Comparative Genomic Analysis of Members of the Genera Methanosphaera and Methanobrevibacter Reveals Distinct Clades with Specific Potential Metabolic Functions

Methanobrevibacter and Methanosphaera species represent some of the most prevalent methanogenic archaea in the gastrointestinal tract of animals and humans and play an important role in this environment. The aim of this study was to identify genomic features that are shared or specific for members of each genus with a special emphasis of the analysis on the assimilation of nitrogen and acetate and the utilization of methanol and ethanol for methanogenesis. Here, draft genome sequences of Methanobrevibacter thaueri strain DSM 11995T, Methanobrevibacter woesei strain DSM 11979T, and Methanosphaera cuniculi strain 4103T are reported and compared to those of 16 other Methanobrevibacter and Methanosphaera genomes, including genomes of the 13 currently available types of strains of the two genera. The comparative genome analyses indicate that among other genes, the absence of molybdopterin cofactor biosynthesis is conserved in Methanosphaera species but reveals also that the three species share a core set of more than 300 genes that distinguishes the genus Methanosphaera from the genus Methanobrevibacter. Multilocus sequence analysis shows that the genus Methanobrevibacter can be subdivided into clades, potentially new genera, which may display characteristic specific metabolic features. These features include not only the potential ability of nitrogen fixation and acetate assimilation in a clade comprised of Methanobrevibacter species from the termite gut and Methanobrevibacter arboriphilus strains but also the potential capability to utilize ethanol and methanol in a clade comprising Methanobrevibacter wolinii strain DSM 11976T, Mbb. sp. AbM4, and Mbb. boviskoreani strain DSM 25824T.


Introduction
The microbial ecology of the intestinal tract of humans and animals has been subject to extensive research in recent years, and it is becoming increasingly evident that commensal intestinal microbes have a strong impact on host physiology and well-being. One microbial group, the methanogenic archaea (methanogens), has been of particular interest in this regard. Methanogens have not only been implicated in greenhouse gas emission from livestock animals [1,2] but several studies have also linked these microorganisms to specific bodyweight phenotypes in humans [3] and to differences in feed conversion efficiency in ruminants [4,5].
The majority of the rumen and intestinal methanogens appear to belong to two of the seven known orders, the Methanobacteriales [6] and the recently described Methanomassiliicoccales [7]. Two genera of the order Methanobacteriales, Methanobrevibacter and Methanosphaera, comprise a large proportion of intestinal and rumen methanogenic archaea [8,9]. Species of the genus Methanobrevibacter have been isolated from not only a wide range of different environments, including invertebrate and vertebrate guts, but also non-host-associated environments. The majority of the members of the genus Methanobrevibacter grow primarily hydrogenotrophically [10], using CO 2 and H 2 as substrates. A few strains and/or species have been shown by cultivation-independent methods to be the predominant in specific intestinal environments, such as Mbb. smithii from the human gut [8] or Mbb. ruminantium, and Mbb. gottschalkii from the rumen of sheep and cows [11]. The former three species have gained considerable attention in the analysis of the human and rumen microbiome, but there are also Mbb. isolates that are less well investigated by cultivation-independent studies, for example, Mbb. thaueri strain DSM 11995 T and Mbb. woesei strain DSM 11976 T . Mbb. thaueri strain DSM 11995 T was isolated from cow feces while Mbb. woesei strain DSM 11976 T had been isolated from goose feces [12]. The 16S rRNA gene of either of the two species shares the highest sequence identity to Mbb. smithii strain DSM 861 T , Mbb. gottschalkii strain DSM 11977 T , and Mbb. millerae strain DSM 16643 T [12][13][14][15]. Both species have been detected in a few cultivation-independent studies on fecal and rumen samples (for Mbb. thaueri, see [16,17] and for Mbb. woesei, see [18][19][20]), but besides their original description, there is very little information on the physiology and ecology of these two species.
The genus Methanosphaera is less well characterized than the genus Methanobrevibacter, and only few species have been isolated or detected by cultivation-independent methods. Two species, Msp. stadtmanae DSM 3091 T and Msp. cuniculi DSM 4103 T , are currently the only formally described Methanosphaera-type species [21,22]. The first genome-sequenced Methanosphaera species, Msp. stadtmanae DSM3091 T , was isolated from human feces and has been shown to be restricted to growth on methanol and hydrogen under in vitro conditions [22]. This substrate restriction can to some extent be considered to be a typical trait of the genus Methanosphaera, but its genetic basis remained poorly understood until the genome of Msp. stadtmanae strain DSM 3091 T was analyzed [23]. Comparative genome analysis revealed that most genes for biosynthesis of the molybdopterin cofactor (Moco), the cofactor of formylmethanofuran dehydrogenase (fmd), are absent from the genome of this methanogen. This explained why Msp. stadtmanae strain DSM3091 T is not able to grow by disproportionation of methanol nor of CO 2 and hydrogen as substrates. Moreover did the analysis reveal that the genome encodes four homologues of each subunit of the methanol : coenzyme-M methyltransferase (mtaABC) [23]. The MtaABC proteins form the key enzyme complex for the utilization of methanol and had before primarily been detected in species of the order Methanosarcinales.
The second genome of a genus Methanosphaera representative, Methanosphaera sp. WGK6, has only recently become available. This methanogen's genome sequence is to large extent nearly identical to that of Msp. stadtmanae DSM 3091 T , but some of the few genomic differences result in phenotypic differences between the two species. This regards mainly two genes, putative alcohol (walC) and aldehyde (walD) dehydrogenases [24]. These two genes were apparently acquired via horizontal gene transfer and seem to enable Msp. sp. WGK6 to utilize ethanol as substrate for methanogenesis (in addition to its ability to grow on methanol and hydrogen) [24]. Homologues of walC and walD genes have also been detected in genomes of other methanogens but have not been investigated systematically.
Here, draft genomes of Mbb. thaueri strain DSM 11995 T , Mbb. woesei strain DSM 11979 T , and Msp. cuniculi strain DSM 4103 T (a draft genome of M. cuniculi DSM 4103 T of similar quality was also recently published by Gilmore et al. [25]) are presented and compared to those of all other currently available Methanosphaera-and Methanobrevibactertype strain genomes. The comparative analyses aim at investigating and identifying some of the distinctive features of the Methanobrevibacter and Methanosphaera genomes that allow differentiating the two closely related genera. The major emphasis was set on key genes that may have large impact on the overall physiology of the analyzed species, such as those involved in substrate utilization and nitrogen and acetate assimilation.  [26] was used to filter low-quality reads and for clipping of adapter contaminations. The assembly was performed with the SPAdes genome assembler software 3.11 [27]. Coverages were determined using QualiMap version 2.1 [27][28][29], and automatic annotation was performed using the software tool PROKKA [30], and data analysis was partly performed using the IMG/ER system (Integrated Microbial Genomes & Microbiomes) [31]. The quality and the completeness of the draft genomes have been validated with CheckM [32].
2.3.1. Analysis of 16S rRNA Genes. Aligned 16S rRNA gene sequences were selected from the ARB-compatible rumen and intestinal methanogen database (RIM-DB) [14,40]. Aligned sequences were exported in PHYLIP format to construct phylogenetic trees using all available base positions. Maximum-likelihood phylogenetic trees based on aligned archaeal 16S rRNA gene sequences were generated using RAxML version 7.3.0 [38]. Unless stated otherwise, the parameters "-m GTRGAMMA -# 500 -f a -× 2 -p 2" were used.
2.4. Nucleotide Sequence Accession Number. The annotated genomes of Mbb. thaueri DSM 11995 T and Mbb. woesei DSM 11979 T have been deposited at DDBJ/EMBL/GenBank under the accession MZGS00000000 and MZGU00000000, respectively. The versions described in this paper are versions MZGS01000000 and MZGU01000000, respectively. The annotated genome of Msp. cuniculi DSM 4103 T has been deposited at DDBJ/EMBL/GenBank under the accession LWMS00000000. The version described in this paper is version LWMS01000000.  Table 1, and results of CheckM analysis (including all genomes used in the analysis) are shown in Table S1. Circular representation of the three genomes is shown in supporting Figures S1 and S2.

Comparative Analysis of Methanobrevibacter Genomes.
Sixteen different Methanobrevibacter and three Methanosphaera genomes were included for the comparative analyses. The Methanobrevibacter strains had been isolated from a range of different intestinal environments, for example, bovine and ovine rumen [12,41], human gut [42], and termite hindguts [43,44], but include also the genome of the non-host-associated Mbb. arboriphilus strain DSM 1125 T [45,46]. Results of the single-and multilocus analyses are shown in Figure 1. Both analyses revealed clear separation between the two genera.
The MLSA also supports distinct clades within the genus Methanobrevibacter ( Figure 1). There is limited association of the observed clades with a specific host origin, for example, some species from the rumen form distinct clades while others cluster with Methanobrevibacter species isolated from other hosts. A BLAST-based analysis of each clade identifies genes specific for clades and genes that are shared between them ( Figure 2). Among the clade-specific genes of clade 1 are catalase, nitrogenase, and also a tRNA-specific endonuclease (VapC), but most other clade-specific genes of clade 1 and the other clades are without functional annotation and await further characterization.
It was also investigated whether certain metabolic traits could be associated with specific clades as pointed out in Figure 1. This concerns mainly the metabolism of alcohols and acetate assimilation. Carbon monoxide/acetyl-CoA-synthetase complex (CODH-ACS) is-in addition to other enzymes-important for acetate assimilation, and its presence is one prerequisite for autotrophic growth. CODH-ACS is found in non-host-associated and autotrophic methanogens, for example, Methanothermobacter species [47][48][49], and is detected in four out of five clade 1 species, but gene homologues of the enzyme appear to be absent from the genomes of methanogens in clades 2-4.
The potential utilization of alcohols, specifically methanol and ethanol, is also less clearly distributed among the 19 genomes (and the four different clades) and there are currently no known Methanobrevibacter species that are restricted to growth on H 2 and methanol/ethanol [10]. Genes for methanol utilization of the Methanosphaera/Methanosarcina type (mtaABC) are distributed broadly within the genus Methanobrevibacter as outlined in Figure 1 and have also already been reported for some strains [46,50]. Physiological characterizations of the strains that harbor mtaABC genes (Mbb. arboriphilus strains DSM 1125 T and ANOR1, Mbb. smithii strain DSM 861 T , Mbb. wolinii strain DSM 11976 T ) is still outstanding to confirm that these species are capable of growth on methanol/methanol-hydrogen.
The utilization of ethanol by Methanobrevibacter species has only recently gained additional attention and it has been shown that Mbb. sp. AbM4 is capable of growth in the absence of hydrogen but in presence of methanol/ethanol [51,52]. Details regarding the exact metabolism remain speculative, but the walC and walD genes recently identified in Msp. WGK6 [24] are also present in Mbb. sp. AbM4 and in the closely related Mbb. boviskoreani strain DSM 25824 T , Mbb. olleyae strain DSM 16632 T , and Mbb. wolinii strain DSM11976 T conferring this metabolic trait potentially to the entire clade. Mbb. olleyae strain DSM 16632 T is currently the only Mbb. species outside this clade that is harboring the walCD genes.
Only few additional genome sequences of Methanobrevibacter isolates other than for Mbb. smithii are currently available, but the clade-distinguishing features appear to be conserved in these isolates as well (see Table S2 for general  genome features and Table S3 for shared genes).

Shared and Distinctive Genome Features in the
Genomes of Methanosphaera Species. The currently known Overall, more than 1000 genes are shared among all three Methanosphaera species (Figure 3), but the comparison with Methanobrevibacter genomes shows that several key genes are missing. Notably, this concerns the lack of genes for molybdopterin cofactor biosynthesis, energy-converting hydrogenase A (ehaA-Q), formate dehydrogenase (fdh), and methyl-coenzyme M reductase isoenzyme 1 mcrABCG (only genes encoding isoenzyme II are present (mrtABDG)).
Despite the presence of formylmethanofuran dehydrogenase genes, it is not possible for Methanosphaera species to produce a functional enzyme due to the absence of the molybdopterin cofactor. The absence of functional formylmethanofuran dehydrogenase leads to the inability of these methanogens to grow hydrogenotrophically or to disproportionate methanol. The absence of Moco biosynthesis also seems to be specific to Methanosphaera species as all of the analyzed Methanobrevibacter genomes are capable of synthesizing this cofactor. One of the noteworthy differences between the known Methanosphaera species is the capability of Msp. WGK6 to  Table S3 for details). Open-reading frames that were classified as pseudogenes were not included in this analysis. See also

Discussion
The presented comparative genome analysis reveals insights into the genome content of different Methanobrevibacter species and how it compares to that of closely related Methanosphaera species. The analysis suggests that phenotypic variation among Methanobrevibacter strains may be larger than previously assumed. The comparative analysis indicates the presence of genes for methanol (in four Mbb. genomes) and ethanol utilization (in four Mbb. genomes) in the sixteen analyzed Methanobrevibacter genomes, with the Mbb. wolinii genome harboring both walCD and mtaABC gene homologues. More physiological analysis will be required to determine if the detected genes are conferring the ability to utilize ethanol/methanol to all these strains. The ability of Methanobrevibacter strains to utilize alcohols may not have been investigated in an in-depth manner previously as this trait is more typically associated with other orders of methanogens, but some studies suggest that the genes may in some cases also not be functional or may have unknown different functions, for example, growth of Mbb. smithii on methanol was not detected despite the presence of mtaABC genes (growth on methanol but not methanol and hydrogen was tested) [59]. It is noteworthy that all three sequenced strains of the Mbb. wolinii clade harbor the genes for ethanol utilization, which could point to a specific ecological role of the species in this clade; however, there are only few studies that have detected significant numbers of either of these three species in a natural environment [5,60] making it currently difficult to determine potential cooccurrences or specific syntrophic interactions with other microorganisms. The presence of genes that could contribute to autotrophic growth, for example, nitrogenase and CODH-ACS in clade 1 containing Mbb. species from termites and in Mbb. arboriphilus strain, has been reported previously [46], and these genes appear to be absent from species clades 2 to 4. It can only be speculated why Mbb. species of clade 1 did not undergo the same loss of these key genes like their counterparts in the clades that have primarily been isolated from the vertebrate intestinal tract. However, auxotrophy of some Mbb. species may be the result of a close symbiotic interaction with other microorganisms (and potentially the host) that provide favorable growth conditions as well as ammonium and acetate for the methanogen. It is also noteworthy that Mbb. curvatus in the "autotrophic clade" has nitrogenase genes, but apparently, no CODH complex genes and may therefore require externally supplied acetate. This could represent an intermediate stage between the potentially autotrophic strains and the other clades and/or may be an adaptation to a specific niche.
In addition to differences between species, there is currently only little information on strain diversity within a species but studies indicate that there may be considerable differences as shown for Mbb. smithii and Mbb. arboriphilus [46,61]. However, undertaking pan-genome approaches, such as the one for Mbb. smithii by Hansen et al. [61], requires substantial cultivation efforts as there are only few strains available for each of the described methanogen species. As development of sequencing technologies continues to advance, it may also become feasible to obtain highquality closed genomes from low amounts of starting DNA or through metagenomic approaches. Having such genomes will allow greater certainty in determining the presence and absence of specific genome features and will also enable detection of small genomic differences between strains that may go unnoticed in draft genomes.
Lastly, the results of our analyses corroborate the hypothesis that the absence of molydopterin cofactor biosynthesis is a characteristic trait shared by members of the genus Methanosphaera, while all sequenced Methanobrevibacter genomes seem to encode genes for Moco biosynthesis. However, it also needs to be considered that Methanosphaera species encode the genes for formylmethanofuran dehydrogenase and other genes required for hydrogenotrophic or methylotrophic methanogenesis [62,63]. The presence of these genes may indicate that Methanosphaera species may still be able to utilize the aforementioned methanogenic pathways, if molybdopterin cofactor is present and taken up by the methanogen (as suggested by Fricke et al. 2006

Conclusion
The presented study includes genomes of all currently available types of strain (and other selected isolate) of Methanobrevibacter and Methanosphaera genomes and allows comprehensive insights into the genera Methanobrevibacter and Methanosphaera. The analyses reveal that distinct clades within the genus Methanobrevibacter exist and that the lack of molybdopterin cofactor biosynthesis may be a specific trait for the genus Methanosphaera. Additional isolates and further physiological and genomic analyses will be required to determine if division of the genus Methanobrevibacter in more than one genus could be justified. The primary use of the type of strains (and other isolates) for the analysis in this study does warrant access of the scientific community to most of the analyzed Methanobrevibacter and Methanosphaera isolates and will facilitate testing of the predicted physiological phenotypes.

Data Availability
The data used to the support the findings of this study are availabe in the supplemental information or -as in the case of the genomes-are downloadable using the accession numbers provided in Section 2.4.

Conflicts of Interest
The authors declare that they have no conflicts of interest.

Supplementary Materials
Supplementary 1. Figure S1:  Supplementary 3. Table S1: CheckM analysis to determine the completeness of genomes. Table S1A shows results from analysis of genomes from the main manuscript while Table S1B lists the results for genomes mainly mentioned in the supporting information. Table S2: general features of additional Methanobrevibacter genomes. General features of genomes mainly mentioned in the supporting information. Table S3: table of shared and unshared Methanobrevibacter and Methanosphaera genes. Highlighted in colors corresponding to those of Figure 1 are genes for CODH-ACS complex, nitrogenase, methanol, and ethanol utilization. Annotations were taken from the Mbb. arboriphilus DSM1125 T genome.