Methanolobus mangrovi sp. nov. and Methanolobus sediminis sp. nov, two novel methylotrophic methanogens isolated from mangrove sediments in China.

Two methylotrophic methanogens, designated strains FTZ2T and FTZ6T, were isolated from mangrove sediment sampled in Futian Mangrove Nature Reserve in Shenzhen, PR China. Cells of strains FTZ2T and FTZ6T were cocci, with diameters of 0.6-1.0 µm and 0.6-0.9 µm, respectively. Both strains grew on methanol, methylamine, dimethylamine and trimethylamine, but not on acetate, formate, H2/CO2, choline, betaine or dimethyl sulphide. Strain FTZ2T grew at 10-37 °C (optimally at 33 °C), pH 5.5-8.0 (optimally at pH 7.0) and 0-1.03 M NaCl (optimally at 0.17 M NaCl). In contrast, strain FTZ6T grew at 15-42 °C (optimally at 37 °C), pH 5.0-7.5 (optimally pH 6.5) and 0-1.03 M NaCl (optimally at 0.17 M NaCl). Both strains required magnesium for growth and were susceptible to sodium dodecyl sulphate. Biotin was required for the growth of strain FTZ2T but not of strain FTZ6T. The genomic G+C contents of strains FTZ2T and FTZ6T were 41.6 and 40.9 mol%, respectively. Phylogenetic analyses revealed that strain FTZ2T was mostly related to Methanolobus psychrotolerans YSF-03T, with 16S rRNA gene similarity of 98.6 %, an average nucleotide identity (ANI) of 82.5 %, and a digital DNA-DNA hybridization (dDDH) of 24.6 %. While strain FTZ6T was mostly related to Methanolobus vulcani PL-12/MT, with 16S rRNA gene similarity of 99.4 %, an ANI of 88.6% and a dDDH of 34.6 %. Based on phenotypic, phylogenetic and genotypic evidence, two novel species of the genus Methanolobus, Methanolobus mangrovi sp. nov. and Methanolobus sediminis sp. nov., are proposed. The type strain of M. mangrovi sp. nov. is FTZ2T (=CCAM 1276T=JCM 39396T) and the type strain of M. sediminis sp. nov. is FTZ6T (=CCAM 1277T=JCM 39397T).

Isolation and physiological properties of methanogenic archaea that degrade tetramethylammonium hydroxide.

Tetramethylammonium hydroxide (TMAH) is a known toxic chemical used in the photolithography process of semiconductor photoelectronic processes. Significant amounts of wastewater containing TMAH are discharged from electronic industries. It is therefore attractive to apply anaerobic treatment to industrial wastewater containing TMAH. In this study, a novel TMAH-degrading methanogenic archaeon was isolated from the granular sludge of a psychrophilic upflow anaerobic sludge blanket (UASB) reactor treating synthetic wastewater containing TMAH. Although the isolate (strain NY-STAYD) was phylogenetically related to Methanomethylovorans uponensis, it was the only isolated Methanomethylovorans strain capable of TMAH degradation. Strain NY-STAYD was capable of degrading methylamine compounds, similar to the previously isolated Methanomethylovorans spp. While the strain was able to grow at temperatures ranging from 15 to 37°C, the cell yield was higher at lower temperatures. The distribution of archaeal cells affiliated with the genus Methanomethylovorans in the original granular sludge was investigated by fluorescence in situ hybridization (FISH) using specific oligonucleotide probe targeting 16S rRNA. The results demonstrated that the TMAH-degrading cells associated with the genus Methanomethylovorans were not intermingled with other microorganisms but rather isolated on the granule's surface as a lone dominant archaeon. KEY POINTS: • A TMAH-degrading methanogenic Methanomethylovorans strain was isolated • This strain was the only known Methanomethylovorans isolate that can degrade TMAH • The highest cell yield of the isolate was obtained at psychrophilic conditions.

Ecology of Methanonatronarchaeia.

Methanonatronarchaeia represents a deep-branching phylogenetic lineage of extremely halo(alkali)philic and moderately thermophilic methyl-reducing methanogens belonging to the phylum Halobacteriota. It includes two genera, the alkaliphilic Methanonatronarchaeum and the neutrophilic Ca. Methanohalarchaeum. The former is represented by multiple closely related pure culture isolates from hypersaline soda lakes, while the knowledge about the latter is limited to a few mixed cultures with anaerobic haloarchaea. To get more insight into the distribution and ecophysiology of this enigmatic group of extremophilic methanogens, potential activity tests and enrichment cultivation with different substrates and at different conditions were performed with anaerobic sediment slurries from various hypersaline lakes in Russia. Methanonatronarchaeum proliferated exclusively in hypersaline soda lake samples mostly at elevated temperature, while at mesophilic conditions it coexisted with the extremely salt-tolerant methylotroph Methanosalsum natronophilum. Methanonatronarchaeum was also able to serve as a methylotrophic or hydrogenotrophic partner in several thermophilic enrichment cultures with fermentative bacteria. Ca. Methanohalarchaeum did not proliferate at mesophilic conditions and at thermophilic conditions it competed with extremely halophilic and moderately thermophilic methylotroph Methanohalobium, which it outcompeted at a combination of elevated temperature and methyl-reducing conditions. Overall, the results demonstrated that Methanonatronarchaeia are specialized extremophiles specifically proliferating in conditions of elevated temperature coupled with extreme salinity and simultaneous availability of a wide range of C1 -methylated compounds and H2 /formate.

Functional Insights of Salinity Stress-Related Pathways in Metagenome-Resolved Methanothrix Genomes.

Recently, methanogenic archaea belonging to the genus Methanothrix were reported to have a fundamental role in maintaining stable ecosystem functioning in anaerobic bioreactors under different configurations/conditions. In this study, we reconstructed three Methanothrix metagenome-assembled genomes (MAGs) from granular sludge collected from saline upflow anaerobic sludge blanket (UASB) reactors, where Methanothrix harundinacea was previously implicated with the formation of compact and stable granules under elevated salinity levels (up to 20 g/L Na+). Genome annotation and pathway analysis of the Methanothrix MAGs revealed a genetic repertoire supporting their growth under high salinity. Specifically, the most dominant Methanothrix (MAG_279), classified as a subspecies of Methanothrix_A harundinacea_D, had the potential to augment its salinity resistance through the production of different glycoconjugates via the N-glycosylation process, and via the production of compatible solutes as Nε-acetyl-ß-lysine and ectoine. The stabilization and reinforcement of the cell membrane via the production of isoprenoids was identified as an additional stress-related pathway in this microorganism. The improved understanding of the salinity stress-related mechanisms of M. harundinacea highlights its ecological niche in extreme conditions, opening new perspectives for high-efficiency methanisation of organic waste at high salinities, as well as the possible persistence of this methanogen in highly-saline natural anaerobic environments. IMPORTANCE Using genome-centric metagenomics, we discovered a new Methanothrix harundinacea subspecies that appears to be a halotolerant acetoclastic methanogen with the flexibility for adaptation in the anaerobic digestion process both at low (5 g/L Na+) and high salinity conditions (20 g/L Na+). Annotation of the recovered M. harundinacea genome revealed salinity stress-related functions, including the modification of EPS glycoconjugates and the production of compatible solutes. This is the first study reporting these genomic features within a Methanothrix sp., a milestone further supporting previous studies that identified M. harundinacea as a key-driver in anaerobic granulation under high salinity stress.

Methanosaeta and "Candidatus Velamenicoccus archaeovorus".

The phylum "Candidatus Omnitrophica" (candidate division OP3) is ubiquitous in anaerobic habitats but is currently characterized only by draft genomes from metagenomes and single cells. We had visualized cells of the phylotype OP3 LiM in methanogenic cultures on limonene as small epibiotic cells. In this study, we enriched OP3 cells by double density gradient centrifugation and obtained the first closed genome of an apparently clonal OP3 cell population by applying metagenomics and PCR for gap closure. Filaments of acetoclastic Methanosaeta, the largest morphotype in the culture community, contained empty cells, cells devoid of rRNA or of both rRNA and DNA, and dead cells according to transmission electron microscopy (TEM), thin-section TEM, scanning electron microscopy (SEM), catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), and LIVE/DEAD imaging. OP3 LiM cells were ultramicrobacteria (200 to 300 nm in diameter) and showed two physiological stages in CARD-FISH fluorescence signals: strong signals of OP3 LiM cells attached to Bacteria and to Archaea indicated many rRNA molecules and an active metabolism, whereas free-living OP3 cells had weak signals. Metaproteomics revealed that OP3 LiM lives with highly expressed secreted proteins involved in depolymerization and uptake of macromolecules and an active glycolysis and energy conservation by the utilization of pyruvate via a pyruvate:ferredoxin oxidoreductase and an Rnf complex (ferredoxin:NAD oxidoreductase). Besides sugar fermentation, a nucleotidyl transferase may contribute to energy conservation by phosphorolysis, the phosphate-dependent depolymerization of nucleic acids. Thin-section TEM showed distinctive structures of predation. Our study demonstrated a predatory metabolism for OP3 LiM cells, and therefore, we propose the name "Candidatus Velamenicoccus archaeovorus" gen. nov., sp. nov., for OP3 LiM. IMPORTANCE Epibiotic bacteria are known to live on and off bacterial cells. Here, we describe the ultramicrobacterial anaerobic epibiont OP3 LiM living on Archaea and Bacteria. We detected sick and dead cells of the filamentous archaeon Methanosaeta in slowly growing methanogenic cultures. OP3 LiM lives as a sugar fermenter, likely on polysaccharides from outer membranes, and has the genomic potential to live as a syntroph. The predatory lifestyle of OP3 LiM was supported by its genome, the first closed genome for the phylum "Candidatus Omnitrophica," and by images of cell-to-cell contact with prey cells. We propose naming OP3 LiM "Candidatus Velamenicoccus archaeovorus." Its metabolic versatility explains the ubiquitous presence of "Candidatus Omnitrophica" 3 in anoxic habitats and gives ultramicrobacterial epibionts an important role in the recycling and remineralization of microbial biomass. The removal of polysaccharides from outer membranes by ultramicrobacteria may also influence biological interactions between pro- and eukaryotes.

Methanococcoides orientis sp. nov., a methylotrophic methanogen isolated from sediment of the East China Sea.

A novel methylotrophic methanogen Methanococcoides orientis sp. nov. was isolated from East China Sea sediment. Type strain LMO-1T of Methanococcoides orientis sp. nov. was irregular 1-2 µm cocci without flagella. Strain LMO-1T could utilize a variety of methylated compounds including methanol, methylamine, dimethylamine and trimethylamine for growth and methanogenesis, while H2/CO2 or acetate could not be used for growth or methanogenesis. Optimum growth temperature was 30-35 °C, optimum pH range for growth was 7.0-7.5, while the optimum salinity spectrum for growth was 1.0%-5.0% NaCl. Based on 16S rRNA gene similarity, strain LMO-1T belongs to Methanococcoides, with the highest sequence similarity to Methanococcoides methylutens DSM 2657T (99.8 %), Methanococcoides vulcani SLH33T(99.4 %), followed by Methanococcoides alaskense AK-5T(98.1 %), Methanococcoides burtonii DSM 6242T (98.0 %). Digital DNA-DNA hybridization also showed highest similarity with Methanococcoides methylutens DSM 2657T, with the value of 58.4 %. The average nucleotide identity between strain LMO-1T and Methanococcoides methylutens DSM 2657T was 94.06 %. In summary, LMO-1T represents a novel species of the genus Methaococcoides, for which the name Methanococcoides orientis sp. nov. is proposed. The type strain is LMO-1T (=MCCC 4K00106T=JCM 39195T).

The Structure of the Dimeric State of IscU Harboring Two Adjacent [2Fe-2S] Clusters Provides Mechanistic Insights into Cluster Conversion to [4Fe-4S].

IscU serves as a scaffold for the de novo assembly of a [2Fe-2S] cluster prior to its delivery to recipient protein. It has also been proposed that on one dimer of bacterial IscU, two [2Fe-2S] clusters can be converted into a single [4Fe-4S] cluster. However, lack of structural information about the dimeric state of IscU has hindered our understanding of the underlying mechanisms. In this study, we determine the X-ray crystal structure of IscU from the thermophilic archaeon Methanothrix thermoacetophila and demonstrate a dimer structure of IscU in which two [2Fe-2S] clusters are facing each other in close proximity at the dimer interface. Our structure also reveals for the first time that Asp40 serves as a fourth ligand to the [2Fe-2S] cluster with three Cys ligands in each monomer, consistent with previous spectroscopic data. We confirm by EPR spectroscopic analysis that in solution two adjacent [2Fe-2S] clusters in the wild-type dimer are converted to a [4Fe-4S] cluster via reductive coupling. Furthermore, we find that the H106A substitution abolishes the reductive conversion to the [4Fe-4S] cluster without structural alteration, suggesting that His106 is functionally involved in this process. Overall, these findings provide a structural explanation for the assembly and conversion of Fe-S clusters on IscU and highlight a dynamic process that advances via association and dissociation of the IscU dimer.

Post-transcriptional regulation is involved in the cold-active methanol-based methanogenic pathway of a psychrophilic methanogen.

The methanol-derived methanogenetic pathway contributes to bulk methane production in cold regions, but the cold adaptation mechanisms are obscure. This work investigated the mechanisms using a psychrophilic methylotrophic methanogen Methanolobus psychrophilus R15. R15 possesses two mtaCB operon paralogues-encoding methanol:corrinoid methyltransferase that is key to methanol-based methanogenesis. Molecular combined methanogenic assays determined that MtaC1 is important in methanogenesis at the optimal temperature of 18°C, but MtaC2 can be a cold-adaptive paralogue by highly upregulated at 8°C. The 5'P-seq and 5'RACE all assayed that processing occurred at the 5' untranslated region (5'-UTR) of mtaC2; reporter genes detected higher protein expression, and RNA half-life experiments assayed prolonged lifespan of the processed transcript. Therefore, mtaC2 5'-UTR processing to move the bulged structure elevated both the translation efficiency and transcript stability. 5'P-seq, quantitative RT-PCR and northern blot all identified enhanced mtaC2 5'-UTR processing at 8°C, which could contribute to the upregulation of mtaC2 at cold. The R15 cell extract contains an endoribonuclease cleaving an identified 10 nt-processing motif and the native mtaC2 5'-UTR particularly folded at 8°C. Therefore, this study revealed a 5'-UTR processing mediated post-transcriptional regulation mechanism controlling the cold-adaptive methanol-supported methanogenetic pathway, which may be used by other methylotrophic methanogens.

Methyl-compounds driven benthic carbon cycling in the sulfate-reducing sediments of South China Sea.

Methane is a potent greenhouse gas; methane production and consumption within seafloor sediments has generated intense interest. Anaerobic oxidation of methane (AOM) and methanogenesis (MOG) primarily occur at the depth of the sulfate-methane transition zone or underlying sediment respectively. Methanogenesis can also occur in the sulfate-reducing sediments through the utilization of non-competitive methylated compounds; however, the occurrence and importance of this process are not fully understood. Here, we combined a variety of data, including geochemical measurements, rate measurements and molecular analyses to demonstrate the presence of a cryptic methane cycle in sulfate-reducing sediments from the continental shelf of the northern South China Sea. The abundance of methanogenic substrates as well as the high MOG rates from methylated compounds indicated that methylotrophic methanogenesis was the dominant methanogenic pathway; this conclusion was further supported by the presence of the methylotrophic genus Methanococcoides. High potential rates of AOM were observed in the sediments, indicating that methane produced in situ could be oxidized simultaneously by AOM, presumably by ANME-2a/b as indicated by 16S rRNA gene analysis. A significant correlation between the relative abundance of methanogens and methanotrophs was observed over sediment depth, indicating that methylotrophic methanogenesis could potentially fuel AOM in this environment. In addition, higher potential rates of AOM than sulfate reduction rates at in situ methane conditions were observed, making alternative electron acceptors important to support AOM in sulfate-reducing sediment. AOM rates were stimulated by the addition of Fe/Mn oxides, suggesting AOM could be partially coupled to metal oxide reduction. These results suggest that methyl-compounds driven methane production drives a cryptic methane cycling and fuels AOM coupled to the reduction of sulfate and other electron acceptors.

Methanolobus halotolerans sp. nov., isolated from the saline Lake Tus in Siberia.

A halotolerant, psychrotolerant and methylotrophic methanogen, strain SY-01T, was isolated from the saline Lake Tus in Siberia. Cells of strain SY-01T were non-motile, cocci and 0.8-1.0 µm in diameter. The only methanogenic substrate utilized by strain SY-01T was methanol. The temperature range of growth for strain SY-01T was from 4 to 40 °C and the optimal temperature for growth was 30 °C. The pH range of growth was from pH 7.2 to 9.0, with optimal growth at pH 8.0. The NaCl range of growth was 0-1.55 M with optimal growth at 0.51 M NaCl. The G+C content of the genome of strain SY-01T was 43.6 mol % as determined by genome sequencing. Phylogenetic analysis revealed that strain SY-01T was most closely related to Methanolobus zinderi SD1T (97.3 % 16S rRNA gene sequence similarity), and had 95.5-97.2 % similarities to other Methanolobus species with valid names. Genome relatedness between strain SY-01T and DSM 21339T was computed using average nucleotide identity and digital DNA-DNAhybridization, which yielded values of 79.7 and 21.7 %, respectively. Based on morphological, phenotypic, phylogenetic and genomic relatedness data presented here, it is evident that strain SY-01T represents a novel species of the genus Methanolobus, and the name Methanolobus halotolerans sp. nov. is proposed. The type strain is SY-01T (=BCRC AR10051T=NBRC 113166 T=DSM 107642T).

Biochemical and phylogenetic characterization of a monomeric isocitrate dehydrogenase from a marine methanogenic archaeon Methanococcoides methylutens.

Monomeric isocitrate dehydrogenase (IDH) stands for a separated subgroup among IDH protein family. Up to now, all reported monomeric IDHs are from prokaryotes. Here, a monomeric IDH from a marine methanogenic archaeon Methanococcoides methylutens (MmIDH) was reported for the first time. BLAST search demonstrated that only a few marine archaea encode the monomeric IDH and all these organisms are methylotrophic. MmIDH shows the highest homology (~ 70%) to the monomeric IDHs from some marine bacteria, suggesting a lateral gene transfer event between marine bacteria and archaea. The monomeric state of MmIDH was determined by size exclusion chromatography. MmIDH is divalent cation-dependent and Mn2+ is the most favored. Kinetic analysis showed that MmIDH is highly specific to NADP+ and cannot utilize the NAD+. The optimal temperature for MmIDH activity is 50 °C and the optimal pH is 8.2. Heat inactivation assay revealed that MmIDH is a mesophilic enzyme. It sustained 50% activity after incubation at 39 °C for 20 min. Moreover, the putative coenzyme binding residues (His590, Arg601, and Arg650) of MmIDH were explored by mutagenesis. The triple mutant H590L/R601D/R650S displayed a 5.93-fold preference for NAD+ over NADP+, indicating that the coenzyme specificity of MmIDH was significantly switched from NADP+ to NAD+ by three key mutations.

Methanogenesis in the Lake Elton saline aquatic system.

Cultivation and molecular approaches were used to study methanogenesis in saline aquatic system of the Lake Elton (southern Russia), the largest hypersaline lake in Europe. The potential rates of hydrogenotrophic, acetoclastic, methylotrophic and methyl-reducing methanogenesis and diversity of the growth-enriched for by adding electron donors methanogenic communities were studied in the sediment slurry incubations at salinity range from 7 to 275 g/L. The most active pathway detected at all salinities was methylotrophic with a dominance of Methanohalobium and Methanohalophilus genera, at salt saturation and moderately halophilic Methanolobus and Methanococcoides at lower salinity. The absence of methane production from acetate, formate and H2/CO2 under hypersaline conditions was most probably associated with the energy constraints. The contribution of hydrogenotrophic, acetoclastic, and methyl-reducing methanogens to the community increases with a decrease in salinity. Temperature might play an important regulatory function in hypersaline habitats; i.e. methylotrophic methanogens and hydrogenotrophic sulfate-reducing bacteria (SRB) outcompeting methyl-reducing methanogens under mesophilic conditions, and vice versa under thermophilic conditions. An active methane production together with negligible methane oxidation makes hypersaline environments a potential source of methane emission.

A CUGGU/UUGGU-specific MazF homologue from Methanohalobium evestigatum.

MazF is a sequence-specific endoribonuclease or mRNA interferase, which cleaves RNA at a specific sequence. Since the expression of a specific gene or a group of specific genes can be regulated by MazF, expanding the repertoire of recognition sequences by MazF mRNA interferases is highly desirable for biotechnological and medical applications. Here, we identified a gene for a MazF homologue (MazFme) from Methanohalobium evestigatum, an extremely halophilic archaeon. In order to suppress the toxicity of MazFme to the E. coli cells, the C-terminal half of the cognate antitoxin MazEme was fused to the N-terminal end of MazFme. Since the fusion of the C-terminal half of MazEme to MazFme was able to neutralize MazFme toxicity, the MazEme-MazFme fusion protein was expressed in a large amount without any toxic effects. After purification of the MazEme, the free MazFme RNA cleavage specificity was determined by primer extension and synthetic ribonucleotides, revealing that MazFme is a CUGGU/UUGGU-specific endoribonuclease.

Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis.

Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an 'all-or-none' pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry.

A peptide adhesive molded by magnesium glues Rubisco's subunits together.

Rubisco enzymes play central roles in carbon fixation, with potential importance in biotechnology, but have eluded a full description of their multistep assembly and function. A new article describes the fascinating discovery that some archaeal Rubiscos contain a built-in assembly domain inserted into an otherwise canonical Rubisco fold, providing a tremendous expansion of our understanding of the diversity of naturally occurring Rubiscos.

A unique structural domain in Methanococcoides burtonii ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts as a small subunit mimic.

The catalytic inefficiencies of the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity. Strategies to engineer more efficient plant Rubiscos have been hampered by evolutionary constraints, prompting interest in Rubisco isoforms from non-photosynthetic organisms. The methanogenic archaeon Methanococcoides burtonii contains a Rubisco isoform that functions to scavenge the ribulose-1,5-bisphosphate (RuBP) by-product of purine/pyrimidine metabolism. The crystal structure of M. burtonii Rubisco (MbR) presented here at 2.6 Å resolution is composed of catalytic large subunits (LSu) assembled into pentamers of dimers, (L2)5, and differs from Rubiscos from higher plants where LSus are glued together by small subunits (SSu) into hexadecameric L8S8 enzymes. MbR contains a unique 29-amino acid insertion near the C terminus, which folds as a separate domain in the structure. This domain, which is visualized for the first time in this study, is located in a similar position to SSus in L8S8 enzymes between LSus of adjacent L2 dimers, where negatively charged residues coordinate around a Mg2+ ion in a fashion that suggests this domain may be important for the assembly process. The Rubisco assembly domain is thus an inbuilt SSu mimic that concentrates L2 dimers. MbR assembly is ligand-stimulated, and we show that only 6-carbon molecules with a particular stereochemistry at the C3 carbon can induce oligomerization. Based on MbR structure, subunit arrangement, sequence, phylogenetic distribution, and function, MbR and a subset of Rubiscos from the Methanosarcinales order are proposed to belong to a new Rubisco subgroup, named form IIIB.

Comparative genomics and physiology of the genus Methanohalophilus, a prevalent methanogen in hydraulically fractured shale.

About 60% of natural gas production in the United States comes from hydraulic fracturing of unconventional reservoirs, such as shales or organic-rich micrites. This process inoculates and enriches for halotolerant microorganisms in these reservoirs over time, resulting in a saline ecosystem that includes methane producing archaea. Here, we survey the biogeography of methanogens across unconventional reservoirs, and report that members of genus Methanohalophilus are recovered from every hydraulically fractured unconventional reservoir sampled by metagenomics. We provide the first genomic sequencing of three isolate genomes, as well as two metagenome assembled genomes (MAGs). Utilizing six other previously sequenced isolate genomes and MAGs, we perform comparative analysis of the 11 genomes representing this genus. This genomic investigation revealed distinctions between surface and subsurface derived genomes that are consistent with constraints encountered in each environment. Genotypic differences were also uncovered between isolate genomes recovered from the same well, suggesting niche partitioning among closely related strains. These genomic substrate utilization predictions were then confirmed by physiological investigation. Fine-scale microdiversity was observed in CRISPR-Cas systems of Methanohalophilus, with genomes from geographically distinct unconventional reservoirs sharing spacers targeting the same viral population. These findings have implications for augmentation strategies resulting in enhanced biogenic methane production in hydraulically fractured unconventional reservoirs.

Increases in temperature and nutrient availability positively affect methane-cycling microorganisms in Arctic thermokarst lake sediments.

Arctic permafrost soils store large amounts of organic matter that is sensitive to temperature increases and subsequent microbial degradation to methane (CH 4 ) and carbon dioxide (CO 2 ). Here, we studied methanogenic and methanotrophic activity and community composition in thermokarst lake sediments from Utqiag˙vik (formerly Barrow), Alaska. This experiment was carried out under in situ temperature conditions (4°C) and the IPCC 2013 Arctic climate change scenario (10°C) after addition of methanogenic and methanotrophic substrates for nearly a year. Trimethylamine (TMA) amendment with warming showed highest maximum CH 4 production rates, being 30% higher at 10°C than at 4°C. Maximum methanotrophic rates increased by up to 57% at 10°C compared to 4°C. 16S rRNA gene sequencing indicated high relative abundance of Methanosarcinaceae in TMA amended incubations, and for methanotrophic incubations Methylococcaeae were highly enriched. Anaerobic methanotrophic activity with nitrite or nitrate as electron acceptor was not detected. This study indicates that the methane cycling microbial community can adapt to temperature increases and that their activity is highly dependent on substrate availability.

Methanolobus psychrotolerans sp. nov., a psychrotolerant methanoarchaeon isolated from a saline meromictic lake in Siberia.

A psychrotolerant, methylotrophic methanogen, strain YSF-03T, was isolated from the saline meromictic Lake Shira in Siberia. Cells of strain YSF-03T were non-motile, irregular cocci and 0.8-1.2 µm in diameter. The methanogenic substrates utilized by strain YSF-03T were methanol and trimethylamine. The temperature range of growth for strain YSF-03T was from 0 to 37 °C. The optimum growth conditions were 30-37 °C, pH 7.0-7.4 and 0.17 M NaCl. The G+C content of the genome of strain YSF-03T was 41.3 mol%. Phylogenetic analysis revealed that strain YSF-03T was most closely related to Methanolobus profundi MobMT (98.15 % similarity in 16S rRNA gene sequence). Genome relatedness between strain YSF-03T and MobMT was computed using the Genome-to-Genome Distance Calculator and average nucleotide identity, which gave values of 23.5 and 79.3 %, respectively. Based on the morphological, phenotypic, phylogenetic and genomic relatedness data presented here, it is evident that strain YSF-03T represents a novel species of the genus Methanolobus, for which the name Methanolobus psychrotolerans sp. nov. is proposed. The type strain is YSF-03T (=BCRC AR10049T=DSM 104044T=NBRC 112514T).

Pyrrolysyl-tRNA Synthetase with a Unique Architecture Enhances the Availability of Lysine Derivatives in Synthetic Genetic Codes.

Genetic code expansion has largely relied on two types of the tRNA-aminoacyl-tRNA synthetase pairs. One involves pyrrolysyl-tRNA synthetase (PylRS), which is used to incorporate various lysine derivatives into proteins. The widely used PylRS from Methanosarcinaceae comprises two distinct domains while the bacterial molecules consist of two separate polypeptides. The recently identified PylRS from Candidatus Methanomethylophilus alvus (CMaPylRS) is a single-domain, one-polypeptide enzyme that belongs to a third category. In the present study, we showed that the PylRS-tRNAPyl pair from C. M. alvus can incorporate lysine derivatives much more efficiently (up to 14-times) than Methanosarcinaceae PylRSs in Escherichia coli cell-based and cell-free systems. Then we investigated the tRNA and amino-acid recognition by CMaPylRS. The cognate tRNAPyl has two structural idiosyncrasies: no connecting nucleotide between the acceptor and D stems and an additional nucleotide in the anticodon stem and it was found that these features are hardly recognized by CMaPylRS. Lastly, the Tyr126Ala and Met129Leu substitutions at the amino-acid binding pocket were shown to allow CMaPylRS to recognize various derivatives of the bulky Nε-benzyloxycarbonyl-l-lysine (ZLys). With the high incorporation efficiency and the amenability to engineering, CMaPylRS would enhance the availability of lysine derivatives in expanded codes.