Research Article | Open Access
The Genome Sequence of Methanohalophilus mahii SLPT Reveals Differences in the Energy Metabolism among Members of the Methanosarcinaceae Inhabiting Freshwater and Saline Environments
Methanohalophilus mahii is the type species of the genus Methanohalophilus, which currently comprises three distinct species with validly published names. Mhp. mahii represents moderately halophilic methanogenic archaea with a strictly methylotrophic metabolism. The type strain SLPT was isolated from hypersaline sediments collected from the southern arm of Great Salt Lake, Utah. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,012,424 bp genome is a single replicon with 2032 protein-coding and 63 RNA genes and part of the Genomic Encyclopedia of Bacteria and Archaea project. A comparison of the reconstructed energy metabolism in the halophilic species Mhp. mahii with other representatives of the Methanosarcinaceae reveals some interesting differences to freshwater species.
Halophilic methanogens contribute significantly to carbon mineralization in marine and hypersaline environments. The preferred substrates for methanogenesis in these habitats are C-1 methylated compounds, for example, methylamines that are constantly provided by the degradation of osmolytes like glycine betaine or membrane compounds such as choline. The preference of methylated C-1 compounds over hydrogen by methanogens thriving in saline environments reflects a competition with sulfate-reducing bacteria that are able to utilize hydrogen more efficiently than methanogens, but usually cannot use methanol or methylamines as substrates . Strain SLPT (= DSM 5219T = ATCC 35705T) is the type strain of the moderately halophilic methanogen Methanohalophilus mahii . SLPT (Salt Lake Paterek) is the only strain of this species available from culture collections and was isolated from anoxic sediments of Great Salt Lake in Utah (USA) .
Although SLPT represents the only described strain of this species, cultivation independent studies indicate that methanogens that are closely related to Mhp. mahii are quite common in anoxic saline environments. The 16S rRNA gene sequence of the nearest neighbor, Methanohalophilus portucalensis strain FDF-1, shares 99.8% sequence identity with SLPT whereas the type strains of all other species in the Methanosarcinales share less than 94.7% with SLPT . Numerous cloned 16S rRNA genes more than 99% identical to the sequence of SLPT were retrieved from hypersaline microbial mats of solar salterns in Guerrero Negro (Baja California Sur, Mexico)  and Eilat (Israel) , an endorheic hypersaline lake in La Macha, Spain (EF031086, unpublished), the deep-sea anoxic brine lake Urania, Eastern Mediterranean Sea (AM268272, unpublished) and an oilfield in Qinghai, China (EF190085, unpublished). In addition, clones of the methyl coenzyme M reductase alpha subunit (McrA) gene were obtained from hypersaline microbial mats in Guerrero Negro ponds (EU585971-EU585973, unpublished) that display high-sequence similarities (>97%) at the amino acid level with the McrA protein gene of Mhp. mahii (Mmah_0612).
Mhp. mahii strain SLPT is the first genome sequenced member of the genus Methanohalophilus representing moderately halophilic, methylotrophic methanogens. The comparison with the genomes of other freshwater and halophilic species belonging to the family Methanosarcinaceae provides novel insights into the genomic adaptations of methanogens to their environments.
2. Material and Methods
2.1. Growth Conditions and DNA Isolation
Mhp. mahii SLPT, DSM 5219T, was grown anaerobically in DSMZ medium 479  at 37°C. DNA was isolated from 1–1.5 g of cell paste using MasterPure Gram Positive DNA Purification Kit (Epicentre MGP04100) according to the manufacturer’s instructions with modification st/DLALM for cell lysis as described in Wu et al. 2009 
2.2. Genome Project History
This organism was selected for sequencing on the basis of its phylogenetic position  and is part of the Genomic Encyclopedia of Bacteria and Archaea project . The genome project is deposited in the Genomes OnLine Database  with the identifier Gc01255 and the complete genome sequence is accessible in GenBank as CP001994. Sequencing, finishing, and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 1 in supplementary material available online at doi 10.1155/2010/690737
2.3. Genome Sequencing and Assembly
The genome of Mhp. mahii SLPT was sequenced using a 6.8 kb Sanger DNA library. All general aspects of library construction and sequencing performed at the JGI can be found at (http://www.jgi.doe.gov). Draft assemblies were based on 12,365 total reads. This library provided 6.8x coverage of the genome. The Phred/Phrap/Consed software package (http://www.phrap.com) was used for sequence assembly and quality assessment [11–13]. After the shotgun sequencing stage reads were assembled with parallel phrap (High Performance Software, LLC). Possible misassemblies were corrected with Dupfinisher . Gaps between contigs were closed by editing in Consed, custom primer walks, or PCR amplification (Roche Applied Science, Indianapolis, IN). A total of 222 primer walk reactions and 5 pcr shatter libraries were necessary to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The completed genome sequence contains 14,767 reads, achieving an average of 8-fold sequence coverage per base with an error rate less than 1 in 100,000.
2.4. Genome Annotation
Genes were identified using Prodigal  as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline . The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform .
2.5. Phylogenetic Analysis
Total genome protein sequences from 13 members of the class “Methanomicrobia”  and one outgroup taxon (Haloterrigena turkmenica , Halobacteriaceae) were retrieved from the IMG website (http://img.jgi.doe.gov/cgi-bin/geba/main.cgi) or from NCBI (Methanocella paludicola SANAE; NC_013665). “Methanomicrobia” appear as a sister group of Halobacteriaceae in a recent comprehensive 16S rRNA tree . All-against-all protein BLAST was performed using mpiBLAST version 1.5 (http://www.mpiblast.org/), a parallel implementation of NCBI BLAST , using soft masking instead of complexity filtering. To determine orthologs, BLAST e-values were transformed using our own reimplementation of the OrthoMCL algorithm  in conjunction with MCL version 08-312 (http://micans.org/mcl/) using an inflation parameter of 2.0. OrthoMCL clusters containing inparalogs were reduced by selecting the most “central” of several sequences from the same genome, that is, the sequence with the highest sum of within-cluster BLAST scores. The reduced OrthoMCL clusters were aligned using MUSCLE version 3.7 . The program scan_orphanerrs from the RASCAL package version 1.3.4  was applied to detect orphan sequences within the alignments. After removal of orphan sequences (if present), poorly aligned columns and divergent regions were eliminated with GBLOCKS version 0.91 b  using a minimum block length of two amino acids and allowing gap positions in all sequences.
Filtered OrthoMCL cluster alignments comprising at least four sequences were concatenated to form a supermatrix for phylogenetic analysis. Maximum likelihood (ML) phylogenetic trees were inferred from the supermatrix with the Pthreads-parallelized RAxML package  version 7.2.5, applying fast bootstrapping with subsequent search for the best tree , the autoMRE bootstopping criterion  and the LG model of amino acid evolution  in conjunction with gamma-distributed substitution rates  and empirical amino acid frequencies. Tree searches under the maximum parsimony (MP) criterion were conducted with PAUP* version 4b10  using 100 rounds of random sequence addition and subsequent TBR branch swapping, saving no more than 10 best trees per round and collapsing potential zero-length branches during tree search. MP bootstrap support was calculated with PAUP* using 1000 replicates with 10 rounds of heuristic search per replicate.
A comprehensive set of 16S rRNA sequences including most described species of Methanosarcinales were aligned using POA version 2.0  and filtered with GBLOCKS. A phylogenetic tree was inferred under ML as described above but using the GTR+GAMMA substitution model.
3. Results and Discussion
3.1. Biology and Evolution of Mhp. mahii
3.1.1. Phenotypic Characteristics
Cells of Mhp. mahii SLPT are irregular cocci with a diameter of 0.8 to 1.8 μm that occur singly or in small clumps (Figure 1). Cells stain Gram-negative and are nonmotile . Suitable salinities for growth of this moderately halophilic strain are in the range between 0.5 and 3.5 M NaCl. The highest growth rates are achieved in medium containing 2.0 M of sodium chloride, but the highest culture density was found at a salinity of 1.2 M NaCl . Besides sodium, the metal ions Mg2+, K+, Ca2+, and Fe2+ are essential for methanogenesis and growth. The pH range for growth is 6.5 to 8.2 with an optimum at pH 7.5 .
Mhp. mahii SLPT is strictly anaerobic and grows heterotrophically using methanol or methylamines as substrates for methanogenesis. No growth occurs lithotrophically with hydrogen and carbon dioxide or with the organic carbon sources acetate, formate, methionine, choline, or betaine . Biotin stimulates growth of this strain in mineral medium with trimethylamine as substrate .
The polar lipid composition of Mhp. mahii strain SLPT was analyzed by Koga et al. . They identified the following lipid components: β-hydroxyarchaeol as core lipid, glucose as glycolipid sugar and myo-inositol, ethanolamine and glycerol as phospholipid-polar head groups. The deduced structure of the polar lipid diethers is in agreement with the current taxonomy that places the genus Methanohalophilus in the family Methanosarcinaceae . Further data about chemotaxonomical traits in the genus Methanohalophilus, including the determination of cytochromes or methanophenazine are currently not available. The classification and general features of Mhp. mahii strain SLPT in accordance with the MIGS recommendations  are summarized in Supplementary Table 2.
3.1.2. Phylogeny and Taxonomy
According to current taxonomy halophilic methanogens are mainly affiliated to several genera within the family Methanosarcinaceae. Most representatives of this clade of halophilic archaea have a coccoid morphology and are methylotrophic and neutrophilic or alkaliphilic. Moderately halophilic methylotrophic species belong to the genera Methanohalophilus and Methanosalsum, slightly halophilic methanogens to the genera Methanolobus and Methanococcoides and extreme halophiles are classified in the genus Methanohalobium. In addition, the slightly halophilic species Methanolobus siciliae was described , but it was later transferred to the genus Methanosarcina . At present the genus Methanohalophilus comprises three species with validly published names: Mhp. mahii (type species), Mhp. halophilus and Mhp. portucalensis. A fourth species, “Mhp. euhalobius”, was described by Obraztsova et al.  and Davidova et al. , but the species epithet has not been validated.
The 16S rRNA-based tree in Figure 2 shows the phylogenetic position of Mhp. mahii strain SLPT among members of the order Methanosarcinales. The genome of strain SLPT contains three copies of the 16S rRNA gene that differ by up to one nucleotide from the previously published 16S rRNA gene sequence generated from the same strain (M59133), which contains 23 ambiguous base calls. The difference between the genome data and the herereported 16S rRNA gene sequence is most likely due to sequencing errors in the previously reported sequence.
In order to obtain a more accurate view on the evolution of halophilic members of the Methanosarcinaceae a phylogenetic reconstruction based on whole-genome data of sequenced representatives of the class “Methanomicrobia”  was performed. The number of aligned and filtered OrthoMCL clusters containing at least four entries (i.e., genes from distinct genomes) was 2344 and their length ranging from 27 to 1732 amino acids (267.45 on average). The concatenated supermatrix thus comprised 626,903 columns, including 577,297 variable and 291,638 parsimony-informative characters. The ML phylogeny inferred from the concatenated gene alignments is shown in Figure 3 together with ML and MP bootstrap support values. The final highest log likelihood obtained was −10,355,411.023, whereas the single best MP tree had a score of 1,907,449 (excluding uninformative sites). ML and MP topologies were identical except for a sister-group relationship between Methanoculleus marisnigri and Methanosphaerula palustris, which was preferred by MP (data not shown). Support was maximum (100%) for all branches under ML, and maximum for all but two branches under MP. The taxonomy was well recovered, and the tree showing the monophyly of all families and orders was included in the sample. However, the branching order within the family Methanosarcinaceae differed between the 16S rRNA- and whole-genome-based tree. Interestingly, the halophilic species Methanococcoides burtonii, Methanosalsum zhilinae and Mhp. mahii, which depend on NaCl for growth (Table 3), form a monophyletic branch in the whole-genome tree, whereas in the reconstructed 16S rRNA tree Methanosalsum zhilinae forms a separate line of descent together with the extremely halophilic species Methanohalobium evestigatum. Thus, it is likely that the tree based on genome-scale data is more accurate than the 16S rRNA tree and for this reason it is in higher agreement with the phenotype. In addition, the whole-genome-based tree indicates that in the evolution of methanogens production of cytochromes originated in the common ancestor of Methanocellales and Methanosarcinales. Originally, it was thought that the presence of cytochromes is restricted to the order Methanosarcinales, but recently genes encoding cytochromes were detected in methanogens from rice rhizosphere representing the newly proposed order Methanocellales .
3.2. Genome Structure and Content
The genome consists of a 2,012,424 bp long chromosome with a 42.6% GC content (Table 1 and Figure 4). Of the 2095 genes predicted, 2032 were protein-coding genes, and 63 RNAs; forty five pseudogenes were also identified. The majority of the protein-coding genes (69.7%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 2.
3.3. Comparative Genome Analysis
Three different types of methanogens that can be mainly distinguished by the phylogenetic analyses of housekeeping genes and by some phenotypic traits have been proposed by Anderson et al. . Class I methanogens belong to the orders Methanobacteriales, Methanococcales and Methanopyrales, class II methanogens are represented by the order Methanomicrobiales and class III methanogens by Methanosarcinales. The species Mhp. mahii belongs to the family Methanosarcinaceae and hence to the class III.
Postulated hallmark traits shared by all class III methanogens include the utilization of methyl compounds as sole substrate for methanogenesis, expression of cytochromes and the presence of genes encoding the following proteins: A, K, and N subunits of reduced coenzyme F420 (F420H2) dehydrogenase, a bacterial-type phosphoglycerate mutase, a bacterial adenylate kinase, phage shock protein A, the nonhistone chromosomal protein MC1 and an elongated variant of the condensin subunit ScpB . The available genomic data set on representatives of the order Methanosarcinales is extended by presenting the complete genome of Mhp. mahii SLPT, thereby allowing a further evaluation of the postulated distribution of signature proteins in class III methanogens.
Furthermore, the adaptation of methanogenic pathways to increasing environmental salt concentration in members of the Methanosarcinaceae can be reconstructed by comparison of the genomes of the saltwater-adapted species Mhp. mahii, Mco. burtonii  and Methanosarcina acetivorans  with the genetic inventory of the freshwater preferring species Msc. mazei  and Msc. barkeri .
3.3.1. Substrate Utilization and Methanogenesis
Strain SLPT is reported to grow exclusively on the C-1 compounds methanol and methyl amines whereas alternative substrates for methanogenesis like hydrogen, formate, and acetate are not utilized as substrates for growth . A schematic drawing of the pathway of methanogenesis in Mhp. mahii is shown in Figure 5. The first step in methylotrophic methanogenesis is the transfer of a methyl group to coenzyme M (CoM). Then, the coenzyme M bound methyl group is reduced with coenzyme B (CoB) to methane, thereby leading to the formation of the CoM-S-S-CoB heterodisulfide. Methyltransferase systems of methanogens generally consist of the following three components that are localized in the cytoplasm: a methyl-accepting corrinoid protein (protein C), a substrate specific methyltransferase that catalyzes methylation of the corrinoid protein (protein B) and a methyltransferase that catalyzes methyl transfer from the methylated corrinoid protein to coenzyme M (MtbA). Methylation of the corrinoid protein requires reduction of the Co(II) corrinoid to the Co(I) state. The ATP-dependent reductive activation of the corrinoid protein is catalyzed in methylotrophic methanogens by the iron-sulfur protein RamA (Mmah_1683) . In Methanosarcina species it was demonstrated that the corrinoid protein and the substrate specific methyltransferase form a tight complex and that the corresponding genes are transcribed in a single operon, whereas the MtbA genes are localized somewhere else in the genome and transcribed separately . This seems also to be the case in the genome of Mhp. mahii strain SLPT, where methyltransferase systems for the following substrates were identified: methanol (Mmah_0975/Mmah_0976), monomethylamine (Mmah_1136/Mmah_1137 and Mmah_1154/Mmah_1155), dimethylamine (Mmah_0498/Mmah_0499 and Mmah_1674/Mmah_1675), and trimethylamine (Mmah_1680/1682). Genes encoding permeases for the uptake of methylamines could be identified in close proximity to the methyltransferase systems: Mmah_1133/1134 (monomethylamine transporter), Mmah_0500 (dimethylamine transporter) and Mmah_1679 (trimethylamine transporter). In addition, six different isozymes of methylcobalamin:coenzyme M methyltransferases (MtbA) could be detected: Mmah_0008, Mmah_0279, Mmah_0518, Mmah_0901, Mmah_0970, and Mmah_1478. In Mhp. mahii as in several other studied Methanosarcina species and Mco. burtonii genes encoding methylamine methyltransferases contain an amber codon (UAG), which causes normally a translation stop, but in this case can be recognized by a specific suppressor tRNA that carries the modified amino acid pyrrolysine . It was shown that this amino acid plays an important role in the catalysis of the methyl transfer to the cognate corrinoid protein. Pyrrolysine is ligated to the amber decoding tRNAPyl by the pyrrolysyl-tRNA synthetase PylS (Mmah_0283).
The reduction of the CoM-bound methyl group to methane is catalyzed by the enzyme methyl-coenzyme M reductase (Mcr) that is exclusively found in methanogens. It is composed of the three subunits: McrA (Mmah_0612), McrB (Mmah_0616), and McrG (Mmah_0613) and contains the nickel porphinoid F430 as coenzyme. The reducing equivalents required for the reduction of the methyl group to methane are generated in strictly methylotrophic methanogens by the oxidation of the methyl group to carbon dioxide. Stoichiometrically, the oxidation of one methyl group to CO2 provides, electrons for the generation of three molecules of methane.
The first step in the oxidative branch of methylotrophic methanogenesis is the transfer of the methyl group from CoM to tetrahydromethanopterin (H4MPT), which is catalyzed by the membrane-bound enzyme complex tetrahydromethanopterin S-methyltransferase (MtrA-H). It has been shown that this multisubunit enzyme couples the exergonic transfer of the methylgroup from tetrahydromethanopterin to coenzyme M with the translocation of two sodium ions . Thus, during methylotrophic methanogenesis this complex requires a sodium motive force to enable the endergonic transfer of the methylgroup from CoM to H4MPT. In Mhp. mahii all subunits of this enzyme are encoded in a single putative operon (Mmah_1776-1783), however, additional paralogs of some genes are found at several sites of the genome.
Subsequently, the enzymes methylene-H4MPT reductase (Mmah_1513) and methylene-H4MPT dehydrogenase (Mmah_0679) oxidize the methyl to a methenylgroup using the deazaflavin cofactor F420 as electron acceptor. The enzyme methenyl-H4MPT cyclohydrolase (Mmah_1138) then catalyzes the hydrolysis of methenyl-H4MPT to formyl-H4MPT. A further oxidation of the formyl group requires the transfer to the cofactor methanofuran (MF) by formyl-MF:H4MPT formyltransferase (Mmah_2027).
Finally, the large enzyme complex formyl-MF dehydrogenase oxidizes the formyl group to CO2 using a ferredoxin as a putative electron acceptor. It was previously shown that two different types of formyl-MF dehydrogenases are encoded in the genomes of Methanosarcina species, which is also observed in Mhp. mahii. Genes for a putative molybdate containing enzyme are arranged in a single unit (Mmah_1266–1271) whereas the presumably tungstate containing enzyme is encoded by genes that are localized at different sites or transcribed in different directions (Mmah_0590, Mmah_0950–952, Mmah_0956, and Mmah_1821). However, no gene encoding the subunit A of the tungstate containing formyl-MF dehydrogenase (fwdA) was identified, which may indicate that this enzyme is not functional in Mhp. mahii. Alternatively, the missing subunit could be replaced by the homologous subunit of the molybdate containing enzyme (FmdA).
Genes encoding enzymes for the utilization of alternative substrates for methanogenesis, like formate dehydrogenase (fdhAB), F420-dependent alcohol dehydrogenases (adf), or energy-conserving [NiFe] hydrogenases (ech) for the uptake of H2 were not detected in the annotated genome sequence, thus confirming the strict methylotrophy of this species. For the activation of acetate for methanogenesis Methanosarcina species use either acetate kinase (ack) and phosphotransacetylase (pta) or an ADP-forming acetyl-coenzyme A synthetase (acdAB). However, none of these genes were identified in the Mhp. mahii genome.
3.3.2. Energy Metabolism
The generation of metabolically useful energy in methylotrophic methanogens strictly depends on the buildup of a chemiosmotic gradient that can be utilized to form ATP by a membrane localized ATP synthase complex. The establishment of an ion-motive force usually requires membrane-bound protein complexes that convert the free energy change of electron transport processes in the translocation of protons or sodium ions outside of the cell. In methanogenic archaea, the energy-conserving electron transfer route is terminated by reduction of the heterodisulfide CoM-S-S-CoB to the corresponding thiol derivates CoM-SH and CoB-SH . Possible electron donors for the electron transport chain in members of the Methanosarcinaceae depend on the substrate and can be molecular hydrogen, F420H2 or reduced ferredoxin . A detailed description of the potential electron transfer routes and membrane-bound complexes involved in Mhp. mahii, including a comparison with the proposed pathways in Methanosarcina species, follows below and is illustrated in Figure 6.
Hydrogenases and Hydrogen Cycling
Recently, a hydrogen-cycling mechanism was postulated for the major mechanism of reducing the heterodisulfide reductase in Msc. barkeri  and Msc. mazei . In these freshwater species the reduced cofactors ferredoxin (Fdred) and F420 (F420H2) are probably oxidized by the membrane-bound Ech hydrogenase and the cytoplasmic Frh F420-hydrogenase, respectively. The produced H2 is in turn oxidized by a membrane-bound methanophenazine-reducing hydrogenase (Vht/Vho). Finally, the reduced methanophenazine (MPH2) is used by the membrane-bound heterodisulfide reductase HdrED for reduction of CoM-S-S-CoB and concomitant proton translocation across the membrane  (Figure 6(a)). Interestingly, no genes encoding functional hydrogenases were identified in the Mhp. mahii genome. This would imply that H2 cannot be used for heterodisulfide reduction during methylotrophic growth. In Table 3 it is shown that genes encoding subunits of [NiFe] hydrogenase are also absent in Mco. burtonii, another halophilic species belonging to the Methanosarcinaceae. Although several types of hydrogenases are encoded in the halotolerant marine species Msc. acetivorans, experimental data have shown that no significant hydrogenase activity is present, so that H2 probably does not play a role as intermediate in methanogenic pathways of this species . Thus, it is possible that the detected hydrogenase genes are silent or have an unknown function . It is obvious from the above mentioned data that Mhp. mahii as well as Msc. acetivorans and Mco. burtonii must have evolved different strategies for the regeneration of reduced cofactors that do not rely on the generation of H2 as intermediate.
Regeneration of F420
In Msc. mazei and Msc. barkeri, an alternative route for the regeneration of F420 is possible that depends on the F420H2 dehydrogenase complex (Fpo) and indicates a branched electron transfer chain in these species. Fpo is a multimeric membrane-bound complex with proton translocation activity and has some similarities with the canonical NADH dehydrogenase I (Nuo) of aerobic bacteria . In Mhp. mahii and other methanogens, this enzyme complex is encoded in a large putative operon (Mmah_1589–1601) with the conserved gene arrangement fpoABCDHIJJKLMNO. Only the gene of subunit FpoF (Mmah_1512) that contains the catalytic active site for F420H2 oxidation is encoded elsewhere. Interestingly, in the case of Mhp. mahii, it is adjacent to the gene of the methylene-H4MPT reductase (Mmah_1513), which represents one of the two enzymes in the oxidative branch of methylotrophic methanogenesis that reduce F420. Like the bacterial NADH dehydrogenase the Fpo F420H2 dehydrogenase of methanogens reduces a lipophilic redox carrier that is localized in the cytoplasmic membrane. Methanophenazine was identified in all hitherto studied species of the Methanosarcinaceae  as a functional analogue of the respiratory lipoquinones that are reduced by the bacterial NADH dehydrogenase. Thus, in Mhp. mahii, the Fpo dehydrogenase is probably responsible for the regeneration of the oxidized F420 cofactor using methanophenazine as electron acceptor. Mhp. mahii encodes also the membrane-bound heterodisulfide reductase HdrED (Mmah_0632/0633) that catalyzes the reduction of the heterodisulfide of CoM-S-S-CoB with MPH2 as reductant.
Regeneration of Oxidized Ferredoxin
Several mechanisms for the regeneration of oxidized ferredoxin without the generation of hydrogen have been recently proposed. A possible pathway involves a membrane-bound protein complex that was shown to be highly expressed in Msc. acetivorans and resembles the Rnf complex of bacteria . It is assumed that in Rhodobacter capsulatus, this complex employs a chemiosmotic gradient to enable the endergonic reduction of ferredoxins with NADH . In Msc. acetivorans electron transfer probably takes place in the reverse direction leading to the exergonic oxidation of ferredoxin with methanophenazine as electron acceptor and concomitant proton or sodium ion translocation . Notably, freshwater species of Methanosarcina that encode an Ech hydrogenase catalyzing ferredoxin oxidation do not contain genes for a Rnf complex whereas in the genomes of Mhp. mahii (Mmah_1689–1696) and Mco. burtonii a putative Rnf operon was detected (Table 3), suggesting that this way of ferredoxin regeneration is the preferred option in halophilic and saltwater-inhabiting species of Methanosarcinaceae (Figure 6(b)).
An alternative route for ferredoxin oxidation was postulated by Buan and Metcalf . Based on their findings obtained with gene-deletion mutants of Msc. acetivorans, they postulate that a cytoplasmic HdrABC complex uses reduced ferredoxin for the direct reduction of the CoM-S-S-CoB heterodisulfide. A possible advantage of this reaction would be that methanophenazine would not be required as an intermediate thereby allowing faster turnover rates for ferredoxin regeneration. Moreover, a possible growth-inhibiting overreduction of the methanophenazine pool by the Rnf complex is avoided. This assumption is supported by the finding that the cytoplasmic heterodisulfide reductase is especially important under conditions allowing fast growth of Msc. acetivorans in which regeneration of CoM-SH with reduced methanophenazine could represent a rate-limiting step . In Mhp. mahii two sets of genes encoding cytoplasmic heterodisulfide reductases were found. One set is organized in a putative transcriptional unit (Mmah_0415–0417) whereas the other set encodes HdrA (Mmah_0955) and HdrBC (Mmah_1786/1787) separately.
ATP Synthase Complex and Ion Motive Force
A common genetic trait of halophilic representatives of Methanosarcinaceae seems to be the presence of genes encoding a multisubunit sodium/proton antiporter (Table 3). Antiporters of this type are encoded by six to seven different genes and were first described in bacteria as multiple-resistance/pH regulation (Mrp) complexes . In Mhp. mahii, the Mrp complex is encoded in a single putative operon (Mmah_0736–0742). It has been proposed that these membrane-associated complexes function as proton-driven pumps for the efflux of sodium ions to achieve cellular homeostasis of pH and sodium . Typically, genes encoding Mrp complexes are arranged as cassettes and often found in combination with distinct enzymes of the electron-transport chain, like formate dehydrogenases or energy converting hydrogenases that are supposed to translocate proton or sodium ions . What could be the reason for the restricted distribution of the Mrp complex in halophilic species of the Methanosarcinaceae? One idea is that the Mrp complex plays a role in energy metabolism when chemiosmotic gradients of Na+ instead of protons are used for ATP synthesis. There is no direct evidence for this assumption, but it was shown by gene expression analyses that utilization of acetate in Msc. acetivorans stimulated production of the ATP synthase complex and the Mrp antiporter [42, 57], which might indicate that the Mrp complex is essential for the proper functioning of the ATP synthase. A tight coupling of electron transport with ATP synthesis is most important during the growth of methanogens on acetate, because the energy yield of this substrate is very small ( kJ/mol CH4) and close to the limit allowing cell growth . A possible explanation for the absence of the Mrp complex in freshwater species of Methanosarcina is that their ATP synthases translocate protons instead of Na+,and hence are less dependent on sodium homeostasis compared to marine Methanosarcina species exposed to varying salinities and pH values.
In contrast to several Methanosarcina species that contain two different types of ATP synthases, Mhp. mahii contains only an ATP synthase of the archaeal A1A0-type that is encoded in a single putative operon (Mmah_1442–1450). Bioinformatic analyses of the ion-translocating proteolipid c-subunit of ATP synthases would indicate that Na+ translocation is used by all Methanosarcina species for the synthesis of ATP . However, laboratory experiments of Pisa et al.  have shown that the ATP synthase of the freshwater species Msc. mazei depends on proton translocation, thus either bioinformatic predictions of the coupling ion are unreliable or ATP synthases of Methanosarcina species can depend on both, protons and sodium ions, depending on the growth conditions.
3.3.3. Biosynthetic Metabolism
Mhp. mahii is able to grow in minimal media with methanol as sole carbon source, which indicates that all biosynthetic precursors can be synthesized from C-1 compounds. The first step of carbon assimilation is catalyzed by the cytoplasmic acetyl-CoA synthase/CO dehydrogenase complex (Mmah_1166–1172) that synthesizes acetyl-CoA from CO2, CoA, and methyl-H4MPT in a reaction that depends on reduced ferredoxin. Alternatively, external acetate may be transported into the cell and then activated via an adenosine 5′-monophosphate (AMP) and pyrophosphate forming acetyl-CoA synthase (Mmah_1375). The gene for a membrane-bound proton-translocating pyrophosphatase (Mmah_1380) is located in close proximity on the genome and may convert the chemical energy of the pyrophosphate bond into a chemiosmotic gradient that could be used for ATP synthesis, thereby regaining some of the energy invested for the activation of acetate. Carbon assimilation further proceeds by the reductive carboxylation of acetyl-CoA to pyruvate that is catalyzed by pyruvate:ferredoxin oxidoreductase (Mmah_0351/0354). The reduced ferredoxin required for carbon assimilation is probably provided by the formyl-MF dehydrogenase complex.
Central Carbon Metabolism
Starting from pyruvate, further carbon skeletons for biosynthesis can be either produced by the citric acid cycle or by gluconeogenesis. In Mhp. Mahii, the enzymes phosphoenolpyruvate synthase (Mmah_0997) and pyruvate phosphate dikinase (Mmah_0650) convert pyruvate into phosphoenolpyruvate (PEP) that can enter the gluconeogenic Embden-Meyerhof-Parnas (EMP) pathway by enolase (Mmah_1993) and phosphoglycerate mutase, which is present in an archaeal (Mmah_0223) and bacterial version (Mmah_1844), as in all studied members of the Methanosarcinales . In Mhp. mahii, all necessary genes are present to operate the EMP pathway also in the glycolytic direction, which indicates that glycogen or starch is probably used as reserve material in this species and degraded under starvation conditions. On the other hand, pyruvate can be directed to the citric acid cycle by the enzyme pyruvate carboxylase (Mmah_0621/0622) that produces oxaloacetate. The important precursor metabolite succinyl-CoA is probably synthesized from oxaloacetate through the reductive branch of the citric acid cycle. Annotated genes representing enzymes of the reductive fork of the citric acid cycle include malate dehydrogenase (Mmah_1107), fumarase (Mmah_0724 and Mmah_1279/1280) and fumarate reductase (Mmah_0725/0726 and Mmah_1949/1950). Although genes for a canonical succinyl-CoA synthetase were not identified, it is possible that such an enzyme is encoded by the genes Mmah_1653 (potential ATPase) and Mmh_1654 (acyl-CoA synthetase), resulting in a complete set of enzymes for the partial reductive citric acid cycle. In Mhp. mahii, as in other members of the Methanosarcinaceae studied so far, 2-oxoglutarate is synthesized from oxaloacetate through the oxidative branch of the citric acid cycle using the enzymes citrate synthase (Mmah_0479), aconitase (Mmah_0478), and isocitrate dehydrogenase (Mmah_0582). It has to be considered that the citrate synthase gene of Mhp. mahii contains an amber stop codon at position 177 of the coding nucleotide sequence, so that it is currently designated as a pseudogene. On the other hand, 2-oxoglutarate is an indispensable precursor for the synthesis of glutamate and other essential amino acids, which leads to the assumption that the UAG stop codon could encode the noncanonical amino acid pyrrolysine as in the case of the methylamine methyltransferases discussed above.
The enzymology of biochemical pathways for the synthesis of methanogenesis-specific cofactors has only recently begun to unveil. However, detailed studies on the synthesis of methanofuran and coenzyme F430 are still missing .
Coenzyme M biosynthesis was studied in Methanocaldococcus jannaschii, and a complete pathway including the involved genes was described . Of the four enzymes catalyzing CoM synthesis in Methanocaldococcus jannaschii only the genes for sulfopyruvate decarboxylase (comDE; Mmah_1754/1755) were identified in the Mhp. mahii genome, which could indicate the existence of multiple pathways for sulfopyruvate production in methanogens. In contrast, several genes could be detected that probably encode enzymes participating in 2-oxoacid elongation steps required for synthesis of the 7-mercaptoheptanoyl chain of coenzyme B, that is, aksA (Mmah_1938), aksD (Mmah_0418), aksE (Mmah_0127), and aksF (Mmah_0129). Likewise, a complete set of genes (cofC, cofD, cofE, and cofH) involved in synthesis of the coenzyme F420 was identified in the Mhp. mahii genome.
All members of Methanosarcinaceae analyzed so far contain methanophenazine and cytochromes as part of their electron transport chains, so that genes encoding enzymes for their synthesis should be also present in Mhp. mahii. Only recently, Ogawa et al.  identified an enzyme in Msc. mazei that is specifically involved in the synthesis of methanophenazine. Methanophenazine contains a C-25 polyprenyl side-chain as lipophilic membrane anchor and an ether-linked redox-active phenazine moiety. It is thought that a specific step in the pathway of methanophenazine biosynthesis is the production of a C-25 prenyl group [(all-) geranylfarnesyl diphosphate] that is subsequently combined with 2-hydroxyphenazine or its precursor. In Msc. mazei the synthesis of all- geranylfarnesyl diphosphate is catalyzed by an (all-) prenyl diphosphate synthase encoded by the gene MM_0789 whereas a homologous enzyme producing the C-20 (all-) geranylgeranyl diphosphate is probably encoded by MM_1767 . C-20 polyprenyl hydrocarbons are generally used in methanogenic archaea for the synthesis of membrane lipids, indicating that the distribution of this enzyme is not restricted to methanogens-producing methanophenazine. Indeed, it was found that highly similar sequences ( values ) of the gene MM_0789 were found only in the genomes of Mhp. mahii (Mmah_1452) and other members of the Methanosarcinales whereas a similarly restricted distribution was not evident for the gene MM_1767. Interestingly, in the genome of Mhp. mahii, a cluster of genes required for heme biosynthesis (Mmah_1457–1461) and the ATP synthase operon are located in close proximity to the tentative (all-) geranylfarnesyl diphosphate synthase gene (Mmah_1452), which could indicate a coordinated regulation of genes involved in electron transport phosphorylation.
3.3.4. Protection against Osmotic and Oxidative Stress
Halophilic or halotolerant microbes adapt to low water activity or varying salt concentrations by accumulating compatible solutes that balance the cell turgor under conditions of osmotic stress. The composition of osmolytes in Mhp. mahii is currently unknown but it was investigated in the phylogenetically related species Mhp. portucalensis. In this species the principal osmotic solutes are glycine betaine, -acetyl-β-lysine, β-glutamine and α-glucosylglycerate . For energetic reasons the uptake of organic compatible solutes is preferred to de novo synthesis, hence transporters of common osmotic solutes should be encoded in the Mhp. mahii genome. Indeed, genes for an ABC-type transporter for glycine betaine (Mmah_0337–0340) were found. In addition, genes for alternative BetT-type choline transporters (Mmah_0372 and Mmah_1398) could be involved in the uptake of betaine or its precursors.
In Methanosarcina species transient osmotic stress is usually compensated by the intracellular potassium (K+) concentration . Accordingly, in Mhp. mahii a K+ uptake system encoded by the genes trkA (Mmah_1614), trkI (Mmah_0056) and trkH (Mmah_2022) was identified and could play a role in a salinity-dependent increase of the cytoplasmic K+ concentration. Under conditions of hypoosmolarity efflux of K+ and Na+ ions could be catalyzed by KefC-type potassium/proton antiporters (Mmah_1757/Mmah_2010) and an NhaP-type sodium/proton antiporter (Mmah_1674), respectively. In addition, it is thought that mechanosensitive ion channels (mscS1-3: Mmah_0364, Mmah_0563, and Mmah_0666) that are gated by membrane tension play a role in the nonspecific efflux of solutes in response to hypoosmotic stress .
The immediate adaptation of cells to an increase of external salt concentrations by the rapid uptake of solutes is usually complemented by the cellular synthesis of organic osmolytes. In Mhp. mahii, the synthesis of -acetyl-β-lysine is catalyzed by lysine-2,3-aminomutase (ablA, Mmah_1439) and β-lysine acetyltransferase (ablB, Mmah_1438) whereas glycine betaine synthesis in methanogens relies on the genes for glycine sarcosine N-methyltransferase (Mmah_0524) and sarcosine dimethylglycine N-methyltransferase (Mmah_0525) .
Cultivation-independent studies have revealed that methanogens closely related to the type strain of Mhp. mahii are abundant in the upper layers of photosynthetically active oxygenic microbial mats of solar salterns . It is likely that these microbial mats are frequently exposed to UV light and oxygen, which lead to the generation of reactive oxygen species (ROS) being extremely toxic for obligately anaerobic cells. Consequently, members of this species must have acquired highly developed mechanisms to cope with oxidative stress. An analysis of the annotated genome sequence of Mhp. mahii indicates that the oxidative stress protection system in this species comprises multiple lines of defense characterized by enzymes reacting directly with oxygen and its derivatives or are involved in the repair of damaged cellular compounds. A whole array of enzymes is putatively involved in primary oxygen detoxification: Molecular oxygen entering the cell is reduced by a predicted F420H2 oxidase (fprA, Mmah_1537). The highly reactive superoxide radical () generated by auto-oxidation of thiols or flavins can be detoxified by two distinct desulfoferrodoxin-type superoxide reductases (Mmah_0553 and Mmah_1131) that reduce superoxide to hydrogen peroxide (H2O2). In turn, H2O2 is reduced to water by several types of peroxidases, including rubrerythrin or rubredoxin peroxidase (rbr, Mmah_0444), a ferritin-like Dps protein (dps, Mmah_0552), and a bacterial-type bifunctional catalase/peroxidase (katG, Mmah_1997).
On the other hand, an effective damage-repair mechanisms is installed that reacts with cellular compounds after they have become oxidized by ROS. Mhp. mahii encodes three different isozymes of 3-Cys thioredoxin peroxidases or peroxiredoxins (Mmah_0555, Mmah_1055, and Mmah_1299) that probably function as alkyl hydroperoxide reductases and repair oxidized lipids of the cytoplasmic membrane. Besides membrane lipids methionine residues of cytoplasmic proteins represent another prime target of ROS. The generation of methionine sulfoxide can lead to an alteration of the protein structure and other deleterious effects, so that it is necessary to reduce the sulfoxide moiety. In Mhp. mahii, the two different epimeric forms of methionine sulfoxide are reduced by the enzymes methionine-R-sulfoxide reductase (msrB, Mmah_0636) and methionine-S-sulfoxide reductase (msrA, Mmah_0990).
The electron transfer chain to desulfoferrodoxins, rubrerythrin, and peroxiredoxins is probably initiated by NADPH:thioredoxin reductase (Mmah_1201) or ferredoxin:thioredoxin reductase (Mmah_0953).
Anderson et al. proposed the classification of methanogens in three different classes based on a phylogenetic analysis of conserved genes and the distribution of signature proteins . Based upon these criteria, Mhp. mahii is affiliated to class III methanogens except that no gene for the phage shock protein A was detected, which has been found so far in all members of the Methanosarcinales.
An interesting outcome of this study is that Mhp. mahii and other halophilic species of the Methanosarcinaceae use different energy-conserving electron routes during methylotrophic methanogenesis compared to freshwater species. Msc. mazei and Msc. barkeri rely on a hydrogen cycle with molecular H2 as intermediate whereas species adapted to saline environments prefer reduced cofactors for the intracellular transfer of reducing power. Obviously, the type of energy metabolism in Methanosarcinaceae does not correlate with the inherited halotolerance or the phylogenetic position, but seems to be the result of an adaptation to the occupied habitat, which explains why only Msc. acetivorans has omitted an H-cycle, but not the halotolerant freshwater species Msc. mazei and Msc. barkeri. It is tempting to speculate that the advantage of installing an H-cycle is the achievement of faster growth rates. Redox reactions of the rapidly diffusing H2 molecule can proceed very fast, because they do not rely on membrane-bound redox carriers that have to interact with large enzyme complexes, which is likely to slow down electron transfer. This would offer a possible explanation for the observation that methanogens without methanophenazines can have doubling times as low as one hour whereas methanogens with a membrane-bound eletron transport chain have generally doubling times above ten hours . On the other hand, utilization of H2 as redox carrier could lead to the loss of reducing power from the cell, if freely diffusing H2 is scavenged by competing microorganisms. The abandonment of molecular hydrogen as intermediate substrate for methanogenesis in halophilic representatives of the Methanosarcinaceae can thus be explained by a competition with sulfate reducing microorganisms, which are usually only highly abundant in sulfate-rich saline environments and consume hydrogen more efficiently than methanogens [53, 72]. The observed effect of habitat salinity on the energy metabolism of methanogens seems to be restricted to members of the Methanosarcinaceae. This is probably based on the approximately tenfold lower hydrogen affinity of cytochrome-containing methanogens compared to methanogens of class I and class II, which are able to thrive by hydrogenotrophic methanogenesis in saline environments .
In summary, the obtained genome data allow the conclusion that the revealed abundance of Mhp. mahii in microbial mats of saline and hypersaline environments is based on a successful competition with other anaerobes, maybe by omitting molecular hydrogen as redox carrier, and by acquiring an oxidative stress protection system that is fully comparable to the system of microaerotolerant sulfate-reducing bacteria .
The authors would like to gratefully acknowledge the help of Maren Schröder for growing Mhp. mahii cells and Susanne Schneider for DNA extraction and quality control (both at DSMZ). This work was performed under the auspices of the US Department of Energy's Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under Contract no. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under contract no. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract no. DE-AC02-06NA25396, UT-Battelle and Oak Ridge National Laboratory under Contract no. DE-AC05-00OR22725. German Research Foundation (DFG) supported DSMZ under INST 599/1-2.
Supplementary Table 1. Genome sequencing project information.
Supplementary Table 2. Classification and general features of Mhp. mahii strain SLPT in accordance with the MIGS recommendations .
Evidence codes: 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). These evidence codes are from the Gene Ontology project .
* In version 4.0 of the BMSB Taxonomic Outline  the class “Methanomicrobia” (including the orders Methanomicrobiales and Methanosarcinales) was created to replace the class Methanococci, whose definition seems to be in conflict with current analyses of complete genome data.
- R. S. Oremland and S. Polcin, “Methanogenesis and sulfate reduction: competitive and noncompetitive substrates in estuarine sediments,” Applied and Environmental Microbiology, vol. 44, pp. 1270–1276, 1982.
- J. R. Paterek and P. H. Smith, “Methanophilus mahii gen. nov., sp. nov., a methylotrophic halophilic methanogen,” International Journal of Systematic Bacteriology, vol. 38, no. 1, pp. 122–123, 1988.
- J. R. Paterek and P. H. Smith, “Isolation and characterization of a halophilic methanogen from Great Salt Lake,” Applied and Environmental Microbiology, vol. 50, no. 4, pp. 877–881, 1985.
- J. Chun, J. H. Lee, Y. Jung et al., “EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences,” International Journal of Systematic and Evolutionary Microbiology, vol. 57, no. 10, pp. 2259–2261, 2007.
- C. E. Robertson, J. R. Spear, J. K. Harris, and N. R. Pace, “Diversity and stratification of archaea in a hypersaline microbial Mat,” Applied and Environmental Microbiology, vol. 75, no. 7, pp. 1801–1810, 2009.
- K. B. Sørensen, D. E. Canfield, A. P. Teske, and A. Oren, “Community composition of a hypersaline endoevaporitic microbial mat,” Applied and Environmental Microbiology, vol. 71, no. 11, pp. 7352–7365, 2005.
- List of growth media used at DSMZ, http://www.dsmz.de/microorganisms/media_list.php.
- D. Wu, P. Hugenholtz, K. Mavromatis et al., “A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea,” Nature, vol. 462, no. 7276, pp. 1056–1060, 2009.
- H. P. Klenk and M. Göker, “En route to a genome-based classification of Archaea and Bacteria?” Systematic and Applied Microbiology, vol. 33, no. 4, pp. 175–182, 2010.
- K. Liolios, I. A. Chen, K. Mavromatis et al., “The Genomes On Line Database (GOLD) in 2009: status of genomic and metagenomic projects and their associated metadata,” Nucleic Acids Research, vol. 38, pp. D346–D354, 2009.
- B. Ewing and P. Green, “Base-calling of automated sequencer traces using phred. II. Error probabilities,” Genome Research, vol. 8, no. 3, pp. 186–194, 1998.
- B. Ewing, L. Hillier, M. C. Wendl, and P. Green, “Base-calling of automated sequencer traces using phred. I. Accuracy assessment,” Genome Research, vol. 8, no. 3, pp. 175–185, 1998.
- D. Gordon, C. Abajian, and P. Green, “Consed: a graphical tool for sequence finishing,” Genome Research, vol. 8, no. 3, pp. 195–202, 1998.
- C. S. Han and P. Chain, “Finishing repeat regions automatically with Dupfinisher,” in Proceeding of the International Conference on Bioinformatics & Computational Biology, R. Hamid, Ed., pp. 141–146, Arabnia and Homayoun Valafar, CSREA Press, June 2006.
- D. Hyatt, G. L. Chen, P. F. Locascio, M. L. Land, F. W. Larimer, and L. J. Hauser, “Prodigal prokaryotic dynamic programming genefinding algorithm,” BMC Bioinformatics, vol. 11, article 119, 2010.
- A. Pati, N. N. Ivanova, N. Mikhailova et al., “GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes,” Nature Methods, vol. 7, no. 6, pp. 455–457, 2010.
- V. M. Markowitz, K. Mavromatis, N. N. Ivanova, I. M. A. Chen, K. Chu, and N. C. Kyrpides, “IMG ER: a system for microbial genome annotation expert review and curation,” Bioinformatics, vol. 25, no. 17, pp. 2271–2278, 2009.
- G. M. Garrity, J. A. Bell, and T. G. Lilburn, “Taxonomic outline of the procaryotes,” in Bergey's Manual of Systematic Bacteriology, Springer, New York, NY, USA, 2nd edition.
- E. Saunders, B. J. Tindall, R. Fähnrich et al., “Complete genome sequence of Haloterrigena turkmenica type strain (4),” Standards in Genomic Sciences, vol. 1, pp. 107–116, 2010.
- P. Yarza, M. Richter, J. Peplies et al., “The all-species living tree project: a 16S rRNA-based phylogenetic tree of all sequenced type strains,” Systematic and Applied Microbiology, vol. 31, no. 4, pp. 241–250, 2008.
- S. F. Altschul, T. L. Madden, A. A. Schäffer et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Research, vol. 25, no. 17, pp. 3389–3402, 1997.
- L. Li, C. J. Stoeckert, and D. S. Roos, “OrthoMCL: identification of ortholog groups for eukaryotic genomes,” Genome Research, vol. 13, no. 9, pp. 2178–2189, 2003.
- R. C. Edgar, “MUSCLE: multiple sequence alignment with high accuracy and high throughput,” Nucleic Acids Research, vol. 32, no. 5, pp. 1792–1797, 2004.
- J. D. Thompson, J. C. Thierry, and O. Poch, “RASCAL: rapid scanning and correction of multiple sequence alignments,” Bioinformatics, vol. 19, no. 9, pp. 1155–1161, 2003.
- J. Castresana, “Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis,” Molecular Biology and Evolution, vol. 17, no. 4, pp. 540–552, 2000.
- A. Stamatakis, “RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models,” Bioinformatics, vol. 22, no. 21, pp. 2688–2690, 2006.
- A. Stamatakis, P. Hoover, and J. Rougemont, “A rapid bootstrap algorithm for the RAxML web servers,” Systematic Biology, vol. 57, no. 5, pp. 758–771, 2008.
- N. D. Pattengale, M. Alipour, O. R. P. Bininda-Emonds, B. M. E. Moret, and A. Stamatakis, “How many bootstrap replicates are necessary?” Lecture Notes in Computer Science, vol. 5541, pp. 184–200, 2009.
- SI. Q. Le and O. Gascuel, “An improved general amino acid replacement matrix,” Molecular Biology and Evolution, vol. 25, no. 7, pp. 1307–1320, 2008.
- Z. Yang, “Maximum-likelihood estimation of phylogeny from DNA sequences when substitution rates differ over sites,” Molecular Biology and Evolution, vol. 10, no. 6, pp. 1396–1401, 1993.
- D. L. Swofford, PAUP∗: Phylogenetic Analysis Using Parsimony (∗and Other Methods), Sinauer Associates, Sunderland, Mass, USA, 2002.
- C. Lee, C. Grasso, and M. F. Sharlow, “Multiple sequence alignment using partial order graphs,” Bioinformatics, vol. 18, no. 3, pp. 452–464, 2002.
- D. R. Boone, I. M. Mathrani, Y. Liu, J. A. G. F. Menaia, R. A. Mah, and J. E. Boone, “Isolation and characterization of Methanohalophilus portucalensis sp. nov. and DNA reassociation study of the genus Methanohalophilus,” International Journal of Systematic Bacteriology, vol. 43, no. 3, pp. 430–437, 1993.
- Y. Koga, M. Akagawa-Matsushita, M. Ohga, and M. Nishihara, “Taxonomic significance of the distribution of component parts of polar ether lipids in methanogens,” Systematic and Applied Microbiology, vol. 16, no. 3, pp. 342–351, 1993.
- Y. Koga and M. Nakano, “A dendrogram of archaea based on lipid component parts composition and its relationship to rRNA phylogeny,” Systematic and Applied Microbiology, vol. 31, no. 3, pp. 169–182, 2008.
- D. Field, G. Garrity, T. Gray et al., “Towards a richer description of our complete collection of genomes and metagenomes: the “Minimum Information about a Genome Sequence” (MIGS) specification,” Nature Biotechnology, vol. 26, pp. 541–547, 2008.
- S. Ni and D. R. Boone, “Isolation and characterization of a dimethyl sulfide-degrading methanogen, Methanolobus siciliae HI350, from an oil well, characterization of M. siciliae T4/M(T), and emendation of M. siciliae,” International Journal of Systematic Bacteriology, vol. 41, no. 3, pp. 410–416, 1991.
- S. Ni, C. R. Woese, H. C. Aldrich, and D. R. Boone, “Transfer of Methanolobus siciliae to the genus Methanosarcina, naming it Methanosarcina siciliae, and emendation of the genus Methanosarcina,” International Journal of Systematic Bacteriology, vol. 44, no. 2, pp. 357–359, 1994.
- A. Y. Obraztsova, O. V. Shipin, L. V. Bezrukova, and S. S. Belyaev, “Properties of the coccoid methylotrophic methanogen, Methanococcoides euhalobius sp. nov,” Microbiology, vol. 56, pp. 523–527, 1987.
- I. A. Davidova, H. J. M. Harmsen, A. J. M. Stams, S. S. Belyaev, and A. J. B. Zehnder, “Taxonomic description of Methanococcoides euhalobius and its transfer to the Methanohalophilus genus,” Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology, vol. 71, no. 4, pp. 313–318, 1997.
- R. K. Thauer, A. K. Kaster, H. Seedorf, W. Buckel, and R. Hedderich, “Methanogenic archaea: ecologically relevant differences in energy conservation,” Nature Reviews Microbiology, vol. 6, no. 8, pp. 579–591, 2008.
- L. Rohlin and R. P. Gunsalus, “Carbon-dependent control of electron transfer and central carbon pathway genes for methane biosynthesis in the Archaean, Methanosarcina acetivorans strain C2A,” BMC Microbiology, vol. 10, article 62, 2010.
- D. R. Boone, “Class II. Methanococci class. nov,” in Bergey's Manual of Systematic Bacteriology, G. M. Garrity, D. R. Boone, and R. W. Castenholz, Eds., vol. 1, pp. 235–294, Springer, New York, NY, USA, 2nd edition, 2001.
- I. Anderson, L. E. Ulrich, B. Lupa et al., “Genomic characterization of methanomicrobiales reveals three classes of methanogens,” PLoS ONE, vol. 4, no. 6, Article ID e5797, 2009.
- N. F. Saunders, T. Thomas, P. M. Curmi et al., “Mechanisms of thermal adaptation revealed from the genomes of the Antarctic archaea Methanogenium frigidum and Methanococcoides burtonii,” Genome Research, vol. 13, pp. 1580–1588, 2003.
- J. E. Galagan, C. Nusbaum, A. Roy et al., “The genome of M. acetivorans reveals extensive metabolic and physiological diversity,” Genome Research, vol. 12, no. 4, pp. 532–542, 2002.
- U. Deppenmeier, A. Johann, T. Hartsch et al., “The genome of Methanosarcina mazei: evidence for lateral gene transfer between bacteria and archaea,” Journal of Molecular Microbiology and Biotechnology, vol. 4, no. 4, pp. 453–461, 2002.
- D. L. Maeder, I. Anderson, T. S. Brettin et al., “The Methanosarcina barkeri genome: comparative analysis with Methanosarcina acetivorans and Methanosarcina mazei reveals extensive rearrangement within methanosarcinal genomes,” Journal of Bacteriology, vol. 188, no. 22, pp. 7922–7931, 2006.
- T. Ferguson, J. A. Soares, T. Lienard, G. Gottschalk, and J. A. Krzycki, “RamA, a protein required for reductive activation of corrinoid-dependent methylamine methyltransferase reactions in methanogenic archaea,” Journal of Biological Chemistry, vol. 284, no. 4, pp. 2285–2295, 2009.
- W. F. Fricke, H. Seedorf, A. Henne et al., “The genome sequence of Methanosphaera stadtmanae reveals why this human intestinal archaeon is restricted to methanol and for methane formation and ATP synthesis,” Journal of Bacteriology, vol. 188, no. 2, pp. 642–658, 2006.
- J. A. Krzycki, “The direct genetic encoding of pyrrolysine,” Current Opinion in Microbiology, vol. 8, no. 6, pp. 706–712, 2005.
- G. Gottschalk and R. K. Thauer, “The N-translocating methyltransferase complex from methanogenic archaea,” Biochimica et Biophysica Acta—Bioenergetics, vol. 1505, no. 1, pp. 28–36, 2001.
- Y. Liu and W. B. Whitman, “Metabolic, phylogenetic, and ecological diversity of the methanogenic Archaea,” in Incredible Anaerobes: From Physiology to Genomics to Fuels, J. Wiegel, R. J. Maier, and M. W. W. Adams, Eds., pp. 171–189, New York Academy of Sciences, Boston, Mass, USA, 2008.
- G. Kulkarni, D. M. Kridelbaugh, A. M. Guss, and W. W. Metcalf, “Hydrogen is a preferred intermediate in the energy-conserving electron transport chain of Methanosarcina barkeri,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 37, pp. 15915–15920, 2009.
- C. Welte, V. Kallnik, M. Grapp, G. Bender, S. Ragsdale, and U. Deppenmeier, “Function of Ech hydrogenase in ferredoxin-dependent, membrane-bound electron transport in Methanosarcina mazei,” Journal of Bacteriology, vol. 192, no. 3, pp. 674–678, 2010.
- A. M. Guss, G. Kulkarni, and W. W. Metcalf, “Differences in hydrogenase Gene expression between Methanosarcina acetivorans and Methanosarcina barkeri,” Journal of Bacteriology, vol. 191, no. 8, pp. 2826–2833, 2009.
- Q. Li, L. Li, T. Rejtar, D. J. Lessner, B. L. Karger, and J. G. Ferry, “Electron transport in the pathway of acetate conversion to methane in the marine archaeon Methanosarcina acetivorans,” Journal of Bacteriology, vol. 188, no. 2, pp. 702–710, 2006.
- N. R. Buan and W. W. Metcalf, “Methanogenesis by Methanosarcina acetivorans involves two structurally and functionally distinct classes of heterodisulfide reductase,” Molecular Microbiology, vol. 75, no. 4, pp. 843–853, 2010.
- U. Deppenmeier, “The membrane-bound electron transport system of Methanosarcina species,” Journal of Bioenergetics and Biomembranes, vol. 36, no. 1, pp. 55–64, 2004.
- S. Bäumer, T. Ide, C. Jacobi, A. Johann, G. Gottschalk, and U. Deppenmeier, “The dehydrogenase from Methanosarcina mazei is a redox-driven proton pump closely related to NADH dehydrogenases,” Journal of Biological Chemistry, vol. 275, no. 24, pp. 17968–17973, 2000.
- K. Saeki and H. Kumagai, “The rnf gene products in Rhodobacter capsulatus play an essential role in nitrogen fixation during anaerobic DMSO-dependent growth in the dark,” Archives of Microbiology, vol. 169, no. 5, pp. 464–467, 1998.
- T. H. Swartz, S. Ikewada, O. Ishikawa, M. Ito, and T. A. Krulwich, “The Mrp system: a giant among monovalent cation/proton antiporters?” Extremophiles, vol. 9, no. 5, pp. 345–354, 2005.
- S. Spring, R. Rachel, A. Lapidus et al., “Complete genome sequence of Thermosphaera aggregans type strain (M11T),” Standards in Genomic Sciences, vol. 2, pp. 245–259, 2010.
- K. Y. Pisa, C. Weidner, H. Maischak, H. Kavermann, and V. Müller, “The coupling ion in the methanoarchaeal ATP synthases: vs. N in the ATP synthase from the archaeon Methanosarcina mazei Gö1,” FEMS Microbiology Letters, vol. 277, no. 1, pp. 56–63, 2007.
- D. E. Graham and R. H. White, “Elucidation of methanogenic coenzyme biosyntheses: from spectroscopy to genomics,” Natural Product Reports, vol. 19, no. 2, pp. 133–147, 2002.
- D. E. Graham, H. Xu, and R. H. White, “Identification of coenzyme M biosynthetic phosphosulfolactate synthase. A new family of sulfonate-biosynthesizing enzymes,” Journal of Biological Chemistry, vol. 277, no. 16, pp. 13421–13429, 2002.
- T. Ogawa, T. Yoshimura, and H. Hemmi, “Geranylfarnesyl diphosphate synthase from Methanosarcina mazei: different role, different evolution,” Biochemical and Biophysical Research Communications, vol. 393, no. 1, pp. 16–20, 2010.
- D. E. Robertson, M. C. Lai, R. P. Gunsalus, and M. F. Roberts, “Composition, variation, and dynamics of major osmotic solutes in Methanohalophilus strain FDF1,” Applied and Environmental Microbiology, vol. 58, no. 8, pp. 2438–2443, 1992.
- K. R. Sowers and R. P. Gunsalus, “Halotolerance in Methanosarcina spp.: role of N(ε)-acetyl-β-lysine, α- glutamate, glycine betaine, and as compatible solutes for osmotic adaptation,” Applied and Environmental Microbiology, vol. 61, no. 12, pp. 4382–4388, 1995.
- M. D. Edwards, I. R. Booth, and S. Miller, “Gating the bacterial mechanosensitive channels: MscS a new paradigm,” Current Opinion in Microbiology, vol. 7, no. 2, pp. 163–167, 2004.
- M. F. Roberts, M. C. Lai, and R. P. Gunsalus, “Biosynthetic pathways of the osmolytes -acetyl-β-lysine, β- glutamine, and betaine in Methanohalophilus strain FDF1 suggested by nuclear magnetic resonance analyses,” Journal of Bacteriology, vol. 174, no. 20, pp. 6688–6693, 1992.
- J. W. Abram and D. B. Nedwell, “Inhibition of methanogenesis by sulphate reducing bacteria competing for transferred hydrogen,” Archives of Microbiology, vol. 117, no. 1, pp. 89–92, 1978.
- C. Marschall, P. Frenzel, and H. Cypionka, “Influence of oxygen on sulfate reduction and growth of sulfate-reducing bacteria,” Archives of Microbiology, vol. 159, no. 2, pp. 168–173, 1993.
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