The Hydrogen Economy of Methanosarcina barkeri: Life in the Fast Lane

Two recent studies (T. D. Mand, G. Kulkarni, and W. W. Metcalf, J. Bacteriol 200:e00342-18, 2018, https://doi.org/10.1128/JB.00342-18, and G. Kulkarni, T. D. Mand, and W. W. Metcalf, mBio 9:e01256-18, 2018, https://doi.org/10.1128/mBio.01256-18) analyzed an impressive array of hydrogenase-deficient mutant strains of Methanosarcina barkeri not only to describe H₂-based growth but also to demonstrate the conservation of energy with intracellular hydrogen cycling, a novel strategy for creating a proton motive force to support ATP synthesis.

ABSTRACT

Two recent studies (T. D. Mand, G. Kulkarni, and W. W. Metcalf, J. Bacteriol 200:e00342-18, 2018, https://doi.org/10.1128/JB.00342-18, and G. Kulkarni, T. D. Mand, and W. W. Metcalf, mBio 9:e01256-18, 2018, https://doi.org/10.1128/mBio.01256-18) analyzed an impressive array of hydrogenase-deficient mutant strains of Methanosarcina barkeri not only to describe H₂-based growth but also to demonstrate the conservation of energy with intracellular hydrogen cycling, a novel strategy for creating a proton motive force to support ATP synthesis.

Jack of all trades, master of none, though often better than a master of one.

—Quote of uncertain origin

TEXT

Methanosarcina barkeri occupies a unique niche among methanogens. Most other intensively investigated methanogens specialize in the use of H₂-formate as the electron donor for methane production (1). Methanothrix (formerly Methanosaeta) species eschew H₂-formate and are highly effective in converting acetate to methane (2). Even some other Methanosarcina species have opted out of metabolizing H₂ and focus on consuming acetate and methyl substrates, such as methanol and methylamines (3).

In contrast, M. barkeri is an omnivore. It can grow on H₂-CO₂, acetate, methanol, and methylamines (3). Further along the path not taken, M. barkeri metabolizes H₂ with a strategy different from that typically employed by the H₂ specialists and conserves energy from acetate metabolism differently than the acetate specialists (1, 2). These unique functions all revolve around how M. barkeri interacts with H₂.

Now in a true tour de force, Thomas D. Mand, Gargi Kulkarni, and William W. Metcalf have made an amazing collection of hydrogenase deletion mutants to further evaluate how M. barkeri consumes and produces, H₂ (4, 5). Every possible combination of hydrogenase mutants was constructed, including conditional strains in which expression of a hydrogenase required for growth under all known conditions could be controlled with an inducer. Mand et al. found that two putative hydrogenases are not important in H₂ production or consumption (5). The remaining three have distinct and important roles. These are the hydrogenases designated Ech, Vht, and Frh (Table 1). Analysis of the growth, gene expression, and methane and H₂ production phenotypes of the mutant strains provide new insights into intracellular H₂ cycling as well as give stronger support for previous concepts about M. barkeri's H₂ metabolism (4, 5).

TABLE 1.

The three functional hydrogenases in Methanosarcina barkeri reported in reference 5 to be required for growth on H₂-CO₂ and their role (4) in intracellular hydrogen cycling^a

Name	Reaction catalyzed	Location	Role in IHC
Ech	Fdred + 2 H+ ↔ Fdox + H2 + (2 H+ pumped out)	Membrane	H2 production
Vht	H2 + MP → 2 H+ + MPH2	Membrane	H2 oxidation
Frh	F420H2 ↔ F420 + H2	Cytoplasm	H2 production

^aFd_red, reduced ferredoxin; Fd_ox, oxidized ferredoxin; MP, methanophenazine.

The results have broad significance. M. barkeri is not just an easy-to-culture “lab rat” microbe. Molecular studies routinely identify Methanosarcina species that are closely related to M. barkeri in diverse methanogenic environments. These include methanogenic soils and sediments that are important sources of atmospheric methane (see reference 6 for a recent example), as well as anaerobic digesters converting organic wastes to methane, an important bioenergy process (7, 8). Thus, the enhanced insights into the mechanisms by which M. barkeri conserves energy from diverse substrates that are available from these new studies (4, 5) are helpful in understanding the ecology and practical applications of this widely distributed methanogen.

One of the most significant questions that Kulkarni, Mand, and Metcalf addressed with their mutants was the possibility that M. barkeri uses hydrogen (H₂, H⁺) cycling as a strategy for energy conservation (4, 5). The general concept of hydrogen cycling, first introduced by Odom and Peck in studies on Desulfovibrio species (9), is that a proton gradient can be generated across the cell membrane by producing H₂ in the cytoplasm and then oxidizing that H₂ on the outer surface of the membrane (Fig. 1). The H₂ production consumes two protons and the H₂ oxidation releases two protons, resulting in a proton gradient across the membrane. ATP synthase captures the energy in this gradient as ATP. This intracellular cycling of hydrogen is much different from the extracellular hydrogen cycling of interspecies H₂ transfer, in which H₂ functions as an external electron and H⁺ carrier between two microorganisms syntrophically metabolizing substrates (10, 11). In order to clearly distinguish the two types of hydrogen cycling, the hydrogen cycling of Odom and Peck origin is referred to here as intracellular hydrogen cycling (IHC) (9).

FIG 1.

Generalized model for energy conservation with internal hydrogen cycling (IHC). red, reduced; ox, oxidized.

The concept of IHC in Desulfovibrio has been contentious. After many years of study, the current consensus is that IHC might take place in some sulfate reducers, but even in those sulfate reducers there are alternative pathways for energy conservation that function concurrently with IHC, muddling the interpretation of almost any experiment (12, 13). The beauty of the studies of IHC in M. barkeri is that there appears to be one growth condition (metabolism of acetate) in which H₂ is the only way out (of the cytoplasm) for electrons (4).

Early studies speculated that H₂ cycling might be a mechanism for energy conservation in Methanosarcina, based on the production and consumption of H₂ during growth on acetate, methanol, and trimethylamine (14). The discovery of the Ech hydrogenase suggested that M. barkeri could produce H₂ from the reduced ferrodoxin generated during acetate metabolism (15). A mutant that lacked the Ech hydrogenase could not grow on acetate, providing in vivo evidence that H₂ production via Ech is essential in order to metabolize acetate (16). However, an essential role or pathway for H₂ uptake had not been demonstrated.

Kulkarni and colleagues found that cells could not grow on acetate in the absence of Vht (4), which oxidizes H₂ external to the membrane with the reduction of the membrane-bound electron carrier methanophenazine (see reference 5 and references therein). Reduced methanophenazine is an electron donor for the membrane-bound heterodisulfide reductase (HdrDE), which carries out the terminal step in electron transport, reducing coenzyme M (CoM)-S-S-coenzyme B (CoB) to regenerate the reduced forms of the two cofactors (Fig. 2). The requirement for Vht is clear evidence that H₂ uptake is necessary for growth on acetate.

FIG 2.

Model for M. barkeri energy conservation with internal hydrogen cycling (IHC) during acetate metabolism, based on results presented elsewhere (4) and previously described (2) Methanosarcina barkeri physiology. Fd_red, reduced ferredoxin; Fd_ox, oxidized ferredoxin; CoA, coenzyme A; MP, methanophenazine; H₄SPT, tetrahydrosarcinapterin.

Thus, the model for IHC during growth on acetate (4) starts with Ech oxidizing the reduced ferrodoxin produced in the initial steps of acetate metabolism (Fig. 2). Ech pumps two protons across the membrane during this reaction, and two additional cytoplasmic protons are consumed in the Ech-catalyzed production of H₂. Vht captures H₂ as it diffuses across the membrane. Vht oxidation of H₂ releases two protons outside the membrane. Vht reduces methanophenazine, consuming two protons from the cytoplasm (Fig. 2). The HdrDE complex oxidizes the reduced methanophenazine with the release of two protons outside the membrane and the reduction of CoM-S-S-CoB (Fig. 2). The initial activation of acetate requires the input of an ATP, but the proton flux during IHC more than compensates for that investment, providing net energy to support growth (Fig. 2).

IHC during growth on methanol is more complex than that during growth on acetate because, in addition to reduced ferrodoxin, the deazaflavin cofactor F₄₂₀ is also reduced in the methanol pathway. The cytoplasmic hydrogenase Frh oxidizes reduced F₄₂₀ with the production of H₂ (see reference 5 and references therein). During growth on methanol, a mutant lacking Frh produced substantially less H₂ than the wild type, suggesting that the H₂ produced by Frh was an intermediate in methanol metabolism (4). H₂ continuously accumulated during methanol metabolism in a conditional Vht strain in the absence of the inducer for Vht expression. This demonstrated the important role of Vht in capturing the H₂ that Frh produced (4). Thus, during growth on methanol there is the additional IHC step in which Frh uses electrons from methanol oxidation to consume protons in the cytoplasm with the production of H₂ and Vht oxidizes the H₂ to release the protons on the outer surface of the membrane.

M. barkeri can grow on methanol without IHC (5). It adapts by employing alternative pathways for oxidizing reduced ferrodoxin and F₄₂₀. Growth of the mutant is poor, indicating that IHC is the preferred route for methanol metabolism (4).

Analysis of growth and methane production confirmed the requirement of all three hydrogenases for the metabolism of H₂-CO₂ (4). Transcriptome sequencing analysis indicated that there was not a compensatory increase in the expression of hydrogenase genes in response to the deletion of other hydrogenase genes, making it possible to roughly assess the relative activity of the three hydrogenases from the activities in various combinations of mutants. Frh may be responsible for up to 75% of the hydrogenase activity, with Ech contributing only ca. 4% and Vht contributing the remainder.

M. barkeri conserves substantially more energy from H₂-CO₂ conversion to methane than H₂ specialists (1). This can be attributed to the cooperative activity of Vht and HdrDE that generates a proton gradient across the cell membrane (Fig. 2) in M. barkeri, whereas H₂ specialists employ a cytoplasmic system for reducing CoM-S-S-CoB with H₂ (1). However, the higher energy conservation comes at a cost: the minimum threshold concentration for M. barkeri uptake of H₂ is about an order of magnitude higher than it is for the H₂ specialists (1). In a similar manner, M. barkeri has higher growth yields on acetate than the acetate specialist Methanothrix because M. barkeri invests less energy in acetate activation, but the threshold concentration at which it can metabolize acetate is much higher (2, 17).

Why does M. barkeri make these trade-offs? M. barkeri is unlikely to be very competitive in low-energy environments in which the methanogens that can consume H₂ or acetate at the lowest concentrations will outcompete M. barkeri with its lower affinity for these substrates. Rather, M. barkeri's omnivorous appetite seems designed for life in the fast lane—when fermentative bacteria are rapidly spitting out H₂ and acetate. Under these conditions, a generalist that can use both H₂ and acetate, as well as any methanol and methylamines released, with high ATP yields is likely to win the day. This may explain why Methanosarcina species are typically abundant in wastewater digesters under stress conditions (7, 8), when H₂ and acetate are likely to be well above the nanomolar (H₂) and micromolar (acetate) steady-state levels found in more well-balanced systems with lower energy inputs, such as some aquatic sediments.

In addition to its high thresholds for H₂ and acetate, M. barkeri engages in IHC, which is not particularly adaptive when extracellular H₂ concentrations are low and M. barkeri is surrounded by competitors with much higher H₂ affinities trying to steal its lunch. However, this disadvantage is minimized in non-steady-state environments where H₂ concentrations are higher. Furthermore, the tendency for M. barkeri and close relatives to self-aggregate may limit H₂ losses (18). Notably, M. acetivorans, a marine isolate that does not aggregate, does not rely on IHC, possibly because of the much lower H₂ concentrations found in most marine sediments (1). The growth rates and yields of M. barkeri and M. acetivorans on acetate, the most important methanogenic substrate in most environments, are comparable (19), suggesting no clear energetic advantage to IHC. One reason why M. barkeri has stuck with IHC may be that if it is going to express hydrogenases anyway, in order to consume H₂ for CO₂ reduction, it is energetically advantageous to use the same enzymes for energy conservation during acetate and methanol metabolism.

Being the generalist that it is, M. barkeri is also able to accept electrons for carbon dioxide reduction through direct interspecies electron transfer (DIET) (20, 21). It is as yet unknown how electrons enter M. barkeri during DIET, but it seems unlikely that hydrogenases are involved in the initial electron uptake into the cell or that IHC is required for energy conservation. The M. barkeri hydrogenase mutants now available (5) will facilitate testing of those assumptions.

One of the mysteries of DIET that needs to be resolved is how electrons that are presumably delivered to the M. barkeri membrane can be transferred to the cytoplasmic electron carrier F₄₂₀. Reduced F₄₂₀ is required for the conversion of carbon dioxide to methane. The Frh-deficient strain that Mand et al. constructed (5) faced the same challenge when asked to produce methane from H₂-CO₂. Although it could not grow on H₂-CO₂, cell suspensions made methane (5). A likely explanation is that Fpo, the membrane-bound F₄₂₀ dehydrogenase that is generally considered to oxidize reduced F₄₂₀, with the reduction of methanophenazine, can run in the reverse direction to produce reduced F₄₂₀ (5). Fpo functioning in this direction could also be a route for cells receiving electrons via DIET to generate reduced F₄₂₀.

Although M. barkeri was discovered over 70 years ago (22, 23), previously unrecognized environmental activities and practical applications for Methanosarcina species are still being proposed, some just within the last couple of months (24–26). The detailed and careful genetic approaches in the recent publications by Kulkarni, Mand, and Metcalf (4, 5) are exemplars for how to dissect these new physiologies.

The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.