Hydrogenotrophic methanogenesis in archaeal phylum Verstraetearchaeota reveals the shared ancestry of all methanogens Bojk A. Berghuisa, Feiqiao Brian Yua,b, Frederik Schulzc, Paul C. Blaineyd,e, Tanja Woykec, and Stephen R. Quakea,b,f,1 aDepartment of Bioengineering, Stanford University, Stanford, CA 94305; bChan Zuckerberg Biohub, San Francisco, CA 94158; cDepartment of Energy Joint Genome Institute, Walnut Creek, CA 94598; dDepartment of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; eBroad Institute of Harvard and MIT, Cambridge, MA 02142; and fDepartment of Applied Physics, Stanford University, Stanford, CA 94305 Contributed by Stephen R. Quake, December 29, 2018 (sent for review September 27, 2018; reviewed by Jared R. Leadbetter and Marc Strous) Methanogenic archaea are major contributors to the global carbon electron donor (1, 11). To conserve energy, hydrogenotrophs couple cycle and were long thought to belong exclusively to the euryarchaeal the WLP to methanogenesis. This coupling is established by N5- phylum. Discovery of the methanogenesis gene cluster methyl- methyl-tetrahydromethanopterin:coenzyme M methyltransferase coenzyme M reductase (Mcr) in the Bathyarchaeota, and thereafter (Mtr; also known as tetrahydromethanopterin S-methyltransferase), the Verstraetearchaeota, led to a paradigm shift, pushing back the which transfers the methyl group from the WLP to coenzyme M. Mtr + evolutionary origin of methanogenesis to predate that of the uses the free energy of methyl transfer to establish a Na -motive Euryarchaeota. The methylotrophic methanogenesis found in force across the membrane (14). Methyl coenzyme M reductase then the non-Euryarchaota distinguished itself from the predomi- reduces methyl-coenzyme M to methane, using coenzyme B as an nantly hydrogenotrophic methanogens found in euryarchaeal orders as the former do not couple methanogenesis to carbon fixation through electron donor. The established disulfide bond between these coen- the reductive acetyl-CoA [Wood–Ljungdahl pathway (WLP)], which zymes is then broken again by heterodisulfide reductase (HdrABC/ was interpreted as evidence for independent evolution of the two mvhADG). This cytoplasmic electron bifurcating complex concomi- tantly generates the reduced ferredoxin required for CO reduction in methanogenesis pathways. Here, we report the discovery of a com- 2 EVOLUTION plete and divergent hydrogenotrophic methanogenesis pathway in a the process (15, 16). thermophilic order of the Verstraetearchaeota, which we have named The Bathyarchaeota, Verstraetearchaeota, and Methano- Candidatus Methanohydrogenales, as well as the presence of the WLP massiliicoccales are, together with some Methanobacteriales and in the crenarchaeal order Desulfurococcales. Our findings support the ancient origin of hydrogenotrophic methanogenesis, suggest that Significance methylotrophic methanogenesis might be a later adaptation of spe- cific orders, and provide insight into how the transition from hydro- Methane-producing microorganisms are thought to be among genotrophic to methylotrophic methanogenesis might have occurred. the earliest cellular life forms colonizing our planet, and are major contributors to the past and present global carbon cycle. methanogenesis | archaea | evolution Currently, all methanogens belong to the archaeal domain of life, and there is compounding evidence for a variety of meth- ll known methanogenic organisms belong exclusively to the anogenic metabolisms among a wide distribution of archaeal Aarchaeal domain of life. Methanogens are typically found in phyla. However, the predominantly hydrogenotrophic (CO2- the oxygen-depleted environments of soils, sediments, and the fixing) Euryarchaeota are distinct from the recently discovered intestinal tract of humans and animals (1). With an estimated methylotrophic (biomass-degrading) noneuryarchaea, making the combined annual production of 500 million tons of the green- shared ancestry and origins of all methanogens unclear. We house gas methane, methanogenic archaea are key contributors discovered hydrogenotrophic methanogenesis in a thermophilic to the global carbon cycle and play an important role in climate order of the Verstraetearchaeota, a noneuryarchaeote. The change (2, 3). Until recently, all known methanogens belonged to the Verstraetearchaeota, hitherto known as methylotrophs, unify Euryarchaeota and were categorized into two classes (class I and class the origins of methanogenesis and shed light on how organisms II). The hypothesis that methane metabolism originated early in the can evolve to adapt from hydrogenotrophic to methylotrophic evolution of the Euryarchaeota (4) has since been challenged fol- methane metabolism. lowing the recent discovery of a putative methane metabolism in the archaeal phyla Bathyarchaeota (formerly the miscellaneous Cren- Author contributions: B.A.B., F.B.Y., and S.R.Q. designed research; B.A.B. and F.B.Y. per- formed research; P.C.B. collected samples; B.A.B., F.S., T.W., and S.R.Q. analyzed data; and archaeota group) (5, 6) and Verstraetearchaeota (7). B.A.B., F.B.Y., F.S., P.C.B., T.W., and S.R.Q. wrote the paper. Three major pathways of methanogenesis are known (8, 9): Reviewers: J.R.L., California Institute of Technology; and M.S., University of Calgary. hydrogenotrophic, methylotrophic, and acetoclastic (Fig. 1A). Conflict of interest statement: S.R.Q. is a shareholder of Fluidigm Corporation. T.W. and The only enzyme that is present in all types of methanogenesis is M.S. are coauthors on a 2017 software assessment paper. methyl-coenzyme M reductase (Mcr), a Ni-corrinoid protein cat- This open access article is distributed under Creative Commons Attribution-NonCommercial- alyzing the last step of methyl group reduction to methane (1, 10, NoDerivatives License 4.0 (CC BY-NC-ND). 11). Hydrogenotrophic methanogenesis is the most widespread Data deposition: We have used the Department of Energy Joint Genome Institute’sIn- pathway (1) and has been suggested to represent the ancestral form tegrated Microbial Genomes and Microbiomes (IMG/M) database to upload our genome of methane production (12). Class I methanogens (Methanopyrales, and metagenome datasets, as well as performing parts of the analysis there. These ge- Methanococcales, and Methanobacteriales), as well as most class II nomes and metagenomes are available for download at https://img.jgi.doe.gov.IMG genome IDs are as follows: 2767802456, 2767802469,and2770939329–2770939439.IMG methanogens (Methanomicrobiales, Methanocellales, and Meth- metagenome IDs are as follows: 3300017482, 3300017562, 3300017696, 3300017469, anosarcinales, with the exception of Methanomassiliicoccales) are and 3300017461. 1 hydrogenotrophs. They reduce CO2 to CH4 in six steps via the re- To whom correspondence should be addressed. Email: [email protected]. ductive acetyl-CoA or Wood–Ljungdahl pathway (WLP). The WLP This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. is one of the most important processes for energy generation and 1073/pnas.1815631116/-/DCSupplemental. carbon fixation (13). Here, H2, or sometimes formate, is used as an Published online February 27, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1815631116 PNAS | March 12, 2019 | vol. 116 | no. 11 | 5037–5044 Downloaded by guest on September 30, 2021 A Hydrogenotrophic Methylotrophic methanogenesis Carbon fixation using methanogenesis archaeal WLP CO CO without methanogenesis 2 2 CO 2 Fd CO reduction red 2 Fd WLP WLP WLP WLP ox Cdh H MPT-CH acetyl-CoA coupled 4 3 biomass biomass with energy Mtr biomass Mtr Mtr Mtr conservation R-CH CoM-S-CH 3 R-CH 3 3 Ferredoxin Fd ox regeneration Mcr Mcr Mcr Mcr Fd related red Ignisphaera, methanogenesis CH CH CH Archaeoglobus, 4 4 4 Bathyarchaeota, Altiarchaeales, Euryarchaeota Bathyarchaeota Methanomassiliicoccales, Hadesarchaea, Methanohydrogenales BA1,BA2 Methanomethyliales Loki-, Thorarchaeota Cdh McrMtrRgy Hco B Korarchaeota OP bin 011 OP bin 015 Thaumarchaeota OP bin 010 OP bin 042 JZ bin 32 BA1 Bathyarchaeota BA2 JdFR-11 JdFR-10 JZ bin 37 JZ bin 38 Methanohydrogenales OP bin 008 JZ bin 30 Methanomediales OP bin 054 V4 Verstraete- V5 archaeota V3 Methanomethyliales V1 V2 OP bin 021 OP bin 046 8 YNP, WY Geoarchaeota 10 GBS, NV OP bin 108 Early Marsarchaeota OP bin 107 7 OP bin 061 Marsarchaeota 11 Thermoproteales Desulfurococcales Ignisphaera Crenarchaeota Acidilobales Sulfolobales 0.1 Fig. 1. WLP coupled to methanogenesis in the Methanohydrogenales. (A) Different configurations for the associated or independent functioning of the archaeal version of the WLP and methanogenesis. Missing enzymatic complexes or pathways are shaded in gray. The following are shown: CO2-reducing methanogenesis as present in the Methanohydrogenales as well as class I and class II methanogens without cytochromes (Left); methanogenesis by reduction of methyl compounds using H2 as inferred in Bathyarchaeota BA1, and a potential link with the WLP in the absence of Mtr (Left Center); methanogenesis by reduction of methyl compounds using H2 as present in the Methanomassiliicoccales and Methanomethyliales (Right Center); and carbon fixation using the archaeal WLP in the absence of methanogenesis, and proposal of a mechanism to generate low potential ferredoxin during sulfate reduction in the case of the Archaeoglobales (Right). Fd, ferredoxin. (B, Left) Genome-based phylogeny of the TACK superphylum genomes found in the OP dataset. The tree was inferred using a concatenated