Methyl-reducing methanogenesis by a thermophilic culture of Korarchaeia Viola Krukenberg, Anthony J Kohtz, Zackary J. Jay, Roland Hatzenpichler This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: http://dx.doi.org/10.1038/s41586-024-07829-8 Made available through Montana State University’s ScholarWorks 1 Methyl-reducing methanogenesis by a thermophilic culture of Korarchaeia Viola Krukenberg1,*,#, Anthony J. Kohtz1,*, Zackary J. Jay1, Roland Hatzenpichler1,2,# 1, Department of Chemistry and Biochemistry, Center for Biofilm Engineering, and Thermal Biology Institute, Montana State University, Bozeman, MT-59717, USA 2, Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT- 59717, USA *, These authors equally contributed to this work #, Correspondence to viola.krukenberg@montana.edu and roland.hatzenpichler@montana.edu Abstract Methanogenesis mediated by archaea is the major source of methane, a strong greenhouse gas, and thus is critical for understanding Earth’s climate dynamics. Recently, the genes encoding the methanogenesis pathway were discovered across multiple archaeal phyla. However, experimental verification of functional and active methanogenic pathways is currently limited to the Euryarchaeota. We here show for the first time the methanogenic capability of Candidatus Methanodesulfokora washburnenis, a deep-branching lineage of archaea found in hot springs that represents a novel group of methanogens in the phylum Thermoproteota. Following enrichment cultivation, we used measurements of metabolic activity and isotope tracer conversion to demonstrate methanol reduction to methane by this archaeon. Analysis of the circular genome revealed unique modifications in the energy conservation pathways linked to methanogenesis, including enzyme complexes involved in the oxidation of hydrogen or reduced sulfur compounds. The cultivation and characterization of this novel group of archaea is critical for a deeper evaluation of the diversity, physiology and biochemistry of methanogens. Introduction Methane is a potent greenhouse gas, and its atmospheric levels contribute to regulating Earth’s climate. The majority of methane (approximately 69%) is generated in anoxic environments by methanogenic archaea1,2 in a strictly anaerobic process called methanogenesis3. These microorganisms play a critical role in the global carbon cycle by catalyzing the final step in organic matter degradation and are of high interest for biotechnological applications including the production of methane as energy source4. Methanogens utilize compounds such as CO2/H2, acetate or methanol and the enzymatic pathways leading to methane formation differ depending on the substrate3,5. However, all methanogens employ the methyl-coenzyme M reductase (MCR) complex for the final conversion of methyl-coenzyme M and coenzyme B into methane and the CoM-S-S-CoB heterodisulfide6. This key enzyme also catalyzes the reversible reaction in the anaerobic oxidation of methane and other alkanes in alkanotrophic archaea6,7. The isolation of methanogens into axenic cultures has been fundamental to decades of research on their physiology and biochemistry8. To date, methanogenesis has exclusively been studied in 2 lineages of the Euryarchaeota, and no methanogen from outside this superphylum has ever been cultured for experimental investigation. Only recently, environmental metagenomic studies have identified genes of the methanogenesis pathway, including those in the MCR complex, to be encoded by archaea across multiple new lineages9–15. These newly proposed groups of methanogenic archaea could have important but so far unrecognized environmental impacts or hold undiscovered biotechnological potential. Candidatus Methanodesulfokora washburnensis, formerly Candidatus Methanodesulfokores washburnensis11,16, is a deeply branching archaeon from the phylum Thermoproteota (formerly the TACK superphylum). It was first identified as a potential methanogen by metagenome- assembled genomes (MAGs) from Washburn Hot Springs in Yellowstone National Park (YNP, WY, USA)9,11,12. Metabolic reconstruction predicted methyl-reducing hydrogen-dependent methanogenesis among other possible respiratory pathways, including anaerobic oxidation of methane and sulfite reduction 9,11,12. The deeply branching phylogenetic position, together with the unique metabolic potential at the intersection of carbon and sulfur cycling9,11,12 make this archaeon a prime candidate for culture-dependent research into the physiology, biochemistry, and metabolic versatility of methanogens. Here, we combined selective cultivation with fluorescence microscopy, activity studies, and metagenome sequencing to demonstrate methyl-reducing methanogenesis by a thermophilic culture of this korarchaeon. Methanogenic enrichments of Korarchaeia We selected Washburn Hot Springs (WHS) and two hot springs in the Lower Culex Basin (LCB003 and LCB058) of YNP as source materials for cultivation. These hot springs ranged in temperature (64-77°C) and pH (6.1-6.5) and varied in the relative abundance of Korarchaeales-related 16S rRNA gene amplicons (0.4-3.6%; Fig. 1A, Extended Data Fig. 1). From each hot spring two anoxic incubations of sediment slurries were conducted at in situ temperature (WHS, 64ºC; LCB003, 77ºC; LCB058, 70°C) and supplied with methanol and hydrogen in the presence or absence of antibiotics. Within 64 to 67 days of incubation we detected a strong increase in the relative abundance of both 16S rRNA and mcrA gene amplicons identified as Ca. M. washburnensis. Ca. M. washburnensis dominated the microbial community of all six incubations, constituting up to 86% relative abundance. Other MCR- encoding archaea affiliated with Methanomethyliales, Archaeoglobales and Methanobacteriales were also detected. All incubations showed methane production (Fig. 1A), with two incubations (LCB003-1A,1B and LCB058-1B) containing near-exclusively mcrA gene sequences related to Ca. M. washburnensis (Korarchaeales, >98.5% relative abundance). The highest methane levels were observed in two incubations (WHS-B and LCB003-A), which, based on 16S rRNA gene amplicon data, were co-enriched in Ca. M. washburnensis and Methanothermobacter sp., a thermophilic, obligate CO2-reducing hydrogenotrophic methanogen previously isolated from geothermal environments of YNP17,18. As inoculum for the further cultivation of Ca. M. washburnensis we selected one of these initial enrichments (LCB003-1B; Fig. 1A), in which methane production of up to 0.6% headspace gas 3 was measured. The microbial community of this culture lacked previously cultured Euryarchaeotal methanogens and was strongly dominated by Ca. M. washburnensis (65% and 99.9% relative abundance of 16S rRNA and mcrA gene amplicons, respectively). For selective cultivation, we designed a medium that matched the growth requirements for hydrogen- dependent methyl-reducing methanogenesis as predicted from metabolic reconstructions of Ca. M. washburnensis MAGs9,11,12. Methanol (10 mM) and hydrogen (50%) were supplied as methanogenic substrates, antibiotics were amended to reduce bacterial growth, and incubation was conducted at in situ temperature of 77°C, allowing the growth of thermophiles. Activity was monitored by measuring methane concentrations in the headspace and cultures were transferred at late exponential phase of methane production. Continuous transfers of the active methanogenic culture into fresh media resulted in a sediment-free culture, designated culture LCB3. During 13 continuous cultivation cycles (i.e., transfers; over 500 days total) the final methane concentration and methane production rate increased steadily from a maximum of 2% to 5% headspace gas, equivalent to about 2 mM of methane and from approximately 63 to 38 days, respectively (Fig. 1B). Importantly, the temperature at which culture LCB3 grows is well above the known upper temperature limit for methanol-utilizing methanogens19,20 and near the boiling point of methanol (84°C at 200 kPa), which likely reduced the amount of methanol effectively available in the liquid media. Figure 1. Methane-producing enrichment cultures from hot spring sediments. A. Relative abundance of Korarchaeales (red) in hot spring sediments (Env) and in initial enrichments (1A, 1B) after 64-67 days inferred by amplicon sequencing of mcrA and 16S rRNA genes. Enrichments were established from three hot springs in the Lower Culex Basin (LCB003, LCB058) and Washburn Hot Springs (WHS). Enrichments were supplied with methanol and hydrogen in the absence (1A) or presence (1B) of antibiotics. Circle sizes are proportional to relative sequence abundance (%). Lineages containing MCR-encoding archaea are depicted on order level, relative abundances >1% are shown. Shading of squares indicates methane concentrations (%) detected in the enrichment headspace. B. Headspace methane development during long-term cultivation. Culture LCB3 was generated from initial enrichment LCB003-1B (shown in A) and was maintained in anoxic media amended with methanol plus hydrogen and antibiotics. Stars indicate timepoints at which metagenome sequencing (blue) and experimental evaluation (orange) of culture LCB3 was performed. 4 Methyl-reducing methanogenesis Fluorescence in situ hybridization (FISH) on culture LCB3 using a probe specific for the 16S rRNA of Ca. M. washburnensis revealed abundant filamentous cells (Fig. 2A, Extended Data Table 1, Fig. 2), similar in morphology to the previously described non-methanogenic Ca. Korarchaeum cryptofilum21. Filaments varied in lengths between approximately 3 µm to >20 µm, and filaments were observed both individually and in cell aggregates. We tracked the growth of Ca. M. washburnensis in culture LCB3 by catalyzed reporter deposition (CARD)- FISH. This showed that its relative cell abundance increased with methane production from approximately 30% during lag phase to 70% during log phase (Fig. 2A). Based on cell counts, the total cell density of culture LCB3 reached 2×107 cells mL-1 with an estimated doubling time of six days. By growing culture LCB3 in the presence of 13C-methanol and measuring the transfer of isotopic label into 13C-methane, we confirmed that methanol was converted to methane (Fig. 2B). Methane production ceased upon addition of bromoethanesulfonate (BES), an inhibitor of the MCR complex. Figure 2. Physiological experiments on culture LCB3. A. Methane production and fraction of Ca. M. washburnensis cells. Relative abundance of Ca. M. washburnensis cells was determined at four time points (day 0, 12, 24, 40) based on the fraction of Ca. M. washburnensis specific FISH counts (red) versus total counts of DAPI stained cells (blue). Error bars indicate the standard deviation of four biological replicates. B. Production of 12C-methane and 13C-methane in cultures amended with 12C-methanol (open symbols, solid line) or 13C- methanol (grey symbols, dashed line). No 13C enrichment of methane was detected in cultures amended with 12C- methanol. BES addition to active cultures resulted in the inhibition of methanogenesis (black circles, dashed line). Error bars indicate the standard deviation of three biological replicates. C. Anabolic activity of cells in culture LCB3 visualized via BONCAT (green), combined with CARD-FISH (red) to identify Ca. M. washburnensis cells. Ca. M. washburnensis cells were translationally active in a methane-producing culture but not in a culture in which methanogenesis was inhibited with BES. In methanogenic cultures BONCAT-labelled cells (green bar) and CARD-FISH labelled cells (red bar) each accounted for 84% of all DAPI stained cells. In non-methanogenic cultures BONCAT-labelled cells accounted for 2% while CARD-FISH labelled cells accounted for 82%. This suggests that anabolic activity of Ca. M. washburnensis cells stops when methanogenesis is inhibited. Error bars indicate the standard deviation of three biological replicates. Scale bars 5 µm. To provide evidence for methanogenesis by Ca. M. washburnensis, we evaluated its activity under methanogenic and non-methanogenic conditions using a combination of bioorthogonal non-canonical amino acid tagging (BONCAT) and FISH. For this, two replicate culture sets 5 were grown to exponential phase, at which point one set was inhibited with BES before both sets were spiked with the amino acid analog L-homopropargylglycine (HPG) to trace translationally active cells (Extended Data Fig. 3). We visualized cells of Ca. M. washburnensis via CARD-FISH and identified anabolically active cells via click chemistry mediated dye- staining. Based on cell counts, Ca. M. washburnensis accounted for approximately 80% of the cells in both culture sets, while BONCAT-labelling revealed cellular activity of Ca. M. washburnensis only under methanogenic conditions (Fig. 2C). This indicated that translational activity is dependent on the activity the MCR complex. Taken together, we present the first experimental evidence that a representative of the Korarchaeia grows by methanogenesis. Circular genome of Ca. M. washburnensis Metagenome sequencing performed on culture LCB3 at two time points (day 180 and 352, Fig. 1B) resulted in the recovery of a complete, closed chromosome that was highly related to previously obtained Ca. M. washburnensis MAGs (NM4, LMO9, and MDKW; Extended Data Table 2)9,11,12. This was apparent in both average nucleotide identity and average amino acid identity (97-98%; Extended Data Fig. 4A), phylogenomic reconstruction (Fig. 3A, Extended Data Table 3, Fig. 5A), and 16S rRNA gene identity and phylogeny (99%; Extended Data Fig. 5B). We thus designate the here described archaeon as Ca. M. washburnensis strain LCB3. The genome of strain LCB3 is similar in size and number of coding sequences to the MAG of Ca. M. washburnensis NM4 and the genome of Ca. K. cryptofilum OPF8, but notably smaller than the MAGs of both Ca. M. washburnensis MDKW and LMO9 (Extended Data Table 2). MAGs NM4, MDKW, and LMO9 were independently recovered from the same metagenome dataset of Washburn Hot Springs (WHS), but processed by different bioinformatic methods 9,11,12. The variation in recovered MAGs from WHS together with considering the size of the genome of strain LCB3, suggests that the Ca. M. washburnensis population in WHS consists of several strains. Based on metagenomic read mapping to the genome of strain LCB3 we estimate its abundance in the environment of WHS, LCB003, and LCB058 between 0.1 to 2.9%, and in culture LCB3 at 62-82% (Fig. 3B, Extended Data Fig. 4B). Other community members in culture LCB3 with >1% relative sequence abundance included Archaeoglobaceae, Thermofilum, and Fervidicoccaceae (Fig. 3B, Extended Data Fig. 5B), and we recovered nearly complete (92-99%) genomes of all three archaea (Extended Data Table 4). Consistently, application of a general archaeal CARD-FISH probe revealed filamentous and cocci shaped cells, while no bacterial cells were observed with general bacterial probes (Fig. 3B, Extended Data Table 1, Fig. 2). Importantly, the only mcr genes (mcrABG) identified in the metagenomes from culture LCB3 at both time points belong to the genome of strain LCB3. Phylogenetic analysis of the single copy of McrA revealed that it is highly related to previously recovered McrA from Korarchaeia MAGs and an McrA gene from a metagenome of hot spring LCB003 (Fig. 3C), the source material from which culture LCB3 was obtained. This, along with the stable isotope data and metabolic activity measurements, is strong evidence that strain LCB3 is the only methanogen in the culture. 6 Figure 3. Phylogenetic affiliation of Ca. Methanodesulfokora washburnensis strain LCB3. A. Phylogenetic reconstruction based on 33 single copy marker genes showing affiliation of strain LCB3 with representative Korarchaeia genomes and MAGs. B. Composition of culture LCB3 based on relative abundance of recovered MAGs, and visualization of Korarchaeal (red) and other archaeal cells (green) by CARD-FISH. Note that due to two central mismatches between the 16S rRNA of Ca. M. washburnensis and the archaea-specific probe (Arch915, position 923 and 930), no dual labelling of Ca. M. washburnensis was observed. C. Phylogenetic tree of McrA showing the close affiliation of McrA from Ca. M. washburnensis strain LCB3 with McrA from MAG MDKW and McrA from the metagenome of hot spring LCB003. Asterisks indicate lineages with availability of isolates (blue) or enrichment cultures (purple), which all belong to the superphylum Euryarchaeota. Circles represent bootstrap support values >95. Methyl-reducing methanogenesis pathway To study the mechanisms involved in methyl-reducing methanogenesis in Ca. M. washburnensis strain LCB3 we analyzed its complete genome and compared it to previously described MAGs. Strain LCB3 encodes the Mcr complex (mcrABGCD) and methanol:coenzyme M methyltransferase (mtaABC) that are both required for the conversion of methanol to methane and a heterodisulfide (CoM-S-S-CoB)3,5. However, they lack the methyltetrahydromethanopterin:coenzyme M methyltransferase (Mtr) complex and the methyl-branch of the Wood-Ljungdahl pathway (Fig. 4, Extended Data Table 5). This supports the idea that methanogenesis in strain LCB3 is based on methyl-reduction rather than methyl- disproportionation into methane and carbon dioxide. Consequently, strain LCB3 relies on the oxidation of an electron donor, such as hydrogen, for the reduction of CoM-S-S-CoB, as previously discussed for related MAGs9,11,12. Importantly, all previous metabolic inferences from Ca. M. washburnensis MAGs suggested an energy conservation mechanism via a soluble electron-bifurcating hydrogenase/heterodisulfide reductase complex (Mvh/Hdr) coupled to a Fpo-like/HdrD complex9,11,12. While the LCB3 genome encodes a Fpo-like complex (Fig. 4, Extended Data Fig. 6B, Table 5) and HdrD subunits, no genes encoding an Mvh/Hdr complex were identified, which suggests an alternative route for linking hydrogen oxidation and CoM- 7 S-S-CoB reduction. Our metabolic reconstruction suggests that in strain LCB3 hydrogen is oxidized by a membrane-bound [NiFe]-hydrogenase of group 1g22, and electrons are transferred via an unknown membrane-bound electron carrier to an integral membrane b-type cytochrome containing heterodisulfide reductase complex (HdrDE) that catalyzes the cytoplasmic reduction of CoM-S-S-CoB (Fig. 4, Extended Data 7B, Table 5). Different from previously characterized group 1 [NiFe]-hydrogenases of the 1k/1j type (or Vht/Vho, respectively) in methanogens, the group 1g [NiFe]-hydrogenase complex encoded in the LCB3 genome does not contain a cytochrome b subunit (Extended Data Fig. 6A). Instead, it includes two proteins with similarity to NrfC and NrfD families (Extended Data Fig. 7A). The encoded NrfD-like subunit is an integral membrane protein that could replace the function of cytochrome b in reducing a membrane-bound electron carrier23. Group 1g [NiFe]- hydrogenases have not been previously linked to methanogenesis and may present a unique Korarchaeia-specific differentiation in the enzymatic machinery of methanogens. Energy conservation during hydrogen-dependent methyl-reducing methanogenesis could thus rely on a simple respiratory chain of membrane-bound group 1g [NiFe]-hydrogenase and b-type cytochrome (HdrE), in which both enzyme complexes would contribute to establishing an ion gradient that subsequently can be used for the synthesis of ATP via a V-type ATP synthase (Fig. 4). Figure 4. Metabolic reconstruction based on the genome of strain LCB3. Depicted are the methanogenesis pathway and possible alternative energy conserving complexes with linkage to methanogenesis. Abbreviations are as follows: ATP Syn, ATP Synthase; Coo, Carbon monoxide dehydrogenase; Dsr, Dissimilatory sulfite reductase; Fd, Ferredoxin; Fpo, F420:Methanophenazine oxidoreductase; Glc, Lactate dehydrogenase; Hdr, Heterodisulfide reductase; Hyd, [NiFe]-Hydrogenase; Mcr, Methyl-coenzyme M reductase; Mta, Methanol:coenzyme M methyltransferase; Mrp, Na+/H+ antiporter; M, Membrane-bound electron carrier; ox, oxidized form; rd, reduced form. Dashed lines represent alternative reactions, dotted lines exemplify CoM-S-S- CoB recycling, color-coding differentiates proteins/protein complexes. Alternative energy-conserving complexes Additional enzyme complexes encoded in the LCB3 genome could facilitate either the internal production of hydrogen or allow for alternative mechanisms of energy conservation during methanogenesis. Strain LCB3 encodes a membrane-bound group 4 [NiFe]-hydrogenase also found in diverse Thermoproteota lineages affiliated with a novel cluster, termed here Ehi (Extended Data Fig. 6A, Table 5). It may function similar to the Ech or Ehb-type hydrogenases characterized in Euryarchaeotal methanogens8, oxidizing hydrogen at the expenditure of an ion gradient to produce reduced ferredoxin24, which could be used at a Fpo-like/HdrD complex for the reduction of CoM-S-S-CoB (Fig. 4). The reverse reaction may play a role in energy 8 conservation, with the oxidation of ferredoxin coupled to the production of hydrogen and ion translocation. Additionally, this Ehi hydrogenase could potentially form a complex with HdrD, allowing for coupling the direct oxidation of hydrogen to the reduction of CoM-S-S-CoB and translocation of ions across the membrane (Fig. 4), functionally similar to the HdrB-associated hydrogenases in other Thermoproteota lineages9,25. Strain LCB3 encodes an additional membrane-bound group 4b [NiFe]-hydrogenase/carbon monoxide dehydrogenase complex that could couple the oxidation of carbon monoxide to the production of hydrogen and translocation of ions across the membrane26,27. This would provide an internal source of hydrogen for methanogenesis and a means of energy conservation. As an alternative to hydrogen, strain LCB3 also encodes the potential to use hydrogen sulfide as electron donor. The encoded cytoplasmic DsrAB complex could catalyze the oxidation of sulfide to sulfite with electron transfer via the DsrC protein to the membrane-bound b-type cytochrome containing DsrMK complex for the reduction of a membrane-bound electron carrier (Fig. 4). This would allow sulfide oxidation to be coupled to CoM-S-S-CoB reduction and energy conservation, a process not known in any other cultured methanogens. In addition, strain LCB3 as well as other MCR-encoding archaea within the Thermoproteota contains adjacent genes encoding HdrD and GlcD, which has homology to lactate dehydrogenase. This suggests that lactate could be used as an electron donor, providing electrons to HdrD for the reduction of CoM-S-S-CoB13,14 coupled to the translocation of ions at either a Fpo-like or Ehi complex, a mechanism also not described in cultured methanogens. In summary, the metabolic reconstruction of Ca. M. washburnensis strain LCB3 is consistent with methyl-reducing hydrogenotrophic methanogenesis from methanol, and indicates that the archaeon possibly uses a diverse and unique set of enzymes to conserve energy. Future work will be required to understand the interplay between carbon, hydrogen, and sulfur cycling in strain LCB3, explore alternative electron donors, and study under which conditions this archaeon might change its energy-conserving strategy entirely. Conclusion The cultivation of the elusive methanogen Ca. M. washburnensis enabled us to gain first insights into its growth and metabolism under laboratory conditions. Thermophilic cultures of strain LCB3 formed methane from methanol and cellular activity of strain LCB3 was reliant on the function of the MCR complex. This demonstrates the capacity for methanogenesis exists in the deepest branching archaeal lineage of the Thermoproteota, which may have important implications for the evolution and diversification of methanogens. Strain LCB3 is a thermophile from a high temperature hot spring and its growth at 77°C extends the upper temperature range of both methyl-reducing and b-type cytochrome-involving methanogenesis. This may impact its niche differentiation, because cytochrome-based energy conservation is more efficient8; however, no such methanogen has been studied in this regard. To date, all members of the Ca. Methanodesulfokora genus have been detected exclusively in terrestrial geothermal systems9,11,12,28 and may have evolved unique mechanisms to thrive in these often extreme and dynamic environments. Strain LCB3 appears to be specialized towards 9 utilizing methanol as substrate for methanogenesis. However, it encodes a diverse set of energy-conserving complexes and the potential to use electron donors other than hydrogen, which is rare in cultured methyl-reducing methanogens5,20. The capacity to exploit a diversity of electron sources, either directly or indirectly linked to methanogenesis, could provide Ca. M. washburnensis with an advantage in geothermal systems, where reduced compounds such as sulfide, hydrogen and carbon monoxide often co-occur28,29. Besides methanogenesis, Ca. M. washburnensis encode alternative energy-conserving pathways such as sulfite reduction and methane oxidation and the potential for a mixotrophic lifestyle11. Thus, methanogenesis, a process with comparatively low energy yield8 (CH3OH + H2 → CH4 + H2O, ΔG0 ~ -112.5 kJ mol-1), may be used in addition to or alternatingly with other metabolisms, a trait rarely observed in Euryarchaeotal methanogens. Notably, the volatilization of methanol in high temperature environments might present a challenge to its use as a substrate, and alternative metabolisms may support the survival of strain LCB3 during periods of methanol starvation. Thus, our genomic and experimental evaluation of strain LCB3 indicates metabolic traits unique to this methanogen and suggests that many yet unresolved aspects of its physiology, biochemistry, cell biology and ecology await discovery. To date, Ca. M. washburnensis, is the second member of the Korarchaeia obtained in an enrichment culture and contrasts the non-methanogenic Ca. K. cryptofilum21. The cultivation of Ca. M. washburnensis opens the possibility for fundamental research on the biology of this archaeon, which will both advance our current knowledge on Korarchaeia and expand our understanding of methanogenesis. Etymology. Methanodesulfokora, methanum (Latin): methane, de (Latin): from, sulfo (Latin): sulfur, kore (Greek): young woman; washburnensis (Latin): pertaining to Washburn Hot Springs. The name implies a methane and sulfur metabolizing member of the class Korarchaeia within the phylum Thermoproteota, which metagenome-assembled genome was first obtained from Washburn Hot Springs in Yellowstone National Park11. The archaeon cultured in this study represents Ca. M. washburnensis strain LCB3. Locality. Enriched from geothermally heated sediment of an unnamed hot spring, catalogued as feature LCB00328, in the Lower Culex Basin of Yellowstone National Park, WY, USA. Diagnosis. Anaerobic, thermophilic, methyl-reducing methanogen of filamentous morphology. 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Sample collection, enrichment, and cultivation Hot spring sediment was retrieved from three geothermal features in Yellowstone National Park (YNP): LCB003 (44.57763, -110.78957; November 2020; 77°C; pH 6.5), LCB058 (44.57039, -110.80521; October 2020; 70°C; pH 6.1) located in the Lower Culex Basin (LCB), and WHS (44.76493, -110.43030; October 2019; 64°C; pH 6.4) located in the Washburn Hot Springs thermal area. A mixture of surface sediments (~1 cm deep) and spring water were collected into glass bottles sealed headspace-free with a butyl rubber stopper. Slurries were stored at in situ temperature for 24 h before transfer to room temperature for long-term storage. Initial enrichments were initiated within 24 h of material retrieval from LCB003 and LCB058 or within two years of material retrieval for WHS, and were set up in sterile serum vials (50 or 70 ml) under a N2/CO2/H2 (90/5/5%) atmosphere. Slurries from LCB058 and WHS were diluted with anoxic media (1:10). Media was prepared as described previously30 and contained KH2PO4, 0.5 g L-1; MgSO4·7H2O, 0.4 g L-1; NaCl, 0.5 g L-1; NH4Cl, 0.4 g L-1; CaCl2·2H2O, 0.05 g L-1; MES, 2.17 g L-1; yeast extract, 0.1 g L-1; and 0.002% (w/v) (NH4)2Fe(SO4)2·6H2O, 5 mM NaHCO3, 1 ml L-1 trace element solution SL-10, 1 ml L-1 Selenite-Tungstate solution, 1 ml L-1 CCM vitamins31, 0.0005% (w/v) resazurin, 10 ml g L-1 of coenzyme-M, 2 ml L-1 sodium dithionite, 1 mM dithiothreitol, 1 mM Na2S·9H2O, with pH adjusted to 6.5 with NaOH. Serum vials were sealed with butyl rubber stoppers and aluminum crimps before the headspace was degassed with N2/CO2 (90/10) for 5 minutes and set to 200 kPa. Hydrogen and methanol were added at final concentration of 50% and 10 mM, respectively. Two incubations were prepared from the slurry of each hot spring. One was amended with the bacterial antibiotics streptomycin (50 mg L-1; inhibitor of protein synthesis) and vancomycin (50 mg L-1; inhibitor of peptidoglycan synthesis) and one was not amended with antibiotics. Additional control incubations were supplied with only methanol or only hydrogen. Incubation was conducted at in situ temperatures. All stock solutions were anoxic and sterilized by filtration or autoclavation. After 64 days of incubation an initial enrichment from LCB003 was selected as inoculum for the cultivation of Ca. M. washburnensis, and transferred into anoxic media (10% v/v), amended with hydrogen, methanol and antibiotics. Cultivation was conducted at 77°C and cultures were maintained by regular transfer into fresh media (10% v/v). Methane measurements Methane concentrations were determined from subsamples of 250 µL headspace gas collected with a gas tight syringe (Hamilton) and injected into 10 mL autosampler vials sealed with grey chlorobutyl septa. Subsamples were injected into a Shimadzu 2020-GC equipped with a GS- CarbonPLOT column (30 m x 0.32 mm; 1.5 µm film thickness; Agilent) and a Rt-Q-BOND column (30 m x 0.32 mm; 1.5 µm film thickness; Restek) and operated with injector, column, 13 and flame ionization detector (FID) maintained at 200°C, 50°C, and 240°C, respectively. Methane concentrations were calculated based on injection of a standard curve. Stable isotope incubation For tracking the conversion of 13C-methanol to 13C-methane, active enrichment cultures were incubated in the presence of 13C-methanol (99% labelled; Cambridge Isotope Laboratories). 30 ml incubations were carried out in 60 ml serum bottles with 20% (v/v) inoculum. Control incubations were performed in the presence of (i) 12C-methanol (100%), (ii) 13C-methanol (100%) plus bromoethanesulfonate (BES) to inhibit methanogenesis, (iii) 13C-methanol (100%) plus paraformaldehyde to inhibit cellular activity, and (iv) 13C-methanol (100%) without inoculum. Substrate and inhibitor were added at a concentration of 10 mM. Headspace samples were collected regularly as described above and analyzed using a Shimadzu QP2020 NX GCMS equipped with a GS-CarbonPLOT column (30 m x 0.35 mm; 30 µm film thickness; Agilent) and operated in Selected Ion Monitoring mode. The instrument was operated as described in Ai et al., 201332. Peak areas corresponding to m/z ratios of 16 for 12C-methane, 17 for 13C-methane were used for quantification. Fluorescence in situ hybridization and cell counts Aliquots of enrichment cultures were fixed in 2% paraformaldehyde (PFA) for 1 h at room temperature, washed twice by centrifugation at 16,000xg, removing the supernatant and resuspending the cells in 1x phosphate buffered saline (PBS), before cell suspensions were stored at 4°C. For direct cell counts, aliquots of fixed cell suspensions were filtered onto polycarbonate filters (0.2 μm pore size, 25 mm diameter, GTTP Millipore, Germany) and air dried before filter pieces were cut, stained with DAPI, embedded in Citifluor-Vectashield, and enumerated under an epifluorescence microscope (Leica DM4B). Relative abundance of target cells was determined from the fraction of CARD-FISH/DOPE-FISH stained cells compared to total cell counts based on DNA stained cells using DAPI (4,6-diamidino-2-phenylindole). A 16S rRNA-targeted oligonucleotide probe (KRmw515, 5’-CCA GCC TTG CCC TCC CCT- 3’) specific for Ca. Methanodesulfokora washburnensis was designed using the probe design tool in the ARB software package33. Probe KRmw515 has one mismatch to the 16S rRNA of non-target organism Ca. Korarchaeum cryptofilum, which was absent from the culture. The horseradish peroxidase (HRP)-labelled oligonucleotide probe was synthesized by Biomers (Ulm, Germany). Stringency was tested in a catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) experiment by increasing the formamide concentration in the hybridization buffer from 0% to 70%. CARD-FISH was performed as described previously34. Cell wall permeabilization was achieved with lysozyme treatment for 30 min at 37°C [10 mg ml-1 lysozyme, lyophilized powder in 0.1 M Tris–HCl, 0.05 M EDTA, pH 8] followed by proteinase K digestion for 10 min at RT [4.5 mU mL−1 proteinase K (Merck) in 0.1 M Tris- HCl, 0.05 M EDTA and 0.5 M NaCl, pH 8]. Endogenous peroxidases were inactivated with 0.15% H2O2 in methanol (30 min, RT). For hybridization, the following oligonucleotide probes were applied at their respective formamide concentration (35% for all): EUB338I-III35, Arch91536, NON33837 , and KRmw515. Catalyzed reporter deposition was performed using tyramides labelled with the fluorochromes Alexa Fluor 594 or Alexa Fluor 488. When 14 performing double hybridizations, the peroxidase enzymes of the first hybridization were inactivated with 0.3% H2O2 in methanol (30 min, RT) prior to the hybridization with the second HRP-labelled probe. DOPE-FISH was performed as previously described38. Samples were stained with DAPI embedded in Citifluor-Vectashield, and visualized using epifluorescence microscopy. BONCAT-FISH experiments The metabolic activity of strain LCB3 under methanogenic versus non-methanogenic conditions was tested via biorthogonal non-canonical amino acid tagging (BONCAT). Replicate incubations of culture LCB3 were initiated with methanol and hydrogen. Inhibited controls were amended with BES at day 0. When methane production reached exponential phase (day 15), triplicate cultures were either amended with BES (5 mM) or remained unamended. After 72 h both sets were spiked with the biorthogonal amino acid HPG (100 µM). Subsamples were retrieved after 24 h, fixed in 2% PFA, washed and stored in 1xPBS at 4°C. Aliquots of fixed samples were spotted on glass slides and BONCAT-FISH was performed as previously described39. The fractions of BONCAT-labelled, FISH-labelled and DAPI-stained cells were enumerated to determine the relative abundance of BONCAT and FISH labelled cells. Amplicon sequencing and analysis DNA was extracted from environmental samples, and from initial enrichment culture (pellet from 0.5 ml) using the FastDNA Spin Kit for Soil (MP Biomedicals, Irvine, CA) following the manufacturer’s guidelines. mcrA genes were amplified with primer set mlas-mod-F/mcrA-rev- R40,41, and archaeal and bacterial 16S rRNA genes were amplified with the updated Earth Microbiome Project primer set 515F and 806R42–44. Amplicon libraries were prepared as previously described45 and sequenced at the Molecular Research Core Facility at Idaho State University (Pocatello, ID) using an Illumina MiSeq platform with 2 x 300 bp (mcrA amplicon library) and 2 x 250 bp (16S rRNA amplicon library) paired end read chemistry. Both 16S rRNA and mcrA gene reads were processed using QIIME 2 version 2020.246. Primer sequences were removed from demultiplexed reads using cutadapt47 with error rate 0.12, reads were truncated (145 bp forward, 145 bp reverse and 260 bp forward, 200 bp reverse for 16S rRNA and mcrA datasets, respectively), filtered, denoised and merged in DADA2 with default settings48. 16S rRNA gene amplicon sequence variants (ASVs) were taxonomically classified with the sklearn method and the SILVA 132 database49. mcrA gene ASVs were assigned a taxonomy using vsearch with a minimum identity of 70% and no consensus classification against a reference database of representative near-full length mcrA genes encompassing the diversity of publicly available mcrA. Contaminants were removed using the R package decontam50. The mcrA gene dataset was curated by removing ASVs £400 bp and non-mcrA gene ASVs as identified by evaluating the top hits of a blastx search against the NCBI NR database. 15 Metagenome sequencing A 50 mL aliquot of culture LCB3 was centrifuged at 16,000xg for 10 min to pellet cells, supernatant was removed, and the pellet stored at -80°C. Genomic DNA was extracted from the pellet according to Zhou et al., 199651, with the following modifications: (i) cells were disrupted with a tissue grinder, (ii) proteinase K treatment was extended to 1 h, and (iii) DNA was precipitated in the presence of 0.7x volumes isopropanol and 0.1x volume of 3 M sodium acetate. DNA extracts were purified using the Zymo clean and concentrator kit (DCC-10, Zymo Research) according to the manufacturer’s instructions. Purified genomic DNA was used for metagenomic library construction with the Illumina DNA Prep kit following the manufacturer’s recommendations and was sequenced on an Illumina NextSeq 2000 platform with 2x151 bp paired-end read chemistry performed at SeqCenter (Pittsburgh, PA). Metagenome assembly, binning, and quality assessment Illumina read quality, linker, and adapter trimming, artifact and common contaminate removal, and error correction were performed using the rqcfilter2 pipeline (maxns=3, maq=3) and bbcms (mincount=2, hcf=0.6) from the BBTools suite v38.94 (Bushnell B. 2014. BBMap: a fast, accurate, splice-aware aligner. https://sourceforge.net/projects/bbmap). Resulting reads were assembled with SPAdes52 v3.15.3 (-k 33,55,77,99,127 –meta –only-assembler) and coverage was determined with bbmap (ambiguous=random). Assembled scaffolds >2000 bp were binned using Maxbin v2.2.753, Concoct v1.0.054, Metabat v2.12.155 (with and without coverage), and Autometa v156 (bacterial and archaeal modes, including the machine learning step) and bins were refined with DAS_Tool v1.1.257 as previously described25. MAG completeness and redundancy were assessed using CheckM v1.1.358. The genome of strain LCB3 was determined to be circular by identifying a 127 bp sequence overlap at the start and end of the assembled contig. The overlapping sequence was removed from the end of the contig, resulting in a closed circular molecule. AAI and ANI values were computed with CompareM v0.0.23 (--fragLen 2000) (https://github.com/dparks1134/CompareM) and fastANI v1.1 (https://github.com/ParBLiSS/FastANI), respectively for available genomes and MAGs of Ca. M. washburnensis (n=4) and selected Ca. Korarchaeum (n=2) reference genomes. The closed genome of strain LCB3 was used to recruit reads from various metagenomes with bbmap (ambiguous=random) to determine the relative abundance of strain LCB3 in culture LCB3 at different timepoints and in different geothermal features of YNP. Annotation and reconstruction of metabolic potential The genome of strain LCB3 was annotated by the IMG/M database59. Catalytic subunits of [NiFe] hydrogenases (PF00374) were initially classified with HydDB and further classification was done by phylogenetic analyses22. Predicted optimal growth temperatures of MAGs and genomes were determined with Tome predOGT60. Phylogenetic analyses of marker genes A set of 33 single-copy marker proteins were collected from Korarchaeia MAGs and reference archaeal genomes. These markers were aligned with MUSCLE61, trimmed with trimAL62 using 16 a 50% gap threshold, and concatenated to produce a final alignment of 7,118 positions. Iqtree263 was used to reconstruct a maximum likelihood phylogenetic tree, using the LG+F+R10 model, 1000 ultrafast bootstraps, and 1000 iterations of the SH-like approximate- likelihood ratio test64. 16S rRNA genes from the metagenome of culture LCB3 and references were aligned and masked with ssu-align, and a maximum-likelihood phylogenetic tree was constructed with fasttree65. McrA from the genome of strain LCB3 and publicly available references were aligned with MAFFT-linsi66, trimmed with trimAL62 with 50% gap threshold and used for maximum likelihood phylogenetic analysis with Iqtree263 with LG+C60+F+G model and 1000 ultrafast bootstraps. Catalytic subunits of group 1 and group 4 [NiFe]-hydrogenases were extracted from the genome, of strain LCB3 aligned against the HydDB22 reference sequences with Mafft-LINSi66 and trimmed with trimAL62 using a 50% gap threshold. 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ISME Journal 16, 2373–2387 (2022). 20 Acknowledgements This study was funded through a NASA Exobiology program award (80NSSC19K1633). V.K. was supported in part by a grant from the NSF (MCB-1817428). A portion of this research was performed under the Facilities Integrating Collaborations for User Science program (proposal 10.46936/fics.proj.2017.49972/6000002) and used resources at the DOE Joint Genome Institute (https://ror.org/04xm1d337), which is a DOE Office of Science User Facility operated under Contract No. DE-AC02-05CH11231. We thank the US National Park Service for permitting work in YNP under permit number YELL-SCI-8010. We thank Luke McKay and Mackenzie Lynes (both Montana State University) for discussions that informed field sampling, cultivation, taxonomy, and manuscript preparation. We thank Paige Schlegel for assistance in cultivation. Author contributions V.K. and R.H. developed the research project. V.K., A.J.K. and R.H. designed experiments. V.K., A.J.K. and Z.J.J. conducted field work. V.K. and A.J.K. performed cultivation. V.K. extracted DNA for amplicon sequencing and performed BONCAT and CARD-FISH experiments. A.J.K. extracted DNA for metagenome sequencing, performed FISH and stable isotope experiments, and developed GC/GCMS protocols. Z.J.J. processed and annotated metagenomic data, assembled all MAGs, analysed abundance and assigned taxonomy. A.J.K., Z.J.J. and V.K. reconstructed and interpreted the metabolic potential. Z.J.J. constructed 16S rRNA gene phylogeny. V.K. conducted phylogenetic analysis of mcrA and processed amplicon data. A.J.K. performed phylogenomic analysis and the classification and annotation of hydrogenases. R.H. was responsible for funding and supervision of the project. V.K. compiled the initial manuscript. All authors contributed to the writing of the final manuscript. Competing interests The authors declare no competing interests. 21 Extended Data Figure 1. Images of hot springs used as source material for cultivation. A. Washburn Hot Springs. B. Hot spring LCB003. C. Hot spring LCB058. All three hot springs are located in Yellowstone National Park (WY, USA). Extended Data Figure 2. Representative fluorescence micrographs of culture LCB3. A. Cells visualized with DAPI (blue). B. Cells visualized by DOPE-FISH using a Ca. M. washburnensis specific 16S rRNA-targeted oligonucleotide probe (KRmw515, red, same field of view as in A). C. Overlay of A and B. D. Cells visualized with DAPI (blue). E. Cells visualized with DAPI (blue, same field of view as in D) combined with dual CARD- FISH using the Ca. M. washburnensis specific 16S rRNA-targeted oligonucleotide probe (KRmw515, red) and a general archaea probe (Arch915, green). Note that because of two mismatches of the Arch915 probe to the 16S rRNA of Ca. M. washburnensis there is no overlap of signal from probes KRmw515 and Arch915. Scale bars 5 µm. Extended Data Figure 3. Methane production in incubations for BONCAT-FISH experiments. Three replicate culture sets were grown to exponential phase. One culture set was amended with BES at day 15 (filled symbols, dashed line). At day 18 this and one additional culture set were amended with HPG (filled symbols) while another culture set was unamended (open symbols), representing growth under standard cultivation conditions. A control was amended with BES at day 0 (open symbols, dashed line) and no methane production occurred in this culture. Incubations were sampled for cell visualization via BONCAT-FISH after 24 h of incubation with HPG (day 19). Error bars indicate standard deviation from three biological replicates. 22 Extended Data Figure 4. Comparison of Korarchaeia genomes and MAGs. A. Pairwise comparisons of average nucleotide (below white line) and amino acid (above white line) identities (%) of Ca. Methanodesulfokora and Ca. Korarchaeum genomes and Washburn Hot Spring MAGs. B. Ca. M. washburnensis strain LCB3 genome and 16S rRNA gene read abundances in environmental metagenomes LCB003, LCB058, and WSH(2019) and culture LCB3 at day 180 and 352. Extended Data Figure 5. Phylogenetic affiliation of Ca. M. washburnensis strain LCB3. A. Maximum likelihood phylogenomic tree of archaea based on 33 single copy marker genes. The tree was constructed with IQtree and the best-fit model LG+F+R10, from the concatenated alignment of conserved arCOGs. The Ca. M. washburnensis strain LCB3 genome is highlighted in red. Circles indicate node support via the SH-like approximate likelihood ratio and ultrafast bootstrap values, respectively. B. Maximum likelihood phylogenetic tree of 16S rRNA genes form culture LCB3. The tree was constructed with fasttree including the 16S rRNA gene from the LCB3 genome (red), 16S rRNA genes in MAGs from culture LCB3 and reference sequences. Bootstrap values are indicated. A B 23 Extended Data Figure 6. Phylogenetic classification of [NiFe]-hydogenases in Ca. M. washburnensis strain LCB3. A. Maximum likelihood phylogenetic tree of group 1 [NiFe]-hydrogenases. The tree was constructed using IQtree2 with the best fit model LG+R10 and 1000 ultrafast bootstraps. Hydrogenase classes were assigned according to the HydDB. The sequence from the LCB3 genome is highlighted in red. Black squares indicate ultrafast bootstrap values >95. B. Maximum likelihood phylogenetic tree of group 4 [NiFe]-hydrogenases. The tree was constructed using IQtree2 with the best fit model LG+R10 and 1000 ultrafast bootstraps. Clades containing sequences from the LCB3 genome are highlighted in red. Black circles indicate ultrafast bootstrap values >95. Extended Data Figure 7. Annotation of enzyme complexes in Ca. M. washburnensis strain LCB3. A. Comparison of group 1 [NiFe]-hydrogenase organization and structure between Ca. M. washburnensis strain LCB3 and other MCR-encoding archaeal lineages. Models of the enzyme arrangement in the membrane with matching colors between the models indicating conserved function. Transmembrane helix (TMH) probabilities for intermembrane subunits. Groups 1j/1k have b-type cytochrome containing subunits with five helices, while the Korarchaeia group 1g [NiFe]-hydrogenase has an NrfD-like subunit (HcaC) with ten helices and lacks b-type cytochromes. B. Annotation of the Ca. M. washburnensis strain LCB3 HdrDE complex. Model representing the arrangement of genes in the membrane. All HdrE subunits analyzed have 5 TMHs. The HdrD subunit in the LCB3 genome has cysteine residues that are conserved in HdrD subunits from other lineages. Transmembrane helices were predicted using the TMHMM 2.0 server. 24 Extended Data Table 1. Oligonucleotide probes used in this study. Probe Probe sequence (5'-3') Specificity Target site1 FA2 Probe label3 Reference KRmw515R CCAGCCTTGCCCTCCCCT Ca. M. washburnensis 499- 515 20 HRP, DOPE This study KR515R CCAGCCTTACCCTCCCCT Ca. K. cryptofilum 499- 515 20 HRP, DOPE Elkins et al., 2008 cKR515R4 CCAGCCTTACCCTCCCCT competitor for Ca. K. crypto. 499- 515 20 none This study KR565R AGTATGCGTGGGAACCCCTC Ca. K. crypto., Ca. M. washb. 546- 565 20 HRP, DOPE Elkins et al., 2008 Arch915 GTGCTCCCCCGCCAATTCCT most archaea 915- 935 35 HRP Stahl&Am ann, 1999 EUB338I GCTGCCTCCCGTAGGAGT most bacteria 338- 355 35 HRP Daims et al., 1999 EUB338II GCAGCCACCCGTAGGTGT EUB338III GCTGCCACCCGTAGGTGT NON338 ACTCCTACGGGAGGCAGC negative control 338- 355 35 HRP Wallner et al., 1993 1, position in E. coli 16S rRNA 2, formamide concentration (v/v) in hybridization buffer 3, probe label used in this study; HRP, horseradish peroxidase; DOPE, double-labelled oligonucleotide probe 4, unlabelled competitor oligonucleotide used together with probe KRmw515 to limit probe hybridization to Ca. K. cryptofilum 25 Extended Data Table 2. Statistics of representative Korarchaeia genomes and Washburn Hot Springs metagenome assembled genomes. Seq, sequence; GC, GC content; CDS, coding sequence; pOGT, predicted optimal growth temperature; Cov, coverage; Compl, completeness; Red, redundancy; Ref, reference. Taxon Seq # Length (Mb) GC (%) CDS pOGT (°C) Comp (%) Red (%) tRNAs CRISPR Ref Ca. M. washburnensis LCB3 1 1.67 43.5 1,834 80.0 93.9 1.9 46 2 This study Ca. M. washburnensis MDKW 179 2.94 42.9 3,199 85.8 95.1 4.3 48 6 McKay et al., 2019 Ca. M. washburnensis NM4 122 1.42 43.4 1,544 80.1 85.5 1.8 44 0 Borrel et al., 2019 Ca. M. washburnensis LMO9 301 2.24 43.2 2,536 83.1 90.2 0.5 47 2 Wang et al., 2019 Ca. K. cryptofilum WS 51 1.77 48.7 1,779 76.1 95.6 3.10 47 4 McKay et al., 2019 Ca. K. cryptofilum OPF8 1 1.59 49.0 1,627 75.1 93.39 2.80 46 2 Elkins et al., 2008 26 Extended Data Table 3. Marker genes used in phylogenomic analysis of strain LCB3. arCOG Gene Description arCOG00785 RpmC Ribosomal protein L29 arCOG01001 Map Methionine aminopeptidase arCOG01227 FtsY Signal recognition particle GTPase arCOG01559 FusA Translation elongation factor G, EF-G (GTPase) arCOG01722 RpsM/rps13p Ribosomal protein S13 arCOG04050 FEN1 5'-3' exonuclease (including N-terminal domain of PolI) arCOG04067 RplB Ribosomal protein L2 arCOG04070 RplC Ribosomal protein L3 arCOG04071 RplD Ribosomal protein L4 arCOG04072 RplW Ribosomal protein L23 arCOG04086 RpmD Ribosomal protein L30 arCOG04087 RpsE Ribosomal protein S5 arCOG04088 RplR Ribosomal protein L18 arCOG04090 RplF/rpl6p Ribosomal protein L6P arCOG04091 RpsH/rps8p Ribosomal protein S8 arCOG04092 RplE/rpl5p Ribosomal protein L5 arCOG04094 RplX/rpl24p Ribosomal protein L24 arCOG04095 RplN/rps14p Ribosomal protein L14 arCOG04096 RpsQ/rps17p Ribosomal protein S17 arCOG04097 RpsC/rps3p Ribosomal protein S3 arCOG04098 RplV/rpl22p Ribosomal protein L22 arCOG04099 RpsS/rps19p Ribosomal protein S19 arCOG04169 SecY Preprotein translocase subunit SecY arCOG04223 SUI1 Translation initiation factor 1 (eIF-1/SUI1) arCOG04239 RpsD/rps4p Ribosomal protein S4 or related protein arCOG04240 RpsK/rps11p Ribosomal protein S11 arCOG04241 RpoA/Rpo1/rpoD DNA-directed RNA polymerase subunit D arCOG04242 RplM/rpl13p Ribosomal protein L13 arCOG04243 RpsI/rps9p Ribosomal protein S9 arCOG04245 RpsB/rps2p Ribosomal protein S2 arCOG04254 RpsG/rps7p Ribosomal protein S7 arCOG04288 RplJ Ribosomal protein L10 arCOG04289 RplA/rpl1p Ribosomal protein L1 Extended Data Table 4. Statistics of other MAGs recovered from culture LCB3. Seq, sequence; GC, GC content; CDS, coding sequence; pOGT, predicted optimal growth temperature; Cov, coverage; Comp, completeness; Red, redundancy; Abd, relative abundance. Taxon Seq # Length (Mb) GC (%) CDS pOGT (°C) Comp (%) Red (%) tRNAs CRISPR Abd (%) Archaeoglobaceae 84 1.65 43.3 1,771 75.8 99.3 0 50 2 13.2 Thermofilum 130 2.09 47.3 2,220 80.4 92.7 6.2 24 15 10.0 Fervidicoccaceae 7 1.35 44.2 1,422 92.0 96.9 3.2 33 2 2.75 Ignisphaera 233 1.72 39.9 1,708 85.2 81.9 0.6 29 1 0.75 Desulfurococcaceae 75 1.49 40.9 1,552 84.8 94.6 1.0 30 0 0.73 27 Extended Data Table 5. Overview of genes identified in the LCB3 genome. Included are genes discussed in the text. Locus tags refer to IMG Genome ID 3300058130 (strain LCB3). Locus tag Gene Annotation Ga0588336_01_1498205_1498459 mcrA methyl-coenzyme M reductase alpha subunit Ga0588336_01_1214849_1216177 mcrB methyl-coenzyme M reductase beta subunit Ga0588336_01_1217207_1217998 mcrG methyl-coenzyme M reductase gamma subunit Ga0588336_01_1216569_1217210 mcrC methyl-coenzyme M reductase subunit C Ga0588336_01_1216180_1216572 mcrD methyl-coenzyme M reductase subunit D Ga0588336_01_1196565_1197617 mtaA methanol:coenzyme M methyltransferase Ga0588336_01_1209784_1210245 mtaA methanol:coenzyme M methyltransferase Ga0588336_01_1208435_1209439 mtaA methanol:coenzyme M methyltransferase Ga0588336_01_1256403_1257407 mtaA methanol:coenzyme M methyltransferase Ga0588336_01_1200192_1201577 mtaB methanol-5-hydroxybenzimidazolylcobamide comethyltransferase Ga0588336_01_1201574_1202422 mtaC methanol corrinoid protein Ga0588336_01_920467_920844 fpoA-like F420-H2 dehydrogenase subunit A Ga0588336_01_919958_920482 fpoB-like F420-H2 dehydrogenase subunit B Ga0588336_01_919495_919971 fpoC-like F420-H2 dehydrogenasesubunit C Ga0588336_01_918336_919505 fpoD-like F420-H2 dehydrogenase subunit D Ga0588336_01_917317_918336 fpoH-like F420-H2 dehydrogenase subunit H Ga0588336_01_916845_917336 fpoI-like F420-H2 dehydrogenase subunit I Ga0588336_01_916379_916861 fpoJ-like F420-H2 dehydrogenase subunit J Ga0588336_01_916071_916376 fpoK-like F420-H2 dehydrogenase subunit K Ga0588336_01_912465_914540 fpoL-like F420-H2 dehydrogenase subunit L Ga0588336_01_914546_916066 fpoM-like F420-H2 dehydrogenase subunit M Ga0588336_01_911068_912468 fpoN-like F420-H2 dehydrogenase subunit N Ga0588336_01_302773_304227 mrpA/mnhA multicomponent Na+:H+ antiporter subunit A Ga0588336_01_297950_299314 mrpD/mnhD multicomponent Na+:H+ antiporter subunit D Ga0588336_01_300872_302779 mrpD/mnhD multicomponent Na+:H+ antiporter subunit D Ga0588336_01_812845_814626 atpA V/A-type H+-transporting ATPase subunit A Ga0588336_01_809780_812845 atpB V/A-type H+-transporting ATPase subunit B Ga0588336_01_808152_809162 atpC V/A-type H+-transporting ATPase subunit C Ga0588336_01_809163_809780 atpD V/A-type H+-transporting ATPase subunit D Ga0588336_01_814627_815247 atpE V/A-type H+-transporting ATPase subunit E Ga0588336_01_815540_815872 atpF V/A-type H+-transporting ATPase subunit F Ga0588336_01_815240_815530 atpH V/A-type H+-transporting ATPase subunit G/H Ga0588336_01_805172_807541 atpI V/A-type H+-transporting ATPase subunit I Ga0588336_01_807605_807925 atpK V/A-type H+-transporting ATPase subunit K Ga0588336_01_1251631_1252734 hdrD heterodisulfide reductase subunit D Ga0588336_01_1221513_1222643 hdrD heterodisulfide reductase subunit D Ga0588336_01_1617244_1618869 hdrD heterodisulfide reductase subunit D Ga0588336_01_1618856_1619773 hdrE heterodisulfide reductase subunit E, cytochrome-b Ga0588336_01_1220248_1221516 glcD putative lactate dehydrogenase Ga0588336_01_307153_307767 cooF anaerobic carbon-monoxide dehydrogenase iron sulfur subunit Ga0588336_01_305228_307153 cooS anaerobic carbon-monoxide dehydrogenase catalytic subunit Ga0588336_01_1313931_1315136 dsrA sulfite reductase subunit A Ga0588336_01_1315152_1316216 dsrB sulfite reductase subunit B Ga0588336_01_1308276_1308596 dsrC sulfite reductase subunit C Ga0588336_01_1311720_1313183 dsrK sulfite reductase subunit K Ga0588336_01_1310918_1311718 dsrM sulfite reductase subunit M Ga0588336_01_1277950_1279725 hyd 1g group 1g [NiFe] hydrogenase, catalytic subunit Ga0588336_01_296409_297953 hyd 4b group 4b [NiFe] hydrogenase, catalytic subunit Ga0588336_01_160718_161899 hyd 4g Ehi group 4g [NiFe] hydrogenase, catalytic subunit Copy Cover Page.pdf Blank Page Blank Page