Formate and hydrogen in hydrothermal vents and their use by extremely thermophilic methanogens and heterotrophs

Extremely thermophilic methanogens in the Methanococci and heterotrophs in the Thermococci are common in deep-sea hydrothermal vents. All Methanococci use H2 as an electron donor, and a few species can also use formate. Most Methanococci have a coenzyme F420-reducing formate dehydrogenase. All Thermococci reduce S0 but have hydrogenases and produce H2 in the absence of S0. Some Thermococci have formate hydrogenlyase (Fhl) that reversibly converts H2 and CO2 to formate or an NAD(P)+-reducing formate dehydrogenase (Nfd). Questions remain if Methanococci or Thermococci use or produce formate in nature, why only certain species can grow on or produce formate, and what the physiological role of formate is? Formate forms abiotically in hydrothermal fluids through chemical equilibrium with primarily H2, CO2, and CO and is strongly dependent upon H2 concentration, pH, and temperature. Formate concentrations are highest in hydrothermal fluids where H2 concentrations are also high, such as in ultramafic systems where serpentinization reactions occur. In nature, Methanococci are likely to use formate as an electron donor when H2 is limiting. Thermococci with Fhl likely convert H2 and CO2 to formate when H2 concentrations become inhibitory for growth. They are unlikely to grow on formate in nature unless formate is more abundant than H2 in the environment. Nearly all Methanococci and Thermococci have a gene for at least one formate dehydrogenase catalytic subunit, which may be used to provide free formate for de novo purine biosynthesis. However, only species with a membrane-bound formate transporter can grow on or secrete formate. Interspecies H2 transfer occurs between Thermococci and Methanococci. This and putative interspecies formate transfer may support Methanococci in low H2 environments, which in turn may prevent growth inhibition of Thermococci by its own H2. Future research directions include understanding when, where, and how formate is used and produced by these organisms in nature, and how transcription of Thermococci genes encoding formate-related enzymes are regulated.


Introduction
It was estimated that 40% of bacterial and archaeal global biomass is found in the rocky portion of the ocean crust below ocean sediments ( Bar-On et al., 2018;Fleming and Wuertz, 2019). These microbes live in cracks and pores of the rocky subseafloor in the absence of sunlight and often in the absence of oxygen and rely on the gases, aqueous compounds (e.g., sulfide, sulfate, and nitrate), organic compounds, and minerals found locally for growth. In high-temperature anoxic environments, H 2 is generally considered to be the primary electron donor and CO 2 the primary carbon source for autotrophic metabolism. However, recently other electron donors and carbon sources such as formate have been considered as alternatives (Windman et al., 2007), especially in high pH environments where dissolved inorganic carbon precipitates as calcium carbonate and is largely unavailable to autotrophs (Lang et al., 2018;McGonigle et al., 2020;Brazelton et al., 2022). There are strong links between formate and H 2 in hydrothermal environments and in the physiology of microbes that consume and produce formate and H 2 .
High-temperature microbes that use formate and H 2 are examined herein, namely methanogens (in the class Methanococci and the class Methanopyri) and heterotrophs (in the class Thermococci). These organisms are found in deepsea hydrothermal vents on or near tectonic plate boundariesboth mid-ocean ridges and subduction zones. Thermophiles and hyperthermophiles are defined as those organisms with optimal growth temperatures above 50 • C and 80 • C, respectively (Stetter, 2006). In this review, the term 'extreme thermophile' will be used to describe organisms with optimal growth temperatures above 65 • C. Extremely thermophilic Methanococci and Thermococci are among the more cosmopolitan and well-studied microbes found in hydrothermal vent environments. All Methanococci and the marine hyperthermophile Methanopyrus kandleri (the sole member of the Methanopyri) use H 2 and CO 2 as energy and carbon sources to produce CH 4 , H 2 O, and biomass (Thauer et al., 2008). All Thermococci use peptides and sugars as carbon and energy sources and reduce zero-valent sulfur (S 0 ) to a sulfide species or reduce 2 H + to H 2 in the absence of S 0 (Wu et al., 2018). However, some extremely thermophilic Methanococci and Thermococci grow using formate as an energy source only or as both energy and carbon sources (Belay et al., 1986;Kim et al., 2010;Lim et al., 2014). This raises questions about which organisms can use formate, when they use formate in nature, and for what purpose. This review describes how formate and H 2 are formed in hydrothermal vents, the concentrations of these compounds in pure hydrothermal fluids, the physiology of extremely thermophilic Methanococci and Thermococci as it relates to formate and H 2 use, transcriptional regulation of formate dehydrogenase and hydrogenase genes, and suggests likely roles for formate use by these organisms in nature.

Abiotic H 2 production in hydrothermal vents
Deep-sea hydrothermal vents provide one of the best access points to the hydrothermally influenced portion of the rocky subseafloor and are ideal starting points for understanding biogeochemical processes in these regions of the crust. Some hydrothermal fluids rise through the crust undiluted, so-called "end-member hydrothermal fluid, " and exit the seafloor at temperatures generally above 300 • C ( Table 1). It can also mix with cold seawater on or below the seafloor creating habitats for extremely thermophilic anaerobes either within the host rock (e.g., basalt) or in metal sulfide mineral precipitates (e.g., black smoker chimneys). Most hydrothermal vent studies are focused on one of three types of sites: ultramafic sites along slow-to-ultraslow tectonic spreading centers, mafic sites along intermediate-to-fast spreading centers, and subduction-influenced sites near tectonic convergence zones (Figure 1).
The host rock in mafic and ultramafic sites have high concentrations of MgO and FeO, but they differ in their silica content, with ultramafic rocks having silica concentrations less than 45% (by weight), while mafic rocks have concentrations above 45%. Most abiotic formation of H 2 in hydrothermal vents occurs by hydrothermal alteration of the ultramafic rock peridotite (i.e., serpentinization) ( Table 1). Serpentinization occurs in environments with limited magma supply where peridotite is present in the rock hosting hydrothermal circulation and is mostly associated with ultramafic sites. Olivine and orthopyroxene, the most abundant minerals in peridotite, are unstable under hydrothermal conditions, which causes dissolution-reprecipitation reactions and the formation of serpentine, magnetite, and H 2 [e.g., 6 (Mg, Fe) 2 SiO 4 + 7 H 2 O → 3(Mg, Fe)Si 2 O 5 (OH) 4 + Fe 3 O 4 + H 2 ] (Klein et al., 2020). Methanococci and Thermococci are common in most ultramafic-influenced hydrothermal sites except at the Lost City hydrothermal vent field (Table 1). At Lost City, the high pH hydrothermal fluids formed by low temperature serpentinization lead to calcium carbonate precipitation and very low dissolved inorganic carbon concentrations. This likely hinders the growth of autotrophs such as Methanococci and Methanopyri unless they can grow on an aqueous carbon source such as formate.
Serpentinization is inhibited by silica and is thus less common in mafic and felsic rocks (felsic rocks are > 65% silica by weight). In mafic (basalt)-hosted hydrothermal systems, the oxidation of ferrous iron minerals, such as pyrrhotite to pyrite (FeS + H 2 S → FeS 2 + H 2 ) and magnetite to hematite (2 Fe 3 O 4 + H 2 O → 3 Fe 2 O 3 + H 2 ), and weathering of the ocean crust by oxygendepleted water in the root zone of a hydrothermal system are also significant sources of H 2 in hydrothermal systems (Klein et al., 2020). H 2 and H 2 S concentrations are controlled by chemical equilibrium between fluid and the pyrite-pyrrhotitemagnetite mineral assemblages present. Most H 2 and H 2 S fluid compositions fall close to the metastable extension of pyritepyrrhotite equilibrium (Klein et al., 2020). H 2 concentrations in mafic hydrothermal fluids also increase significantly following a volcanic eruption as circulating fluids interact with newly injected rock (Lilley et al., 2003;Seewald et al., 2003;Von Damm and Lilley, 2004). Mafic hydrothermal vent sites generally tend to have Thermococci and Methanococci present (Table 1), especially following volcanic eruptions (Holden et al., 1998;Huber et al., 2002;Meyer et al., 2013), but Methanococci can become rare or undetectable during quiescent periods between eruptions when H 2 concentrations decrease or in low H 2 hydrothermal vents (Ver Eecke et al., 2009. In contrast, hydrothermal vents that form along volcanic arcs at convergent plate boundaries have host rock with hydrous Frontiers in Microbiology 02 frontiersin.org The pH and concentrations of H2 and formate are for end-member (zero-Mg 2 + ) hydrothermal fluid while the microbial data represent presence at the site in low-temperature fluids (F) and mineral samples (M). ND, not detected; -, not analyzed. a Sometimes reported as mmol/kg or µmol/kg, respectively. b Takai et al. (2004b), Gallant and Von Damm (2006), Kumagai et al. (2008),  Gamo et al. (2004) and Nakagawa et al. (2006). Global map of the hydrothermal vents in Table 1. Hydrothermal vents hosted in ultramafic rock are shown in red; basalt, in black; and in subduction zones, in yellow-brown.
minerals, silica accumulation in aging oceanic crust, and more felsic character, such as dacite and andesite. The hydrothermal fluids from these rocks tend to have lower pH and lower H 2 ( Table 1). While Thermococci are generally present at these sites, Methanococci tend to be rare or undetectable ( (e.g., shallow vent sites) and radiolysis of water (Klein et al., 2020). Biotic sources of H 2 at extremely thermophilic temperatures by Thermococci are described in Section "4. H 2 production by Thermococci."

Hydrogenases in Methanococci and Methanopyri
The whole genome sequences of 10 extremely thermophilic Methanococci plus M. kandleri were analyzed for known hydrogenases (see Supplementary materials). All 11 of the Methanococci and Methanopyri in the genome survey have at least one of the following hydrogenase genes (see Greening et al., 2016 for a review): (1) eha and ehb operons, which encode for membrane-bound multimeric hydrogenases that couple H 2 oxidation to ferredoxin reduction and are H + /Na + driven for anaplerotic (Eha) and anabolic (Ehb) purposes (Porat et al., 2006;Lie et al., 2012); (2) an frh operon, which encodes for a soluble complex that directly couples H 2 oxidation to coenzyme F 420 reduction ; (3) an hmd gene, which encodes a soluble methylenetetrahydromethanopterin dehydrogenase that couples oxidation of H 2 to the reduction of methenyltetrahydromethanopterin in the archaeal Wood-Ljungdahl CO 2 fixation pathway ; and (4) a vhu operon, which encodes for soluble heterodisulfide reductase-linked complexes that bifurcate electrons from H 2 to heterodisulfide (coenzyme M-coenzyme B) and ferredoxin (Kaster et al., 2011). These hydrogenases are described and listed in Figure 2,  (Thauer et al., 2008). Extremely thermophilic Methanococci and Methanopyri will often have two or three copies of the genes encoding these enzymes (

Growth of Methanococci on H 2
The growth of natural assemblages of extremely thermophilic Methanococci in hydrothermal vent fluids from Axial Seamount is largely dependent on H 2 availability and temperature (Topçuoglu et al., 2016). The Monod kinetic half-saturation value (K s ) for growth of extremely thermophilic methanogens was 27-66 µM with maximum methane production rates of 24-43 fmol CH 4 produced cell −1 h −1 (Ver Eecke et al., 2012;Stewart et al., 2019). Methanocaldococcus jannaschii and Methanothermococcus thermolithotrophicum were shown to grow by interspecies H 2 transfer when grown in co-culture with Thermococcus celer, Thermococcus stetteri, and Pyrococcus furiosus (Bonch-Osmolovskaya and Stetter, 1991). When M. jannaschii was grown in monoculture at high (80-83 µM) and low (15-27 µM) H 2 concentrations and in co-culture with the hyperthermophilic H 2 producer Thermococcus paralvinellae (representing very low H 2 flux), growth and cell-specific CH 4 production rates decreased with decreasing H 2 availability (Topçuoglu et al., 2019). However, the number of cells produced per mole of CH 4 produced (i.e., cell yield) increased six-fold with decreasing H 2 indicating increased growth efficiency when growth was limited by H 2 (Topçuoglu et al., 2019). Relative to high H 2 concentrations, isotopic fractionation of CO 2 to CH 4 was 16 larger for cultures grown at low H 2 concentrations and 45-56 larger in co-culture suggesting reversal of the Wood-Ljungdahl pathway during methanogenesis with low H 2 flux (Valentine et al., 2004;Topçuoglu et al., 2019). While all four types of hydrogenases were synthesized by M. jannaschii with high and low H 2 flux, transcript levels of hmd and eha decreased with decreasing H 2 availability (Topçuoglu et al., 2019).

Hydrogenases in Thermococci
The whole genome sequences of 30 Thermococci were analyzed for known hydrogenases (see Supplementary materials). All 30 Thermococci analyzed have at least one of the following hydrogenase operons: (1) An mbh operon, which encodes for a membrane-bound hydrogenase that couples oxidation of ferredoxin to H 2 evolution with concomitant H + /Na + translocation across the membrane using antiporters (Sapra et al., 2003); (2) an sh operon, which encodes for a soluble sulfhydrogenase that couples oxidation of H 2 oxidation to the reduction of NAD(P) + (Van Haaster et al., 2008); (3) an frh operon, which encodes for cytoplasmic coenzyme F 420 reducingtype hydrogenase that oxidizes H 2 and passes electrons to a thioredoxin reductase (Jung et al., 2020); and (4) a codh operon, which encodes for a membrane-bound hydrogenase that couples oxidation of CO to H 2 evolution with concomitant H + /Na + translocation across the membrane using antiporters (Bae et al., 2012;Moon et al., 2012). These hydrogenases are described and listed in Figure 3, Table 3, and Supplementary Table 2.
All Thermococci have at least one mbh operon and all but one have at least one sh operon (Table 3). These enzymes are the core hydrogenases for Thermococci (Schut et al., 2012;Boyd et al., 2014). Twelve of the 30 Thermococci in the survey have frh operons. Five of the 30 Thermococci have codh operons. It was shown that the growth of Thermococcus sp. strain AM4 and Thermococcus onnurineus can be supported by CO with concomitant H 2 production (Sokolova et al., 2004;Bae et al., 2012;Moon et al., 2012), although the physiological role of this enzyme in Thermococcus is yet to be determined for growth in its natural environment.
The number of circles per column represents the number of times the gene(s) for that complex appears in the organism's genome.  Formate dehydrogenase (Fdh), hydrogenase, and formate transporter proteins and their reactions that are found in Methanococci and Methanopyri. Fdh catalyzes the following formate oxidation reactions: cytoplasmic reduction of coenzyme F 420 (F 420 ) and cytoplasmic reduction of coenzyme M

Growth of Thermococci with and without S 0
In Thermococci, the reduction of S 0 is the preferred route for electron disposal over the reduction of H + to H 2 . In P. furiosus, the presence of S 0 in growth media resulted in decreases in Mbh and Sh hydrogenase specific activities, each by an order of magnitude . There was an immediate downregulation of mbh and an upregulation of mbs (membrane-bound sulfane reductase) (Wu et al., 2018) and nsr (NAD(P)H:S 0 reductase) in P. furiosus when S 0 was added to growth medium (Schut et al., , 2007. A sulfur response regulator protein (SurR) was identified as the transcription factor regulating hydrogenase and sulfur responsive genes (Lipscomb et al., 2009(Lipscomb et al., , 2017). The proposed model suggests that SurR contains a redox-active cysteine disulfide that can reduce S 0 to H 2 S (Yang et al., 2010). SurR is reduced   in a redox cascade involving NAD(P)H-dependent thioredoxin reductase (TrxR) and protein disulfide oxidoreductase (Pdo) as the electron donors (Lim et al., 2017). In the absence of S 0 , SurR remains reduced, binds to GTTn 3 AAC(n 5 GTT), promotes the transcription of mbh and sh genes, and represses the expression of mbs and nsr genes (Lipscomb et al., 2009). Thermococci with the Frh hydrogenase also can reduce TrxR using H 2 as the electron donor (Jung et al., 2020).

Formate production in hydrothermal fluids
Abiotic formation of formate, carbon monoxide, methane, and hydrocarbons in hydrothermal vents is of interest as potential growth substrates for microbes. Methane and hydrocarbons in vents were suggested to form through Fischer-Tropsch type reactions [(2n + 1)H 2 + nCO → C n H 2n+2 + nH 2 O] or leach from fluid inclusions in plutonic rocks (Berndt et al., 1996;Horita and Berndt, 1999;McCollom and Seewald, 2001;McDermott et al., 2015). In contrast to hydrocarbons, there is a strong thermodynamic drive toward rapid C-H-O equilibrium in hydrothermal fluids within hours to days. Kinetic barriers preclude the formation of CH 4 in this equilibrium (Shock, 1990). This permits the creation of metastable formate species (H 2 + CO 2 ↔ HCOOH), CO (HCOOH ↔ CO + H 2 O), formaldehyde (HCOOH + H 2 ↔ CH 2 O + H 2 O), and methanol (CH 2 O + H 2 ↔ CH 3 OH) through the sequential reduction of CO 2 using H 2 as the reductant (Seewald et al., 2006).
The abundance of formate in chemical equilibrium with dissolved inorganic carbon is strongly dependent on H 2 concentration, pH, and temperature (McCollom and Seewald, 2003;Seewald et al., 2006). In a gold-titanium reaction cell, HCOO − was formed from CO 2 at 300 • C and 350 bar in less than 24 h from H 2 generated from hydrothermal alteration of olivine serving as the reductant (McCollom and Seewald, 2001). In a separate study, incubation of a 175 mmol/kg HCOOH solution at 300 • C and 350 bar in the gold reaction cell led to near complete conversion to H 2 and CO 2 within 48 h, CO reached 0.83 mmol/kg, and HCOO − + HCOOH (or HCOOH) decreased to 0.38 mmol/kg (Seewald et al., 2006). Reducing the temperature to 200 • C and then to 150 • C each led to an increase in HCOOH, a decrease in CO, and C-H-O equilibrium within 115 h and 71 h, respectively. Injection of 172 mmol/kg CO led to production of H 2 , CO 2 , and HCOOH, and decreasing CO. Alkaline conditions favored the formation of HCOOH, HCO 3 − , and CO 3 2− (Seewald et al., 2006). Therefore, the abundance of formate, CO, and CH 3 OH in seafloor hydrothermal systems will be regulated by the residence times of fluids in reactions zones, and physical and chemical conditions in the subsurface environments. Formate is also generated across a pH gradient of more than three pH units using a mineral precipitate bridge at the interface of two fluids (Hudson et al., 2020). This may be relevant to the formation of formate on the early Earth or possibly in extraterrestrial oceans where high pH serpentinized fluids are Formate dehydrogenase, hydrogenase, and formate transporter proteins and their reactions that are found in Thermococci. Formate hydrogenlyase (Fhl) catalyzes membrane-bound oxidation of formate to H 2 and CO 2 . NAD(P)H: formate dehydrogenase (Nfd) catalyzes cytoplasmic oxidation of formate coupled with reduction of NAD(P) + . The hydrogenases catalyze the following reactions: membrane-bound oxidation of ferredoxin (Fd) coupled with H 2 production (Mbh), cytoplasmic H 2 oxidation coupled with reduction of NAD(P) + (Sh), and cytoplasmic H 2 oxidation (Frd) coupled with reduction of thioredoxin reductase (TrxR). Fd and NAD(P)H are cytoplasmic electron carriers. TrxR is part of the redox cascade for sulfur response regulation using SurR. Created with BioRender.com. emitted into an acid ocean. Under standard conditions, the generation of formate from H 2 and CO 2 is not thermodynamically favorable. However, H 2 in synthetic alkaline vent fluid (pH 12.3) passed electrons to dissolved CO 2 in a synthetic acid ocean (pH 3.9) at 25 • C through a Fe(Ni)S mineral interface generating 1.5 µM HCOO − in the ocean fluid (Hudson et al., 2020). Isotopic labeling showed that protonation occurred using H 2 O on the ocean side of the interface, not H 2 on the vent side. Weakening the pH gradient led to decreased concentrations of HCOO − produced. Nickel in the precipitate is a crucial part of the reduction mechanism as HCOO − yield dropped below detection without Ni in the ocean precipitation fluid.

Formate concentrations in hydrothermal fluids
There have been very few measurements of formate in natural hydrothermal fluids due in part to the analytical difficulty of measuring formate at low concentrations (Schink et al., 2017). Formate has been measured mostly at sites with high H 2 concentrations such as at the Lost City, Von Damm, and Piccard hydrothermal vent sites and were 36-669 µM ( Table 1). Formate and H 2 were also measured at Snake Pit and TAG hydrothermal vents, which are mafic hydrothermal vents on the Mid-Atlantic Ridge, where formate concentrations were 1-2 nM and H 2 concentrations were 0.08-2.4 µM (Konn et al., 2022). At ultramafic sites, formate concentrations are generally 10-100 fold lower than that of H 2 at the same site (Lang et al., 2010;McDermott et al., 2015) while at mafic sites the formate concentration is often more than 1,000 fold lower than the H 2 concentration (McDermott et al., 2018;Konn et al., 2022). 6. Formate use by methanogens 6.1. Free formate use for de novo purine biosynthesis Methanocaldococcus jannaschii was shown to incorporate 14 C-formate into biomass during growth (Sprott et al., 1993), which may be used in part for de novo purine biosynthesis. Inosine monophosphate (IMP) is a precursor for adenine and guanine synthesis for purine biosynthesis and is made from ribose-5-phosphate (Figure 4). In most organisms, the pathway intermediates glycinamide-ribose-5-phosphate (GAR) and aminoimidazole carboxamide-ribose-5-phosphate (AICAR) are formylated using N 10 -formyl-tetrahydrofolate as the formyl donor. However, the genes for these enzymes are absent in Methanococci and Methanopyri and are replaced with genes that encode for enzymes that use free formate and energy from ATP to formylate their substrates (White, 1997;Brown et al., 2011). These enzymes are formylglycinamide-ribose-5-phosphate synthetase (PurT) and forminido imidazole carboxamide-ribose-5phosphate synthetase (PurP) (Figure 4). M. jannaschii was shown to have PurP activity and that it produced free 13 C-formate in the cell when incubated with H 2 and H 13 CO 3 (Ownby et al., 2005). Herein, a genome survey of the eleven extremely thermophilic methanogens showed that all the organisms have homologs for purP and all but M. kandleri have homologs for purT ( Table 2 and  Supplementary Table 1). This suggests that these organisms have a mechanism for formate synthesis.

Formate dehydrogenases in Methanococci and Methanopyri
Nine of the 11 Methanococci and Methanopyri genomes have genes that encode for a cytoplasmic formate dehydrogenase ( Table 2 and Supplementary Table 1). Formate dehydrogenases catalyze the reversible oxidation of formate to CO 2 using various electron acceptors. The catalytic α subunit (FdhA) contains tungsten, selenocysteine, and a (Fe 4 -S 4 ) cluster as cofactors while the β subunit (FdhB) contains three (Fe 4 -S 4 ) clusters (Niks and Hille, 2019). FdhAB in Methanococci and Methanopyri is homologous to two formate dehydrogenases in the mesophilic methanogen Methanococcus maripaludis, also a Methanococci, that use coenzyme F 420 as their redox partner (Figure 2; Wood et al., 2003;Lupa et al., 2008). M. maripaludis grows hydrogenotrophically on H 2 and CO 2 but also grows on formate in their absence (Jones et al., 1983b). When fdhA1 was mutated in M. maripaludis, the organism was unable to grow on formate and formate dehydrogenase activity in cell extracts was undetectable (Lupa et al., 2008). Observations with hydrogenase mutants in M. maripaludis suggest that coenzyme F 420 is an intermediate in formate-to-H 2 conversion (Lupa et al., 2008). An M. maripaludis fdhA1 fdhA2 double mutant grown in purinefree defined medium grew as well as the wild-type strain suggesting that formate dehydrogenase is not essential for de novo purine biosynthesis (Wood et al., 2003). The absence of fdhAB genes in Methanocaldococcus infernus and Methanofervidicoccus abyssi ( Table 2 and Supplementary Table 1) also supports the idea that formate dehydrogenase is not essential for purine biosynthesis. However, it is likely that H 2 and coenzyme F 420 are electron donors for formate production and can help meet the cellular demand for formate for purine biosynthesis.
The formate dehydrogenase (FdhA1B1) from M. maripaludis also forms an enzyme complex with heterodisulfide reductase, the soluble hydrogenase Vhu, and formylmethanofuran dehydrogenase (Figure 2; Costa et al., 2010). It was necessary for the organism's growth on formate but not on H 2 (Costa et al., 2010). Therefore, in addition to coenzyme F 420 reduction, this formate dehydrogenase also oxidizes formate to reduce the heterodisulfide coenzyme M-coenzyme B and ferredoxin through electron bifurcation. Coenzyme M, coenzyme B, and ferredoxin are cytoplasmic electron carriers in these methanogens. Expression of the second formate dehydrogenase gene (fdhA2) in M. maripaludis increased when cells were grown under H 2 limited conditions but was unchanged under formate limited conditions (Costa et al., 2013) and was not required for growth on formate (Lupa et al., 2008) suggesting that this isoenzyme may have a separate physiological function.

Formate transporters in Methanococci
For extremely thermophilic methanogens, it appears that a formate transporter is required for growth on formate. Formate transporters import or export formate across the cytoplasmic membrane and require co-translocation of a H + (Figure 2). Three thermophilic methanogens in our survey (Methanotorris formicicus, Methanothermococcus okinawensis, and Methanothermococcus thermolithotrophicus) grew on formate in the absence of H 2 and CO 2 but not any of the other methanogens examined ( Table 2). Each of these methanogens that grew on formate has a gene that encodes for a membrane-bound formate transporter (fdhC) in its genome, which is absent in all other methanogens examined, except for Methanocaldococcus fervens which was not tested for growth on formate ( Table 2 and  Supplementary Table 1). M. maripaludis has an fdhC gene in an operon with fdhA1B1 (Sattler et al., 2013). In M. fervens and M. okinawensis, the formate transporter gene fdhC appears to be in the same operon as fdhAB suggesting they are co-transcribed (Supplementary Table 1).

Free formate use for de novo purine biosynthesis
Like Methanococci, all Thermococci lack the enzymes that use N 10 -formyl-tetrahydrofolate as the formyl donor for de novo purine biosynthesis (Brown et al., 2011). Instead, most Thermococci use formate-dependent enzymes (PurT and PurP) for de novo purine biosynthesis (Figure 4, Table 3, and Supplementary  Table 2). Therefore, they depend on a source of free formate in the cell for de novo synthesis. However, some Thermococcus species (T. paralvinellae, T. barophilus CH5, T. onnurineus, and T. gorgonarius) lack most or all the genes for the purine biosynthesis pathway (Brown et al., 2011) and likely rely on environmental sources of purines.

Formate dehydrogenases in Thermococci
All 30 Thermococci genomes have at least one copy of the gene that encodes for the catalytic α subunit of formate dehydrogenase (FdhA) either in the form of formate hydrogenlyase, NAD(P) +dependent formate dehydrogenase, or the catalytic subunit alone ( Table 3 and Supplementary Table 2). The phylogeny of FdhA in extremely thermophilic Methanococci, Methanopyri, and Thermococci showed one clade for Methanococci and Methanopyri and five clades among the Thermococci (Figure 5). In Thermococci, hydrogenase operons often flank fdhA-containing operons in the genome (Figure 6 and Supplementary Table 2) suggesting a close association between formate and H 2 in these organisms. In Groups 1 and 2 in Figure 5, fdhA was encoded in an operon with a formate transporter gene. For Group 1, in nearly all instances, Biochemical pathway for de novo purine biosynthesis using free formate as the source of the formyl group (after Brown et al., 2011). the fdhA-containing operon was immediately downstream from an frh operon and immediately upstream from one or two mbh operons on the same DNA strand suggesting that they may be co-transcribed (Figure 6). In Group 1A, fdhA was encoded in a formate hydrogenlyase (fhl) operon (Kim et al., 2010;Topçuoglu et al., 2018;Le Guellec et al., 2021; Table 3;  Supplementary Table 2). This enzyme reversibly couples formate oxidation to H 2 evolution on the cytoplasmic membrane with concomitant H + /Na + translocation across the membrane via antiporter modules (Figure 4; Kim et al., 2010;Lim et al., 2014). In Group 1B, fdhA was encoded in a NAD(P) + -dependent formate dehydrogenase (nfd) operon (Figure 6). This soluble enzyme catalyzes the reversible oxidation of formate using NAD(P) + or ferredoxin as its redox partner (Le Guellec et al., 2021;Yang et al., 2022;Figure 4). In Group 2, fdhA was encoded in an nfd operon but neighbored an sh operon in the genome instead of frh and mbh operons (Figure 6). These nfd and sh operons are transcribed in opposite directions from the same intergenic spacer region.
The fdhA from Groups 3 and 4 are in fhl operons that lack a formate transporter gene. In Group 3, the fhl operon was next to an sh operon (Figure 6). These fhl and sh operons are transcribed in opposite directions from the same intergenic spacer region. In Group 4, the fhl operon did not neighbor any hydrogenase operons in the genome, and in Group 5 the fdhA gene was the only formate dehydrogenase-related gene present in the genome (Figure 6). Often these solo genes in Group 5 are near the purine biosynthesis genes in genome sequences (Supplementary Table 2). In T. sibiricus, nearly all the genes for de novo purine biosynthesis (purFCMTEDPSQL) and fdhA are next to each other in the genome, although they are not all on the same DNA strand (Figure 6). In these organisms, it is unknown if fdhA alone encodes for a functional formate dehydrogenase or what the redox partner is for this putative enzyme. However, it is plausible that it might be used to produce formate for purine biosynthesis when other formate dehydrogenases and formate transport proteins are absent.

Formate transporters in Thermococci
Under defined growth conditions, 11 of the 30 Thermococci strains analyzed either oxidized added formate as an energy source (plus trace levels of organic compounds as a carbon source) and produced H 2 (Kim et al., 2010;Topçuoglu et al., 2018) or secreted formate when grown on organic compounds in the presence of high background H 2 and the absence of added formate (Hensley et al., 2016;Topçuoglu et al., 2018;Le Guellec et al., 2021). These 11 strains are the only Thermococci in the survey that have a formate transporter gene ( Table 3). The other 19 Thermococci lack this gene and were unable to grow on formate or secrete formate (Kim et al., 2010;Le Guellec et al., 2021). Therefore, it appears that a formate transporter is required for Thermococci to secrete formate or, like Methanococci, for growth of Thermococci on formate. The presence of a formate transporter gene or transcript should be a criterion when determining if Methanococci or Thermococci are potentially using or producing formate in their natural habitat.

Formate production versus consumption by Thermococci in nature
The standard Gibbs energy for interconversion between formate and H 2 + CO 2 is small; therefore, the direction of the reaction is highly dependent upon the relative concentrations of formate and H 2 in the environment (Schink et al., 2017;Le Guellec et al., 2021). Le Guellec et al. (2021) calculated that CO 2 reduction to formate using H 2 is thermodynamically more favorable than formate oxidation to H 2 and CO 2 at Phylogenetic tree based on catalytic subunit alpha (FdhA) for the various formate dehydrogenases found in extremely thermophilic Methanococci, Methanopyri, and Thermococci. The phylogeny of FdhA was inferred by using a maximum likelihood method and Jones-Taylor-Thornton (JTT) matrix-based modeling (Jones et al., 1992). After 1000 bootstrap constructions, the tree with the highest log likelihood (-31,270.37) is shown, with values next to nodes indicating the percentage of reconstructions in which the topology was preserved (values < 70% are omitted for clarity). There were a total of 736 positions in the final dataset. Branch lengths are to scale and indicate the number of substitutions per site. GenBank/EMBL/DDBJ open reading frame numbers are included in parentheses. Evolutionary analyses were conducted in MEGA11 (Tamura et al., 2021). Clade associations with operon arrangements on the genomes and the presence of a formate transporter or putative regulatory elements are shown.
Lost City, Von Damm, Rainbow, Lucky Strike, Snake Pit, and Ashadze 1 hydrothermal vent sites based on relative formate and H 2 concentrations in hydrothermal fluids. The physiological response of Thermococcus is in keeping with this idea. Growth of T. paralvinellae on a sugar or peptides when sparged with H 2 led to higher levels of fhl1 expression and higher formate secretion relative to cultures sparged with N 2 (Topçuoglu et al., 2018). It was concluded that fhl and nfd expression in Thermococci is primarily for the purpose of ameliorating H 2 inhibition rather than for growth on formate (Topçuoglu et al., 2019;Le Guellec et al., 2021). Thermococci would require an environment where formate concentrations exceed H 2 concentrations to grow on formate. The formate produced by Thermococci may supplement the growth of Methanococci even when Thermococci produce H 2 , as is observed with fermentermethanogen relationships in mesophilic environments (Schink et al., 2017  Operon and gene maps for Thermococci containing operons for formate hydrogenlyase (fhl), NAD(P)H: formate dehydrogenase (nfd), membrane hydrogenase (mbh), soluble hydrogenase (sh), and F 420 -reducing-like hydrogenase (frh) (Groups 1-4) and genes for the catalytic subunit of formate dehydrogenase (fdhA), and purine biosynthesis (pur) (Group 5). Also shown are the locations of SurR binding sites (S), the tetR gene for transcriptional regulation, and the operons containing a formate transporter (FT) gene. The top scale bar is for the Group 1-4 operons; the bottom scale bar, the genes for Group 5.
formate or H 2 regulates fdhAB expression in these organisms. The thermophilic methanogen Methanobacterium thermoformicicum grows on H 2 and CO 2 as well as separately on formate. It has a formate transporter gene (fdhC) directly upstream of its formate dehydrogenase genes (fdhAB) (Nolling and Reeve, 1997). Transcripts of fdhCAB were present in M. thermoformicicum at all growth stages when grown on formate. When grown on H 2 and CO 2 , fdhCAB transcripts were barely detectable in early exponential growth phase but increased dramatically as cells approached late exponential growth phase in a closed batch system when H 2 became more limiting. Similarly, fdh expression in M. maripaludis was controlled by the presence of H 2 and not formate (Wood et al., 2003). Using fdhC-lacZ gene fusions, βgalactosidase activity increased in M. maripaludis cells grown on H 2 and CO 2 as they approached late exponential growth phase, again when H 2 became limiting. When grown on formate, βgalactosidase activity was higher in cells with N 2 and CO 2 in the headspace relative to those with H 2 and CO 2 in the headspace. βgalactosidase activity increased in cells grown on formate plus H 2 and CO 2 after the H 2 and CO 2 was replaced mid-growth phase with N 2 and CO 2 . In M. maripaludis, genes for a putative response regulator and a histidine kinase are directly upstream of fdhC, which is three genes upstream of fdhA1B1 and part of a putative five-gene operon (Sattler et al., 2013). Random mutagenesis showed that disruption of this putative response regulator led to slower growth of M. maripaludis on formate relative to the wild type. It also led to increased fdhA1 transcriptional abundance regardless of whether H 2 and CO 2 or formate was the growth substrate. Impairment of derepression of the fdhC-fdhA1B1 operon is a plausible explanation (Sattler et al., 2013). Therefore, H 2 present at high concentrations may interact with the histidine kinase and activate the response regulator in a two-component regulatory system that represses fdhC-fdhA1B1 expression, which is derepressed when H 2 levels are low or absent.

Transcriptional regulation in Thermococci
Very little is known about transcriptional regulation of the fhl and nfd operons in Thermococci. Group 1 Thermococci genomes (Figure 6) encode syntenic frh, either fhl or nfd, and mbh operons with a formate transporter gene encoded in the fhl or nfd operon (Figure 6). These frh, fhl, nfd, and mbh operons each have GTTn 3 AAC(n 5 GTT) in their promoter region just upstream of BRE/TATA RNA polymerase binding sites suggesting they are also regulated and promoted by the sulfur response regulator protein SurR (see Section "4.2. Growth of Thermococci with and without S 0 "). Furthermore, Frh was shown to oxidize H 2 and reduce TrxR (Jung et al., 2020), which reduces SurR via Pdo, suggesting that it might serve as a regulatory hydrogenase that promotes frh, fhl, nfd, and mbh expression when H 2 concentrations increase in the cell. Therefore, like Methanococci, H 2 abundance appears to regulate formate use in Thermococci. A remaining question is whether formate also regulates gene expression in Thermococci. In T. paralvinellae, expression of the Group 1A fhl operon containing the formate transporter gene increased when cells were grown on formate relative to growth on maltose or peptides while expression of mbh either remained unchanged or decreased (Topçuoglu et al., 2018). This suggests that in addition to SurR regulation, formate either directly or indirectly regulates gene expression in T. paralvinellae as well. Validation and the mechanism of this putative regulation is yet to be determined.
None of the promoter regions for the nfd, fhl, or sh operons in Groups 2-4 had a SurR nucleotide binding sequence. All but one of the Group 4 fhl operons have a gene encoding for a TetR/AcrR family transcriptional regulator that is ∼350 nucleotides upstream of and transcribed in the same direction as the fhl operon (Supplementary Table 2). TetR/AcrR family transcriptional regulators are one-component systems where a single protein contains both a sensory domain and a DNAbinding domain (Cuthbertson and Nodwell, 2013). They are widely associated with antibiotic resistance and the regulation of genes encoding small molecule exporters and are usually encoded alongside target operons (Colclough et al., 2019). In T. paralvinellae, expression of the Group 4 fhl operon decreased when cells were grown on formate relative to growth on maltose or peptides (Topçuoglu et al., 2018). The mechanism for regulation of Group 2-5 formate dehydrogenase-related genes is unknown.

Conclusion
Formate and H 2 are linked both in hydrothermal vent environments and in the metabolisms of extremely thermophilic Methanococci and Thermococci. Methanococci prefer H 2 oxidation to formate oxidation but appear to switch to the latter when H 2 is limiting. Similarly, Thermococci appear to prefer H 2 production to formate production but switch to the latter when H 2 is excessive and inhibitory. H 2 is typically far more abundant than formate in hydrothermal vent fluids suggesting that in high H 2 environments formate is unlikely to be used by Methanococci and Methanopyri for growth. However, in hydrothermal environments that are very low H 2 environments but rich in organic compounds, Thermococci may produce H 2 and formate that are then used to support the growth of extremely thermophilic methanogens. Understanding where, when, and how formate is used by extreme thermophiles in nature is largely unknown and an area of future research. Furthermore, our understanding of transcriptional regulation of fhl and nfd in Thermococci is nascent. A key question is if and how formate influences gene expression, especially in concert with SurR regulation of hydrogenases and sulfur responsive genes.

Author contributions
JH and HS contributed to the conceptualization, original draft preparation, review, and editing of the manuscript. JH conducted bioinformatic analyses and data compilation. Both authors read and agreed to the published version of the manuscript.

Funding
This research was provided by the NASA Exobiology grant 80NSSC21K1240 and USDA National Institute of Food and Agriculture grant MAS00550 to JH.