Proteomic and transcriptomic analysis of selenium utilization in Methanococcus maripaludis.

Methanococcus maripaludis utilizes selenocysteine- (Sec-) containing proteins (selenoproteins), mostly active in the organism's primary energy metabolism, methanogenesis. During selenium depletion, M. maripaludis employs a set of enzymes containing cysteine (Cys) instead of Sec. The genes coding for these Sec-/Cys-containing isoforms were the only genes known of which expression is influenced by the selenium status of the cell. Using proteomics and transcriptomics, approx. 7% and 12%, respectively, of all genes/proteins were found differentially expressed/synthesized in response to the selenium supply. Some of the genes identified involve methanogenesis, nitrogenase functions, and putative transporters. An increase of transcript abundance for putative transporters under selenium depletion indicated the organism's effort to tap into alternative sources of selenium. M. maripaludis is known to utilize selenite and dimethylselenide as selenium sources. To expand this list, a selenium-responsive reporter strain was assessed with nine other, environmentally relevant selenium species. While the effect of some was very similar to that of selenite, others were effectively utilized at lower concentrations. Conversely, selenate and seleno-amino acids were only utilized at unphysiologically high concentrations and two compounds were not utilized at all. To address the role of the selenium-regulated putative transporters, M. maripaludis mutant strains lacking one or two of the putative transporters were tested for the capability to utilize the different selenium species. Of the five putative transporters analyzed by loss-of-function mutagenesis, none appeared to be absolutely required for utilizing any of the selenium species tested, indicating they have redundant and/or overlapping specificities or are not dedicated selenium transporters. IMPORTANCE: While selenium metabolism in microorganisms has been studied intensively in the past, global gene expression approaches have not been employed so far. Furthermore, the use of different selenium sources, widely environmentally interconvertible via biotic and abiotic processes, was also not extensively studied before. Methanococcus maripaludis JJ is ideally suited for such analyses, thanks to its known selenium usage and available genetic tools. Thus, an overall view on the selenium regulon of M. maripaludis was obtained via transcriptomic and proteomic analyses, which inspired further experimentation. This led to demonstrating the use of selenium sources M. maripaludis was previously not known to employ. Also, an attempt-although so far unsuccessful-was made to pinpoint potential selenium transporter genes, in order to deepen our understanding of trace element utilization in this important model organism.

Pyruvate-dependent growth of Methanosarcina acetivorans.

Methanogenesis is a key step during anaerobic biomass degradation. Methanogenic archaea (methanogens) are the only organisms coupling methanogenic substrate conversion to energy conservation. The range of substrates utilized by methanogens is limited, with acetate and H2+CO2 being the ecologically most relevant. The only single methanogenic energy substrate containing more carbon-carbon bonds than acetate is pyruvate. Only the aggregate-forming, freshwater methanogen Methanosarcina barkeri Fusaro was shown to grow on this compound. Here, the pyruvate-utilizing capabilities of the single-celled, marine Methanosarcina acetivorans were addressed. Robust pyruvate-dependent, methanogenic, growth could be established by omitting CO2 from the growth medium. Growth rates which were independent of the pyruvate concentration indicated that M. acetivorans actively translocates pyruvate across the cytoplasmic membrane. When 2-bromoethanesulfonate (BES) inhibited methanogenesis to more than 99%, pyruvate-dependent growth was acetogenic and sustained. However, when methanogenesis was completely inhibited M. acetivorans did not grow on pyruvate. Analysis of metabolites showed that acetogenesis is used by BES-inhibited M. acetivorans as a sink for electrons derived from pyruvate oxidation and that other, thus far unidentified, metabolites are produced.IMPORTANCEThe known range of methanogenic growth substrates is very limited and M. acetivorans is only the second methanogenic species for which growth on pyruvate is demonstrated. Besides some commonalities, analysis of M. acetivorans highlights differences in pyruvate metabolism among Methanosarcina species. The observation that M. acetivorans probably imports pyruvate actively indicates that the capabilities for heterotrophic catabolism in methanogens may be underestimated. The mostly acetogenic growth of M. acetivorans on pyruvate with concomitant inhibition of methanogenesis confirms that energy conservation of methanogenic archaea can be independent of methane formation.

Genetic and Physiological Probing of Cytoplasmic Bypasses for the Energy-Converting Methyltransferase Mtr in Methanosarcina acetivorans.

Methanogenesis is a unique energy metabolism carried out by members of the domain Archaea. Unlike most other methanogens, which reduce CO2 to methane with hydrogen as the electron donor, Methanosarcina acetivorans is able to grow on methylated compounds, on acetate, and on carbon monoxide (CO). These substrates are metabolized via distinct yet overlapping pathways. For the use of any single methanogenic substrate, the membrane-integral, energy-converting N5-methyl-tetrahydrosarcinapterin (H4SPT):coenzyme M (HS-CoM) methyltransferase (Mtr) is required. It was proposed that M. acetivorans can bypass the methyl transfer catalyzed by Mtr via cytoplasmic activities. To address this issue, conversion of different energy substrates by an mtr deletion mutant was analyzed. No significant methyl transfer from H4SPT to HS-CoM could be detected with CO as the electron donor. In contrast, formation of methane and CO2 in the presence of methanol or trimethylamine was indicative of an Mtr bypass in the oxidative direction. As methane thiol and dimethyl sulfide were transiently produced during methylotrophic methanogenesis in the mtr mutant, involvement in this process of methyl sulfide-dependent methyltransferases (Mts) was analyzed in a strain lacking both the Mts system and Mtr. It could be unequivocally demonstrated that the Mts system is not involved in bypassing Mtr, thereby ruling out previous proposals. Conversion of [13C]methanol indicated that in the absence of Mtr M. acetivorans provides the reducing equivalents for methyl-S-CoM reduction to methane by oxidizing (an) intracellular compound(s) to CO2 rather than disproportioning the source of methyl groups. Thus, no in vivo Mtr bypass appears to exist in M. acetivorans. IMPORTANCE Methanogenic archaea possess only a limited number of chemiosmotic coupling sites in their respiratory chains. Among them, N5-methyl-H4SPT:HS-CoM methyltransferase (Mtr) is the most widely distributed. Previous observations led to the conclusion that Methanosarcina acetivorans is able to bypass this reaction via methyl sulfide-dependent methyltransferases (Mts). However, strains lacking Mtr are not able to produce methane from CO. Also, these strains are unable to oxidize methylated substrates to CO2, in contrast to observations in the close relative Methanosarcina barkeri. The results also highlight the sole function of the Mts system in methyl sulfide metabolism. Thus, no in vivo Mtr bypass appears to exist in M. acetivorans.

In vivo probing of SECIS-dependent selenocysteine translation in Archaea.

Cotranslational insertion of selenocysteine (Sec) proceeds by recoding UGA to a sense codon. This recoding is governed by the Sec insertion sequence (SECIS) element, an RNA structure on the mRNA, but size, location, structure determinants, and mechanism differ for Bacteria, Eukarya, and Archaea. For Archaea, the structure-function relation of the SECIS is poorly understood, as only rather laborious experimental approaches are established. Furthermore, these methods do not allow for quantitative probing of Sec insertion. In order to overcome these limitations, we engineered bacterial ß-lactamase into an archaeal selenoprotein, thereby establishing a reporter system, which correlates enzyme activity to Sec insertion. Using this system, in vivo Sec insertion depending on the availability of selenium and the presence of a SECIS element was assessed in Methanococcus maripaludis Furthermore, a minimal SECIS element required for Sec insertion in M. maripaludis was defined and a conserved structural motif shown to be essential for function. Besides developing a convenient tool for selenium research, converting a bacterial enzyme into an archaeal selenoprotein provides proof of concept that novel selenoproteins can be engineered in Archaea.

Deconstructing Methanosarcina acetivorans into an acetogenic archaeon.

The reductive acetyl-coenzyme A (acetyl-CoA) pathway, whereby carbon dioxide is sequentially reduced to acetyl-CoA via coenzyme-bound C1 intermediates, is the only autotrophic pathway that can at the same time be the means for energy conservation. A conceptually similar metabolism and a key process in the global carbon cycle is methanogenesis, the biogenic formation of methane. All known methanogenic archaea depend on methanogenesis to sustain growth and use the reductive acetyl-CoA pathway for autotrophic carbon fixation. Here, we converted a methanogen into an acetogen and show that Methanosarcina acetivorans can dispense with methanogenesis for energy conservation completely. By targeted disruption of the methanogenic pathway, followed by adaptive evolution, a strain was created that sustained growth via carbon monoxide-dependent acetogenesis. A minute flux (less than 0.2% of the carbon monoxide consumed) through the methane-liberating reaction remained essential, indicating that currently living methanogens utilize metabolites of this reaction also for anabolic purposes. These results suggest that the metabolic flexibility of methanogenic archaea might be much greater than currently known. Also, our ability to deconstruct a methanogen into an acetogen by merely removing cellular functions provides experimental support for the notion that methanogenesis could have evolved from the reductive acetyl-coenzyme A pathway.

Characterization of Blf4, an Archaeal Lytic Virus Targeting a Member of the Methanomicrobiales.

Today, the number of known viruses infecting methanogenic archaea is limited. Here, we report on a novel lytic virus, designated Blf4, and its host strain Methanoculleus bourgensis E02.3, a methanogenic archaeon belonging to the Methanomicrobiales, both isolated from a commercial biogas plant in Germany. The virus consists of an icosahedral head 60 nm in diameter and a long non-contractile tail of 125 nm in length, which is consistent with the new isolate belonging to the Siphoviridae family. Electron microscopy revealed that Blf4 attaches to the vegetative cells of M. bourgensis E02.3 as well as to cellular appendages. Apart from M. bourgensis E02.3, none of the tested Methanoculleus strains were lysed by Blf4, indicating a narrow host range. The complete 37 kb dsDNA genome of Blf4 contains 63 open reading frames (ORFs), all organized in the same transcriptional direction. For most of the ORFs, potential functions were predicted. In addition, the genome of the host M. bourgensis E02.3 was sequenced and assembled, resulting in a 2.6 Mbp draft genome consisting of nine contigs. All genes required for a hydrogenotrophic lifestyle were predicted. A CRISPR/Cas system (type I-U) was identified with six spacers directed against Blf4, indicating that this defense system might not be very efficient in fending off invading Blf4 virus.

Characterization of the lytic archaeal virus Drs3 infecting Methanobacterium formicicum.

Viruses are ubiquitous in the biosphere and greatly affect the hosts they infect. It is generally accepted that members of every microbial taxon are susceptible to at least one virus, and a plethora of bacterial viruses are known. In contrast, knowledge of the archaeal virosphere is still limited. Here, a novel lytic archaeal virus is described, designated "Drs3", as well as its host, Methanobacterium formicicum strain Khl10. This hydrogenotrophic methanogenic archaeon and its virus were isolated from the anaerobic digester of an experimental biogas plant in Germany. The tailed virus has an icosahedral head with a diameter of approximately 60 nm and a long non-contractile tail of approximately 230 nm. These structural observations suggest that the new isolate belongs to the family Siphoviridae, but it could not be assigned to an existing genus. Lysis of the host Khl10 was observed 40-44 h after infection. Lysis of the type strain Methanobacterium formicicum DSMZ 1535 was not observed in the presence of Drs3, pointing towards resistance in the type strain or a rather narrow host range of this newly isolated archaeal virus. The complete 37-kb linear dsDNA genome of Drs3 contains 39 open reading frames, only 12 of which show similarity to genes with predicted functions.

Identification of a unique Radical SAM methyltransferase required for the sp3-C-methylation of an arginine residue of methyl-coenzyme M reductase.

The biological formation of methane (methanogenesis) is a globally important process, which is exploited in biogas technology, but also contributes to global warming through the release of a potent greenhouse gas into the atmosphere. The last and methane-releasing step of methanogenesis is catalysed by the enzyme methyl-coenzyme M reductase (MCR), which carries several exceptional posttranslational amino acid modifications. Among these, a 5-C-(S)-methylarginine is located close to the active site of the enzyme. Here, we show that a unique Radical S-adenosyl-L-methionine (SAM) methyltransferase is required for the methylation of the arginine residue. The gene encoding the methyltransferase is currently annotated as "methanogenesis marker 10" whose function was unknown until now. The deletion of the methyltransferase gene ma4551 in Methanosarcina acetivorans WWM1 leads to the production of an active MCR lacking the C-5-methylation of the respective arginine residue. The growth behaviour of the corresponding M. acetivorans mutant strain and the biophysical characterization of the isolated MCR indicate that the methylated arginine is important for MCR stability under stress conditions.

Selenoprotein synthesis and regulation in Archaea.

BACKGROUND: The major biological form of selenium is that of the co-translationally inserted amino acid selenocysteine (Sec). In Archaea, the majority of proteins containing Sec, selenoproteins, are involved in methanogenesis. However, the function of this residue is often not known because selenium-independent homologs of the selenoproteins can be employed, sometimes even in one organism. SCOPE OF REVIEW: This review summarizes current knowledge about the selenoproteins of Archaea, the metabolic pathways where they are involved, and discusses the (potential) function of individual Sec residues. Also, what is known about the "archaeal" way of selenoprotein synthesis, and the regulatory mechanism leading to the replacement of the selenoproteins with selenium-independent homologs, will be presented. Where appropriate, similarities with (and differences to) the respective steps employed in the other two domains, Bacteria and Eukarya, will be emphasized. MAJOR CONCLUSIONS: Genetic and biochemical studies guided by analysis of genome sequences of Sec-encoding archaea has revealed that the pathway of Sec synthesis in Archaea and Eukarya are principally identical and that Sec insertion in Eukarya probably evolved from an archaeal mechanism employed prior to the separation of the archaeal and eukaryal lines of decent. GENERAL SIGNIFICANCE: In light of the emerging close phylogenetic relationship of Eukarya and Archaea, archaeal models may be highly valuable tools for unraveling "eukaryotic" principles in molecular and cell biology.

Selenium-dependent gene expression in Methanococcus maripaludis: Involvement of the transcriptional regulator HrsM.

BACKGROUND: The archaeon Methanococcus maripaludis strain JJ employs several selenocysteine (Sec)-containing proteins in its primary energy metabolism, methanogenesis. Upon selenium deprivation, or when the pathway for selenoprotein synthesis is disrupted, they are replaced by cysteine (Cys)-containing isoforms, thus allowing for selenium-independent growth. METHODS: Expression of a fusion of the promoter region of frcA (encoding a subunit of the selenium-independent hydrogenase Frc) and bla [encoding ß-lactamase (Bla)] in M. maripaludis JJ was assessed in response to the selenium supply, growth substrate, and growth phase. Random transposon mutants of the reporter strain were screened for deregulated bla expression, which identified HrsM, a LysR-type transcriptional regulator (LTTR). Its involvement in selenium-dependent gene regulation was further assessed by analyzing in vivo transcription, synthesis of selenoproteins and of HrsM, and by analyzing in vitro binding of HrsM to DNA. RESULTS: HrsM, which is not required for selenoprotein synthesis, acts as a positive effector of selenoprotein gene expression and as a negative effector of Cys-encoding isogene expression, but its own expression is independent of the selenium availability. Specific binding in vitro of HrsM to a promoter region under in vivo HrsM control verified its role in selenium-dependent gene regulation. CONCLUSIONS: HrsM exerts a key role in regulating expression of selenoprotein genes and their Cys-encoding isogenes in M. maripaludis in a selenium-dependent fashion. However, this activity is not achieved via autoregulation but probably by a mechanism, which modulates the DNA-binding of HrsM. GENERAL SIGNIFICANCE: Although LTTRs are abundant in Bacteria, HrsM represents only the second characterized member of this group in Archaea.

Complete Genome Sequence of the Methanococcus maripaludis Type Strain JJ (DSM 2067), a Model for Selenoprotein Synthesis in Archaea.

Methanococcus maripaludis type strain JJ (DSM 2067) is an important organism because it serves as a model for primary energy metabolism and hydrogenotrophic methanogenesis and is amenable to genetic manipulation. The complete genome (1.7 Mb) harbors 1,815 predicted protein-encoding genes, including 9 encoding selenoproteins.

Methanogens: biochemical background and biotechnological applications.

Since fossil sources for fuel and platform chemicals will become limited in the near future, it is important to develop new concepts for energy supply and production of basic reagents for chemical industry. One alternative to crude oil and fossil natural gas could be the biological conversion of CO2 or small organic molecules to methane via methanogenic archaea. This process has been known from biogas plants, but recently, new insights into the methanogenic metabolism, technical optimizations and new technology combinations were gained, which would allow moving beyond the mere conversion of biomass. In biogas plants, steps have been undertaken to increase yield and purity of the biogas, such as addition of hydrogen or metal granulate. Furthermore, the integration of electrodes led to the development of microbial electrosynthesis (MES). The idea behind this technique is to use CO2 and electrical power to generate methane via the microbial metabolism. This review summarizes the biochemical and metabolic background of methanogenesis as well as the latest technical applications of methanogens. As a result, it shall give a sufficient overview over the topic to both, biologists and engineers handling biological or bioelectrochemical methanogenesis.

Identification of HcgC as a SAM-Dependent Pyridinol Methyltransferase in [Fe]-Hydrogenase Cofactor Biosynthesis.

Previous retrosynthetic and isotope-labeling studies have indicated that biosynthesis of the iron guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase requires a methyltransferase. This hypothetical enzyme covalently attaches the methyl group at the 3-position of the pyridinol ring. We describe the identification of HcgC, a gene product of the hcgA-G cluster responsible for FeGP cofactor biosynthesis. It acts as an S-adenosylmethionine (SAM)-dependent methyltransferase, based on the crystal structures of HcgC and the HcgC/SAM and HcgC/S-adenosylhomocysteine (SAH) complexes. The pyridinol substrate, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol, was predicted based on properties of the conserved binding pocket and substrate docking simulations. For verification, the assumed substrate was synthesized and used in a kinetic assay. Mass spectrometry and NMR analysis revealed 6-carboxymethyl-3,5-dimethyl-4-hydroxy-2-pyridinol as the reaction product, which confirmed the function of HcgC.

Influence of DNA isolation method on the investigation of archaeal diversity and abundance in biogas plants.

Various methods are available for DNA isolation from environmental samples. Because the chemical and biological composition of samples such as soil, sludge, or plant material is different, the effectiveness of DNA isolation can vary depending on the method applied and thus, have a substantial effect on the results of downstream analysis of the microbial community. Although the process of biogas formation is being intensely investigated, a systematic evaluation of kits for DNA isolation from material of biogas plants is still lacking. Since no DNA isolation kit specifically tailored for DNA isolation from sludge of biogas plants is available, this study compares five commercially available kits regarding their influence on downstream analyses such denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR). The results show that not all kits are equally suited for the DNA isolation from samples of different biogas plants, but highly reproducible DGGE fingerprints as well as qPCR results across the tested samples from biogas reactors using different substrate compositions could be produced using selected kits.

Assessment of hydrogen metabolism in commercial anaerobic digesters.

Degradation of biomass in the absence of exogenous electron acceptors via anaerobic digestion involves a syntrophic association of a plethora of anaerobic microorganisms. The commercial application of this process is the large-scale production of biogas from renewable feedstock as an alternative to fossil fuels. After hydrolysis of polymers, monomers are fermented to short-chain fatty acids and alcohols, which are further oxidized to acetate. Carbon dioxide, molecular hydrogen (H2), and acetate generated during the process are converted to methane by methanogenic archaea. Since many of the metabolic pathways as well as the syntrophic interactions and dependencies during anaerobic digestion involve formation, utilization, or transfer of H2, its metabolism and the methanogenic population were assessed in various samples from three commercial biogas plants. Addition of H2 significantly increased the rate of methane formation, which suggested that hydrogenotrophic methanogenesis is not a rate-limiting step during biogas formation. Methanoculleus and Methanosarcina appeared to numerically dominate the archaeal population of the three digesters, but their proportion and the Bacteria-to-Archaea ratio did not correlate with the methane productivity. Instead, hydrogenase activity in cell-free extracts from digester sludge correlated with methane productivity in a positive fashion. Since most microorganisms involved in biogas formation contain this activity, it approximates the overall anaerobic metabolic activity and may, thus, be suitable for monitoring biogas reactor performance.

Methanosarcina flavescens sp. nov., a methanogenic archaeon isolated from a full-scale anaerobic digester.

A novel, strictly anaerobic, methanogenic archaeon, strain E03.2T, was isolated from a full-scale biogas plant in Germany. Cells were non-motile sarcina-like cocci, occurring in aggregates. Strain E03.2T grew autotrophically on H2 plus CO2, and additionally cells could utilize acetate, methanol, moni-, di- and trimethylamine as carbon and energy sources; however, growth or methanogenesis on formate was not observed. Yeast extract and vitamins stimulated growth but were not mandatory. The optimal growth temperature of strain E03.2T was approximately 45 °C; maximal growth rates were obtained at about pH 7.0 in the presence of approximately 6.8 mM NaCl. The DNA G+C content of strain E03.2T was 41.3 mol%. Phylogenetic analyses based on 16S rRNA gene and mcrA sequences placed strain E03.2T within the genus Methanosarcina. Based on 16S rRNA gene sequence similarity strain E03.2T was related to seven different species of the genus Methanosarcina, but most closely related to Methanosarcina thermophila TM-1T. Phenotypic, physiological and genomic characteristics indicated that strain E03.2T represents a novel species of the genus Methanosarcina, for which the name Methanosarcina flavescens sp. nov. is proposed. The type strain is E03.2T ( = DSM 100822T = JCM 30921T).

Selenocysteine-independent suppression of UGA codons in the archaeon Methanococcus maripaludis.

BACKGROUND: Proteins containing selenocysteine (sec) are found in Bacteria, Eukarya, and Archaea. While selenium-dependence of methanogenesis from H(2)+CO(2) in the archaeon Methanococcus maripaludis JJ is compensated by induction of a set of cysteine-containing homologs, growth on formate is abrogated in the absence of sec due to the dependence of formate dehydrogenase (Fdh) on selenium. Despite this dependence, formate-dependent growth occurs after prolonged incubation of M. maripaludis mutants lacking sec. METHODS: To study this phenomenon, a M. maripaludis strain with only one Fdh isoform and an FdhA selenoprotein C-terminally tagged for affinity enrichment was constructed. Factors required for sec synthesis were deleted in this strain and translation of UGA in fdhA was analyzed physiologically, enzymatically, immunologically, and via mass spectrometry. RESULTS: M. maripaludis JJ mutants lacking sec synthesis grew at least five times more slowly than the wild type on formate due to a 20-35-fold reduction of Fdh activity. The enzyme in the mutant strains lacked sec but was still produced as a full-length protein. Peptide mass spectrometry revealed that both cysteine (cys) and tryptophan (trp) were inserted at the UGA encoding sec without apparent mutations in tRNA(cys) or tRNA(trp), respectively. CONCLUSIONS: We demonstrate that M. maripaludis has the inherent capacity to translate UGA with cys and trp; other mechanisms to replace sec with cys in the absence of selenium could thereby be ruled out. GENERAL SIGNIFICANCE: This study exemplifies how an organism uses the inherent flexibility in its canonical protein synthesis machinery to recover some activity of an essential selenium-dependent enzyme in the absence of sec.

Cadmium-induced formation of sulphide and cadmium sulphide particles in the aquatic hyphomycete Heliscus lugdunensis.

Freshwater fungi which can survive under metal exposure receive increasing scientific attention. Enhanced synthesis of sulphide and glutathione but no phytochelatin synthesis in response to cadmium (up to 80 µM Cd(2+) in the medium) was measured in the aquatic hyphomycete Heliscus lugdunensis. Up to 25 µmol g(-1) dry mass the fungus formed sulphide in an exponentially Cd(2+)-concentration-dependent manner. Using light microscopy, precipitates were observed outside of the hyphae which could be determined as amorphous particles by X-ray diffraction (XRD). Energy dispersive X-ray spectroscopy (EDS) analysis indicated that these particles were mainly composed of Cd and S with an atomic ratio of 1:1, but some elements of the culture medium such as P and Cl were also present. Fungal cells exposed to Cd(2+) accumulated 12-28 µmol metal g(-1) dry mass over a period of 7-28 days. The results may indicate that sulphide could sequester excess Cd(2+) under oxygen deprived conditions and thereby reduce its toxicity via an additional avoidance mechanism of this fungus.

Methanobacterium aggregans sp. nov., a hydrogenotrophic methanogenic archaeon isolated from an anaerobic digester.

A novel, strictly anaerobic, hydrogenotrophic methanogen, strain E09F.3T, was isolated from a commercial biogas plant in Germany. Cells of E09F.3T were Gram-stain-negative, non-motile, slightly curved rods, long chains of which formed large aggregates consisting of intertwined bundles of chains. Cells utilized H2+CO2 and, to a lesser extent, formate as substrates for growth and methanogenesis. The optimal growth temperature was around 40 °C; maximum growth rate was obtained at pH around 7.0 with approximately 6.8 mM NaCl. The DNA G+C content of strain E09F.3T was 39.1 mol%. Phylogenetic analyses based on 16S rRNA and mcrA gene sequences placed strain E09F.3T within the genus Methanobacterium. On the basis of 16S rRNA gene sequence similarity, strain E09F.3T was closely related to Methanobacterium congolense CT but morphological, physiological and genomic characteristics indicated that strain E09F.3T represents a novel species. The name Methanobacterium aggregans sp. nov. is proposed for this novel species, with strain E09F.3T ( = DSM 29428T = JCM 30569T) as the type strain.