Methanosarcina Research Articles

Crystal Structure of the Apo-Form of NADPH-Dependent Thioredoxin Reductase from a Methane-Producing Archaeon.

The redox regulation of proteins via reversible dithiol/disulfide exchange reactions involves the thioredoxin system, which is composed of a reductant, a thioredoxin reductase (TR), and thioredoxin (Trx). In the pyridine nucleotide-dependent Trx reduction pathway, reducing equivalents, typically from reduced nicotinamide adenine dinucleotide phosphate (NADPH), are transferred from NADPH-TR (NTR) to Trx and, in turn, to target proteins, thus resulting in the reversible modification of the structural and functional properties of the targets. NTR enzymes contain three functional sites: an NADPH binding pocket, a non-covalently bound flavin cofactor, and a redox-active disulfide in the form of CxxC. With the aim of increasing our knowledge of the thioredoxin system in archaea, we here report the high-resolution crystal structure of NTR from the methane-generating organism Methanosarcina mazei strain Gö1 (MmNTR) at 2.6 Å resolution. Based on the crystals presently described, MmNTR assumes an overall fold that is nearly identical to the archetypal fold of authentic NTRs; however, surprisingly, we observed no electron density for flavin adenine dinucleotide (FAD) despite the well-defined and conserved FAD-binding cavity in the folded module. Remarkably, the dimers of the apo-protein within the crystal were different from those observed by small angle X-ray scattering (SAXS) for the holo-protein, suggesting that the binding of the flavin cofactor does not require major protein structural rearrangements. Rather, binding results in the stabilization of essential parts of the structure, such as those involved in dimer stabilization. Altogether, this structure represents the example of an apo-form of an NTR that yields important insight into the effects of the cofactor on protein folding.

Engineering and manipulation of a mevalonate pathway in Escherichia coli for isoprene production.

Isoprene is a useful phytochemical with high commercial values in many industrial applications including synthetic rubber, elastomers, isoprenoid medicines, and fossil fuel. Currently, isoprene is on large scale produced from petrochemical sources. An efficient biological process for isoprene production utilizing renewable feedstocks would be an important direction of research due to the fossil raw material depletion and air pollution. In this study, we introduced the mevalonate (MVA) pathway genes/acetoacetyl-coenzyme A thiolase (mvaE) and MVA synthase (mvaS) from Enterococcus faecalis (E. faecalis); MVA kinase (mvk) derived from Methanosarcina mazei (M. mazei); and phosphomevalonate kinase (pmk), diphosphomevalonate decarboxylase (mvaD), and isopentenyl diphosphate isomerase (idi) from Streptococcus pneumoniae (S. pneumoniae) to accelerate dimethylallyl diphosphate (DMAPP) accumulation in Escherichia coli (E. coli). Together with a codon-optimized isoprene synthase (ispS) from Populus alba (P. alba), E. coli strain succeeded in formation of isoprene. We then manipulated the heterologous MVA pathway for high-level production of isoprene, by controlling the gene expression levels of the MVA pathway genes. We engineered four E. coli strains which showed different gene expression levels and different isoprene productivities, and we also characterized them with quantitative real-time PCR and metabolite analysis. To further improve the isoprene titers and release the toxicity to cells, we developed the extraction fermentation by adding dodecane in cultures. Finally, strain BL2T7P1TrcP harboring balanced gene expression system produced 587 ± 47mg/L isoprene, with a 5.2-fold titer improvement in comparison with strain BL7CT7P. This work indicated that a balanced metabolic flux played a significant role to improve the isoprene production via MVA pathway.

Gut Colonization with Methanogenic Archaea Lowers Plasma Trimethylamine N-oxide Concentrations in Apolipoprotein e\u2212/\u2212 Mice

A mechanistic link between trimethylamine N-oxide (TMAO) and atherogenesis has been reported. TMAO is generated enzymatically in the liver by the oxidation of trimethylamine (TMA), which is produced from dietary choline, carnitine and betaine by gut bacteria. It is known that certain members of methanogenic archaea (MA) could use methylated amines such as trimethylamine as growth substrates in culture. Therefore, we investigated the efficacy of gut colonization with MA on lowering plasma TMAO concentrations. Initially, we screened for the colonization potential and TMAO lowering efficacy of five MA species in C57BL/6 mice fed with high choline/TMA supplemented diet, and found out that all five species could colonize and lover plasma TMAO levels, although with different efficacies. The top performing MA, Methanobrevibacter smithii, Methanosarcina mazei, and Methanomicrococcus blatticola, were transplanted into Apoe−/− mice fed with high choline/TMA supplemented diet. Similar to C57BL/6 mice, following initial provision of the MA, there was progressive attrition of MA within fecal microbial communities post-transplantation during the initial 3 weeks of the study. In general, plasma TMAO concentrations decreased significantly in proportion to the level of MA colonization. In a subsequent experiment, use of antibiotics and repeated transplantation of Apoe−/− mice with M. smithii, led to high engraftment levels during the 9 weeks of the study, resulting in a sustained and significantly lower average plasma TMAO concentrations (18.2 ± 19.6 μM) compared to that in mock-transplanted control mice (120.8 ± 13.0 μM, p < 0.001). Compared to control Apoe−/− mice, M. smithii-colonized mice also had a 44% decrease in aortic plaque area (8,570 μm [95% CI 19587–151821] vs. 15,369 μm [95% CI [70058–237321], p = 0.34), and 52% reduction in the fat content in the atherosclerotic plaques (14,283 μm [95% CI 4,957–23,608] vs. 29,870 μm [95% CI 18,074–41,666], p = 0.10), although these differences did not reach significance. Gut colonization with M. smithii leads to a significant reduction in plasma TMAO levels, with a tendency for attenuation of atherosclerosis burden in Apoe−/− mice. The anti-atherogenic potential of MA should be further tested in adequately powered experiments.

Methanomethylophilus alvus Mx1201 Provides Basis for Mutual Orthogonal Pyrrolysyl tRNA/Aminoacyl-tRNA Synthetase Pairs in Mammalian Cells.

Genetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded noncanonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei (Mma PylRS/PylT) being the most active and versatile to date. We found a pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that this PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its noncognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

An Evolved Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase/tRNA Pair Is Highly Active and Orthogonal in Mammalian Cells.

We recently characterized a new class of pyrrolysyl-tRNA synthetase (PylRS)/PyltRNA pairs from Methanomassiliicocales that are active and orthogonal in Escherichia coli. The aminoacyl-tRNA synthetases (aaRSs) of these pairs lack the N-terminal domain that is essential for tRNA recognition and in vivo activity in the Methanosarcina mazei (Mm) PylRS but share a homologous active site with MmPylRS; this facilitates the transplantation of mutations discovered with existing PylRS systems into the new PylRS systems to reprogram their substrate specificity for the incorporation of noncanonical amino acids (ncAAs). Several of the new PylRS/PyltRNA pairs, or their evolved variants [including Methanomethylophilus alvus (Ma) PylRS/MaPyltRNA(6)CUA], are mutually orthogonal to the MmPylRS/MmPyltRNA pair, and the active sites of the Mm pair and Ma pair can be diverged to enable the incorporation of distinct ncAAs in response to distinct codons via orthogonal translation in E. coli. Here we demonstrate that MaPylRS/MaPyltRNA(6)CUA is orthogonal to the aaRSs and tRNAs in mammalian cells and directs efficient incorporation of ncAAs into proteins. Moreover, we confirm that the MaPylRS/MaPyltRNA(6) and MmPylRS/MmPyltRNA pairs are mutually orthogonal in mammalian cells and demonstrates that these pairs can be used to encode distinct ncAAs into a protein in mammalian cells. Thus, the MaPylRS/MaPyltRNA(6)CUA pair provides an additional pair that is orthogonal in both E. coli and mammalian systems and is mutually orthogonal to the most widely used system for genetic code expansion. Our results provide a foundation for expanding the scope of genetic code expansion and may also facilitate strategies for proteome-wide ncAA tagging with mutually orthogonal systems.

16s rRNA gene sequencing and radioisotopic analysis reveal the composition of ammonia acclimatized methanogenic consortia

Different mesophilic and thermophilic methanogenic consortia were acclimatised and enriched to extreme total ammonia (9.0 and 5.0 g NH4+-N L−1, respectively) and free ammonia (1.0 and 1.4 g NH3-N L−1, respectively) levels in this study. [2-14C] acetate radioisotopic analyses showed the dominance of aceticlastic methanogenesis in all enriched consortia. According to 16S rRNA gene sequencing result, in mesophilic consortia, methylotrophic Methanomassiliicoccus luminyensis was predominant, followed by aceticlastic Methanosarcina soligelidi. A possible scenario explaining the dominance of M. luminyensis includes the use of methylamine produced by Tissierella spp. and biomass build-up by metabolizing acetate. Nevertheless, further studies are needed to pinpoint the exact metabolic pathway of M. luminyensis. In thermophilic consortia, aceticlastic Methanosarcina thermophila was the sole dominant methanogen. Overall, results derived from this study demonstrated the efficient biomethanation ability of these ammonia-tolerant methanogenic consortia, indicating a potential application of these consortia to solve ammonia toxicity problems in future full-scale reactors.

Cross-cleavage activity of Cas6b in crRNA processing of two different CRISPR-Cas systems in Methanosarcina mazei Gö1

ABSTRACTThe clustered regularly interspaced short palindromic repeat (CRISPR) system is a prokaryotic adaptive defense system against foreign nucleic acids. In the methanoarchaeon Methanosarcina mazei Gö1, two types of CRISPR-Cas systems are present (type I-B and type III-C). Both loci encode a Cas6 endonuclease, Cas6b-IB and Cas6b-IIIC, typically responsible for maturation of functional short CRISPR RNAs (crRNAs). To evaluate potential cross cleavage activity, we biochemically characterized both Cas6b proteins regarding their crRNA binding behavior and their ability to process pre-crRNA from the respective CRISPR array in vivo. Maturation of crRNA was studied in the respective single deletion mutants by northern blot and RNA-Seq analysis demonstrating that in vivo primarily Cas6b-IB is responsible for crRNA processing of both CRISPR arrays. Tentative protein level evidence for the translation of both Cas6b proteins under standard growth conditions was detected, arguing for different activities or a potential non-redundant role of Cas6b-IIIC within the cell. Conservation of both Cas6 endonucleases was observed in several other M. mazei isolates, though a wide variety was displayed. In general, repeat and leader sequence conservation revealed a close correlation in the M. mazei strains. The repeat sequences from both CRISPR arrays from M. mazei Gö1 contain the same sequence motif with differences only in two nucleotides. These data stand in contrast to all other analyzed M. mazei isolates, which have at least one additional CRISPR array with repeats belonging to another sequence motif. This conforms to the finding that Cas6b-IB is the crucial and functional endonuclease in M. mazei Gö1.Abbreviations:sRNA: small RNA; crRNA: CRISPR RNA; pre-crRNAs: Precursor CRISPR RNA; CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR associated; nt: nucleotide; RNP: ribonucleoprotein; RBS: ribosome binding site

The Methanosarcina mazei MM2060 Gene Encodes a Bifunctional Kinase/Decarboxylase Enzyme Involved in Cobamide Biosynthesis.

Cobamides (Cbas) are synthesized by many archaea, but some aspects of Cba biosynthesis in these microorganisms remain unclear. Here, we demonstrate that open reading frame MM2060 in the archaeum Methanosarcina mazei strain Gö1 encodes a bifunctional enzyme with l-threonine- O-3-phosphate (l-Thr-P) decarboxylase (EC 4.1.1.81) and l-Thr kinase activities (EC 2.7.1.177). In Salmonella enterica, where Cba biosynthesis has been extensively studied, the activities mentioned above are encoded by separate genes, namely, cobD and pduX, respectively. The activities associated with the MM2060 protein ( MmCobD) were validated in vitro and in vivo. In vitro, MmCobD used ATP and l-Thr as substrates and generated ADP, l-Thr-P, and ( R)-1-aminopropan-2-ol O-phosphate as products. Notably, MmCobD has a 111-amino acid C-terminal extension of unknown function, which contains a putative metal-binding motif. This C-terminal domain alone did not display activity either in vivo or in vitro. Although the C-terminal MmCobD domain was not required for l-Thr-P decarboxylase or l-Thr kinase activities in vivo, its absence negatively affected both activities. In vitro results suggested that this domain may have a regulatory or substrate-gating role. When purified under anoxic conditions, MmCobD displayed Michaelis-Menten kinetics and had a 1000-fold higher affinity for ATP and a catalytic efficiency 1300-fold higher than that of MmCobD purified under oxic conditions. To the best of our knowledge, MmCobD is the first example of a new class of l-Thr-P decarboxylases that also have l-Thr kinase activity. An archaeal protein with l-Thr kinase activity had not been identified prior to this work.

A Semi-Rationally Engineered Bacterial Pyrrolysyl-tRNA Synthetase Genetically Encodes Phenyl Azide Chemistry.

The site-specific incorporation of non-canonical amino acids (ncAAs) at amber codons requires an aminoacyl-tRNA synthetase and a cognate amber suppressor tRNA (tRNACUA ). The archaeal tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii and the pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei have been extensively engineered to accept a versatile set of ncAAs. The PylRS/tRNACUA pair from the bacterium Desulfitobacterium hafniense is functional in Escherichia coli, however, variants of this PylRS have not been reported yet. In this study, the authors describe a bacterial PylRS from Desulfitobacterium hafniense, which the authors engineered for the reactive ncAA para-azido-l-phenylalanine (DhAzFRS) using a semi-rational approach. DhAzFRS preferred para-azido-l-phenylalanine to the canonical l-phenylalanine as the substrate. In addition, the authors demonstrate the functionality in E. coli of a hybrid DhAzFRS carrying the first 190 N-terminal amino acids of the Methanosarcina mazei PylRS. These results suggest that bacterial and archaeal PylRSs can be "mixed and matched" to tune their substrate specificity.

Site-Specific Incorporation of Selenocysteine by Genetic Encoding as a Photocaged Unnatural Amino Acid.

Selenocysteine (Sec) is a naturally occurring amino acid that is also referred to as the 21st amino acid. Site-specific incorporation of Sec into proteins is attractive, because the reactivity of a selenol group exceeds that of a thiol group and thus allows site-specific protein modifications. It is incorporated into proteins by an unusual enzymatic mechanism which, in E. coli and other organisms, involves the recognition of a selenocysteine insertion sequence (SECIS) in the mRNA of the target protein. Reengineering of the natural machinery for Sec incorporation at arbitrary sites independent of SECIS elements, however, is challenging. Here we demonstrate an alternative route, whereby a photocaged selenocysteine (PSc) is incorporated as an unnatural amino acid in response to an amber stop codon, using a mutant Methanosarcina mazei pyrrolysyl-tRNA synthetase, Mm PCC2RS, and its cognate tRNACUA. Following decaging by UV irradiation, proteins synthesized with PSc are readily tagged, e.g., with NMR probes to study ligand binding by NMR spectroscopy. The approach provides a facile route for genetically encoded Sec incorporation. It allows the production of pure selenoproteins and the Sec residue enables site-specific covalent protein modification with reagents that would usually react first with naturally occurring cysteine residues. The much greater reactivity of Sec residues allows their selective alkylation in the presence of highly solvent-exposed cysteine residues.

Methanogenic degradation of tetramethylammonium hydroxide by Methanomethylovorans and Methanosarcina

This study evaluated the methanogens responsible for methanogenic degradation of tetramethylammonium hydroxide (TMAH) in a continuous flow bioreactor. The enriched methanogens attained an estimated maximum specific TMAH degradation rate and half-saturation constant of 39.5 mg TMAH/gVSS/h and 820 mg/L, following the Monod-type kinetic expression for methanogenic TMAH degradation. Presence of sulfide more than 20 mg/L significantly extended lag period and slowed down specific TMAH degradation rates. The results of terminal restriction fragment length polymorphism (T-RFLP), cloning/sequencing, and quantitative real-time PCR analyses targeting on the methyl coenzyme M reductase alpha subunit (mcrA) genes retrieved from the bioreactor and batch experiments indicated that Methanomethylovorans species were the dominant methanogens responsible for methanogenic degradation of TMAH. The isolated TMAH-degrading methanogen from the bioreactor, however, was identified closely related to Methanosarcina mazei. It is likely that a very low TMAH environment in the bioreactor favored the growth of Methanomethylovorans hollandica, while the much higher TMAH in the isolation growth medium proliferated Methanosarcina mazei.

Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs.

Genetically encoding distinct non-canonical amino acids (ncAAs) into proteins synthesized in cells requires mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The pyrrolysyl-tRNA synthetase/PyltRNA pair from Methanosarcina mazei (Mm) has been engineered to incorporate diverse ncAAs and is commonly considered an ideal pair for genetic code expansion. However, finding new aaRS/tRNA pairs that share the advantages of the MmPylRS/MmPyltRNA pair and are orthogonal to both endogenous aaRS/tRNA pairs and the MmPylRS/MmPyltRNA pair has proved challenging. Here we demonstrate that several ΔNPylRS/PyltRNACUA pairs, in which PylRS lacks an N-terminal domain, are active, orthogonal and efficiently incorporate ncAAs in Escherichia coli. We create new PylRS/PyltRNA pairs that are mutually orthogonal to the MmPylRS/MmPyltRNA pair and show that transplanting mutations that reprogram the ncAA specificity of MmPylRS into the new PylRS reprograms its substrate specificity. Finally, we show that distinct PylRS/PyltRNA-derived pairs can function in the same cell, decode distinct codons and incorporate distinct ncAAs.

Methanosarcina mazei sRNA41 is the first archaeal sRNA that interacts with several ribosome binding sites within a polycistronic mRNA. This 120 nt regulatory RNA is highly expressed under N‐sufficiency and is antisense to the translation initation region of multiple target genes as shown in a multi‐sequence alignment (top). The detailed interaction of sRNA41 with MM2089 (encoding for acetyl‐CoA decarbonylase/synthase) is

Molecular MicrobiologyVolume 107, Issue 5 p. i-i Front CoverFree Access Methanosarcina mazei sRNA41 is the first archaeal sRNA that interacts with several ribosome binding sites within a polycistronic mRNA. This 120 nt regulatory RNA is highly expressed under N-sufficiency and is antisense to the translation initation region of multiple target genes as shown in a multi-sequence alignment (top). The detailed interaction of sRNA41 with MM2089 (encoding for acetyl-CoA decarbonylase/synthase) is illustrated (bottom). For details, see the article by Buddeweg et al. on pp. 595–609 of this issue. First published: 20 February 2018 https://doi.org/10.1111/mmi.13786AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume107, Issue5March 2018Pages i-i RelatedInformation

SRNA41 affects ribosome binding sites within polycistronic mRNAs in Methanosarcina mazei Gö1.

Several noncoding RNAs potentially involved in nitrogen (N)-regulation have been detected in Methanosarcina mazei, however, targets have been identified only for one of them. Here, we report on the function of sRNA41 , highly expressed under N-sufficiency. Comprising 120 nucleotides, sRNA41 shows high sequence and structural conservation within draft genomes of numerous Methanosarcina species. In silico target prediction revealed several potential targets, including genes of two homologous operons encoding for acetyl-CoA-decarbonylase/synthase complexes (ACDS) representing highly probable target candidates. A highly conserved single stranded region of sRNA41 was predicted to mask six independent ribosome binding sites of these two polycistronic mRNAs and was verified in vitro by microscale thermophoresis. Proteome analysis of the respective sRNA41 -deletion mutant showed increased protein expression of both ACDS complexes in the absence of sRNA41 , whereas no effect on transcript levels was detected, arguing for sRNA41 -mediated post-transcriptional fine-tuning of ACDS expression. We hypothesize that the physiological advantage of downregulating sRNA41 under N-limiting conditions is the resulting increase of ACDS protein levels. This provides sufficient amounts of amino acids for nitrogenase synthesis as well as reducing equivalents and energy for N2 -fixation, thus linking the carbon and N-metabolism.

Cell-Free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 Deletion E. coli Strains.

Cell-free protein synthesis (CFPS) is an effective method for the site-specific incorporations of noncanonical amino acids (ncAAs) into proteins. The nature of in vitro synthesis enables the use of experimental conditions that are toxic or reduce cellular uptake during in vivo site-specific incorporations of ncAAs. Using the Escherichia coli cell extract (S30) from the highly reproductive RF-1 deletion strains, B-60.∆A::Z and B-95.∆A, with orthogonal tRNA and aminoacyl-tRNA synthetase (aaRS) pairs from Methanosarcina mazei, we have developed CFPS methods for the highly productive and efficient multiple incorporation of ncAAs. In this chapter, we describe our methods for the preparation of the S30 and the orthogonal tRNAPyl and PylRS pair, and two CFPS protocols for ncAA incorporation.

Structural Basis for Genetic-Code Expansion with Various Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase

Pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl have been extensively used for genetic-code expansion. A Methanosarcina mazei PylRS mutant bearing the Y306A and Y384F mutations (PylRS(Y306A/Y384F)) encodes various bulky non-natural lysine derivatives by UAG. In this study, we examined how PylRS(Y306A/Y384F) recognizes many amino acids. Among 17 non-natural lysine derivatives, Ne-(benzyloxycarbonyl)lysine (ZLys) and 10 ortho/meta/para-substituted ZLys derivatives were efficiently tRNAPyl ligated and protein incorporated by PylRS(Y306A/Y384F). We determined crystal structures of 14 non-natural lysine derivatives bound to the PylRS(Y306A/Y384F) catalytic fragment. The meta- and para-substituted ZLys derivatives are snugly accommodated in the productive mode. In contrast, ZLys and the unsubstituted or ortho-substituted ZLys derivatives exhibited an alternative binding mode in addition to the productive mode. PylRS(Y306A/Y384F) displayed a high aminoacylation rate for ZLys, indicating that the double binding mode minimally affects aminoacylation. These precise substrate recognition mechanisms by PylRS(Y306A/Y384F) may facilitate the structure-based design of novel non-natural amino acids.

Methanosarcina Spherical Virus, a Novel Archaeal Lytic Virus Targeting Methanosarcina Strains.

A novel archaeal lytic virus targeting species of the genus Methanosarcina was isolated using Methanosarcina mazei strain Gö1 as the host. Due to its spherical morphology, the virus was designated Methanosarcina spherical virus (MetSV). Molecular analysis demonstrated that MetSV contains double-stranded linear DNA with a genome size of 10,567 bp containing 22 open reading frames (ORFs), all oriented in the same direction. Functions were predicted for some of these ORFs, i.e., such as DNA polymerase, ATPase, and DNA-binding protein as well as envelope (structural) protein. MetSV-derived spacers in CRISPR loci were detected in several published Methanosarcina draft genomes using bioinformatic tools, revealing a potential protospacer-adjacent motif (PAM) motif (TTA/T). Transcription and expression of several predicted viral ORFs were validated by reverse transcription-PCR (RT-PCR), PAGE analysis, and liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Analysis of core lipids by atmospheric pressure chemical ionization (APCI) mass spectrometry showed that MetSV and Methanosarcina mazei both contain archaeol and glycerol dialkyl glycerol tetraether without a cyclopentane moiety (GDGT-0). The MetSV host range is limited to Methanosarcina strains growing as single cells (M. mazei, Methanosarcina barkeri and Methanosarcina soligelidi). In contrast, strains growing as sarcina-like aggregates were apparently protected from infection. Heterogeneity related to morphology phases in M. mazei cultures allowed acquisition of resistance to MetSV after challenge by growing cultures as sarcina-like aggregates. CRISPR/Cas-mediated resistance was excluded since neither of the two CRISPR arrays showed MetSV-derived spacer acquisition. Based on these findings, we propose that changing the morphology from single cells to sarcina-like aggregates upon rearrangement of the envelope structure prevents infection and subsequent lysis by MetSV.IMPORTANCE Methanoarchaea are among the most abundant organisms on the planet since they are present in high numbers in major anaerobic environments. They convert various carbon sources, e.g., acetate, methylamines, or methanol, to methane and carbon dioxide; thus, they have a significant impact on the emission of major greenhouse gases. Today, very little is known about viruses specifically infecting methanoarchaea that most probably impact the abundance of methanoarchaea in microbial consortia. Here, we characterize the first identified Methanosarcina-infecting virus (MetSV) and show a mechanism for acquiring resistance against MetSV. Based on our results, we propose that growth as sarcina-like aggregates prevents infection and subsequent lysis. These findings allow new insights into the virus-host relationship in methanogenic community structures, their dynamics, and their phase heterogeneity. Moreover, the availability of a specific virus provides new possibilities to deepen our knowledge of the defense mechanisms of potential hosts and offers tools for genetic manipulation.

Magnetite production and transformation in the methanogenic consortia from coastal riverine sediments.

Minerals that contain ferric iron, such as amorphous Fe(III) oxides (A), can inhibit methanogenesis by competitively accepting electrons. In contrast, ferric iron reduced products, such as magnetite (M), can function as electrical conductors to stimulate methanogenesis, however, the processes and effects of magnetite production and transformation in the methanogenic consortia are not yet known. Here we compare the effects on methanogenesis of amorphous Fe (III) oxides (A) and magnetite (M) with ethanol as the electron donor. RNA-based terminal restriction fragment length polymorphism with a clone library was used to analyse both bacterial and archaeal communities. Iron (III)-reducing bacteria including Geobacteraceae and methanogens such as Methanosarcina were enriched in iron oxide-supplemented enrichment cultures for two generations with ethanol as the electron donor. The enrichment cultures with A and non-Fe (N) dominated by the active bacteria belong to Veillonellaceae, and archaea belong to Methanoregulaceae and Methanobacteriaceae, Methanosarcinaceae (Methanosarcina mazei), respectively. While the enrichment cultures with M, dominated by the archaea belong to Methanosarcinaceae (Methanosarcina barkeri). The results also showed that methanogenesis was accelerated in the transferred cultures with ethanol as the electron donor during magnetite production from A reduction. Powder X-ray diffraction analysis indicated that magnetite was generated from microbial reduction of A and M was transformed into siderite and vivianite with ethanol as the electron donor. Our data showed the processes and effects of magnetite production and transformation in the methanogenic consortia, suggesting that significantly different effects of iron minerals on microbial methanogenesis in the iron-rich coastal riverine environment were present.

SRNA154 a newly identified regulator of nitrogen fixation in Methanosarcina mazei strain Gö1

ABSTRACTTrans-encoded sRNA154 is exclusively expressed under nitrogen (N)-deficiency in Methanosarcina mazei strain Gö1. The sRNA154 deletion strain showed a significant decrease in growth under N-limitation, pointing toward a regulatory role of sRNA154 in N-metabolism. Aiming to elucidate its regulatory function we characterized sRNA154 by means of biochemical and genetic approaches. 24 homologs of sRNA154 were identified in recently reported draft genomes of Methanosarcina strains, demonstrating high conservation in sequence and predicted secondary structure with two highly conserved single stranded loops. Transcriptome studies of sRNA154 deletion mutants by an RNA-seq approach uncovered nifH- and nrpA-mRNA, encoding the α–subunit of nitrogenase and the transcriptional activator of the nitrogen fixation (nif)-operon, as potential targets besides other components of the N-metabolism. Furthermore, results obtained from stability, complementation and western blot analysis, as well as in silico target predictions combined with electrophoretic mobility shift-assays, argue for a stabilizing effect of sRNA154 on the polycistronic nif-mRNA and nrpA-mRNA by binding with both loops. Further identified N-related targets were studied, which demonstrates that translation initiation of glnA2-mRNA, encoding glutamine synthetase2, appears to be affected by sRNA154 masking the ribosome binding site, whereas glnA1-mRNA appears to be stabilized by sRNA154. Overall, we propose that sRNA154 has a crucial regulatory role in N-metabolism in M. mazei by stabilizing the polycistronic mRNA encoding nitrogenase and glnA1-mRNA, as well as allowing a feed forward regulation of nif-gene expression by stabilizing nrpA-mRNA. Consequently, sRNA154 represents the first archaeal sRNA, for which a positive posttranscriptional regulation is demonstrated as well as inhibition of translation initiation.

Phylogenomic proximity and metabolic discrepancy of Methanosarcina mazei Go1 across methanosarcinal genomes

Methanosarcina mazei Go1 is a heterotrophic methanogenic archaean contributing a significant role in global methane cycling and biomethanation process. Phylogenomic relatedness and metabolic discrepancy of this genome were described herein by comparing its whole genome sequence, intergenomic distance, genome function, synteny homologs and origin of replication, and marker genes with very closely related genomes, Methanosarcina acetivorans and Methanosarcina barkeri. Phylogenomic analysis of this study revealed that genome functional feature and metabolic core of M. mazei and M. barkeri could be originated from M. acetivorans. The metabolic core of these genomes shares a common evolutionary origin to perform the metabolic activity at different environmental niches. Genome expansion, dynamics and gene collinearity were constrained and restrained the conservation of the metabolic core genes by duplication events occurring across methanosarcinal genomes. The Darwinian positive selection was an evolutionary constraint to purify the function of core metabolic genes. Using genome-wide metabolic survey, we found the existence of four novel putative metabolic pathways such as complete methanogenesis from acetate, indole-3-acetate biosynthesis V, 4-aminobutyrate degradation III, galactosamine biosynthesis I and siroheme biosynthesis. Overall, the present study would provide a stand point to revisit its phylogenomic status in order to understand the origin and evolution history of this organism.