Intergenomic evolution and metabolic cross-talk between rumen and thermophilic autotrophic methanogenic archaea

Accepted Manuscript Intergenomic Evolution and Metabolic Cross-talk between Rumen and Thermophilic Autotrophic Methanogenic Archaea M. Bharathi, P. Chellapandi PII: DOI: Reference:

S1055-7903(16)30339-6 http://dx.doi.org/10.1016/j.ympev.2016.11.008 YMPEV 5667

To appear in:

Molecular Phylogenetics and Evolution

Received Date: Revised Date: Accepted Date:

30 March 2016 17 September 2016 13 November 2016

Please cite this article as: Bharathi, M., Chellapandi, P., Intergenomic Evolution and Metabolic Cross-talk between Rumen and Thermophilic Autotrophic Methanogenic Archaea, Molecular Phylogenetics and Evolution (2016), doi: http://dx.doi.org/10.1016/j.ympev.2016.11.008

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Intergenomic Evolution and Metabolic Cross-talk between Rumen and Thermophilic Autotrophic Methanogenic Archaea Bharathi M and Chellapandi P* Molecular Systems Engineering Lab, Department of Bioinformatics, School of Life Sciences, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India Tel: +91-431-2407071 Fax:+91-431-2407045 Email: [email protected] *Corresponding author

Running title: Intergenomic evolution and metabolic

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Intergenomic Evolution and Metabolic Cross-talk between Rumen and Thermophilic Autotrophic Methanogenic Archaea Abstract Methanobrevibacter ruminantium M1 (MRU) is a rumen methanogenic archaean that can be able to utilize formate and CO2/H2 as growth substrates.

Extensive analysis on the

evolutionary genomic contexts considered herein to unravel its intergenomic relationship and metabolic adjustment acquired from the genomic content of Methanothermobacter thermautotrophicus H. We demonstrated its intergenomic distance, genome function, synteny homologs and gene families, origin of replication, and methanogenesis to reveal the evolutionary

relationships

between

Methanobrevibacter

and

Methanothermobacter.

Comparison of the phylogenetic and metabolic markers was suggested for its archaeal metabolic core lineage that might have evolved from Methanothermobacter. Orthologous genes involved in its hydrogenotrophic methanogenesis might be acquired from intergenomic ancestry

of

Methanothermobacter

via

Methanobacterium

formicicum.

Formate

dehydrogenase (fdhAB) coding gene cluster and carbon monoxide dehydrogenase (cooF) coding gene might have evolved from duplication events within MethanobrevibacterMethanothermobacter lineage, and fdhCD gene cluster acquired from bacterial origins. Genome-wide metabolic survey found the existence of four novel pathways viz. L-tyrosine catabolism, mevalonate pathway II, acyl-carrier protein metabolism II and glutathione redox reactions II in MRU. Finding of these pathways suggested that MRU has shown a metabolic potential to tolerate molecular oxygen, antimicrobial metabolite biosynthesis and atypical lipid composition in cell wall, which was acquainted by metabolic cross-talk with mammalian bacterial origins. We conclude that coevolution of genomic contents between Methanobrevibacter and Methanothermobacter provides a clue to understand the metabolic adaptation of MRU in the rumen at different environmental niches. Keywords: Methanobrevibacter; Methanothermobacter; Comparative genomics; Functional markers; Molecular evolution; Formate dehydrogenase; Methanogenesis; Phylogeny 1. Introduction Gut methanogenic archaea contributes around 40% of global methane emission via enteric fermentation process (Lin et al. 1998). About 18% of the methane produced in the rumen is derived from formate (Schauer et al. 1982). Several methane mitigation interventions have been developed to curtail the enteric methane emission from ruminants (for example, coat, sheep, cow etc.) (Hook et al. 2010; Leahy et al. 2010; Leahy et al. 2013; Kumar et al. 2014). 2

Outgrowth of gut methanogenic archaea and their unbalanced molecular pairing with syntrophic bacteria or hosts have been reported to reduce the feeding efficiency in farm animals (Hook et al. 2010; Carberry et al. 2014) and to cause the gastrointestinal disorders in human (Samuel et al. 2007; Scanlan et al. 2008; Holmes et al. 2012; Triantafyllou et al. 2014). Formate and hydrogen concentrations are maintained to be low in a rumen gut by contrary requirements of syntrophic bacteria and gut methanogenic archaea (Dolfing et al. 2008). Such syntrophic nature of them has gained an importance to study their metabolite and energy coupling across species (Stams and Plugge 2009). Methanobrevibacter ruminantium M1 (MRU), Methanobrevibacter smithii ATCC 35061 (MSI) and Methanobacterium formicicum BRM9 (MFO) are the most prevalent species of Methanobrevibacter found in the ruminants (Sirohi et al. 2010; Kumar et al. 2012; Cersosimo et al. 2015). Methanobrevibacter and Methanothermobacter genera have shown methanotrophic and autotrophic growth capabilities, which has provided a clue to probe their exploratory metabolic revision and intergenomic lineage (Berg et al. 2010; Chellapandi 2013). Coexistence of both genera in the rumen of Indian buffalo was recently evidenced by assessing their taxonomic and metabolic profiles (Kima et al. 2013; Singh et al. 2015). Several phylogenetic studies have been conducted for the identification of rumen methanogenic archaea from different ecosystems and to reveal their evolutionary status in the tree (Dighe et al. 2004; Tatsuoka et al. 2004; Skillman et al. 2006; Snelling et al. 2014). However, accumulation of substantial quantity of genome sequencing data would be enforced in depth study to understand their genomic and metabolic adaptations. MRU and MSI share a common phylogenetic origin and both organisms are able to convert formate and H2/CO2 to methane (Smith and Hungate 1958; Leahy et al. 2010; Kelly et al. 2011). MSI genome consists of genes required to produce a compatible solute ectoine that is not produced by other methanogenic archaea. It has a gene encoded for [Fe]hydrogenase dehydrogenase which links the methenyl-tetrahydro methanopterin reduction, which is not found in other members of the Methanobacteriaceae (Kelly et al. 2011). Genes coding for non-ribosomal peptide synthetases are exclusively found in MRU, provided evidence for the biosynthesis of antimicrobial metabolites (Leahy et al. 2010). Methanothermobacter thermoautotrophicum H (MTH) is the closest ancestor of Methanobrevibacter and is extensively diverged from Methanococcus jannaschii for the location of orthologous genes. Genes involved in DNA metabolism, transcription, and translation of MTH are more similar to eucaryal sequences, but 42% of genes are derived

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from bacterial origins (Smith et al. 1997). Molecular docking studies revealed the structural motion of enzymes involved in methanogenesis that direct its reverse methanogenesis (Chellapandi 2013). Intensely, MTH modulates its cell membrane lipid composition to cope with energy and nutrient availability in dynamic environments by a lipid regulatory mechanism (Yoshinaga et al. 2015). Since, a metabolic similarity/dissimilarity is expected to be found even within closely related species. Intergenomic evolution can affect the distribution of genes involved in energetic metabolism across methanogenic archaeal genomes (Lane 2014). Studies on intergenomic evolution among haloarchaeal rRNA genes (Boucher et al. 2004), archaeal tryptophan synthase (Merkel et al. 2007) and archaeal conjugative plasmids (Basta et al. 2009) provided evidences for the occurrence of intergenomic co-adaptation within archaeal species. A severity of the metabolic cross-talk across species is being environment-dependent (Kim and Copley 2012). Using comparative genomic analysis, we demonstrated how intergenomic evolutionary constraints are subjected to be enforced on the metabolic functions and adjustment across the Methanobrevibacter and Methanothermobacter. Interorganismal crosstalk between Methanobrevibacter and Methanothermobacter described herein for better understanding of their genomic and metabolic evolution. Current genomic and metabolic information allow us gaining in importance of a metabolic cross-talk of gut methanogenic archaea to their syntrophic partners, intergenic organisms or host at the systems level (Samuel et al. 2007; Chellapandi 2013). 2. Materials and methods 2.1 Evolutionary genomic analyses Complete genome sequences of methanogenic archaea were retrieved for the construction of whole genome tree by CVTree server with randomly selected out-group genomes (Xu and Hao 2009). Genomic correlation coefficient of MRU was calculated by searching COG functional profiles (Tatusov et al. 2003) of its phylogenomic neighbors using IMG-M genome comparison tool (Markowitz et al. 2012). Intergenomic distance was calculated between MRU and its phylogenomic neighbors using GGDC server based on their DNADNA hybridization (Meier-Kolthoff et al. 2013). A syntenic dot plot was generated for finding syntenic blocks and orthologs by comparing its coding genes from MSI and MTH using tBLASTX program (Altschul and Koonin 1998). Collinear sets of genes shared between MRU and MSI; MRU and MTH were identified by CoGe: SynMap using DAGChainer algorithm with relative gene order of microbes (Tang et al. 2011). QuotaAlign is a post-processing step to merge adjacent syntenic blocks for downstream analysis of 4

genome rearrangements. Synonymous substitution rates of syntenic coding sequences in each genome were calculated by CodeML and then represented as a syntenic histogram. OriCs and genes encoding for DNA replication proteins were detected from each genome by Ori Finder 2.0 (Gao and Zhang 2008). Escherichia coli genome was chosen as a template to find out specific DnaA boxes pattern TTATCCACA. For finding a sequence-specific conservation pattern across these genomes, the predicted oriCs and DNA replication protein sequences were aligned separately by ClustalX 2.0 software (Thompson et al. 1997). 2.2 Evolutionary genetic analysis of marker genes Phylogenetic and metabolic markers were identified from selected genomes by text mining approach (Chellapandi 2013). Nucleotide sequences of phylogenetic markers and amino acid sequences

of

metabolic

markers

were

retrieved

from

NCBI

server

(http://www.ncbi.nlm.nih.gov/). Similarity hits for these sequences were searched out from NCBI by Blastn and Blastp tools (Altschul et al. 1997). Multiple sequence alignment was performed separately with ClustalX 2.0 software (Thompson et al. 1997) and edited manually to remove improperly aligned sequences. Phylogenetic tree was constructed separately for each marker from aligned sequences by MEGA 5.0 software using neighbor joining algorithm (Tamura et al. 2011). The robustness of the phylogenetic tree was estimated by bootstrap replication of 1000 at uniform rate among sites. Kimura 2-parameter (Kimura 1980) and Jones-Taylor-Thornton matrix (Jones et al. 1992) were chosen as evolutionary models for the construction of phylogenetic marker-based trees and metabolic marker-based tress, respectively. Secondary structures of rRNA were modeled with MFold server and calculated loop energy represented in ΔG plot (Zuker 2003). Since mcrA is a key metabolic marker of all methanogenic archaea, its three dimensional structure was modeled from a structural template (pdb id: 1HBN) by using Swiss-Model (Biasini et al. 2014). The modeled protein structures were aligned and superimposed by 3D-SS for calculating the structural deviations (Sumathi et al. 2006). Segregation sites, mutation rate and nucleotide diversity across the clusters were computed separately for each marker using MEGA. A marker which does not fit the neutral theory model of equilibrium between mutation and genetic drift was examined by Tajima's neutrality static (D) (Tajima 1989). A negative Tajima's D signifies an excess of low frequency polymorphisms relative to expectation (purufying selection) whereas a positive Tajima's D signifies low levels of both low and high frequency polymorphisms (balancing selection).

5

2.3 Comparative metabolic analysis An intensive bioinformatics analysis was employed to investigate a common metabolic sharing between Methanobrevibacter and Methanothermobacter (Chellapandi 2015). Using genome-wide text mining approach, metabolic features including pathways, metabolites, metabolic enzymes and transporters etc. were manually collected from MetaCyc database (Caspi et al. 2014). A complete set of genes and enzymes for different species of these genera were retrieved from Halolex browser (Pfeiffer et al. 2008). Enzymes involved in the central metabolism and methanogenesis were listed out from MetaCyc database. The overlap of resulting gene and enzyme lists were depicted as Venn diagram (Pirooznia et al. 2007). A complete set of metabolic pathways were compared together to identify unique genes in MRU. Uncovered pathways were detected from MRU by locating indentified unique genes in the respecting metabolic networks of MetaCyc database. 3. Results 3.1 Phylogenomic analysis A genome-scale tree of this study demonstrates the current phylogenomic status of MRU among methanogenic archaea as depicted in Fig. 1. Mesophilic methanogenic archaea, Methanocaldococcus and Methanococcus are phylogenomically related to the thermophilic methanogenic archaea, Methanothermococcus. Methanobrevibacter is one of the major groups of rumen methanogenic archaea from which MRU and MSI are grouped with thermophilic autotrophic methanogenic archaea, Methanothermobacter and then clustered with heterotrophic methanogenic archaea such as Methanosaeta and Methanosarcina in the tree. Methanobacterium AL21 serves as an out-group genome to obtain an optimal tree. 3.2 Genomic correlation The first closest genomic neighbour of MRU is found to be MSI whereas the second closest genomes are MTH and MMG as represented in Fig. 2. Genomic function (COG functional profiles) of MRU is more correlated with MTH and MMG than MSI. Also, MRU has shown a genomic relatedness with diverse methanogenic archaea having different growth capabilities at a small extent. 3.3 Genome synteny of homologs The results of genome synteny explicate that a colinear set of genes shared between MRU and MSI is higher than that shared between MRU and MTH (Fig. 3). Syntenic blocks may adjust to maintain a collinearity of either gene orders or gene families at the location of syntenic out-paralogs. MRU was shared more syntenic orthologs with MTH at low synonymous substitution rate than MSI. Syntenic out-paralogs across these genomes are 6

derived from whole genome duplication event (α-event) at different synonymous substitution rate. 3.4 OriC and replication genes Z-curve analysis has predicted three oriCs in upstream of cdc6 genes (mru_0001; mru_0423; mru_0259) from MRU as shown in Fig. 4. MSI shows only one oriC in upstream of cdc6 gene (msm_1264) whereas MTH exhibits two oriCs in upstream of cdc6 genes (MTH_1412; MTH_1599). Archaeal ATPase gene (mru_2140) of MRU is found at the genomic position of 2809226-2810446. AAA+ ATPase genes (msm_0671; msm_1646) are detected from MSI. The sequence of cdc6 genes from MRU shows significant identity to MSI (77%) and MTH (70%). Sequence alignment result indicates that not only cdc6 gene sequence, but also oriCs sequences of MRU are closely matched with MTH. 3.5 Genetic diversity of marker genes Phylogenetic markers responsible for the process of DNA replication and repair (dnaG, mutS, polB, radA), transcription (nusA, nusG, rpoB, rpoC, TFB), translation (aEF-2, fusA, glyS, infB, leuS, rnhB, serS, spoVC, tgtA, truB), and post translational modification (dnaJ, engA, hsp20, mreB, obg/cgtA, secY) were identified from Methanobrevibacter. In our study, metabolic markers considered as evolutionary measures to divulge the metabolic pairing between Methanobrevibacter and Methanothermobacter (Table 1; Supplemental Fig. S1S11). A phylogenetic marker with low number of segregation sites signifies the importance in evolutionary genetic analysis of methanogenic archaea. Molecular diversity analysis indicates that 23s rRNA, pheS, glyS, murD and pyrG genes have more number of segregation sites in their sequences (Fig. 5; Supplementary Fig. S12). Metabolic markers contain average number of segregation sites, reflecting the conservation of genes-associated metabolic pathways across the methanogenic archaea. A population-scaled mutation rate is an important evolutionary constraint that acts on nucleotide diversity of metabolic markers leaading to the functional expansion or purification across methanogenic archaea. Nucleotide diversity of 16s rRNA, 23s rRNA, ispD and mcrA genes are not notably expanded by mutation rate, which reflects neutral evolution acting on their sequence diversity. 3.6 Metabolic features Genome size of MRU is not correlated with number of coding genes compared with MSI and MTH, indicates its metabolic diversity seems to be low (Supplementary Table. S1). MRU and MSI contain 10-20 additional pathways in comparison to the MTH. This may be due to autotrophic growth capability and low requirements of co-factors and prosthetic groups for 7

energetic metabolism of MTH. Methanobrevibacter has a typical system for growing in a rumen ecosystem as its metabolic reactions and substrate efflux systems are higher than that existing in the MTH. Comparison of genome-wide properties demonstrates that MRU consists of 63 unique enzymes and 91 common enzymes across the methanogenic archaea (Supplementary Fig. 13.). MTH contains 31 unique enzymes for central metabolism. It also represents 104 metabolic genes, 148 enzymes for central metabolism and 7 enzymes for methanogenesis which are all shared across the methanogenic archaea. Some unique enzymes in the Methanobrevibacter are found to contribute in the cell wall biosynthesis and amino acid metabolism. 3.7 Functional evolution of methanogenesis Even if mcrA gene is being more closely related in all methanogenic archaea, its protein structure from Methanobrevibacter is speculated from that structure of MTH. The N-terminal domain of mcrA from Methanobrevibacter is shared together whereas C-terminal domain is diverged from that structure of MTH (Supplementary Fig. S14). Formate oxidation and CO2 reduction are interconvertible processes catalyzed by sequential action of fdhAB, frhA, mtd and mer coding enzymes. Phylogenetic analysis suggests the fdhAB gene cluster arose from duplication

events

within

the

Methanobrevibacter-Methanothermobacter

lineage

(Supplementary fig. S15). FdhA (mru_0333; mru_2074), fdhB (mru_0334; mru_2075), fdhC (mru_0332) and fdhD (mru_0681; mru_1939) are important genes involved in formate oxidation process of MRU (Supplementary Fig. S16-S18). The evolutionary link between MRU/MSI and MRU/MTH for the functional divergence of fdhCAB and cooF gene clusters is presented in Fig. 6. Mutation rate was observed as an evolutionary constraint that drastically impacted on the nucleotide diversity of fdhABCD genes (Fig. 5). Across methanogenic archaea, fdhAB gene function may have evolved by acting positive selection whereas fdhC gene may have evolved by purifying selection. Comparative genomic analysis shows that cooF genes from Thermococcus paralvinellae and Thermofilum carboxyditrophus are expected to be evolutionary origins for functional divergence of fdhC gene in the Methanobrevibacter. A purifying selection is acting on the functional evolution of fdhD gene across methanogenic archaea and Proteobacteria. FrhB1GDA gene cluster is identical to each other and appeared in reverse strand. FrhB1 genes from MRU and MTH are duplicated within the same gene cluster and frhB2 from MTH duplicated along with the gene cluster required for cell wall biosynthesis.

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3.8 Discovery of uncovered pathways L-Tyrosine catabolism, mevalonate pathway II, acyl-carrier protein metabolism II and glutathione redox reactions II are exclusive pathways discovered from MRU (Fig. 7). The presence of phenylalanine 4-monooxygenase (EC 1.14.16.1) encoding gene confirms the reaction for conversion of L-phenylalanine to L-tyrosine in tyrosine catabolism. In this reaction, tetrahydrobiopterin acts as a cofactor and N10-formyltetrahydrofolate serves as a donor for formyl group. This reaction is active only in the presence of molecular oxygen. Similarly, a unique gene encoding for glutathione-disulfide reductase (EC 1.8.1.7) is also found in the MRU. This enzyme is solely involved in glutathione redox reactions II that plays a vital role in preventing its genome from oxidative damage. MRU consists of a unique gene encoding for holo-[acyl-carrier-protein] synthase (EC 2.7.8.7) that is required for acyl-carrier protein metabolism II linking with adenosine ribonucleotides de nova biosynthesis. Acylcarrier protein is an important component in both fatty acid and polyketide biosynthesis of many archaea. MRU and MTH contain unique genes encoding for isopentenyl phosphate kinase (EC 2.7.4.26) and isopentenyl-diphosphate Δ-isomerase (EC 5.3.3.2). These enzymes consecutively convert isopentenyl phosphate into dimethylallyl diphosphate, an essential building block of isoprenoid compounds. 4. Discussion In the whole genome tree, we found two major lineages such as MethanococcusMethanothermococcus and Methanobrevibacter-Methanothermobacter. It revealed an evolutionary lineage from thermophilic methanogenic archaea to mesophilic methanogenic archaea. The ancestors of this lineage for mesophilic adaptation in the methanogenic archaea may be recipients of massive horizontal gene transfer from bacterial origins (Foster et al. 2009; López-García et al. 2015). MRU has shown a distinct phylogenomic status in which it has

genomic

equivalency

with

Methanobrevibacter

as

quite

equal

to

the

Methanothermobacter. As similar to whole genome tree, different phylogenetic markers of this study provide a strong support to explicit their evolutionary liaison in accordance to earlier works (Dighe et al. 2004; Tatsuoka et al. 2004; Skillma et al. 2006; Snelling et al. 2014).

Methanobrevibacter (Balch and Wolfe 1981) and Methanothermobacter

(Wasserfallen et al. 2000) are belonging to Methanobacteriaceae family, which are found in diverse anoxic environments including the gastrointestinal tracts of animals (Santana et al. 2012; Oren 2014). Accordingly, intergenomic evolution is expected to share metabolic content across Methanobrevibacter and Methanothermobacter in a rumen ecosystem.

9

Genomic correlation results of our study indicated that even intergenomic distance of MRU was very near to the MSI, its genome functional homology resembles to Methanothermobacter. Lurie-Weinberger et al. (2012) found over 15% of the coding genes of MSI inferred to be bacterial origins. It is expected that lateral gene acquisition from bacterial origins might have attributed in functional evolution of MRU. Consequently, the gene– phenotype relationships and rearrangements of gene family may expand its genome size for a new function, which may have evolved from ancestral sequences of Methanothermobacter (Abby and Daubin, 2007; Novichkov et al. 2009; Gabaldón and Koonin 2013). Homologous gene family evolution and origin of gene family duplicates are deduced from synteny map. In our study, synonymous substitution is a common evolutionary event for syntenic orthologs and duplication (-event) of syntenic out-paralogs of MRU and MTH. Synonymous substitution is a major constraint for collinearity of orthologous gene sets and duplication (-event) of syntenic-out paralogs of MRU and MSI. Orthologs and paralogs are types of homologs in the MRU, which may be derived from speciation divergence and duplication, respectively (Peterson et al. 2009; Gabaldón and Koonin 2013). Disruption of synteny during evolution of Methanobrevibacter and Methanothermobacter characteristically showed a clear and striking pattern with an X-shape as seen in the dot plots. It may be appeared due to a degree of synteny depending on a balance between intensity of recombination, domain rearrangement, horizontal gene flux, and operon/gene cluster disruption (Koonin and Wolf 2008). DNA replication machinery in archaea is structurally and functionally similar to eukaryotic replication system, but it can be functional with unique archaeal features (Kelman and Kelman 2004; Foster 2005; Sarmiento et al. 2014). Eubacterial species usually exhibit only one oriC, but archaeal species can possess one or multiple oriC in upstream of cdc6 gene (Sarmiento et al. 2014). Cdc6 gene coding protein is an essential regulator for DNA replication initiation that requires AAA+ATPase or archaeal ATPase. Similarly, MRU contains several oriCs found in upstream of cdc6 and one archaeal ATPase at different genomic locations compared to the MSI and MTH. Sequence analysis results indicated that DNA replication proteins and oriCs are highly similar to MTH, suggesting a probability of intergenomic evolution across them for sharing DNA replication machinery (Zhang and Zhang 2002; 2004). Gene duplication is a major force in evolution for acquiring new genes from closely related organisms, facilitating the functional divergence and organismal diversity (Jaina et al.

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2002; Magadum et al. 2013). As a result, some of the markers genes are duplicated as one or two more copies in the MRU and are varied even closely related genomes. For example, copy number of 5s rRNA genes detected from MRU is not equal to that copy number of MSI. Besides, optimal loop energy in secondary structure of 5s rRNA from MRU is slightly speckled from MSI and MTH. One more loop in MSI and four more loops in MTH may bring disparity in their secondary structures and mismatches in stem and loop conversion (Supplementary Fig. S19). In some cases, the metabolic rate is directly proportional to the copy number of corresponding metabolic genes (Parkesa et al. 2012; Pei et al. 2012; Noller 2014). Similarly, we observed the existence of many duplicated metabolic genes for energetic metabolism of MRU as quite equivalent to the MSI and MTH. Comparative genome analysis described that major genes in central molecular dogma found to be common between Methanobrevibacter and Methanothermobacter, which may be derived from euryarchaeal origin (Gribaldo and Brochier-Armanet 2006; Ochs et al. 2012). MRU contains a complex gene regulatory system as it posses more transcriptional units compared from MSI and MTH. Protein coding sequences in MMG, 91 coding sequences have a counterpart only in the MRU, MSI and MTH (Kaster et al. 2011). In our study, coding sequences of Methanobrevibacter and Methanothermobacter have shown more biosynthetic function than catabolic function in concurrent to the previous work (Kaster et al. 2011). Bacterial domain-like gene products from MRU found to be involved in the biosynthesis of cofactor and prosthetic groups, transport, nitrogen metabolism, global regulatory functions and syntrophic association with bacteria. It was agreed with earlier works for MSI and MTH (Smith et al. 1999; Vitreschak et al. 2003; Kelly et al. 2014). Enzymes involved in amino acid, nucleotide and vitamin biosynthetic pathways of MRU are bacterial homologs similar to MTH (Smith et al. 1997). A positive Tajima's (D) of signifies the function of many metabolic genes is still to be purified by gene shuffling or gene duplication events. Therefore, we suggested that intergenomic cross-talk is a major constraint to evolve the metabolic genes or gene clusters from ancestry of Methanothermobacter for steady establishment of metabolic systems/pathways as the requirements in rumen environment. Methanogenesis was an ancestral form of energy metabolism in the very first freeliving archaea. Explicating genome content and determining gene ancestry are decisive factors to infer the major events in the evolution of methanogenesis (Kelly et al. 2011). The synthesis of the enzymes, coenzymes, and prosthetic groups involved in hydrogenotrophic methanogenesis and energy conservation system are phylogenetically conserved within Methanothermobacter, and metabolically shared within Methanobacteriaceae (Kaster et al. 11

2011). Accordingly, energetic metabolism may have evolved by intergenomic adjustments of Methanobrevibacter with Methanothermobacter via concomitant loss/gain of genes involved in methanogenesis. MRU contains 6 gene coding proteins for the reduction of CO2 to methane as similar to a metabolic system found in the MTH (Supplementary Fig. S20) (Sakai et al. 2011). Several genes are found in the MTH and MSI for acetoclastic methanogenesis, but related pathway is incomplete. It suggested the existence of complete pathway for hydrogenotrophic

methanogenesis

and

F420

biosynthesis

from

formate

in

the

Methanobrevibacter likely to the Methanothermobacter. Formate is a main methanogenic substrate derived from rumen anaerobic bacteria requiring for hydrogenotrophic methanogenesis and F420 biosynthesis. It is first oxidized to CO2 by fdhAB coding enzymes with concomitant production of F420H2, which is further oxidized by frhA coding enzyme to oxidized F420 and molecular hydrogen (Samuel et al. 2006; Rea et al. 2007; Costa et al. 2013). MTH strain Z-245 can use formate, but MTH strain ΔH cannot since fdhCAB gene cluster is absent in the later strain (Nolling and Reeve 1997; Wood et al. 2003; Sattler et al. 2013). Similarly, we found two set of fdhCAB gene clusters in the Methanobrevibacter that may be appeared by concomitant gain of genes involving in the formate oxidation. The first cluster found to be identical in MRU and MSI and second cluster of MSI duplicated in reverse orientation by gene inversion event. A gene cluster consisting of mvhD2-fdhB2-fdhA2 and fdhB1 in the Methanobrevibacter might have evolved from ancestral gene cluster of MTH via MFC, a closest ancestor of MRU. FrhA gene diverged into pyruvate-formate lyase encoding gene pfl1 of pathogenic Clostridium and NAD-dependent fdhA of Carboxydothermus hydrogenoformans. Probably, NAD-dependent fdhA from MMG might be a recent ancestor for evolution of fdhA1 in the Methanobrevibacter by vertical transfer event. Our phylogenetic analysis indicated that early Methanobrevibacter may have evolved from H2-dependent Methanothermobacter species (Samuel et al. 2007). Apart from that, biphasic kinetics demonstrated the formate transporter fdhC plays a significant role in the kinetics of H2 production in Methanococcus maripaludis (Lupa et al. 2008). FdhD is an accessory protein for improving formate dehydrogenase activity, which is essential to increase the reaction rate of CO2 reduction to methane (Kaster et al. 2011; Soboh et al. 2011). FdhCD genes are acquired from thermophilic bacterial origins and gradually established in the genomes of Methanobrevibacter. Exclusive establishment of fdhCD genes suggested the genomic and metabolic adjustment of Methanobrevibacter to enhance their growth capability on formate in accordance to earlier works (Samuel et al. 2007; Lupa et al. 2008; Poehlein et al. 2012). 12

Several autotrophic methanogenic archaea exhibit carbon monoxide dehydrogenases (cooF) that can oxidize carbon monoxide in the presence of hydrogen. This is coupled to numerous respiratory processes and methanogenesis (Sipma et al. 2004; Oelgeschläger and Rother 2008). In our study we found porCDAB-cooF1-cooF2 gene cluster in the MRU, which might be subsequently evolved from cooF2-cooF1-porBAC gene cluster of MTH by concomitant gain of porD gene. Also, concomitant loss of PorBC genes and gain of porG gene found to be appeared in the MSI. Thus, such metabolic potential of Methanobrevibacter might be derived from Methanothermobacter by gene duplication event. The presence of heme-dependent catalase encoding gene and exogenous hemin confer an effective antioxidative defense in the Methanobrevibacter (Leadbetter et al. 1996; Shima et al. 2001; Brioukhanov and Netrusov, 2012). Metabolic survey of our study stated that unlike MTH, MRU possess a novel route for the conversion of L-tyrosine to L- phenylalanine by consecutive enzymatic reactions. MRU consist of a phenylalanine 4-monooxygenase coding gene that directly converts L- phenylalanine to L-tyrosine in the presence of molecular oxygen in concurrent to earlier works (Fitzpatrick 1999; Steventon and Mitchell, 2009). The existence of such oxygen tolerance system prevents its genome from the oxidative damage in accordance to mammalian and bacterial systems (Pember et al. 1987; Mitchell et al. 2011; Flydal et al. 2012). Phenylalanine 4-monooxygenase reduces the excess level of phenylalanine in human phenylketonuria individuals by biosynthesis of tyrosine in the presence of tetrahydrobiopterin as a cofactor (Mitchell et al. 2011). It is also reported in a skin pathogenic bacterium Chromobacterium violaceum (Pember et al. 1987) and Legionella pneumophila causing Legionnaires’ disease and other non-pneumonic infections in human (Flydal et al. 2012). The second one is symbiotically present in aquatic-borne amoebae and infects accidentally human macrophages (Winiecka-Krusnell and Linder 1999; Escoll et al. 2013). Based on these investigations, we assume that this enzyme encoded gene might have evolved from corresponding gene may be originated from the bacterial origins where a crosstalk may occur for utilization of excessive phenylalanine from mammalian host. Oxidative damage to proteins often results in the formation of mixed disulfides within polypetides. A primary defense against this damage is mediated by the action of glutathionedisulfide reductase (Chung et al. 1991). Atypical disulfide oxidoreductase was found as a new antioxidant system of Sulfolobus solfataricus (Limauro et al. 2014). Similarly, MRU contains glutathione-disulfide reductase coding gene that was acquired from aerobic bacterial origins, suggesting it as a cellular antioxidant for scavenging reactive oxygen species.

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A complete mevalonate pathway II is not functionally active in the MSI, MMG and MTH since isopentenyl phosphate kinase (EC 2.7.4.26) and isopentenyl-diphosphate Δisomerase are absent in these genomes unlike MRU (Smit et al. 2000; Barkle et al. 2004; Lombard and Moreira 2010). It revealed that MRU may possess unique membrane lipids derived from this pathway, leading to have a distinct cell wall composition from closely related species. Holo-[acyl-carrier-protein] synthase coding gene was identified from MRU, which can be involved in the post translational modification (modulation) of non-ribosomal peptide synthetases (Flugel et al. 2000; Beld et al. 2014). The non-ribosomal peptide synthetases exclusively found in the MRU, supporting its metabolic potential to produce the antimicrobial metabolites (Leahy et al. 2010). 5. Conclusions Methanobrevibacter is the most abundant methanogenic archaea found in the gut of ruminants. Comparative genomic analysis of this study can pave an account to analyze the evolutionary relevance, metabolic pairing and distribution of metabolic genes of MRU across Methanobrevibacter and Methanothermobacter. The present evolutionary conclusion should certainly help our understanding of its molecular origin and metabolic diversity in rumen environment. Adventitious interactions in microbial metabolic networks found to evolve a new metabolic pathway in the environments (Kim and Copley 2012). Coevolution and intergenomic evolution shape gut microbiome-host crosstalk leading to diverse cellular and metabolic changes such as harvesting energy, shaping host immune system, and metabolic signalling (Rodionov et al. 2002; Martin et al. 2009; McCutcheon and Moran 2010; Hooper et al. 2012). Accordingly, we stated that emergence of its novel metabolic pathways may require a variety of genome adaptations and metabolic and regulatory networks modulation. Coevolution of MRU with its phylogenomic neighbours (MSI and MTH) would define a metabolic cross-talk, which in turn to monitor the health status of veterinary animals. Nevertheless, its colonization and metabolic adaptation in the gut of ruminants is influenced by many factors including type of animal feeding and widely used antibiotic therapy. Experimental studies are further warranted to properly elucidate its metabolic and global regulatory networks connected with closely related methanogenic archaea, bacterial symbionts and ruminants, and to identify the vaccines and chemogenomic targets for new methane mitigation interventions world widely. Acknowledgements The corresponding author is thankful to the University Grants Commission-Research Award, (F. 30-1/2013(SA-II)/RA-2012-14-SC-TAM-1768), India for the financial assistance. 14

University Grants Commission (42-864/2013(SR)), India is duly acknowledged to provide fellowship to the first author. Conflict of Interest The authors declare that there is no conflict of interest Abbreviations aEF-2: Translation elongation factor cdc6: Cell Division Cycle 6 COG: Clusters of Orthologous Group cooF1/F2: Carbon-monoxide dehydrogenase iron sulfur subunit dnaG/J: DNA primase engA: Ribosome-associated GTPase fdhA/B: Formate dehydrogenase alpha/beta subunit fdhC/D: Formate transporter frhABGDA: Factor F420-reducing hydrogenase fusA: Translation elongation factor GGDC: Genome-to-Genome Distance Calculator glyS: Glycyl-tRNA synthetase hemC: Porphobilinogen deaminas hsp20: Heat shock protein infB: Translation initiation factor IF-2 ispD: 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase leu: 2-isopropylmalate synthase mcrA: Methyl cytosine restriction enzyme mer: F420-dependent methylene-H4MPT reductase metK: Methionine adenosyltransferase MFC: Methanobacterium formicicum BRM9 MMG: Methanothermobacter marburgensis Marburg mreB: Rod shape-determining protein MRU: Methanobrevibacter ruminantium M1 MSI: Methanobrevibacter smithii ATCC 35061 mtd: F420-dependent methylene-H4MPT dehydrogenase MTH: Methanothermobacter thermautotrophicus H murD: UDP-N-acetylmuramoylalanine--D-glutamate ligase

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mutS: DNA mismatch repair ATPase MutS family mvhD2: Methyl viologen-reducing hydrogenase delta subunit nifD: Nitrogenase molybdenum-iron protein nusA: Transcription elongation factor NusA-like protein obg/cgtA: GTPase/ obg family GTPase ogt: Methylated-DNA-[protein]-cysteine S-methyltransferase OriC: Origin of replication OriCs: Origin of replications polB1/B2: DNA polymerase family B porCDABG: Pyruvate ferredoxin oxidoreductase purM: Phosphoribosylformylglycinamidine cyclo-ligase pyrG: CTP synthase radA: DNA repair and recombination protein rnhB: Ribonuclease HII rpoB/C: DNA-directed RNA polymerase subunit secY: Preprotein translocase serS: Serine-tRNA ligase spoVC: Guanosine-3',5'-bis(Diphosphate) 3'-pyrophosphohydrolase TFB: Transcription initiation factor tgtA: 7-cyano-7-deazaguanine tRNA-ribosyltransferase tpiA: Triosephosphate isomerase truB: tRNA pseudouridine synthase B References Abby S, Daubin V. 2007. Comparative genomics and the evolution of prokaryotes. Trends Microbiol. 15:135–141. Basta T, Smyth J, Forterre P, Prangishvili D, Peng X. 2009. Novel archaeal plasmid pAH1 and its interactions with the lipothrixvirus AFV1. Mol Microbiol. 71:23–34. Beld

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Fig. legends Fig. 1 Phylogenomic tree constructed by compositional vectors algorithm using the whole genome sequences of MRU and other closely related methanogenic archaea. Branch lengths are proportional to evolutionary distances. Out-group organism was selected randomly for the classification. Fig. 2 Calculation of Pearson correlation coefficient and intergenomic distance for finding genomic proximity of MRU with its phylogenomic neighbours MRU: Methanobrevibacter ruminantium M1; MSI: Methanobrevibacter smithii ATCC 35061; MEW: Methanobacterium sp. SWAN-1; MVN: Methanococcus vannielii SB; MCJ: Methanosaeta concilii GP6; MPL: Candidatus Methanosphaerula palustris E1-9c; MMG: Methanothermobacter marburgensis Marburg; MTH: Methanothermobacter thermautotrophicus deltaH; MOK: Methanothermococcus okinawensis IH1; MFV: Methanothermus fervidus DSM 2088.

Fig. 3 Syntenic dot plot and synonymous substitution histogram of MRU and MSI (above); MRU and MTH (below). Syntenic gene pairs were identified by DAGChainer and colored based on their synonymous substitution rate as calculated by CodeML. Non-syntenic matches are shown in grey dots. MRU: Methanobrevibacter ruminantium M1; MSI: Methanobrevibacter smithii ATCC 35061; MTH: Methanothermobacter thermautotrophicus deltaH.

Fig. 4 Z-curve (AT, GC, RY and MK disparity curves) representation for comparing the location of DNA replication proteins and OriC across MRU, MTH and MTH. Short vertical red line indicates the location of DNA replication proteins. The black arrows pointing below are the oriCs with origin recognition box sequences. OriC sequence alignment is shown in below left. MRU: Methanobrevibacter ruminantium M1; MSI: Methanobrevibacter smithii ATCC 35061; MTH: Methanothermobacter thermautotrophicus deltaH.

Fig. 5 Estimates of genetic diversity and Darwinian selection of phylogenetic and metabolic markers * The genes fdhAB are not considered as phylogenetic and metabolic markers. Fig. 6 Evolution history for the establishment of fdhCAB and cooF gene clusters in MRU to mediate methanogenesis from formate Abbreviations used for this figures: MRU: Methanobrevibacter ruminantium M1; MSI: Methanobrevibacter smithii ATCC 35061; MTH: Methanothermobacter thermautotrophicus deltaH; MJA: Methanocaldococcus jannaschii DSM 2661; MFC: Methanobacterium formicicum BRM9; MMG: Methanothermobacter marburgensis Marburg; TPA: Thermococcus paralvinellae; TCA: Thermofilum carboxyditrophus; FdhC: formate transporter; FdhA1/A2: formate dehydrogenase alpha subunit; FdhB1/B2: formate dehydrogenase beta subunit; MvhD2: methyl viologen-reducing hydrogenase delta subunit; Fwd: tungsten formylmethanofuran

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dehydrogenase; PorCDABG: pyruvate ferredoxin oxidoreductase; CooF1/F2: carbon-monoxide dehydrogenase iron sulfur subunit; Fwd: tungsten formylmethanofuran dehydrogenase; FrhA: coenzyme F420 hydrogenase subunit alpha

Fig. 7 The uncovered pathways discovered in MRU based on the unique genes (red color).

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Fig. 1 Methanocaldococcus FS406 M.s jannaschii DSM 2661 Methanocaldococcus M. fervens AG86 Methanothermococcus okinawensis IH1 M. voltae A3 M. vannielii SB Methanococcus M. maripaludis S2 M. maripaludis X1 M. marburgensis Marburg Methanothermobacter M. thermautotrophicus Delta H M. ruminantium M1 M. smithii ATCC 35061 Methanobrevibacter Methanobrevibacter AbM4 Methanoregula formicicum SMSP M. concilii GP6 Methanosaeta M. thermophila PT Methanococcoides burtonii DSM 6242 M. barkeri Fusaro M. acetivorans C2A Methanosarcina M. mazei Go1

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Fig. 2

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Fig. 3

29

Fig. 4

30

Fig. 5

31

Fig. 6

32

Fig. 7

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Table 1 Comparison of metabolic markers between RM and TAM RM: Rumen methanogenic archaea; TAM: Thermophilic autotrophic methanogenic archaea; MRU: Methanobrevibacter ruminantium M1; MSI: Methanobrevibacter smithii ATCC 35061; MTH: Methanothermobacter thermautotrophicus deltaH; MOK: Methanothermococcus okinawensis IH1

Gene

Marker name

RM MRU

TAM MSI

MTH

EC

Metabolism

MOK

ahaD

F1Fo ATPase beta subunit

0703

0433

-

-

3.6.3.14

Oxidative phosphorylation

coaE

Dephospho-CoA kinase

1224

0141

-

-

2.7.1.24

Pantothenate and CoA biosynthesis

fdhD

Format dehydrogenase accessory protein

0681|1914

0295|1392

1139|1140|

1073

-

Methanogenesis

0592

2.5.1.61

Heme biosynthesis

2.7.7.60

Terpenoid backbone biosynthesis

|1939

1552

hemC

Porphobilinogen deaminase

1746

0881

874

ispD

2-C-Methyl-D-erythritol 4-phosphate cytidylyltransferase

1056

0377|1542

mcrA

Methyl-coenzyme M reductase alpha subunit

1924

0902|1015

1164

0956

2.8.4.1

Methanogenesis

metk

S-Adenosylmethionine synthetase

0125

1340

1376

0334

2.5.1.6

Cysteine and methionine metabolism | Amino acids biosynthesis

murD

UDP-N-acetylmuramoylalanine--D-glutamate ligase

1118|2092

0118|1191

-

-

6.3.2.9

D-Glutamine and D-glutamate metabolism | Peptidoglycan biosynthesis

murE

UDP-N-acetylmuramyl-tripeptide synthetase

2091

1190

734

-

6.3.2.13

Lysine biosynthesis | Peptidoglycan biosynthesis

nifD

Nitrogenase molybdenum-iron protein alpha subunit

1466

1707

1563

-

1.18.6.1

Nitrogen metabolism

purM

Phosphoribosylformylglycinamidine cyclo-ligase

1979

1039

1204

0360

6.3.3.1

Purine metabolism

pyrG

CTP synthase (UTP-ammonia lyase)

1237

0147

419

0276

6.3.4.2

Pyrimidine metabolism

tpiA

Triosephosphate isomerase

1822

0919

1041

1494

5.3.1.1

Inositol phosphate metabolism | Carbon metabolism | Amino acids biosynthesis

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Graphical abstract

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Highlights  Intergenomic evolutionary link between Methanobrevibacter and Methanothermobacter describes to understand the genome dynamics and metabolic adaptation of MRU in the rumen environment.  Metabolic cross-talk with mammalian bacterial origins supports its oxygen tolerance, antimicrobial metabolite biosynthesis and atypical lipid composition in cell wall.  Identified tyrosine and mevalonate pathway supports to identify therapeutic targets for reducing energy harvest in obese human and farm animals, and to curtail of the global methane emission.

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