Complete genome sequence of Methanospirillum hungatei type strain JF1

Methanospirillum hungatei strain JF1 (DSM 864) is a methane-producing archaeon and is the type species of the genus Methanospirillum, which belongs to the family Methanospirillaceae within the order Methanomicrobiales. Its genome was selected for sequencing due to its ability to utilize hydrogen and carbon dioxide and/or formate as a sole source of energy. Ecologically, M. hungatei functions as the hydrogen- and/or formate-using partner with many species of syntrophic bacteria. Its morphology is distinct from other methanogens with the ability to form long chains of cells (up to 100 μm in length), which are enclosed within a sheath-like structure, and terminal cells with polar flagella. The genome of M. hungatei strain JF1 is the first completely sequenced genome of the family Methanospirillaceae, and it has a circular genome of 3,544,738 bp containing 3,239 protein coding and 68 RNA genes. The large genome of M. hungatei JF1 suggests the presence of unrecognized biochemical/physiological properties that likely extend to the other Methanospirillaceae and include the ability to form the unusual sheath-like structure and to successfully interact with syntrophic bacteria.


Introduction
Strain JF1 (DSM 864 = ATCC 2790D-5) [1] is the type species for M. hungatei and represents the first isolated member of the Methanospirillaceae within the order Methanomicrobiales [2]. The species epithet derives from the Latin and honors Dr. R. E. Hungate, the inventor of methodologies for modern isolation and cultivation of strictly anaerobic bacteria and archaea [3,4]. M. hungatei strain JF1 was isolated from a secondary anaerobic sewage treatment digestor in Urbana, Illinois, as part of a study of anaerobic aromatic hydrocarbon metabolism [5].
Here, we describe the genome sequence of M. hungatei strain JF1, a hydrogen-and formate-utilizing, methaneproducing archaean. The genomic data provide insight towards defining the unique genes needed for anaerobic syntrophy [6], which occurs within a phylogenetically diverse range of bacteria, and for classifying genes identified by environmental DNA sequencing projects.

Morphology and physiology
Cells of Methanospirillum hungatei strain JF1 are narrow, curved rods (i.e., spirillum shaped) that measurẽ 0.5 μm by~7 μm in size (Fig. 1, Table 1). The cells are contained within a sheath-like structure that contain one or more cells; the sheath may extend to over 100 μm in length depending on the nutritional conditions [1,7]. Individual cells stain Gram-negative and are weakly motile by polar tufts of flagella. Cells also possess polyphosphate bodies or granules located at opposing cell ends [8]. Growth and metabolism is strictly anaerobic where hydrogen plus carbon dioxide and/or formate serve as the methanogenic substrate. Acetate is required as the major supply for cell carbon [1,7]. Cells have no other organic nutritional requirements although addition of Casamino Acids or other plant/animal hydrolysis products speeds growth [1]. Temperature range for growth is 20-40°C (optimum at 37°C).
Biogenic methane production is important in the global carbon cycle and is used to treat sewage and other organic wastes and to produce biofuel from biomass [9,10]. The degradation of fatty and aromatic acids is often the ratelimiting step in methanogenesis [6]. Fatty and aromatic acid degradation is thermodynamically favorable only when hydrogenotrophic methanogens such as M. hungatei strain JF1 maintain very low levels of hydrogen and/or formate in a process called syntrophy [10,11]. Members of the genus Methanospirillum are often detected in ecosystems where syntrophy is essential [1,12] and M. hungatei strain JF1 is the model partner in syntrophic cocultures of the propionate degrader Syntrophobacter wolinii [13], the butyrate degrader Syntrophomonas wolfei [14], and the benzoate degraders Syntrophus buswellii and Syntrophus aciditrophicus [15,16].

Classification and features
The phylogenetic neighborhood of M. hungatei strain JF1 is shown in Fig. 2 for representative archaeal 16S rRNA sequences belonging to the order Methanomicrobiales. The four described Methanospirillum species form a well-defined cluster distinct from the other genera within the order where Methanospirillum lacunae and Methanospirillum psychrodurum form one subgroup and M. hungatei plus Methanospirillum stamsii form another. All strains of the genus Methanospirillum synthesize methane from hydrogen and carbon dioxide, though the ability to use formate is variable. None are able to ferment or respire by using other electron acceptors (i.e., with sulfate, nitrate, or iron). Certain species of other genera within the Methanomicrobiales also use formate, and some are reported to also metabolize short chain alcohols.
The analysis of the four 16S rRNA genes present in the M. hungatei JF1 genome reveled nearly identical nucleotide sequences but they differ from one another at two positions (nucleotide positions 937 and 1382) across the 1466 nucleotide length. The previously-published 16S rRNA gene sequences (AY196683 and AB517987) used in phylogenetic investigations were incomplete, i.e., 1271 and 1259 nucleotides, respectively [17,18].

Chemotaxonomic data
The cell envelope of this Gram-negative cell wall type includes a surface layer coat, also known as a surface layer protein, which surrounds the cytoplasmic membrane, and an outermost sheath structure that encapsulates multiple cells, which are arranged in chains up to 0.1 mm in length [1,8,19]. Cytoplasmic membrane Fig. 1 Electron micrograph of M. hungatei strain JF1 cells and associated sheath structure. Scale bar corresponds to 100 nm lipids are composed primarily of biphytanyldiglycerol tetraether glycolipids [20]. M. hungatei strain JF1 lacks b-or c-type hemes, quinones, and methanophenazine (this study). The DNA G + C content was previously reported with 45 mol % [1].

Genome project history
The M. hungatei strain JF1 genome was selected by DOE in 2004 as JGI sequencing project 364479 based on its phylogenetic position, its role in anaerobic decomposition of organic matter, and its ability to grow in co-culture with many syntrophic bacterial species [6]. The genome project is deposited in the Genomes OnLine Database [21] as project Id:Gc00350, and the complete genome sequence is deposited in GenBank. Sequencing, finishing, and annotation of the M. hungatei genome were performed by the DOE Joint Genome Institute [22]. A summary of the project information is shown in Table 2.
Growth conditions and genomic DNA preparation M. hungatei strain JF1 was grown in basal medium under anaerobic conditions at 37°C as previously described [1]. High molecular weight genomic DNA was isolated from cell pellets (DSM 864 = ATCC 2790D-5) using the CTAB method described at the JGI's web site [22]. These evidence codes are from the Gene Ontology project [52] IDA Inferred from Direct Assay, TAS Traceable Author Statement (i.e., a direct report exists in the literature); NAS Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence) a Evidence codes

Genome sequencing and assembly
The genome was sequenced at the Joint Genome Institute using a combination of 3 kb, 8 kb, and 40 kb DNA libraries.
All general aspects of library construction and sequencing performed are described at the JGI's web site [22]. The Phred/Phrap/Consed software package [23] was used to assemble all three libraries and to assess quality [24,25]. Possible miss-assemblies were corrected and gaps between contigs were closed by editing in Consed, custom primer walks, or PCR amplification (Roche Applied Science, Indianapolis, IN). The error rate of completed genome sequence of M. hungatei is less than 1 in 50,000. The sequence of M. hungatei can be accessed using the GenBank accession number CP000254.

Genome annotation
Genes were identified using Prodical [26] as part of the Oak Ridge National Laboratory genome annotations pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [27,28]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information nonredundant database, and the UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was preformed within the Integrated Microbial Genomes-Expert Review platform [29,30]. Membrane transport protein analysis was done by IMG with additional analysis by TransportDB [31] TCDB [32] databases. Transcription factor analysis and prediction was by assisted by TBD database [33].

Genome properties
The genome statistics are provided in Table 3 and Fig. 3   Insights from the genome sequence

Methanogenesis pathway
The M. hungatei JF1 ORFs were organized into pathways where most pathways considered essential for viability of a typical archaeal cell were detected. The methanogenic pathway from hydrogen and carbon dioxide is highly conserved in methanogens and the genes for all the enzymes in the central methanogenic pathway were identified, including a soluble-type heterodisulfide reductase only (Fig. 4)   The reaction catalyzed by a soluble-type Hdr is likely an electron bifurcation, which couples the energetically favorable reduction of CoM-S-SCoB by formate and/or H 2 with the energetically unfavorable reduction of ferredoxin by formate and/or H 2 [34].

Transporters, ion movement, and ATP synthesis
M. hungatei JF1 has 352 genes involved in membrane transport as determined by IMG/ER, which constitute 10.64 % of the genome. These include 34 multi-component ATP-binding cassette or ABC-type transporter genes plus related but unlinked genes (152 genes in total), sixty genes encoding secondary transporters, twelve genes for ion channels, seven genes for P-ATPases, one H + translocating pyrophosphatase (Mvp, H + PPase; Mhun_2414) gene, and four type II secretion systems. A highly unusual feature of the M. hungatei genome is the presence of three H + or Na + -translocating AoA 1 -type ATP synthetase gene clusters encoded by 27 genes (Aha1, Mhun_1177-1185; Aha2, Mhun_1757-1765, and Aha3, Mhun_1768-1775). The gene order is conserved relative to the corresponding Aha complex in Methanosarcina acetivorans [35]. Although it is unknown whether these systems utilize protons or sodium ions, the M. acetivorans ortholog is believed to use sodium ions [35]. Likewise, the membrane-bound H 4 MPT Smethyltransferase (Mtr) is predicted to be sodium dependent. Three genes encode Na +/ H + antiporters (Mhun_0680, Mhun_0841, Mhun_2803) that might maintain ion balance where the last differs by also possessing a Trk domain.

Cell biosynthesis
The genome of M. hungatei encodes an acetyl-CoA synthase/CO dehydrogenase complex (Cdh; Mhun_0686-0690). The role of Cdh is undefined at this time because M. hungatei must acquire acetate supplied in the medium for growth rather than synthesizing acetylCoA

Not in COGs
The total is based on the total number of protein coding genes in the genome from CO 2 , which is the usual role of Cdh in hydrogenotrophic methanogens. Uptake of acetate for incorporation into cell material is predicted to occur by the Mhun_0634 aceP gene product [35]. Five acetyl-CoA synthetase genes are present that could activate acetate to acetyl-CoA. Mhun_0352, Mhun_0567, and Mhun_1721 share > 62 % identity at the amino acid level with each other, but only share < 34.2 % amino acid identity with Mhun_0592 and Mhun_2392.
There are few genomic clues regarding the composition of the M. hungatei cell envelope. The genome contains a large number of PDK domain-containing genes (31 genes) as well as TRP domain-containing genes (41 genes). Many of these have transmembrane and/or SP signal elements that would suggest cell envelope associations but it is unknown if any of the proteins are significantly expressed. There are no clear protein candidates for the morphologically defined cell envelope structures containing a surface layer, sheath, and plugs [1,8].

Regulation and signal transduction
The M. hungatei genome contains a typical set of archaeal RNA polymerase genes and one BRE recognition factor analogous to eukaryotic transcription initiating factor B (Mhun_2481; Tfb) plus two TATA-box binding proteins or TBP's that confer promoter recruitment and specificity (Tbp1, Mhun_0568 and Tbp2, Mhun_0593). There are~65 DNA-binding transcription factors identified that modulate gene expression. These belong to a variety of protein families common to bacteria but include few regulatory proteins typical of eukaryotes (e.g., homeodomain-like, zinc finger, SRF-like, or p53like proteins). There are numerous bacterial-type twocomponent regulatory systems including 82 histidine kinase-type sensor transmitters, 41 response regulatory proteins, and 18 receiver-only domain proteins. Of the 82 histidine kinases, 55 are soluble and 27 are membraneassociated. They are generally unlinked genetically and thus do not suggest an interacting partner in sensory transduction.
Multiple genes (~11 paralogs) are also present in the M. hungatei JF1 genome for archaeal-type pili like those seen in Methanococcus maripaludis, Haloferax volcanii, and Sulfolobus acidocaldarius [39]. These archaeal proteins, distinct from the bacterial pili-type proteins, were previously annotated as hypothetical genes (e.g., Mhun_0297). The H. volcanii pili proteins provide adhesion to surfaces and the orthologs in M. hungatei JF1 may function in cell-cell adhesion or in cell-cell communication, although such appendages have not been previously observed in EM micrographs. All but one of the eleven M. hungatei JF1 paralogs are in clusters of 2 to 3 genes each and often with ABCtype transport genes.
Comparison to other archaeal genomes When M. hungatei ORFs were compared pair-wise to individual microbial genomes [40,41], best reciprocal BLAST hits revealed closest associations to the taxonomically related archaea: Methanoculleus marisnigri (1395 reciprocal gene hits), Methanosarcina acetivorans (1203), and Methanosarcina barkeri (1150), and extending to Haloquadratum walsbyi (657) (Additional file 1: Figure S1). Thus, approximately 650 to 1,200 genes are similar and well-conserved across these 17 archaeal species whereby the remaining genes (ca. 1700 genes) represent a novel complement within the M. hungatei genome. Interestingly, seven of the next thirteen closest matches are bacterial species among which are many syntrophic microorganisms that likely grow in close association with M. hungatei. Strikingly, Syntrophobacter fumaroxidans strain MPOB exhibited 634 best reciprocal BLAST hits.
In another comparison, the best BLAST hit to any microbial gene product was determined (Additional file 2: Figure S2) and showed 1; 167; 277; and 142 ORFs closest hits in the genomes of Methanoculleus marisnigri, Methanocorpusculum labreanum, and Methanosarcina barkeri, respectively. Notably three bacterial genomes, Syntrophus aciditrophicus, Syntrophobacter fumaroxidans, and Nostoc spp. gave 21-19 best BLAST hits each, suggesting the possibility of lateral gene transfer events from these potential syntrophic partners. The occurrence of Nostoc-related genome sequences raises interesting questions concerning microbial interactions and lateral gene transfer with methanogens present in complex microbial communities [42].

Extended insights
The large genome of M. hungatei JF1 suggests the presence of unrecognized biochemical/physiological properties that likely extend to the other Methanospirillaceae and include the ability to form the unusual sheath-like structure and the ability to successfully interact with syntrophic bacteria. A number of genes may have been acquired by lateral gene transfer from its syntrophic partners or other microorganisms present in complex microbial communities. Also of particular note are multiple genes for archaeal type IV pili that may function in cell-cell adhesion or cell-cell communication and genes for multiple hydrogenases and formate dehydrogenases to metabolize hydrogen and formate generated by its syntrophic partners. The core machinery of M. hungatei to produce methane from hydrogen and carbon dioxide and/or formate is typical of other hydrogenotrophic methanogens, except that M. hungatei has genes for three H + or Na + -translocationg A O A 1 -type ATP synthases. M. hungatei has four 16S ribosomal RNA genes that each differ at two positions. Further understanding of the novel compliment of M. hungatei genes will likely provide a more thorough understanding of the multispecies interactions involved in syntrophy and the synthesis of complex structures such as the M. hungatei sheath, which is shared by multiple cells.

Conclusions
We report here an inventory of the genomic features of the methane-producing anaerobic archaeon, Methanospirillum hungatei strain JF1 (DSM 864), and describe its phylogenetic relationship to its neighbors. We further identify from the sizable genome of M. hungatei examples of genes involved in anaerobic syntrophy, and as the type strain of the Methanospirillum, suggest potential universal qualities of this genus. We hope this report aids and stimulates further study of this fascinating organism.