electrons to Hdr for electron bifurcation. During growth on H₂, these enzymes are the F₄₂₀-reducing hydrogenases Fru and Frc and the Hdr-associated hydrogenases Vhu and Vhc. During growth on formate, both kinds of hydrogenases are unnecessary and Fdh performs both functions. Biochemical experiments have demonstrated CoM-S-S-CoB-dependent reduction of a clostridial Fd with H₂ (6), but are still needed to prove that electrons flow from H₂ or formate to Fwd concurrent with flow to Hdr. Nevertheless, in previous work, we showed that in M. maripaludis, Fdh as well as Vhu exist in a complex with Hdr, and that Fwd is in this complex as well (7). Hence, an enzyme complex exists that is suited for electron bifurcation with either H₂ or formate. Fdh, either complexed or existing in an isolated form, also generates F₄₂₀H₂. Through electron bifurcation at Hdr and F₄₂₀ reduction, formate provides the reducing equivalents to all four reductive steps of methanogenesis. A separate electron pool supports anabolism, which depends on electrons from H₂ entering through Ehb and its associated Fd (8, 9). These isolated inputs keep the electron pools for catabolism and anabolism separated and under different regulatory control. Overlap between the two electron pools occurs when electrons from H₂ enter methanogenesis through Eha. When low CoM-S-S-CoB concentrations limit the reduction of CO₂ to formyl-MFR by electron bifurcation, Eha recharges methanogenesis.

Materials and Methods

Construction and Growth of Strains.

Unless otherwise stated, strains were grown in medium containing casamino acids and acetate, with H₂ or formate as the electron donor (11). Strains and plasmids are shown in Table S1. Strain Mm901 was used as the wild-type strain unless otherwise stated. To construct plasmids pCRupt∆fruneo, pCRupt∆frcneo, and pCRupt∆hmdneo, insert DNA from plasmids pCRprt∆fruneo, pCRprt∆frcneo, and pCRprt∆hmdneo (11) was recloned into pCRuptneo (7). To construct plasmids pCRupt∆fruGBneo, pCRupt∆frcGBneo, and pCRupt∆ehbNneo, ∼0.5 kb of DNA upstream and downstream of the designated loci was obtained by PCR from genomic DNA and cloned into pCRuptneo. Primers are shown in Table S2. In each case, the resulting plasmid contained an in-frame deletion consisting of a start codon and a stop codon with intervening codons contained within an AscI site. Plasmid DNA was used to make deletions using a markerless mutagenesis method with neomycin for positive selection and 6-azauracil for negative selection as described (7, 20). When formate was present as electron donor, neomycin was increased to 5 mg/mL on liquid or solid medium (7). MM1290 (Mm901ΔfruAGBΔfrcAGBΔhmd, Δ3H₂ase) was constructed by sequential deletion of frcA, hmd, fruA, fruG and B, and frcG and B. The first deletion was constructed using H₂ as the growth substrate, and the remaining deletions used formate. MM1313 (MM901∆fruA∆frcA∆vhuAU∆vhcA, Δ4H₂ase) was constructed from Mm1272 (MM901∆vhuAU∆vhcA) (7) by deletion of fruA and frcA using formate. MM1289 (MM901∆fruA∆frcA∆hmd∆vhuAU∆vhcA, Δ5H₂ase) was then constructed by deleting hmd. Finally, MM1284 (MM901∆fruA∆frcA∆hmd∆vhuAU∆vhcA∆ehbN, Δ6H₂ase) was constructed by deleting ehbN. During construction of the Δ5H₂ase and Δ6H₂ase mutants, cultures were grown with formate and H₂. All deletions were confirmed by PCR.

Essentiality of Eha.

Merodiploids were constructed containing deletions of the ehaHIJ genes and ehbF (Fig. S3 and Table S1), and the generation of deletion mutants by recombination-based resolution of the merodiploids was attempted using wild-type strain Mm900 as described (20). Among the resulting clones, those containing deletion mutations and wild-type alleles were distinguished by PCR. A trans-complementing plasmid (pMEV1nif::ehaHIJ) (Table S1) was constructed on a replicative vector with ehaHIJ under control of the nitrogen-regulated nif promoter (21, 22). To test the effect of trans-complementation, merodiploids containing this plasmid were plated with alanine or ammonia, and the merodiploids were resolved. In preliminary experiments, ehaHIJ deletion strains could be obtained with either nitrogen source, and ammonia was used henceforth.

H₂ and CH₄ Production.

To measure H₂ production by cell suspensions, cultures (5 mL) were grown on formate to OD₆₆₀ between 0.5 and 0.6, and cells were pelleted, washed, and resuspended anaerobically in the same volume of assay buffer (modified from ref. 5, 50 mM Mops pH 7.0, 400 mM NaCl, 20 mM KCl, 20 mM MgCl₂, 1 mM CaCl₂, 5 mM DTT, and 1 mM bromoethanesulfonate). Tubes were then flushed with N₂ for 10–30 min to remove residual H₂. At time = 0 min, an initial gas sample was taken, then the assay was initiated by addition of 40 mM (final concentration) sodium formate (pH 7) or NaCl and incubated at 37 °C with shaking. At 15-min and 30-min time points, the headspace was sampled and transferred to butyl rubber stoppered 5-mL vials, mouth ID 13 mm × OD 20 mm (Wheaton; catalog no. 223685) preflushed with N₂. H₂ was analyzed with a SRI Instruments gas chromatography (GC) model 8610C equipped with a 6 foot × one-eighth inch stainless steel Molecular Sieve 5A packed column and a reduced gas detector. The carrier gas was He (20 psi), oven temperature was 130 °C, and detector temperature was 290 °C. CH₄ production by cell suspensions was measured as above except cells were grown to mid-log phase (OD₆₆₀ ∼0.25), bromoethanesulfonate was omitted from the assay buffer, and the headspace was directly analyzed on a Buck Scientific model 910 GC equipped with a flame ionization detector provided with air (16 psi) and H₂ (26 psi). The carrier gas was He (24 psi). To measure CH₄ production by cell-free extracts, cells were washed and suspended in buffer (modified from ref. 4) 100 mM Trizma base, 15 mM MgCl₂, 5 mM ATP, 2 mM 2-mercaptoethanol, 500 μM FAD⁺, with or without 50 mM formic acid, and pH adjusted to 7.1 with 1 M HCl). The suspension was sonicated with a Misonix XL-2000 series sonicator to disrupt the cells. Debris was removed by centrifugation at 16,000 × g, and 200 μL supernatant was placed in a 5-mL serum vial and preincubated for 10 min at room temperature. To start the assay, the headspace was flushed with N₂:CO₂ (80:20, for formate) or H₂:CO₂ (80:20), and 1.5 mM CH₃-S-CoM was added (4). Methanogenesis was monitored as described above.