sign in sign up
<<
Home , Euryarchaeota, Ferroplasma, Methanobacteria, Methanococci, Methanococcoides, Methanocorpusculum

The ISME Journal (2016) 10, 2478–2487 © 2016 International Society for Microbial All rights reserved 1751-7362/16 www.nature.com/ismej ORIGINAL ARTICLE Chasing the elusive class WSA2: reveal a uniquely fastidious methyl- reducing

Masaru Konishi Nobu1,2, Takashi Narihiro2, Kyohei Kuroda1,3, Ran Mei1 and Wen-Tso Liu1 1Department of Civil and Environmental Engineering, University of Illinois, Urbana-Champaign, Urbana, IL, USA; 2Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan and 3Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Japan

The ecophysiology of one candidate methanogen class WSA2 (or Arc I) remains largely uncharacterized, despite the long history of research on Euryarchaeota . To expand our understanding of methanogen diversity and evolution, we metagenomically recover eight draft genomes for four WSA2 populations. Taxonomic analyses indicate that WSA2 is a distinct class from

other Euryarchaeota. None of genomes harbor pathways for CO2-reducing and aceticlastic methanogenesis, but all possess H2 and CO oxidation and energy conservation through H2-oxidizing electron confurcation and internal H2 cycling. As the only discernible methanogenic outlet, they consistently encode a methylated thiol methyltransferase. Although incomplete, all draft genomes point to the proposition that WSA2 is the first discovered methanogen restricted to methanogenesis through methylated thiol reduction. In addition, the genomes lack pathways for , nitrogen fixation and biosynthesis of many amino acids. , malonate and propionate may serve as carbon sources. Using methylated thiol reduction, WSA2 may not only bridge the carbon and sulfur cycles in eutrophic methanogenic environments, but also potentially

compete with CO2-reducing and even sulfate reducers. These findings reveal a remarkably unique methanogen ‘Candidatus Methanofastidiosum methylthiophilus’ as the first insight into the sixth class of methanogens ‘Candidatus Methanofastidiosa’. The ISME Journal (2016) 10, 2478–2487; doi:10.1038/ismej.2016.33; published online 4 March 2016

Introduction characterize these ecologically, evolutionarily and metabolically important organisms using cultivation-, As an essential component of the global carbon biochemistry- and genomics-based tools. cycle, methanogenic produce approximately Recent cultivation- and metagenomics-based studies one billion tons of CH4 per year (Thauer et al., 2008). ‘ ’ on Euryarchaeota methanogens discover novel metha- These methanogens , uniquely affiliated to the nogenic lineages, including permafrost-associated archaeal Euryarchaeota, facilitate anaerobic ‘Ca. Methanoflorentaceae’ (also known as rice cluster conversion of H2, formate, acetate, methyl com- II; Mondav et al., 2014) and gut-associated Thermo- pounds and simple alcohols to CH4 and CO2.Ina plasmata order Methanomassiliicoccales (formerly variety of anaerobic ecosystems, including both ‘Ca. Methanoplasmatales’;Dridiet al., 2012, Paul natural and engineered, fermenters, syntrophs and et al., 2012; Iino et al., 2013). However, one potentially , metabolically interact with methanogens class-level Euryarchaeota clade thought to be capable to accomplish holistic mineralization of organic of methanogenesis, WSA2 (or Arc 1), (Hugenholtz compounds (Nobu et al., 2015). Methanogens also 2002; Chouari et al., 2005) remains uncharacterized, date back to 3.5 billion years ago based on isotopic despite the identification through 16S ribosomal RNA signatures of Paleoarchean bubbles locked (rRNA) sequencing more than 15 years ago (Dojka in the Dresser formation (Ueno et al., 2006). Thus, et al., 1998). Moreover, members of this clade have researchers have made continuous efforts to been observed in a wide range of natural and engineered environments (for example, freshwater Correspondence: W-T Liu, Department of Civil and Environmental and marine sediments, contaminated groundwater Engineering, University of Illinois, Urbana-Champaign, 205N. and bioreactors; Dhillon et al., 2005; Cheng et al., Mathews Ave., Urbana, IL 61801, USA. 2012; Saito et al., 2015; Wilkins et al., 2015). To fill E-mail: [email protected] Received 22 October 2015; revised 22 January 2016; accepted this gap in our understanding of methanogen 29 January 2016; published online 4 March 2016 phylogeny and WSA2’s potential roles in anaerobic Novel methanogenic Euryarchaeota class WSA2 MK Nobu et al 2479 biogeochemical cycles, we construct the first WSA2 constructed in ARB using the neighbor-joining genomes through metagenomics of methanogenic bior- algorithm with 5000 bootstrap replications (Ludwig eactors treating wastewater. Using these genomes, we et al., 2004). The mcrA and mts genes from WSA2 and characterize the physiology and methanogenic potential publically available methanogen genomes were of the sixth class of methanogens and ninth class of aligned and put into a phylogenetic tree using Euryarchaeota, ‘Candidatus Methanofastidiosa.’ ClustalW in the MEGA package (Tamura et al., 2007). A representative for each Euryarch- aeota was used to construct a phylogenomic Materials and methods tree including WSA2 genomes using PhyloPhlAn (Segata et al., 2013). To further compare the phyloge- Metagenome sequencing, assembly and binning netic relationship of these Euryarchaeota, core gen- We collected samples from three anaerobic waste- omes were predicted for each genus and family with water treatment samples: one full-scale anaerobic more than one genome publically available by digester (reactor ADurb) and full- and lab-scale identifying genes with 450% amino acid similarity bioreactors treating wastewater from purified ter- and 480% alignment coverage across all genomes of ephthalate process (Bfssc and U1lsi, respectively). that clade using the BLAST+ package (Camacho et al., For ADurb and U1lsi, three samples were taken at 2009), based on criteria used in a core genome different time points, October, November and prediction software (Miele et al., 2011). Core genome December 2013 and February, April and May 2014, overlap between genera and families were determined correspondingly. For Bfssc, samples were taken using the same criteria. through sampling ports at different depths of the reactor along the sludge bed. Metagenomic DNA was extracted using FastDNA SPIN Kit for Soil kit (MP Biomedicals, Santa Ana, CA, USA). Sequencing Results and Discussion libraries were prepared with Kapa Library Prepara- Phylogeny of WSA2 based on 16S rRNA, McrA and tion kit (Kapa Biosystems, Wilmington, MA, USA) phylogenomics with a genomic DNA fragment size ranging between Metagenomics collectively generated genomes for four 300 and 750 bp. These libraries were sequenced on WSA2 populations from a full-scale anaerobic digester HiSeq2500 with TruSeq SBS Rapid Sequencing kit treating a mixture of waste activated sludge and (Illumina, San Diego, CA, USA), generating paired- primary sludge in Urbana, IL, USA (population end reads up to 165 bp each. The generated reads ADurb1213_Bin02801) and lab-scale (U1lsi0214_- were trimmed using Trimmomatic v0.30 with a Bin055 and U1lsi0214_Bin089) and full-scale quality cutoff of 30, sliding window of 6 bp and (B15fssc0709_Meth_Bin003) methanogenic bior- minimum length cutoff of 75 bp (Bolger et al., 2014); eactors treating purified terephthalate wastewater digitally normalized and partition using the khmer (Supplementary Table 1). For the Urbana anaerobic package (Brown et al., 2012; Pell et al., 2012); and digester, we sequenced metagenomes in 1-month assembled using SPAdes v.3.5.0 (Bankevich et al., intervals over 3 months. As for the full-scale 2012). The assembled contigs from three metagen- reactor, microbial community samples were taken omes corresponding to the same reactor were binned at separate sampling ports along the depth of the comparatively using MaxBin2.0 (Wu et al., 2014). reactor sludge bed. Only one high-quality bin was Genes were then predicted using Prodigal v2.5 (Hyatt recovered for each WSA2 population identified in et al., 2010) and annotated using Prokka (Seemann, the lab-scale reactor. In total, the eight draft 2014). We manually curated these bins and their genomes reconstructed from seven distinct sequen- annotations as described in our previous metage- cing efforts range between 1.50 and 1.99 Mb in size nomic study (Nobu et al., 2015). For interpretation of and 32.9–35.4% G+C content with estimated com- physiology and metabolism, WSA2 draft genomes pleteness of 85.0–92.5% based on 40 universal recovered from different samples of the same envir- marker genes (Ciccarelli et al., 2006; Sorek et al., onment (that is, reactor ADurb time points or Bfssc 2007). Draft genomes from the same environment depths) were collectively analyzed as a pangenome. (that is, digester and full-scale reactor) with at least 85% overlap of predicted genes with a 99% similarity cutoff were combined, generating popu- Phylogenetic analysis lation pangenomes for populations ADurb1213_- Genes annotated as small subunit 16S rRNA gene, Bin02801 and B15fssc0709_Meth_Bin003. 16S methyl coenzyme M reductase alpha subunit (McrA) rRNA gene classification, McrA phylogeny and and methylated thiol coenzyme M methyltransferase phylogenomics clearly indicate that these organisms (Mts) were extracted for phylogenetic analysis. The belong to the uncharacterized Euryarchaeota clade 16S rRNA genes were aligned against the Greengenes WSA2 falling between super-classes Methanomicro- and Silva v123 databases using PYNAST and SINA, bia–Halobacteria– and Methanobac- respectively (Caporaso et al., 2010; McDonald et al., teria– (Figure 1a and Supplementary 2012; Pruesse et al., 2012; Quast et al., 2013). Figure 1 and 2). WSA2 is distinct from related Phylogenetic trees for 16S rRNA genes were Euryarchaeota classes and uncultivated clades based

The ISME Journal Novel methanogenic Euryarchaeota class WSA2 MK Nobu et al 2480 Med. Interclade Family Order Class Similarity (%) 78 80 82

Halobacteria

“Ca. Methanomethylophilus alvus Mx1201”, CP004049.1 “Ca. Methanogranum caenicola”, AB749767.1 “Ca. Methanoplasma termitum”, CP010070.1 Methanomassiliicoccales “Ca. Methanomassiliicoccus intestinalis Issoire−Mx1”, CP005934.1 Methanomassiliicoccus luminyensis B10, HQ896499.1 Marine sediment clone SBAK-shallow-02, DQ640155.1 Hypersaline microbial mat clone GNA03C1, EU731612.1 CCA47 Mud volcano microbial mat clone 113A73, EF687639.1 Ocean surface water clone HF130_33C02, DQ156411.1 Ocean surface water clone HF200_25F07, DQ156437.1 Ocean surface water clone clone HF130_27A09, DQ156405.1 Marine Group II Hydrothermal deposits clone YS18As48, AB329815.1 Marine abyssal sediment clone SPG12_213_223_A78, FJ487464.1 Thermoplasmata Salt marsh sediment clone SAT_10E3, FJ655611.1 Salt marsh sediment clone SAT_3A6, FJ655648.1 DHVEG-1 Marine gas hydrate-associated clone HydGC-83-585A, AM229247.1 Freshwater pond sediment clone MVP−7A−32, DQ676252.1 Deep-sea water clone CTD005−33A, AY856361.1 Ocean surface water clone HF10_21C05, DQ156479.1 Marine Group III Deep-sea water clone A3_75_A2-28, FJ002870.1 Hydrothermal deposits clone YS16As27, AB329792.1 volcanium str. GSS1, AF339746.1 sp. str. JTC3, AY830840.1 777.37.3 oshimae str. Kaw2/2, X84901.1 Hot spring clone SK154, AY882711.3 Hot spring clone SK529, DQ833890.1 boonei str. T469, DQ451875.1 Hydrsaline anoxic sediment clone UBBA−104, AY226377.3 Hypersaline anoxic sediment clone DBBA−136, AY226367.3 DL1A-100 Sediment clone SFH1C091, FN391284.1 MSBL1 Deep−sea sediment clone, AY627495.1 Sinkhole clone LP30MA64, FJ902718.1 SAGMEG-1 pMC1-MSBL1 777.67.6 Natural gas field clone MOB4−11, DQ841217.1 hydrothermal fluid clone Papm3A41, AB213097. Hydrothermal deposits clone YS18As79, AB329822.1 pMC1 Hydrothermal fluid clone Papm3A05, AB213087.1 Hydrothermal sediment clone, GU137351.1 Mesophilic UASB sludge granules clone YkA78, AB266909.1 Denitrifying granular sludge clone E−A3, FJ971744.1 Manure sludge clone S38, EU662685.1 Oil-contaminated groundwater clone KuA13, AB077223.1 Landfill leachate clone GZK72, AJ576239.1 779.79.7 Manure sludge clone P41, EU662673.1 Municipal wastewater sludge clone 69−1, AF424763. Methanethiol-fed anaerobic bioreactor clone VII−D1, EF376988.1 Denitrifying granular sludge clone, FJ167435.1 Municipal wastewater sludge clone 44A−1, AF424765.1 WCHA1-57 ADurb1213_Bin02801 B15fssc0709_Meth_Bin003 U1lsi0528_Bin055 880.40.4 Mesophilic anaerobic digester clone, CU917052.1 WSA2 (Arc 1) Municipal wastewater treatment clone 059G05_A_SD_P93, CT573618.1 “Candidatus Mesophilic anaerobic digester clone, CU915939.1 Mesophilic anaerobic digester clone, CU915930.1 Methanofastidiosa” Municipal wastewater treatment plant clone 015F11_A_SD_P15, CT573523.1 Municipal wastewater treatment plant clone 014D11_A_SD_P15, CT573590.1 Municipal wastewater sludge clone 46−1, AF424764.1 Hydrothermal sediment clone 4E09, AY835427.2 Hydrothermal sediments Guaymas Basin clone, AY835426.2 Kazan-3A-21 881.71.7 Active submarine mud volcano clone, FJ712390.1 Mud volcano clone SYNH02_ew01A-001, JQ245675.1 Z7ME43 Hydrothermal sediment clone, AY835425.2 Brackish−marine sediments Aarhus B clone, FN428818.1 19c-33 Hydrothermal sediment clone AT_R040, AF419656.1 Subseafloor sediment clone MD-178-10-3261_T2-P_S150_01A07, JQ817857.1 Fe-A-9 81.5 0.10 Methanococci 78 80 82 Figure 1 16S rRNA-based phylogeny of WSA2 (red) compared with closely related Euryarchaeota classes and clusters (pMC1 and MSBL1). The tree was constructed using ARB neighbor-joining algorithm and GreenGenes 16S rRNA gene database with 5000 bootstrap replications, sequences at least 1200 bp in length, and Methanobacteria as the outgroup. Bootstrap values 490% (black), 75% (gray) and 50% (white) are indicated. The median sequence similarity of the Euryarchaeota classes and clusters (WSA2, pMC1 and MSBL1) are shown as arcs connecting the cluster labels with horizontal heights indicating similarity (right).

The ISME Journal Novel methanogenic Euryarchaeota class WSA2 MK Nobu et al 2481 on phylogenetic tree topology with high-bootstrap outside of Euryarchaeota (11–13%). A notable amount support, regardless of the reference 16S rRNA of genes relate to bacterial clades (28–35%) as also alignments used (Greengenes 2013 and Silva v123). previously observed for other Euryarchaeota (Ruepp Moreover, the median sequence similarities of WSA2 et al., 2000). However, these WSA2 genes have low to neighboring classes (76.63–81.7%) are lower than or average amino acid similarity to genes of known comparable to the similarity between Methanobacteria organisms (44–49%; Supplementary Figure 3). The and Methanococci (81.5%), confirming that WSA2 is core genome of all four WSA2 genomes (defined indeed a class-level clade in Euryarchaeota (Figure 1). by 480% amino acid similarity conserved between Based on tree topology and this median sequence all genomes) contains 842 genes, which has a similar similarity criterion, uncultivated Euryarchaeota clades phylogenetic gene distribution (Figure 2c). pMC1 and MSBL1 may form another class (tentatively To further define WSA2’s physiology and relation- termed pMC1-MSBL1) distinct from WSA2 with ship with other Euryarchaeota beyond this taxonomic phylogenetic radiation between WSA2 and superclass analysis, we compare the core genomes of each clade Methanomicrobia–Halobacteria–Thermoplasmata. (at genus and class level). Genera of similar phylogeny Similarly, genome content also suggests distant have pronounced core genome overlap; however, relationship with known methanogens and other the WSA2 core genome has low overlap with Euryarchaeota as only 25–28% and 50–58% of genes both methanogenic (6–15%) and non-methanogenic in each WSA2 genome, respectively, relate to methano- (2–15%) Euryarchaeota genera (Figure 2d). Moreover, gens and Archaea based on top blastp hits (Figure 2c). even though distantly related methanogenic genera in The remaining Archaea-related genes hit Methanomicrobia, Methanobacteria and Methano- (12–16%), Archaeoglobi (3–4%) and other clades cocci share many core genes (152–257 genes average),

Euryarchaeota phylogenomic tree Methanogen core genome overlap WSA2 top BLAST hits (by gene number) Methanomicrobia

All Genes

Methanogens Thermococci Core Archaeoglobi Genes Other Archaea Archaeoglobi Eukarya Halobacteria

Euryarchaeota genus-level core genome overlap (by percentage)

Thermoplasmata

WSA2

Methanobacteria

Methanococci

Methanopyri Thermococci

Figure 2 (a) Euryarchaeota phylogenomics tree of representative genera with as the outgroup. Nodes are marked for bootstrap values 475% (filled) and 50% (open). For each genus, one representative is shown on the tree. Corresponding to each genus, the presence of the gene/pathway in all (white) or only some (gray, size proportional to percentage) of the genomes of that genus are shown. Genes/pathways are shown for carbon fixation (reductive acetyl-CoA pathway—rACP; reductive pentose phosphate pathway— rPPP; and reductive tricarboxylic acid cycle—rTCA), nitrogen fixation (Nif), type I coenzyme M (CoM) synthesis (comABC), type II CoM synthesis (cysteate synthase—CS; cysteate aminotransferase—CA), and final CoM synthesis step (sulfopyruvate decarboxylase—comDE). (b) The overlap of core genomes between methanogenic genera, including WSA2. The intensity of the red color corresponds to the number of overlapping genes. (c) The phylogenetic distribution of WSA2 genes in the entire genome and only core genomes. All four genomes are shown as individual rings. (d) The overlap of core genomes between all Euryarchaeota genera. The intensity of the red color corresponds to the percent overlap of core genes in reference to the genome on the vertical axis.

The ISME Journal Novel methanogenic Euryarchaeota class WSA2 MK Nobu et al 2482 WSA2 shares much fewer genes with these methano- metabolizing and Methanomassilii- gens (69–111 genes average; Figure 2b). The genes coccales, (b) non-methyl-metabolizing methanogens or shared between WSA2 with Euryarchaeota genera (c) both (Figure 3). We suspect that clusters found in primarily consist of housekeeping genes, including non-methyl-metabolizing methanogens are not catabolic ribosomal proteins, biosynthesis genes and Archaea- and rather serve physiological roles in methanogens. specific genes (for example, histone, thermosome and ATP synthase). WSA2 and methanogenic clades all Catabolism share genes encoding methanogenesis marker proteins H+ or Na+ H+ or Na+ and Mcr, responsible for the terminal step of metha- nogenesis (Supplementary Table 2). Interestingly, WSA2 shares the methylmalonyl-CoA pathway with non-methanogenic genera, which no known methano- ATP EhbA-Q gen genomes harbor. These findings of Mcr, unique synthase genetic composition and unusual methylmalonyl-CoA pathway suggest that WSA2 may perform methano- genesis and yet have distinct capabilities from typical + methanogens. 2H2 4H Fdox Fdred ATP ADP Pi CoM-S-S-CoB Uniquely restricted methanogenic metabolism MvhA HdrA Even though the WSA2 genomes harbor Mcr, all MvhD HdrC eight draft genomes from the seven independent- MvhG HdrB CoB-SH McrABG sequencing efforts lack conventional CO2 reduction CoM-SH CH4

to CH4 and acetyl-CoA synthase pathway, indicating CO CO2 that neither CO2 nor acetate can serve as substrates. CoM-S-CH3 Moreover, while many methanogens missing CODH MtsA these pathways respire and (for example, and Methano- sphaera), we could not identify corresponding Fdox Fdred CH3-SR HS-R methyltransferases essential for such metabolism in any of the WSA2 draft genomes. Strikingly, the WSA2 Anabolism Mdc: Malonate decarboxylase CO Acs: Acetyl-CoA synthetase draft genomes consistently encode methylated thiol 2 Por: Pyruvate:Fd oxidoreductase Acetate Malonate Pyr: Pyruvate carboxylase Mts homologs for funneling C1 compounds into the ATP Mdc Oad: Oxaloacetate decarboxylase reductive arm of methanogenesis (Supplementary Acs Table 2). Thus, WSA2 may only be capable of Acetyl-CoA Acetyl-CoA Tricarboxylic Acid Cycle demethylation of methylated thiols for CH4 genera- Por Citrate tion, which would be the first example of such Pyruvate Oxaloacetate Isocitrate

restricted methanogenic catabolism. Moreover, the CO2 Acetyl- CO2 lack of the methanogenic C1 pathway indicates that Pyc or Oad CoA CoA WSA2 likely use methylated thiols as electron Malate Glyoxylate acceptors rather than donor. Methanol and methy- CO2 lated amine metabolism are observed in methanogens Succinyl-CoA CO associated with sediments or gut environments as 2 Succinate produce methanol and methylated amines CoA Propionate derive from eukaryotic lipids and osmoregulators; Methylmalonyl-CoA pathway thus, it may be logical that the studied anaerobic digester WSA2 populations lack such metabolism. Figure 3 WSA2 (a) catabolism and (b) anabolism. (a)WSA2has Perhaps other WSA2 class members associated with genes for H2 oxidation through electron-bifurcating hydrogenase – marine environments or sediments may possess (HdrABC MvhDGA) and H2 cycling by energy-converting hydro- genase (EhbA-Q); CO oxidation by carbon monoxide dehydrogenase catabolic capacity beyond methylated thiols. (CODH); and methylated thiol reduction and methanogenesis by Mts substantially vary in specificity to methylated methylated thiol Coenzyme M methyltransferase corrinoid fusion thiol substrates, such as methanethiol (MeSH), protein (MtsA) and methyl coenzyme M reductase (McrABG). The dimethylsulfide (DMS), 3-methylmercaptopropionate proton motive force (or cation gradient) generated by Ehb can (MMPA) and 3-mercaptopropionate (MPA; Tallant support ATP production by ATP synthase. (b) Malonate decarbox- ylase and acetyl-CoA synthetase can convert malonate and acetate and Krzycki, 1997; Fu and Metcalf, 2015). Thus, into acetyl-CoA for downstream co-assimilation with CO2 (bolded) accurate annotation of WSA2’s methanogenic capa- through pyruvate:ferredoxin oxidoreductase, pyruvate carboxylase city necessitates systemic determination of the specific and tricarboxylic acid (TCA) cycle. As identified for other function of the two Mts homologs conserved among heterotrophic methanogens, WSA2 does not encode the glyoxylate shunt for acetate assimilation (gray with dotted line). The WSA2 genomes. Phylogenetic analysis of all publically methylmalonyl-CoA pathway can facilitate co-assimilation of

available methanogen Mts-related methyltransferase propionate and CO2 also into the TCA cycle. WSA2 can use key sequences reveals clusters related to (a) methyl- TCA cycle intermediates (pink) as building blocks for biosynthesis.

The ISME Journal Novel methanogenic Euryarchaeota class WSA2 MK Nobu et al 2483 Clusters related to methyl-metabolizing methanogens Borrel et al., 2014) analogous to the were categorized into protein families, of which three cytochrome-dependent H2 cycling (Kulkarni et al., have representatives with biochemical or transcriptomic 2009). Although WSA2 also encodes F420-reducing characterization. A recent genetic and transcriptomic hydrogenase, none of the draft genomes encode other study on Methanosarcina acetivorans C2A shows F420-oxidizing enzymes related to methanogenesis so evidence that MtsD, MtsF and MtsH (family IV), the physiology function of F420 remains unclear. The respectively, degrade DMS, MeSH and both and MptA WSA2 genomes lack genes encoding cytochromes and (family VII) is critical for MMPA metabolism (Moran methanophenazine synthesis, suggesting that they et al., 2008; Bose et al., 2009; Oelgeschläger and perform cytochrome-independent methanogenesis like Rother, 2009; Fu and Metcalf, 2015). M. barkeri MtsA all non-Methanosarcinales methanogens (Thauer et al., (family VIII) has also been demonstrated to facilitate 2008). methyl transfer from DMS, MMPA and MPA, also MeSH to a much lesser extent (LeClerc and Grahame, 1996, Tallant et al., 2001). In WSA2, we identify an Physiology Mts fusion protein encoding a methyltransferase and Archaea are known to utilize a variety of autotrophic corrinoid protein, both of which relate to family VIII carbon fixation pathways (Berg et al., 2010), yet MtsA and MtsB, respectively (Supplementary Figure 4); methanogenic Euryarchaeota specifically possess thus, WSA2 may utilize this MtsA and Mcr to perform reductive acetyl-CoA pathway, pentose phosphate methanogenesis through reduction of multi-carbon pathway (rPPP; or Calvin-Benson cycle), and/or methylated thiols. tricarboxylic acid cycle (Figure 2a). Methanosarci- Many uncharacterized Mts homologs fall into five nales, Methanocellales, ‘Ca. Methanoflorens,’ Metha- other families affiliated with methyl-metabolizing nobacteriales, , and methanogens (I, II, III, V and VI). Although these members encode reductive acetyl-CoA pathway; groups have no experimentally studied representa- members either possess both tives, their exclusive affiliation with methyl- reductive acetyl-CoA pathway and rPPP or only degrading methanogens suggests that these Mts rPPP; and only several phylogenetically scattered homologs relate to methanogenic methyl metabo- species encode reductive tricarboxylic acid cycle. lism. Family I is unlikely to support MeSH, DMS or Interestingly, the Methanomicrobiales members only MMPA metabolism as Methanosarcina acetivorans encoding rPPP all require exogenous organic carbon C2A does not express MA0847 during growth on for growth, suggesting that the rPPP ribulose-1,5- these substrates (Bose et al., 2009; Fu and Metcalf, bisphosphate carboxylase/oxygenase in these organ- 2015). Thus, the metabolic function of family isms participates in AMP metabolism rather than I-related WSA2 Mts-like protein (MtlA) is unclear. carbon fixation (Sato et al., 2007). Likewise, WSA2 Interestingly, MtlA associates with a corrinoid-CoM may require organic carbon sources for growth (that methyltransferase (MtlB), corrinoid protein (MtlC) is, heterotrophic) as they lack any discernable carbon and methyltransferase reductive activase (MtlD; fixation pathway (Figure 2a). In agreement, known Supplementary Table 2). Notably, MtlC is phylogen- methanogens missing such pathways require acetate etically distinct from other known Mts corrinoid or complex exogenous nutrients (for example, yeast proteins (Supplementary Figure 4). Although known extract or rumen fluid) for growth (Whitman et al., Mts can single-handedly catalyze the two-stage 1982; Miller and Wolin, 1985; Tanner and Wolfe, methyl transfer from substrate to CoM, methanol and 1988; Zellner et al., 1989; Miller and Lin, 2002). In methyltransferases require two subunits addition, unlike most methanogens, the WSA2 (Tallant et al., 2001); therefore, MtlAB may have genomes are also deficient in nitrogen fixation genes substrates and biochemical properties distinct from (that is, Nif) though they do encode non-nitrogen- known Mts. However, the presence of MtlA in other fixing NifD- and NifH-like genes (NflDH;Figure2a; methyl-degrading methanogens warrants further Staples et al., 2007). Thus, the draft genomes suggest experimental investigation to expand our understand- that WSA2 may be incapable of both autotrophy and ing of diversity in methanogenic methyl metabolism. nitrogen fixation, a rarity among known methanogens. To complement the reductive methylated thiol This genomic analysis also revealed that several gut- metabolism, the WSA2 genomes also possess H2-and associated (Methanobrevibater sp. CO-oxidizing pathways for donating reducing power JH1 and stadtmanae DSM3091) also into methanogenesis (Supplementary Table 2). For H2 cannot fix carbon and nitrogen. metabolism, WSA2 can perform (i) electron-bifurcating In order to determine the carbon source require- H2 oxidation using a cytosolic methylviologen- ment for WSA2, we genomically evaluate their reducing hydrogenase complexed with a heterodisul- biosynthetic capacity. All WSA2 genomes have genes fide reductase and (ii) proton-pumping H2 generation for gluconeogenesis and tricarboxylic acid (TCA) mediated by a membrane-bound energy-converting cycle, but only two genomes encode a complete PPP hydrogenase (Figure 4). WSA2 may pair these two (Supplementary Table 3). The presence of TCA cycle pathways to accomplish energy-conserving internal and malonate decarboxylase suggests that they can H2 cycling similar to Methanosphaera stadtmaniae utilize acetate as a carbon source and also generate and Methanomassiliicoccales spp. (Thauer et al., 2008; acetate from malonate. They encode a pyruvate:

The ISME Journal Novel methanogenic Euryarchaeota class WSA2 MK Nobu et al 2484 ferredoxin oxidoreductase and pyruvate carboxylase oxidoreductase with either pyruvate carboxylase or or acetate-CoA assimilation. Similarly, heterotrophic oxaloacetate decarboxlyase for acetate assimilation. methanogens encode pyruvate:ferredoxin Unique from other methanogens, WSA2 encode

648055466 Metev_0794 ( evestigatum Z-7303) I 648055929 Metev_1287 (Methanohalobium evestigatum Z-7303) 646706713 Mmah_0657 ( mahii DSM 5219) 2509663950 Metho_2301 (Methanomethylovorans hollandica DSM 15978) 638175599 MA0847 (Methanosarcina acetivorans C2A) 2596417227 IE18DRAFT_02309 (Methanosarcina sp. DSM 11855) APG09_00715 (ADurb1213_Bin02801) APG10_01477 (B03fssc0709_Meth_Bin005) AMQ22_01393 (U1lsi0528_Bin055) AMQ74_01089 (U1lsi0528_Bin089) 2565995199 BP07DRAFT_1155 (Methermicoccus shengliensis DSM 18856) II 2518907835 (Methanomassiliicoccus luminyensis B10) 2518908409 (Methanomassiliicoccus luminyensis B10) III 2509662013 Metho_0364 (Methanomethylovorans hollandica DSM 15978) 2502870330 Mzhil_0354 ( zhilinae WeN5) 648056339 Metev_1709b (Methanohalobium evestigatum Z-7303) IV 2502871809 Mzhil_1795b (Methanosalsum zhilinae WeN5) 646707531 Mmah_1481b (Methanohalophilus mahii DSM 5219) 2509662912 Metho_1263b (Methanomethylovorans hollandica DSM 15978) 638175611 MA0859b (Methanosarcina acetivorans C2A) MtsD DMS 638179461 MA4558b (Methanosarcina acetivorans C2A) MtsF MeSH 638179284 MA4384b (Methanosarcina acetivorans C2A) MtsH DMS 2540666870 MMALV_04210 (Ca. Methanomethylophilus alvus Mx1201) / MeSH 2555937261 H729_02130 (Ca. Methanomassiliicoccus intestinalis Issoire-Mx1) 2540643570 BN140_1356 ( bourgensis MS2) 640116860 Memar_2152 (Methanoculleus marisnigri JR1) 2579682829 EI28_12585 (Methanoculleus sp. MH98A) 2507145897 Metli_0374 ( liminatans GKZPZ) 640867319 Mboo_0104 ( boonei 6A8) 2509037192 Metfor_0231 (Methanoregula formicica SMSP) 2609087959 Mpt1_c05730 (Ca. Methanoplasma termitum strain MpT1) V 2565995584 BP07DRAFT_1543 (Methermicoccus shengliensis DSM 18856) 2518906948 (Methanomassiliicoccus luminyensis B10) 2555937687 MMINT_08950 (Ca. Methanomassiliicoccus intestinalis Issoire-Mx1) 2609087962 Mpt1_c05760 (Ca. Methanoplasma termitum strain MpT1) 2555937678 MMINT_08870 (Ca. Methanomassiliicoccus intestinalis Issoire-Mx1) 2518908577 (Methanomassiliicoccus luminyensis B10) 648195559 Mpet_2558 ( petrolearius DSM 11571) 640868266 Mboo_1040 (Methanoregula boonei 6A8) 2565994707 BP07DRAFT_0661 (Methermicoccus shengliensis DSM 18856) 637958739 Mbur_0957 ( burtonii DSM 6242) 646706069 Mmah_0020 (Methanohalophilus mahii DSM 5219) 2540643434 BN140_1221 (Methanoculleus bourgensis MS2) 648055745 Metev_1081 (Methanohalobium evestigatum Z-7303) 646707526 Mmah_1476 (Methanohalophilus mahii DSM 5219) 638179254 MA4354 (Methanosarcina acetivorans C2A) 2509661996 Metho_0347 (Methanomethylovorans hollandica DSM 15978) 2502870890 Mzhil_0898 (Methanosalsum zhilinae WeN5) 643569939 Mpal_1305 (Methanosphaerula palustris E1-9c) 2540666882 MMALV_04320 (Ca. Methanomethylophilus alvus Mx1201) VI 640787989 Maeo_0598 ( aeolicus Nankai-3) 2519842293 F555DRAFT_00934 ( thermolithotrophicus DSM 2095) 650856365 Metig_0677 ( igneus Kol 5) 2563557093 MMP1150 (Methanococcus maripaludis S2) 640787991 Maeo_0600 (Methanococcus aeolicus Nankai-3) 2519842291 F555DRAFT_00932 (Methanothermococcus thermolithotrophicus DSM 2095) 650856363 Metig_0675 (Methanotorris igneus Kol 5) 640166339 MmarC5_0839 (Methanococcus maripaludis C5) 641284325 MmarC6_1826 (Methanococcus maripaludis C6) 640792597 MmarC7_0076 (Methanococcus maripaludis C7) 2563556772 MMP0830 (Methanococcus maripaludis S2) 2511671703 GYY_04815 (Methanococcus maripaludis X1) 646860021 Mvol_1575 (Methanococcus voltae A3) 646860022 Mvol_1576 (Methanococcus voltae A3) 640166336 MmarC5_0836 (Methanococcus maripaludis C5) 640792600 MmarC7_0079 (Methanococcus maripaludis C7) 641284322 MmarC6_1823 (Methanococcus maripaludis C6) 2563556776 MMP0834 (Methanococcus maripaludis S2) 2511671707 GYY_04835 (Methanococcus maripaludis X1) VII 638179053 MA4165 (Methanosarcina acetivorans C2A) MptA MMPA 637702416 Mbar_A3585 ( Fusaro) VIII Q48924 MTSA_METBA (Methanosarcina barkeri MS) MtsA DMS 638166986 mtsA (Methanosarcina mazei Goe1) / MeSH 2540564346 MmTuc01_2485 (Methanosarcina mazei Tuc01) 2578263266 M886DRAFT_1930 (Methanosarcina sp. SMA-21) 2518908040 (Methanomassiliicoccus luminyensis B10) 2518908806 (Methanomassiliicoccus luminyensis B10) 2540667798 MMALV_13470 (Ca. Methanomethylophilus alvus Mx1201) APG09_00949 (ADurb1213_Bin02801) APG11_00978 (B15fssc0709_Meth_Bin003) MtaA MtbA 0.16

The ISME Journal Novel methanogenic Euryarchaeota class WSA2 MK Nobu et al 2485 methylmalonyl-CoA pathway that integrate propio- methoxylated compounds may yield sufficient nate into the TCA cycle. TCA-based propionate DMS to support methanogenesis (Kadota and assimilation could either split into the oxidative and Ishida, 1972; Zinder and Brock, 1978; Finster et al., reductive arms or strictly go through oxidative TCA 1990; Kiene and Hines, 1995). Thus, we suspect that given acetate or malonate is available as an acetyl-CoA this novel archaeon is adapted to performing source. Thus, WSA2 may utilize acetate, malonate or methanogenesis in eutrophic anaerobic environ- propionate with CO2 as carbon sources. However, all ments and bridging the carbon and sulfur cycles. In genomes lack complete biosynthesis pathways for addition, the existence of a dedicated H2-oxidizing glycine, homocysteince, homoserine, methionine, methylated thiol reducing methanogen can signifi- proline, threonine and tryptophan and some genomes cantly change our thermodynamic understanding of lack full pathways for isoleucine, leucine, phenylala- anaerobic ecosystems. Typical CO2-reducing metha- − nine and tyrosine, suggesting severe auxotrophy nogenesis (4H2+HCO3 → CH4+3H2O: ΔG°ʹ =- − −1 (Supplementary Table 3). To complement this, they 135.56 kJ mol CH4 ) rapidly becomes encode amino acid and peptide transporters. thermodynamically unfavorable as H2 concentration We identify pathways for synthesis of methanogen- decreases due to the high stoichiometry of input H2 to esis cofactors: Coenzyme B, F420,F430 and cobamide generated CH4 (4:1) in contrast with the low stoichio- (partial; Supplementary Table 4). The WSA2 genomes metry (1:1) of WSA2-like methanogenesis (for → also consistently harbor F420 modification genes. example, H2+DMS CH4+CH3SH: Δ ʹ − − 1 Interestingly, they also possess F420 alpha-L-glutamate G° = 161.14 kJ mol CH4 ). For example, at ligase (CofF) that have only been observed in 10 Pa H2,CO2 reduction would only yield − −1 Methanosarcinales and Methanococcales previously 18.48 kJ mol CH4 , while DMS reduction would yied −1 (Li et al., 2003). On the other hand, the WSA2 − 140.04 kJ mol CH4 (50 kPa CH4,100μM thiols, 50 mM genomes are missing CoM synthesis pathways. bicarbonate). Similarly, several phylogenetically scattered metha- In theory, WSA2-like methyl reduction could thrive nogen isolates also lack such genes. All of these under low H2 concentrations and maintain H2 lower methanogens are either dependent on rumen fluid than CO2-reducing methanogenesis to support H2 for growth or strongly stimulated by CoM supple- producers (that is, syntrophs and fermenters) and 2− mentation. Although whether WSA2 and these also compete against sulfate reducers (4H2+SO4 + + → − Δ ʹ − − 1 methanogens possess an unidentified CoM synthesis H HS +4H2O: G° = 152.2 kJ mol ). Thus, WSA2 pathway is unclear, WSA2 is likely adapted to may have an essential and overlooked ecological role availability of exogenous CoM produced by other for syntrophy in methanogenic environments (for methanogens in situ. This suggests that WSA2 can example, anaerobic digestion) and competitive metha- only inhabit ecosystems with other active methano- nogenesis in sulfate-reducing environments (for exam- gens. As for methanogens that do encode CoM ple, marine sediments). biosynthesis, Methanomicrobia and Methanomassilii- Besides these unique features, WSA2 has signa- coccales employ the Methanosarcina-type pathway, tures of cytochrome-independent methanogenesis while Methanobacteria, Methanococci and Methano- (heterodisulfide reductase–methylviologen-reducing pyri employ type (Graupner hydrogenase energy conservation), Methanosarci- et al., 2000; Graham et al., 2002, 2009). nales and Methanococcales F420 modification, Taxonomic classification, core genome comparison, and Methanosphaera and Methanomassiliicoccales unique methylated thiol-specific methanogenic metabo- internal H2 cycling. In addition, characterization of lism and obligate heterotrophy all agree that WSA2 is a WSA2 expands the phylogenetic range of methanogen novel Euryarchaeota class with distinct features from heterotrophy. In conclusion, we present the first detailed other methanogens. Their heterotrophic, auxotrophic description of the sixth methanogen class WSA2 as a and ammonia-dependent nature is unique and yet significant step forward in understanding methanogen appropriate for the organic-rich anaerobic environments phylogeny, metabolic diversity and contribution to they are often associated with (that is, wastewater global biogeochemical cycles with provisional class treatment sludge and marine sediments) where and species assignment of ‘Candidatus Methanofasti- fermentation and methanogenesis produce the diosa’ class. nov. (Me.tha.no.fas.tid.i.o'sa. N.L. pref. required carbon sources (acetate, propionate and methano-, pertaining to methane; N.L. f. adj. fastidiosa, CO2), amino acids, ammonia (Blair and Carter, 1992; highly critical; referring to the nutritional fastidiousness Lettinga, 1995) and possibly free CoM. Moreover, of the organism, particularly on primary isolation) and fermentative degradation of methionine and ‘Candidatus Methanofastidiosum methylthiophilus’

Figure 4 Phylogeny of methylated thiol Coenzyme M methyltransferase (Mts) homologs with methanol- (MtaA) and methylamine- (MtbA) specific methlyltransferases as the outgroup. The tree was constructed using Clustalw with default parameters for alignment and neighbor-joining clustering method for tree construction with 5000 bootstrapped replications. Family classifications are assigned to clusters only containing Mts homologs from methyl-metabolizing methanogens (Methanosarcinales—blue, Methanomassiliicoccales— orange) and/or WSA2 (red and bolded). Only three families contain representatives biochemically or transcriptomically characterized in Methanosarcina acetivorans and M. barkeri (bolded). For those Mts, their predicted substrates are noted. Nodes are marked for bootstrap values 490% (black), 75% (gray with outline) and 50% (gray).

The ISME Journal Novel methanogenic Euryarchaeota class WSA2 MK Nobu et al 2486 gen.nov.sp.nov.(Me.tha.no.fas.tid.i.o'sum. N.L. pref. Chouari R, Le Paslier D, Daegelen P, Ginestet P, Weissenbach J, methano-, pertaining to methane; N.L. neut. adj. Sghir A. (2005). Novel predominant archaeal and fastidiosum, highly critical; referring to the nutritional bacterial groups revealed by molecular analysis of fastidiousness of the organism, particularly on pri- an anaerobic sludge digester. Environ Microbiol 7: – mary isolation/me.thyl.thi.o'phi.lus.M.L.n. methyl,the 1104 1115. methyl group; thi.o′phi.lus. Gr. n. thion sulfur; Ciccarelli FD, Doerks T, von Mering C, Creevey CJ, Snel B, Gr. adj. phylos loving; N.L. neut. adj. methylthiophilus Bork P. (2006). Toward automatic reconstruction of a highly resolved tree of . Science 311: 1283–1287. methyl- and sulfur- loving). Dhillon A, Lever M, Lloyd KG, Albert DB, Sogin ML, Teske A. (2005). Methanogen diversity evidenced by molecular characterization of methyl coenzyme M reductase A Conflict of Interest (mcrA) genes in hydrothermal sediments of the Guaymas Basin. Appl Environ Microbiol 71: 4592–4601. The authors declare no conflict of interest. Dojka MA, Hugenholtz P, Haack SK, Pace NR. (1998). Microbial diversity in a hydrocarbon- and chlorinated- Acknowledgements solvent-contaminated aquifer undergoing intrinsic bior- emediation. Appl Environ Microbiol 64:3869–3877. The Roy J Carver Biotechnology Center High-Throughput Dridi B, Fardeau ML, Ollivier B, Raoult D, Drancourt M. Sequencing and Genotyping Unit DNA services team provided (2012). Methanomassiliicoccus luminyensis gen. nov., helpful support for sequencing projects. We thank Professor sp. nov., a methanogenic archaeon isolated from William W Metcalf at University of Illinois, Urbana-Champaign human faeces. Int J Syst Evol Microbiol 62: 1902–1907. Department of Microbiology for valuable discussion. We Finster K, King GM, Bak F. (1990). Formation of methyl- acknowledge the Energy Bioscience Institute for funding mercaptan and dimethylsulfide from methoxylated through project number OO4J12. aromatic compounds in anoxic marine and fresh water sediments. FEMS Microbiol Ecol 74: 295–301. Fu H, Metcalf WW. (2015). Genetic basis for metabolism of methylated sulfur compounds in Methanosarcina References species. J Bacteriol 197: 1515–1524. Graham DE, Xu H, White RH. (2002). Identification Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, of coenzyme M biosynthetic phosphosulfolactate Kulikov AS et al. (2012). SPAdes: a new genome synthase: a new family of sulfonate-biosynthesizing assembly algorithm and its applications to single- enzymes. J Biol Chem 277: 13421–13429. sequencing. J Comput Biol 19: 455–477. Graham DE, Taylor SM, Wolf RZ, Namboori SC. (2009). Berg IA, Kockelkorn D, Ramos-Vera WH, Say RF, Zarzycki J, Convergent evolution of coenzyme M biosynthesis in the Hugler M et al. (2010). Autotrophic carbon fixation in Methanosarcinales: cysteate synthase evolved from an archaea. Nat Rev Microbiol 8:447–460. ancestral threonine synthase. Biochem J 424:467–478. Blair NE, Carter WD Jr. (1992). The carbon isotope Graupner M, Xu H, White RH. (2000). Identification of the biogeochemistry of acetate from a methanogenic marine gene encoding sulfopyruvate decarboxylase, an enzyme sediment. Geochim Cosmochim Acta 56: 1247–1258. involved in biosynthesis of coenzyme M. JBacteriol182: Bolger AM, Lohse M, Usadel B. (2014). Trimmomatic: 4862–4867. a flexible trimmer for Illumina sequence data. Bioin- Hugenholtz P. (2002). Exploring prokaryotic diversity in formatics 30: 2114–2120. the genomic era. Genome Biol 3: reviews0003. Borrel G, Parisot N, Harris HM, Peyretaillade E, Gaci N, Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Tottey W et al. (2014). Comparative genomics highlights Hauser LJ. (2010). Prodigal: prokaryotic gene recogni- the unique biology of Methanomassiliicoccales,a tion and translation initiation site identification. BMC Thermoplasmatales-related seventh order of methano- Bioinformatics 11: 119. genic archaea that encodes . BMC Genomics Iino T, Tamaki H, Tamazawa S, Ueno Y, Ohkuma M, 15: 679. Suzuki K et al. (2013). Candidatus Methanogranum Bose A, Kulkarni G, Metcalf WW. (2009). Regulation of caenicola: a novel methanogen from the anaerobic putative methyl-sulphide methyltransferases in Metha- digested sludge, and proposal of Methanomassiliicoc- nosarcina acetivorans C2A. Mol Microbiol 74: 227–238. caceae fam. nov. and Methanomassiliicoccales ord. Brown CT, Howe AC, Zhang Q, Pyrkosz AB, Brom TH. nov., for a methanogenic lineage of the class Thermo- (2012), A reference-free algorithm for computational plasmata. Microbes Environ 28: 244–250. normalization of shotgun sequencing data arXiv Kadota H, Ishida Y. (1972). Production of volatile sulfur 1203.4802 [q-bio.GN]. compounds by . Annu Rev Microbiol Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, 26: 127–138. Bealer K et al. (2009). BLAST+: architecture and Kiene RP, Hines ME. (1995). Microbial formation of applications. BMC Bioinformatics 10:421. dimethyl sulfide in anoxic Sphagnum peat. Appl Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Environ Microbiol 61: 2720–2726. Bushman FD, Costello EK et al. (2010). QIIME allows Kulkarni G, Kridelbaugh DM, Guss AM, Metcalf WW. (2009). analysis of high-throughput community sequencing data. is a preferred intermediate in the energy- Nat Methods 7:335–336. conserving electron transport chain of Methanosarcina Cheng TW, Chang YH, Tang SL, Tseng CH, Chiang PW, barkeri. Proc Natl Acad Sci USA 106: 15915–15920. Chang KT et al. (2012). Metabolic stratification driven LeClerc GM, Grahame DA. (1996). Methylcobamide: by surface and subsurface interactions in a terrestrial coenzyme M methyltransferase isozymes from Metha- mud volcano. ISME J 6: 2280–2290. nosarcina barkeri. Physicochemical characterization,

The ISME Journal Novel methanogenic Euryarchaeota class WSA2 MK Nobu et al 2487 cloning, sequence analysis, and heterologous gene mcrA and 16S rRNA gene phylogenetic analyses. expression. J Biol Chem 271: 18725–18731. J Water Envir Tech 13: 279–289. Lettinga G. (1995). Anaerobic digestion and wastewater Sato T, Atomi H, Imanaka T. (2007). Archaeal type III treatment systems. Antonie van Leeuwenhoek 67: RuBisCOs function in a pathway for AMP metabolism. 3–28. Science 315: 1003–1006. Li H, Xu H, Graham DE, White RH. (2003). Glutathione Seemann T. (2014). Prokka: rapid prokaryotic genome synthetase homologs encode alpha-L-glutamate ligases annotation. Bioinformatics 30: 2068–2069. for methanogenic coenzyme F420 and tetrahydrosarci- Segata N, Bornigen D, Morgan XC, Huttenhower C. (2013). napterin biosyntheses. Proc Natl Acad Sci USA 100: PhyloPhlAn is a new method for improved phyloge- 9785–9790. netic and taxonomic placement of microbes. Nat Ludwig W, Strunk O, Westram R, Richter L, Meier H, kumar Commun 4: 2304. Yadhu et al. (2004). ARB: a software environment for Sorek R, Zhu Y, Creevey CJ, Francino MP, Bork P, Rubin EM. sequence data. Nucleic Acids Res 32:1363–1371. (2007). Genome-wide experimental determination of bar- McDonald D, Price MN, Goodrich J, Nawrocki EP, DeSantis TZ, riers to . Science 318:1449–1452. Probst A et al. (2012). An improved Greengenes Staples CR, Lahiri S, Raymond J, Von Herbulis L, Mukho- with explicit ranks for ecological and phadhyay B, Blankenship RE. (2007). Expression and evolutionary analyses of bacteria and archaea. ISME J association of group IV nitrogenase NifD and NifH – 6: 610 618. homologs in the non-nitrogen-fixing archaeon Methano- Miele V, Penel S, Duret L. (2011). Ultra-fast sequence caldococcus jannaschii. J Bacteriol 189: 7392–7398. clustering from similarity networks with SiLiX. BMC Tallant TC, Krzycki JA. (1997). Methylthiol:coenzyme M Bioinformatics 12: 116. methyltransferase from Methanosarcina barkeri,an Miller TL, Wolin MJ. (1985). Methanosphaera stadtmaniae gen. enzyme of methanogenesis from dimethylsulfide and nov., sp. nov.: a species that forms methane by reducing – – methylmercaptopropionate. J Bacteriol 179:6902 6911. methanol with hydrogen. Arch Microbiol 141: 116 122. Tallant TC, Paul L, Krzycki JA. (2001). The MtsA subunit of Miller TL, Lin C. (2002). Description of the methylthiol:coenzyme M methyltransferase of gottschalkii sp. nov., Methanobrevibacter thaueri sp. nov., Methanosarcina barkeri catalyses both half-reactions Methanobrevibacter woesei sp. nov. and Methanobrevibac- – of corrinoid-dependent dimethylsulfide: coenzyme M ter wolinii sp. nov. IntJSystEvolMicrobiol52:819 822. methyl transfer. J Biol Chem 276: 4485–4493. Mondav R, Woodcroft BJ, Kim EH, McCalley CK, Hodgkins SB, Tamura K, Dudley J, Nei M, Kumar S. (2007). MEGA4: Crill PM et al. (2014). Discovery of a novel methanogen Molecular evolutionary genetics analysis (MEGA) soft- prevalent in thawing permafrost. Nat Commun 5: 3212. ware version 4.0. Mol Biol Evol 24: 1596–1599. Moran JJ, House CH, Vrentas JM, Freeman KH. (2008). Tanner RS, Wolfe RS. (1988). Nutritional requirements of Methyl sulfide production by a novel carbon monoxide mobile. Appl Environ Microbiol metabolism in Methanosarcina acetivorans. Appl 54: 625–628. Environ Microbiol 74: 540–542. Thauer RK, Kaster AK, Seedorf H, Buckel W, Hedderich R. Nobu MK, Narihiro T, Rinke C, Kamagata Y, Tringe SG, (2008). Methanogenic archaea: ecologically relevant Woyke T et al. (2015). Microbial dark matter ecoge- differences in energy conservation. Nat Rev Microbiol nomics reveals complex synergistic networks in a – methanogenic bioreactor. ISME J 9: 1710–1722. 6: 579 591. Oelgeschläger E, Rother M. (2009). In vivo role of three Ueno Y, Yamada K, Yoshida N, Maruyama S, Isozaki Y. (2006). Evidence from fluid inclusions for microbial methanogen- fused corrinoid/methyl transfer proteins in Methano- – sarcina acetivorans. Mol Microbiol 72: 1260–1272. esis in the early Archaean era. Nature 440: 516 519. Paul K, Nonoh JO, Mikulski L, Brune A. (2012). 'Methano- Whitman WB, Ankwanda E, Wolfe RS. (1982). Nutrition and carbon metabolism of Methanococcus voltae. plasmatales,' Thermoplasmatales-related archaea in ter- – mite guts and other environments, are the seventh order of J Bacteriol 149: 852 863. methanogens. Appl Environ Microbiol 78:8245–8253. Wilkins D, Lu XY, Shen Z, Chen J, Lee PK. (2015). Pell J, Hintze A, Canino-Koning R, Howe A, Tiedje JM, Pyrosequencing of mcrA and archaeal 16S rRNA genes Brown CT. (2012). Scaling metagenome sequence reveals diversity and substrate preferences of metha- assembly with probabilistic de Bruijn graphs. Proc nogen communities in anaerobic digesters. Appl – Natl Acad Sci USA 109: 13272–13277. Environ Microbiol 81: 604 613. Pruesse E, Peplies J, Glockner FO. (2012). SINA: accurate high- Wu YW, Tang YH, Tringe SG, Simmons BA, Singer SW. throughput multiple sequence alignment of ribosomal (2014). MaxBin: an automated binning method to recover RNA genes. Bioinformatics 28:1823–1829. individual genomes from metagenomes using an Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P expectation-maximization algorithm. Microbiome 2: 26. et al. (2013). The SILVA ribosomal RNA gene database Zellner G, Stackebrandt E, Messner P, Tindall BJ, Conway project: improved data processing and web-based tools. de Macario E, Kneifel H et al. (1989). Methanocorpus- Nucleic Acids Res 41:D590–D596. culaceae fam. nov., represented by Methanocorpuscu- Ruepp A, Graml W, Santos-Martinez ML, Koretke KK, lum parvum, sinense spec. nov. Volker C, Mewes HW et al. (2000). The genome and Methanocorpusculum bavaricum spec. nov. Arch sequence of the thermoacidophilic scavenger Thermo- Microbiol 151: 381–390. plasma acidophilum. Nature 407: 508–513. Zinder SH, Brock TD. (1978). Methane, , Saito Y, Aoki M, Hatamoto M, Yamaguchi T, Takai K, and production from the terminal Imachi H. (2015). Presence of a novel methanogenic methiol group of methionine by anaerobic lake sedi- archaeal lineage in anaerobic digesters inferred from ments. Appl Environ Microb 35: 344–352.

Supplementary Information accompanies this paper on The ISME Journal website (http://www.nature.com/ismej)

The ISME Journal