Diverse methylotrophic methanogenic archaea cause high methane emissions from seagrass meadows.

Marine coastlines colonized by seagrasses are a net source of methane to the atmosphere. However, methane emissions from these environments are still poorly constrained, and the underlying processes and responsible microorganisms remain largely unknown. Here, we investigated methane turnover in seagrass meadows of Posidonia oceanica in the Mediterranean Sea. The underlying sediments exhibited median net fluxes of methane into the water column of ca. 106 µmol CH4 ⋅ m-2 ⋅ d-1 Our data show that this methane production was sustained by methylated compounds produced by the plant, rather than by fermentation of buried organic carbon. Interestingly, methane production was maintained long after the living plant died off, likely due to the persistence of methylated compounds, such as choline, betaines, and dimethylsulfoniopropionate, in detached plant leaves and rhizomes. We recovered multiple mcrA gene sequences, encoding for methyl-coenzyme M reductase (Mcr), the key methanogenic enzyme, from the seagrass sediments. Most retrieved mcrA gene sequences were affiliated with a clade of divergent Mcr and belonged to the uncultured Candidatus Helarchaeota of the Asgard superphylum, suggesting a possible involvement of these divergent Mcr in methane metabolism. Taken together, our findings identify the mechanisms controlling methane emissions from these important blue carbon ecosystems.

Ecogenomics Reveals Microbial Metabolic Networks in a Psychrophilic Methanogenic Bioreactor Treating Soy Sauce Production Wastewater.

An ecogenomic analysis of the methanogenic microbial community in a laboratory-scale up-flow anaerobic sludge blanket (UASB) reactor treating soy sauce-processing wastewater revealed a synergistic metabolic network. Granular sludge samples were collected from the UASB reactor operated under psychrophilic (20°C) conditions with a COD removal rate >75%. A 16S rRNA gene amplicon sequencing-based microbial community analysis classified the major microbial taxa as Methanothrix, Methanobacterium, Pelotomaculaceae, Syntrophomonadaceae, Solidesulfovibrio, and members of the phyla Synergistota and Bacteroidota. Draft genomes of dominant microbial populations were recovered by metagenomic shotgun sequencing. Metagenomic- and metatranscriptomic-assisted metabolic reconstructions indicated that Synergistota- and Bacteroidota-related organisms play major roles in the degradation of amino acids. A metagenomic bin of the uncultured Bacteroidales 4484-276 clade encodes genes for proteins that may function in the catabolism of phenylalanine and tyrosine under microaerobic conditions. Syntrophomonadaceae and Pelotomaculaceae oxidize fatty acid byproducts presumably derived from the degradation of amino acids in syntrophic association with aceticlastic and hydrogenotrophic methanogen populations. Solidesulfovibrio organisms are responsible for the reduction of sulfite and may support the activity of hydrogenotrophic methanogens and other microbial populations by providing hydrogen and ammonia using nitrogen fixation-related proteins. Overall, functionally diverse anaerobic organisms unite to form a metabolic network that performs the complete degradation of amino acids in the psychrophilic methanogenic microbiota.

Genomic Insights into the Ecological Role and Evolution of a Novel Thermoplasmata Order, "Candidatus Sysuiplasmatales".

Recent omics studies have provided invaluable insights into the metabolic potential, adaptation, and evolution of novel archaeal lineages from a variety of extreme environments. We utilized a genome-resolved metagenomic approach to recover eight medium- to high-quality metagenome-assembled genomes (MAGs) that likely represent a new order ("Candidatus Sysuiplasmatales") in the class Thermoplasmata from mine tailings and acid mine drainage (AMD) sediments sampled from two copper mines in South China. 16S rRNA gene-based analyses revealed a narrow habitat range for these uncultured archaea limited to AMD and hot spring-related environments. Metabolic reconstruction indicated a facultatively anaerobic heterotrophic lifestyle. This may allow the archaea to adapt to oxygen fluctuations and is thus in marked contrast to the majority of lineages in the domain Archaea, which typically show obligately anaerobic metabolisms. Notably, "Ca. Sysuiplasmatales" could conserve energy through degradation of fatty acids, amino acid metabolism, and oxidation of reduced inorganic sulfur compounds (RISCs), suggesting that they may contribute to acid generation in the extreme mine environments. Unlike the closely related orders Methanomassiliicoccales and "Candidatus Gimiplasmatales," "Ca. Sysuiplasmatales" lacks the capacity to perform methanogenesis and carbon fixation. Ancestral state reconstruction indicated that "Ca. Sysuiplasmatales," the closely related orders Methanomassiliicoccales and "Ca. Gimiplasmatales," and the orders SG8-5 and RBG-16-68-12 originated from a facultatively anaerobic ancestor capable of carbon fixation via the bacterial-type H4F Wood-Ljungdahl pathway (WLP). Their metabolic divergence might be attributed to different evolutionary paths. IMPORTANCE A wide array of archaea populate Earth's extreme environments; therefore, they may play important roles in mediating biogeochemical processes such as iron and sulfur cycling. However, our knowledge of archaeal biology and evolution is still limited, since the majority of the archaeal diversity is uncultured. For instance, most order-level lineages except Thermoplasmatales, Aciduliprofundales, and Methanomassiliicoccales within Thermoplasmata do not have cultured representatives. Here, we report the discovery and genomic characterization of a novel order, "Ca. Sysuiplasmatales," within Thermoplasmata in extremely acidic mine environments. "Ca. Sysuiplasmatales" are inferred to be facultatively anaerobic heterotrophs and likely contribute to acid generation through the oxidation of RISCs. The physiological divergence between "Ca. Sysuiplasmatales" and closely related Thermoplasmata lineages may be attributed to different evolutionary paths. These results expand our knowledge of archaea in the extreme mine ecosystem.

Comparison of microbial community structures between mesophilic and thermophilic anaerobic digestion of vegetable waste.

The anaerobic digestion performance correlates with the functional microbial community. Mesophilic and thermophilic digestions of vegetable waste were conducted, and dynamics of the microbial community were investigated. The mesophilic and thermophilic collapsed stages occurred at organic loading rates of 1.5 and 2.0 g VS/(L d) due to the accumulation of volatile fatty acids with final concentrations of 2276 and 6476 mg/L, respectively. A high concentration of volatile fatty acids caused the severe inhibition of methanogens, which finally led to the imbalance between acetogenesis and methanogenesis. The mesophilic digestion exhibited a higher microbial diversity and richness than the thermophilic digestion. Syntrophic acetate-oxidizing coupled with hydrogenotrophic methanogenesis was the dominant pathway in the thermophilic stable system, and acetoclastic methanogenesis in the mesophilic stable system. The dominant acidogens, syntrophus, and methanogens were unclassified_f__Anaerolineaceae (8.68%), Candidatus_Cloacamonas (19.70%), Methanosaeta (6.10%), and Methanosarcina (4.08%) in the mesophilic stable stage, and Anaerobaculum (12.59%), Syntrophaceticus (4.84%), Methanosarcina (30.58%), and Methanothermobacter (3.17%) in thermophilic stable stage. Spirochaetae and Thermotogae phyla were the characteristic microorganisms in the mesophilic and thermophilic collapsed stages, respectively. These findings provided valuable information for the deep understanding of the difference of the microbial community and methane-producing mechanism between mesophilic and thermophilic digestion of vegetable waste.

Distinctive distributions of halophilic Archaea across hypersaline environments within the Qaidam Basin of China.

Halophilic Archaea are widely distributed globally in hypersaline environments. However, little is known of how dominant halophilic archaeal genera are distributed across environments and how they may co-associate across ecosystems. Here, the archaeal community composition and diversity from hypersaline environments (> 300 g/L salinity; total of 33 samples) in the Qaidam Basin of China were investigated using high-throughput Illumina sequencing of 16S rRNA genes. The archaeal communities (total of 3,419 OTUs) were dominated by the class Halobacteria (31.7-99.6% relative abundances) within the phylum Euryarchaeota (90.8-99.9%). Five predominant taxa, including Halorubrum, Halobacterium, Halopenitus, Methanothrix, and Halomicrobium, were observed across most samples. However, several distinct genera were associated with individual samples and were inconsistently distributed across samples, which contrast with previous studies of hypersaline archaeal communities. Additionally, co-occurrence network analysis indicated that five network clusters were present and potentially reflective of interspecies interactions among the environments, including three clusters (clusters II, III, and IV) comprising halophilic archaeal taxa within the Halobacteriaceae and Haloferacaceae families. In addition, two other clusters (clusters I and V) were identified that comprised methanogens. Finally, salinity comprising ionic concentrations (in the order of Na+ > Ca2+ > Mg2+) and pH were most correlated with taxonomic distributions across sample sites.

Thermoplasmata and Nitrososphaeria as dominant archaeal members in acid mine drainage sediment of Malanjkhand Copper Project, India.

Acid mine drainage (AMD) harbors all three life forms in spite of its toxic and hazardous nature. In comparison to bacterial diversity, an in-depth understanding of the archaeal diversity in AMD and their ecological significance remain less explored. Archaeal populations are known to play significant roles in various biogeochemical cycles within the AMD ecosystem, and it is imperative to have a deeper understanding of archaeal diversity and their functional potential in AMD system. The present study is aimed to understand the archaeal diversity of an AMD sediment of Malanjkhand Copper Project, India through archaea specific V6 region of 16S rRNA gene amplicon sequencing. Geochemical data confirmed the acidic, toxic, heavy metal-rich nature of the sample. Archaea specific V6-16S rRNA gene amplicon data showed a predominance of Thermoplasmata (BSLdp215, uncultured Thermoplasmata, and Thermoplasmataceae) and Nitrososphaeria (Nitrosotaleaceae) members constituting ~ 95% of the archaeal community. Uncultured members of Bathyarchaeia, Group 1.1c, Hydrothermarchaeota, and Methanomassiliicoccales along with Methanobacteriaceae, Methanocellaceae, Haloferaceae, Methanosaetaceae, and Methanoregulaceae constituted the part of rare taxa. Analysis of sequence reads indicated that apart from their close ecological relevance, members of the Thermoplasmata present in Malanjkhand AMD were mostly involved in chemoheterotrophy, Fe/S redox cycling, and with heavy metal resistance, while the Nitrososphaeria members were responsible for ammonia oxidation and fixation of HCO3- through 3-hydroxypropionate/4-hydroxybutyrate cycle at low pH and oligotrophic environment which subsequently played an important role in nitrification process in AMD sediment. Overall, the present study elucidated the biogeochemical significance of archaeal populations inhabiting the toxic AMD environment.

Archaeal roots of intramembrane aspartyl protease siblings signal peptide peptidase and presenilin.

Signal peptides help newly synthesized proteins reach the cell membrane or be secreted. As part of a biological process key to immune response and surveillance in humans, and associated with diseases, for example, Alzheimer, remnant signal peptides and other transmembrane segments are proteolyzed by the intramembrane aspartyl protease (IAP) enzyme family. Here, we identified IAP orthologs throughout the tree of life. In addition to eukaryotes, IAPs are encoded in metabolically diverse archaea from a wide range of environments. We found three distinct clades of archaeal IAPs: (a) Euryarchaeota (eg, halophilic Halobacteriales, methanogenic Methanosarcinales and Methanomicrobiales, marine Poseidoniales, acidophilic Thermoplasmatales, hyperthermophilic Archaeoglobus spp.), (b) DPANN, and (c) Bathyarchaeota, Crenarchaeota, and Asgard. IAPs were also present in bacterial genomes from uncultivated members of Candidate Phylum Radiation, perhaps due to horizontal gene transfer from DPANN archaeal lineages. Sequence analysis of the catalytic motif YD…GXGD (where X is any amino acid) in IAPs from archaea and bacteria reveals WD in Lokiarchaeota and many residue types in the X position. Gene neighborhood analysis in halophilic archaea shows IAP genes near corrinoid transporters (btuCDF genes). In marine Euryarchaeota, a putative BtuF-like domain is found in N-terminus of the IAP gene, suggesting a role for these IAPs in metal ion cofactor or other nutrient scavenging. Interestingly, eukaryotic IAP family members appear to have evolved either from Euryarchaeota or from Asgard archaea. Taken together, our phylogenetic and bioinformatics analysis should prompt experiments to probe the biological roles of IAPs in prokaryotic secretomes.

Association of fibre degradation with ruminal dissolved hydrogen in growing beef bulls fed with two types of forages.

The present study investigated the association between fibre degradation and the concentration of dissolved molecular hydrogen (H2) in the rumen. Napier grass (NG) silage and corn stover (CS) silage were compared as forages with contrasting structures and degradation patterns. In the first experiment, CS silage had greater 48-h DM, neutral-detergent fibre (NDF) and acid-detergent fibre degradation, and total gas and methane (CH4) volumes, and lower 48-h H2 volume than NG silage in 48-h in vitro incubations. In the second experiment, twenty-four growing beef bulls were fed diets including 55 % (DM basis) NG or CS silages. Bulls fed the CS diet had greater DM intake (DMI), average daily gain, total-tract digestibility of OM and NDF, ruminal dissolved methane (dCH4) concentration and gene copies of protozoa, methanogens, Ruminococcus albus and R. flavefaciens, and had lower ruminal dH2 concentration, and molar proportions of valerate and isovalerate, in comparison with those fed the NG diet. There was a negative correlation between dH2 concentration and NDF digestibility in bulls fed the CS diet, and a lack of relationship between dH2 concentration and NDF digestibility with the NG diet. In summary, the fibre of CS silage was more easily degraded by rumen microorganisms than that of NG silage. Increased dCH4 concentration with the CS diet presumably led to the decreased ruminal dH2 concentration, which may be helpful for fibre degradation and growth of fibrolytic micro-organisms in the rumen.

Evidence for non-methanogenic metabolisms in globally distributed archaeal clades basal to the Methanomassiliicoccales.

Recent discoveries of mcr and mcr-like genes in genomes from diverse archaeal lineages suggest that methane metabolism is an ancient pathway with a complicated evolutionary history. One conventional view is that methanogenesis is an ancestral metabolism of the class Thermoplasmata. Through comparative genomic analysis of 12 Thermoplasmata metagenome-assembled genomes (MAGs) basal to the Methanomassiliicoccales, we show that these microorganisms do not encode the genes required for methanogenesis. Further analysis of 770 Ca. Thermoplasmatota genomes/MAGs found no evidence of mcrA homologues outside of the Methanomassiliicoccales. Together, these results suggest that methanogenesis was laterally acquired by an ancestor of the Methanomassiliicoccales. The 12 analysed MAGs include representatives from four orders basal to the Methanomassiliicoccales, including a high-quality MAG that likely represents a new order, Ca. Lunaplasma lacustris ord. nov. sp. nov. These MAGs are predicted to use diverse energy conservation pathways, including heterotrophy, sulfur and hydrogen metabolism, denitrification, and fermentation. Two lineages are widespread among anoxic, sedimentary environments, whereas Ca. Lunaplasma lacustris has thus far only been detected in alpine caves and subarctic lake sediments. These findings advance our understanding of the metabolic potential, ecology, and global distribution of the Thermoplasmata and provide insight into the evolutionary history of methanogenesis within the Ca. Thermoplasmatota.

Pan-genome analysis and ancestral state reconstruction of class halobacteria: probability of a new super-order.

Halobacteria, a class of Euryarchaeota are extremely halophilic archaea that can adapt to a wide range of salt concentration generally from 10% NaCl to saturated salt concentration of 32% NaCl. It consists of the orders: Halobacteriales, Haloferaciales and Natriabales. Pan-genome analysis of class Halobacteria was done to explore the core (300) and variable components (Softcore: 998, Cloud:36531, Shell:11784). The core component revealed genes of replication, transcription, translation and repair, whereas the variable component had a major portion of environmental information processing. The pan-gene matrix was mapped onto the core-gene tree to find the ancestral (44.8%) and derived genes (55.1%) of the Last Common Ancestor of Halobacteria. A High percentage of derived genes along with presence of transformation and conjugation genes indicate the occurrence of horizontal gene transfer during the evolution of Halobacteria. A Core and pan-gene tree were also constructed to infer a phylogeny which implicated on the new super-order comprising of Natrialbales and Halobacteriales.

Lower methane emissions were associated with higher abundance of ruminal Prevotella in a cohort of Colombian buffalos.

BACKGROUND: Ruminants burp massive amounts of methane into the atmosphere and significantly contribute to the deposition of greenhouse gases and the consequent global warming. It is therefore urgent to devise strategies to mitigate ruminant's methane emissions to alleviate climate change. Ruminal methanogenesis is accomplished by a series of methanogen archaea in the phylum Euryarchaeota, which piggyback into carbohydrate fermentation by utilizing residual hydrogen to produce methane. Abundance of methanogens, therefore, is expected to affect methane production. Furthermore, availability of hydrogen produced by cellulolytic bacteria acting upstream of methanogens is a rate-limiting factor for methane production. The aim of our study was to identify microbes associated with the production of methane which would constitute the basis for the design of mitigation strategies. RESULTS: Moderate differences in the abundance of methanogens were observed between groups. In addition, we present three lines of evidence suggesting an apparent higher abundance of a consortium of Prevotella species in animals with lower methane emissions. First, taxonomic classification revealed increased abundance of at least 29 species of Prevotella. Second, metagenome assembly identified increased abundance of Prevotella ruminicola and another species of Prevotella. Third, metabolic profiling of predicted proteins uncovered 25 enzymes with homology to Prevotella proteins more abundant in the low methane emissions group. CONCLUSIONS: We propose that higher abundance of ruminal Prevotella increases the production of propionic acid and, in doing so, reduces the amount of hydrogen available for methanogenesis. However, further experimentation is required to ascertain the role of Prevotella on methane production and its potential to act as a methane production mitigator.

Hikarchaeia demonstrate an intermediate stage in the methanogen-to-halophile transition.

Halobacteria (henceforth: Haloarchaea) are predominantly aerobic halophiles that are thought to have evolved from anaerobic methanogens. This remarkable transformation most likely involved an extensive influx of bacterial genes. Whether it entailed a single massive transfer event or a gradual stream of transfers remains a matter of debate. To address this, genomes that descend from methanogen-to-halophile intermediates are necessary. Here, we present five such near-complete genomes of Marine Group IV archaea (Hikarchaeia), the closest known relatives of Haloarchaea. Their inclusion in gene tree-aware ancestral reconstructions reveals an intermediate stage that had already lost a large number of genes, including nearly all of those involved in methanogenesis and the Wood-Ljungdahl pathway. In contrast, the last Haloarchaea common ancestor gained a large number of genes and expanded its aerobic respiration and salt/UV resistance gene repertoire. Our results suggest that complex and gradual patterns of gain and loss shaped the methanogen-to-halophile transition.

Several ways one goal-methanogenesis from unconventional substrates.

Methane is the second most important greenhouse gas on earth. It is produced by methanogenic archaea, which play an important role in the global carbon cycle. Three main methanogenesis pathways are known: in the hydrogenotrophic pathway H2 and carbon dioxide are used for methane production, whereas in the methylotrophic pathway small methylated carbon compounds like methanol and methylated amines are used. In the aceticlastic pathway, acetate is disproportionated to methane and carbon dioxide. However, next to these conventional substrates, further methanogenic substrates and pathways have been discovered. Several phylogenetically distinct methanogenic lineages (Methanosphaera, Methanimicrococcus, Methanomassiliicoccus, Methanonatronarchaeum) have evolved hydrogen-dependent methylotrophic methanogenesis without the ability to perform either hydrogenotrophic or methylotrophic methanogenesis. Genome analysis of the deep branching Methanonatronarchaeum revealed an interesting membrane-bound hydrogenase complex affiliated with the hardly described class 4 g of multisubunit hydrogenases possibly providing reducing equivalents for anabolism. Furthermore, methylated sulfur compounds such as methanethiol, dimethyl sulfide, and methylmercaptopropionate were described to be converted into adapted methylotrophic methanogenesis pathways of Methanosarcinales strains. Moreover, recently it has been shown that the methanogen Methermicoccus shengliensis can use methoxylated aromatic compounds in methanogenesis. Also, tertiary amines like choline (N,N,N-trimethylethanolamine) or betaine (N,N,N-trimethylglycine) have been described as substrates for methane production in Methanococcoides and Methanolobus strains. This review article will provide in-depth information on genome-guided metabolic reconstructions, physiology, and biochemistry of these unusual methanogenesis pathways. KEY POINTS: • Newly discovered methanogenic substrates and pathways are reviewed for the first time. • The review provides an in-depth analysis of unusual methanogenesis pathways. • The hydrogenase complex of the deep branching Methanonatronarchaeum is analyzed.

The ambiguity of the basic terms related to eukaryotes and the more consistent etymology based on eukaryotic signatures in Asgard archaea.

The endosymbiosis theory most widely accepted variant surmises the engulfment of a bacterial cell by an archaeal cell. For decades, this scenario was reputed to be an unconfirmed hypothesis, and only recently it has obtained an indirect proof in Asgard archaea environmental DNA encoding eukaryotic signatures - actin cytoskeleton, small GTPases, and ESCRT complex. In view of growing interest to this aspect of the endosymbiosis theory, it seemed timely to revisit the basic terms eukaryotic cell/eukaryotes/nucleated organisms. The article highlights the ambiguous applications of these terms, and seeks for their consistency with regard to phylogeny and taxonomy. Additionally, new name Caryosignifera is proposed for the phylum represented by: (1) the underexplored Asgard archaea manifested by above-mentioned environmental DNA; (2) cultured species of engulfing Asgard archaea; (3) eukaryotic host cells in nucleated organisms (protists, algae, plants, fungi, and animals).

Sex-dependent associations between addiction-related behaviors and the microbiome in outbred rats.

BACKGROUND: Multiple factors contribute to the etiology of addiction, including genetics, sex, and a number of addiction-related behavioral traits. One behavioral trait where individuals assign incentive salience to food stimuli ("sign-trackers", ST) are more impulsive compared to those that do not ("goal-trackers", GT), as well as more sensitive to drugs and drug stimuli. Furthermore, this GT/ST phenotype predicts differences in other behavioral measures. Recent studies have implicated the gut microbiota as a key regulator of brain and behavior, and have shown that many microbiota-associated changes occur in a sex-dependent manner. However, few studies have examined how the microbiome might influence addiction-related behaviors. To this end, we sought to determine if gut microbiome composition was correlated with addiction-related behaviors determined by the GT/ST phenotype. METHODS: Outbred male (N=101) and female (N=101) heterogeneous stock rats underwent a series of behavioral tests measuring impulsivity, attention, reward-learning, incentive salience, and locomotor response. Cecal microbiome composition was estimated using 16S rRNA gene amplicon sequencing. Behavior and microbiome were characterized and correlated with behavioral phenotypes. Robust sex differences were observed in both behavior and microbiome; further analyses were conducted within sex using the pre-established goal/sign-tracking (GT/ST) phenotype and partial least squares differential analysis (PLS-DA) clustered behavioral phenotype. RESULTS: Overall microbiome composition was not associated to the GT/ST phenotype. However, microbial alpha diversity was significantly decreased in female STs. On the other hand, a measure of impulsivity had many significant correlations to microbiome in both males and females. Several measures of impulsivity were correlated with the genus Barnesiella in females. Female STs had notable correlations between microbiome and attentional deficient. In both males and females, many measures were correlated with the bacterial families Ruminocococcaceae and Lachnospiraceae. CONCLUSIONS: These data demonstrate correlations between several addiction-related behaviors and the microbiome specific to sex.

Deciphering Pathways for Carotenogenesis in Haloarchaea.

Bacterioruberin and its derivatives have been described as the major carotenoids produced by haloarchaea (halophilic microbes belonging to the Archaea domain). Recently, different works have revealed that some haloarchaea synthetize other carotenoids at very low concentrations, like lycopene, lycopersene, cis- and trans-phytoene, cis- and trans-phytofluene, neo-ß-carotene, and neo-α-carotene. However, there is still controversy about the nature of the pathways for carotenogenesis in haloarchaea. During the last decade, the number of haloarchaeal genomes fully sequenced and assembled has increased significantly. Although some of these genomes are not fully annotated, and many others are drafts, this information provides a new approach to exploring the capability of haloarchaea to produce carotenoids. This work conducts a deeply bioinformatic analysis to establish a hypothetical metabolic map connecting all the potential pathways involved in carotenogenesis in haloarchaea. Special interest has been focused on the synthesis of bacterioruberin in members of the Haloferax genus. The main finding is that in almost all the genus analyzed, a functioning alternative mevalonic acid (MVA) pathway provides isopentenyl pyrophosphate (IPP) in haloarchaea. Then, the main branch to synthesized carotenoids proceeds up to lycopene from which ß-carotene or bacterioruberin (and its precursors: monoanhydrobacterioriberin, bisanhydrobacterioruberin, dihydrobisanhydrobacteriuberin, isopentenyldehydrorhodopsin, and dihydroisopenthenyldehydrorhodopsin) can be made.

Diversity and Distribution of Methanogenic Community Between Two Typical Alpine Ecosystems on the Qinghai-Tibetan Plateau.

Alpine permafrost regions are important sources of biogenic CH4 and methanogens play an important role in the methane-producing process. The alpine permafrost on the Qinghai-Tibetan plateau comprises about one-sixth of China's land area, and there are various types of alpine ecosystems. However, the methanogenic communities in the typical alpine ecosystems are poorly understood. In this study, the active layers and permafrost layers of the natural ecosystem of alpine grassland (DZ2-1) and alpine swamp meadow (DZ2-5) were selected to investigate the diversity and abundance of methanogenic communities. Methanobacterium (63.65%) are overwhelmingly dominant in the active layer of the alpine grassland (DZ2-1A). ZC-I cluster (26.13%), RC-I cluster (19.56%), and Methanobacterium (15.02%) are the dominant groups in the permafrost layer of the alpine grassland (DZ2-1P). Methanosaeta (32.92%), Fen cluster (29.59%), Methanosarcina (16.33%), and Methanobacterium (13.95%) are the dominant groups in the active layer of the alpine swamp meadow (DZ2-5A), whereas the Fen cluster (50.85%), ZC-I cluster (27.63%), and RC-I cluster (14.15%) are relatively abundant in the permafrost layer of the alpine swamp meadow (DZ2-5P). qPCR data showed that the abundance of methanogens was higher in the natural ecosystem of alpine swamp meadow than in alpine grassland. We found that the community characteristics of methanogens were related to environmental factors. Pearson correlation analyses indicated that the relative abundance of Methanobacterium had a significantly positive correlation with hydrogen concentration (P < 0.01), while the relative abundances of Methanosaeta and Methanosarcina were positively correlated with acetate concentration (P < 0.05). This study will help us to understand the methanogenic communities and their surrounding environments in alpine ecosystems.

The absence of intact polar lipid-derived GDGTs in marine waters dominated by Marine Group II: Implications for lipid biosynthesis in Archaea.

The marine pelagic archaeal community is dominated by three major groups, the marine group I (MGI) Thaumarchaeota, and the marine groups II and III (MGII and MGIII) Euryarchaeota. Studies of both MGI cultures and the environment have shown that the MGI core membrane lipids are predominantly composed of glycerol dibiphytanyl glycerol tetraether (GDGT) lipids and the diether lipid archaeol. However, there are no cultured representatives of MGII and III archaea and, therefore, both their membrane lipid composition and potential contribution to the marine archaeal lipid pool remain unknown. Here, we show that GDGTs present in suspended particulate matter of the (sub)surface waters of the North Atlantic Ocean and the coastal North Sea are derived from MGI archaea, and that MGII archaea do not significantly contribute to the pool of GDGTs and archaeol. This implies, in contrast to previous suggestions, that their lipids do not affect the widely used sea surface temperature proxy TEX86. These findings also indicate that MGII archaea are not able to produce any known archaeal lipids, implying that our understanding of the evolution of membrane lipid biosynthesis in Archaea is far from complete.

Salinigranum halophilum sp. nov., isolated from marine solar salterns.

Three halophilic archaeal strains, YJ-53T, ZS-5 and DYF38, were isolated from marine solar salterns located in different provinces of China. The three strains formed a single cluster (99.7-99.8 and 97.9-99.2 % similarities, respectively) that was separate from the current two members of Salinigranum (96.7-98.0 and 89.8-92.9 % similarities, respectively) on the basis of 16S rRNA and rpoB' gene sequence comparisons and phylogenetic analysis. Diverse phenotypic characteristics differentiated strains YJ-53T, ZS-5 and DYF38 from Salinigranum rubrum GX10T and Salinigranum salinum YJ-50-S2T. The major polar lipids of isolated strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and two major glycolipids chromatographically identical to mannosyl glucosyl diether and sulfated mannosyl glucosyl diether, detected in the current members of Salinigranum. The OrthoANI and in silico DNA-DNA hybridization (DDH) values between the three strains were in the range of 97.7-98.4 % and 80.3-86.1 %, respectively, much higher than the threshold values proposed as species boundaries (average nucleotide identity 95-96 % and in silico DDH 70 %), revealing that the three strains represent one species. Results of comparative OrthoANI and in silico DDH analyses of the strains described in this study with validly described members of the genus Salinigranum supported that strains YJ-53T (=CGMCC 1.12860T=JCM 30238T), ZS-5 (=CGMCC 1.12867=JCM 30240) and DYF38 (=CGMCC 1.13779=JCM 33557) represent a novel species of the genus Salinigranum, for which the name Salinigranum halophilum sp. nov. is proposed.

Tripartite Symbiosis of an Anaerobic Scuticociliate with Two Hydrogenosome-Associated Endosymbionts, a Holospora-Related Alphaproteobacterium and a Methanogenic Archaeon.

A number of anaerobic ciliates, unicellular eukaryotes, intracellularly possess methanogenic archaea and bacteria as symbiotic partners. Although this tripartite relationship is of interest in terms of the fact that each participant is from a different domain, the difficulty in culture and maintenance of those host species with symbiotic partners has disturbed both ecological and functional studies so far. In this study, we obtained a stable culture of a small anaerobic scuticociliate, strain GW7. By transmission electron microscopic observation and fluorescent in situ hybridization with domain-specific probes, we demonstrate that GW7 possesses both archaeal and bacterial endosymbionts in its cytoplasm. These endosymbionts are in dependently associated with hydrogenosomes, which are organelle producing hydrogen and ATP under anaerobic conditions. Clone library analyses targeting prokaryotic 16S rRNA genes, fluorescent in situ hybridization with endosymbiont-specific probes, and molecular phylogenetic analyses revealed the phylogenetic affiliations and intracellular localizations of these endosymbionts. The endosymbiotic archaeon is a methanogen belonging to the genus Methanoregula (order Methanomicrobiales); a member of this genus has previously been described as the endosymbiont of an anaerobic ciliate from the genus Metopus (class Armophorea), which is only distantly related to strain GW7 (class Oligohymenophorea). The endosymbiotic bacterium belongs to the family Holosporaceae of the class Alphaproteobacteria, which also comprises several endosymbionts of various aerobic ciliates. For this endosymbiotic bacterium, we propose a novel candidate genus and species, "Candidatus Hydrogenosomobacter endosymbioticus."IMPORTANCE Tripartite symbioses between anaerobic ciliated protists and their intracellular archaeal and bacterial symbionts are not uncommon, but most reports have been based mainly on microscopic observations. Deeper insights into the function, ecology, and evolution of these fascinating symbioses involving partners from all three domains of life have been hampered by the difficulties of culturing anaerobic ciliates in the laboratory and the frequent loss of their prokaryotic partners during long-term cultivation. In the present study, we report the isolation of an anaerobic scuticociliate, strain GW7, which has been stably maintained in our laboratory for more than 3 years without losing either of its endosymbionts. Unexpectedly, molecular characterization of the endosymbionts revealed that the bacterial partner of GW7 is phylogenetically related to intranuclear endosymbionts of aerobic ciliates. This strain will enable future genomic, transcriptomic, and proteomic analyses of the interactions in this tripartite symbiosis and a comparison with endosymbioses in aerobic ciliates.