A methane-forming enrichment culture was originally obtained from a 9-year-old Romney wether sheep in New Zealand grazing a ryegrass-clover pasture diet. The enrichment culture contained the methanogenic archaeon, ISO4-H5, and a Gram-negative bacterium, subsequently identified as being closely related to Succinivibriodextrinosolvens and designated as strain H5. The methanogenic archaeon ISO4-H5 grows slowly and requires 3 to 4 days to generate detectable methane in the culture headspace. The optical density of cultures after maximal methane formation is very low and ISO4-H5 cells cannot be visualized via fluorescence microscopy at 420 nm due to the apparent lack of the fluorescent 8-hydroxy-5-deazaflavin cofactor, known as F420. The organism has only a thin bi-layer cell membrane, and no S-layer or cell wall was observed in electron micrographs of thin sections of ISO4-H5 cells (Fig. 1). The 16S ribosomal RNA gene of ISO4-H5 is 96 % identical to "Candidatus Methanomethylophilus alvus" Mx1201 enriched from human feces, and 95 % identical to Thermoplasmatales archaeon BRNA1 enriched from bovine rumen (Fig. 2). All three are members of the order Methanomassiliicoccales, but potentially each represent different species. The general features of methanogenic archaeon ISO4-H5 are shown in Table 1 and Additional file 1: Table S1.
To gain insight into the role of methylotrophic methanogens in the rumen environment, the genome of the methanogenic archaeon isolate ISO4-H5 was sequenced. Methanogenic archaeon isolate ISO4-H5 represents the first genome sequence of a member of the order Methanomassiliicoccales isolated from the ovine rumen. A summary of the genome project information is shown in Table 2.
The initial enrichment cultures were obtained by inoculation of sheep rumen contents into BY medium upplemented with (final concentrations), SL10 trace elements solution (1 mL/L), selenite/tungstate solution (1 mL/L), sodium acetate (20 mM), sodium formate (60 mM), methanol (20 mM), vitamin 10 solution (0.1 ml per 10 mL culture tube), and coenzyme M (CoM) (10 muM). The last two additives were added to the sterilized medium from filter-sterilized stock solutions. Hydrogen (H2) was supplied as the energy source by pumping the culture vessels to 180 kPa over pressure with an 80:20 mixture of H2: carbon dioxide (CO2). ISO4-H5 was enriched in tubes receiving sheep rumen contents diluted by a factor of 16,384,000. Several approaches were used to reduce the bacteria in the enrichment culture, including a 10-fold dilution, the addition of antibiotics (combinations of streptomycin, ampicillin, bacitracin at 10 mug/mL each, and vancomycin at 86.7 mug/mL), heat treatment of the enrichment culture at 50  C for 10 to 30 min, and application of lysozyme (2.5 mg/mL). These approaches produced a limited diversity enrichment culture containing ISO4-H5 and S. dextrinosolvens H5, which was verified by phase contrast epifluorescence microscopy and bacterial and archaeal 16S rRNA gene sequencing. Genomic DNA was extracted from cells harvested from a freshly grown (7 d incubation time) 2 L enrichment culture using a modified version of a liquid N2 freezing and grinding method, in which treatment with 2.5 mg lysozyme/mL and 0.8 mg proteinase K/mL replaced the 1 % w/v sodium dodecyl sulfate step, before a Genomic-tip 500/G (Qiagen, Germany) was used, following the manufacturer's instructions, in place of the phenol/chloroform extraction steps.
The DNA extracted from the ISO4-H5 enrichment culture was sequenced via pyrosequencing of a 3 kb mate paired-end sequence library using the 454 GS FLX platform with Titanium chemistry (Macrogen, Korea). Pyrosequencing reads provided 43.8x coverage of the combined ISO4-H5 and Succinivibriodextrinosolvens H5 genomes, and were assembled using the Newbler assembler version 2.7 (Roche 454 Life Sciences, USA). The Newbler assembly resulted in 176 Succinivibriodextrinosolvens H5 contigs across 28 scaffolds and 47 ISO4-H5 contigs in a single scaffold. The assignment of scaffolds to genomes was based on G + C content analysis and identification of the methanogenesis marker gene, methyl coenzyme M reductase (mrtA). Sequence gap closure was managed using the Staden package and gaps were closed using standard PCR techniques with Sanger sequencing. A total of 163 additional sequencing reactions were used to close gaps and to improve the quality of the genome sequence, ensuring correct assembly and to resolve base conflicts.
Genome annotation was carried out as previously described and the ISO4-H5 genome sequence was prepared for NCBI submission using Sequin. The guanosine residue of the start codon of the Cdc6-1 replication initiation protein gene (AR505_0001) was chosen as the first base for the ISO4-H5 genome. The nucleotide sequence of the ISO4-H5 chromosome has been deposited in Genbank under accession number CP014214.
The genome of ISO4-H5 consists of a single, 1,937,882 bp, circular chromosome with a G + C content of 54 %. A total of 1,817 protein-coding genes were predicted, representing 90.2 % of the total genome sequence. A Cluster of Orthologous Groups category was assigned to 1,434 of the protein-coding genes, and the properties of the genome are summarized in Tables 3 and 4.
ISO4-H5 is predicted to contain two Cdc6 genes. Cdc6.1 (AR505_0001) is adjacent to two origin recognition box (ORB) motifs downstream, while Cdc6.2 (AR505_1205) is located 661 kb away from the Cdc6.1 gene and is not associated with any ORB motif. Therefore, Cdc6.1 is predicted to be the origin of replication for ISO4-H5 (Fig. 3). The presence of multiple origins of replications is a feature also observed in the genome sequences of other members of Methanomassiliicoccales, including BRNA1 (TALC00001, TALC00716, 645 kb apart), Mx1201 (MMALV_00010, MMALV_10400, 637 kb apart), Mx1-Issoire (H729_00005, H729_08750, 90 kb apart), and B10 (WP_019178385, WP_019178317). The ISO4-H5 genome contains genes predicted to be integrases (AR505_0313, 0669, 0931, 1543, 1570, 1640, 1697), as well as several Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) genes (AR505_1089 - 1095) associated with a CRISPR region containing 35 repeats (bases 1,153,894 to 1,155,995). There is evidence of a mobile element in the ISO4-H5 genome (AR505_0313-AR505_0358) which excised and segregated from the chromosome over several passages between the sequencing of the genome and subsequent analyses of the annotated locus. The 32 kb mobile element harbors 37 hypothetical protein genes, three adhesin-like protein genes, three DNA-cytosine methyltransferase genes, one phage integrase gene, one DNA mismatch endonuclease gene and one Membrane Occupation and Recognition Nexus (MORN) repeat-containing protein. No plasmids were identified in the ISO4-H5 genome. The genome contains a predicted toxin/antitoxin module (AR505_0857, 0858) and a death-on-curing family protein (AR505_1566), although the latter lacks an identifiable gene encoding a partner toxin.
The genomes of several members of Methanomassiliicoccales are publically available, including M. luminyensis B10 isolated from a human source, "Candidatus Methanomethylophilus alvus" Mx1201 and "Candidatus Methanomassiliicoccus intestinalis" Mx1-Issoire enriched from human sources, "Candidatus Methanoplasma termitum" MpT1 enriched from termite gut, and Thermoplasmatales archaeon BRNA1 enriched from the bovine rumen. These genomes were compared with ISO4-H5 (Table 5). ISO4-H5 is very similar in genome size to the other members of Methanomassiliicoccales, with M. luminyensis B10 being the exception, with a genome 35 % larger than ISO4-H5. The genomic G + C content of the Methanomassiliicoccales range from 49 to 60 %, with "Candidatus Methanomassiliicoccus intestinalis" Mx1-Issoire being different to the rest with a genomic G + C content of 41 %. The organization of genes within the ISO4-H5 genome shows best synteny with "Candidatus Methanomethylophilus alvus" Mx1201 and Thermoplasmatales archaeon BRNA1 (Fig. 4), its two closest genome-sequenced relatives.
Members of the order Methanomassiliicoccales rely solely on hydrogen-dependent methylotrophic methanogenesis to produce energy. However, they use only part of the pathway reported for other methylotrophic methanogens (Fig. 5), such as members of the genera Methanosarcina and Methanosphaera. Methanosarcina spp. disproportionate methanol by electron bifurcation, oxidizing one mole to produce CO2 while generating reducing potential to reduce three further moles to methane. The methanogenesis pathway in ISO4-H5 lacks the genes encoding the enzymes required to oxidize methanol to CO2, and is predicted to only reduce methylated compounds directly to methane. Functionally, this is similar to Methanosphaera stadtmanae MCB-3, which encodes all the genes for the enzymes needed to oxidize methanol to CO2 but does not use this pathway due to the lack of genes encoding synthesis of molybdopterin, a cofactor required for formation of an active formylmethanofuran dehydrogenase. ISO4-H5 is predicted to use a heterodisulfide reductase (HdrABC) and a methyl-viologen hydrogenase (MvhADG) to recycle CoM, using reducing equivalents generated from the hydrogenase. However, unlike M. stadtmanae, the Hdr and Mvh complexes in ISO4-H5 are not predicted to be coupled to an energy-converting-hydrogenase complex, but rather are coupled to a F420-dehydrogenase Fpo-like complex to generate the membrane potential necessary for energy formation via ATP synthase. The energy converting-hydrogenase complex identified in M. luminyensis B10 and "Candidatus Methanomassiliicoccus intestinalis" Mx1-Issoire could possibly have an anaplerotic role. Based on the lack of the corresponding genes, the ISO4-H5 Fpo-like complex lacks the FpoF and FpoO subunits, which in other methanogens contain the iron-sulfur centers likely responsible for interacting with coenzyme F420 and methanophenazine, respectively . This is expected, as ISO4-H5 cells do not fluoresce when illuminated at 420 nm, suggesting that coenzyme F420 is not present in this organism. Furthermore, the genome does not contain genes for cytochrome biosynthesis, which suggests that methanophenazine is also absent. A hypothetical protein (AR505_1626) in the Fpo operon, between fpoK (AR505_1625) and fpoJ (AR505_1627) genes, is predicted to be a transmembrane protein and shares 49.5, 54.4 and 45.9 % amino acid identity to MMALV_02020 of Mx1201, TALC_00216 of BRNA1 and Mpt1_c12590 of MpT1 respectively. In addition, this gene is also located in an operon whose organization is similar to those encoding BRNA1, Mx1201, and MpT1, and is possibly a subunit of the Fpo-like complex.
ISO4-H5 is predicted to have essentially the same methane formation pathway as "Candidatus Methanoplasma termitum" and likely pumps only one ion across the cell membrane for every two methanes formed, to generate a membrane gradient. This is in contrast toM. stadtmanae, which has the same general metabolic stoichiometry but pumps two ions per methane formed. Since ATP synthesis in all of these methanogens is via a membrane-bound ATP synthase, ISO4-H5 is predicted to have a have a much lower ATP (and growth) yield than Methanosphaera spp. which is consistent with the very low culture densities observed when the isolate is grown in the laboratory. However, it can be expected to have a lower threshold for hydrogen, using the same rationale proposed by Lang et al. (2015) for "Candidatus Methanoplasma termitum". This therefore differentiates it ecologically from Methanosphaera, and suggests that Methanosphaera spp. and members of Methanomassiliiococcales, both of which occur in the rumen, occupy different niches.
Interestingly, the cysteate synthase, cysteate aminotransferase (serC) and sulfopyruvate decarboxylase (comDE) genes required for the synthesis of CoM are absent from the ISO4-H5 genome. This suggests that ISO4-H5 cannot synthesize CoM, and requires an external supply of CoM to survive within the rumen, similar to Methanobrevibacter ruminantium M1 and MpT1. This explains the requirement for CoM supplementation in the initial enrichments of ISO4-H5. ISO4-H5 also possesses only a subset of methanogenesis marker genes: 1-8, 11, 13, 15-17 (AR505_1391, 0786, 1390, 1417, 1388, 1389, 1385, 1203, 1637, 0362, 1387, 0724, and 1386 respectively). This suggests that the remaining methanogenesis marker genes (mmp 9, 10, 12 and 14) are not required for the truncated methyl-reducing pathway used by ISO4-H5.
ISO4-H5 possesses a complete operon predicted to encode the genes required for the biosynthesis of pyrrolysine and for aminoacylation of a transfer RNA (tRNA) to pyrrolysine (Fig. 6), enabling read-through of the amber stop codon, UAG. Pyrrolysine is produced from two molecules of lysine by the gene products PylBCD. Methylornithine synthase (PylB) converts L-lysine to (3R)-3-methyl-D-ornithine, which in turn is ligated with a second molecule of L-lysine to produce (2R, 3R)-3-methylornithyl-N6 lysine, catalysed by (2R,3R)-3-methylornithyl-N6-lysine synthase (PylC); pyrrolysine synthase (PylD) converts (2R,3R)-3-methylornithyl-N6-lysine to pyrrolysine. Pyrrolysine-tRNA ligase (PylS) catalyses the aminocylation of tRNA (CUA) which itself is encoded by pylT. The operon organization is conserved across the Methanomassiliicoccales (Fig. 6), suggesting pyrrolysine use is important for members of this order. The in-frame amber codon occurs in 46 ISO4-H5 genes, including the genes encoding methylamine use; trimethylamine:corrinoid methyltransferase, mttB (AR505_0772); methanol corrinoid protein, mtaC (AR505_0952); monomethylamine methyltransferase, mtmB (AR505_1327, 1328); and dimethylamine:corrinoid methyltransferase, mtbB (AR505_1332). The amber codon is also found in the mmp 8 gene, a predicted nitrogenase gene (AR505_1289), an adenylate kinase gene (AR505_1784) involved in purine biosynthesis, a bifunctional phosphoglucose/phosphomannose isomerase gene (AR505_0560) involved in the last step of gluconeogenesis, two geranylgeranyl reductase genes (AR505_1433, AR505_1618) that are likely involved in cell membrane lipid biosynthesis, and the CRISPR-associated endonuclease Cas3 gene (AR505_1089) that is involved in acquired immunity against foreign DNA. Additionally, 17 genes encoding hypothetical proteins, one adhesin-like protein gene, and 10 insertion sequence elements have amber codons. Similar findings have been reported in the genomes of members of Methanomassiliicoccales of human origin and it has been suggested that pyrrolysine synthesis is a particular feature of this order and an important marker in the evolution of methanogenic archaea.