Isolation of an archaeon at the prokaryote-eukaryote interface.

The origin of eukaryotes remains unclear1-4. Current data suggest that eukaryotes may have emerged from an archaeal lineage known as 'Asgard' archaea5,6. Despite the eukaryote-like genomic features that are found in these archaea, the evolutionary transition from archaea to eukaryotes remains unclear, owing to the lack of cultured representatives and corresponding physiological insights. Here we report the decade-long isolation of an Asgard archaeon related to Lokiarchaeota from deep marine sediment. The archaeon-'Candidatus Prometheoarchaeum syntrophicum' strain MK-D1-is an anaerobic, extremely slow-growing, small coccus (around 550 nm in diameter) that degrades amino acids through syntrophy. Although eukaryote-like intracellular complexes have been proposed for Asgard archaea6, the isolate has no visible organelle-like structure. Instead, Ca. P. syntrophicum is morphologically complex and has unique protrusions that are long and often branching. On the basis of the available data obtained from cultivation and genomics, and reasoned interpretations of the existing literature, we propose a hypothetical model for eukaryogenesis, termed the entangle-engulf-endogenize (also known as E3) model.

Promethearchaeum syntrophicum gen. nov., sp. nov., an anaerobic, obligately syntrophic archaeon, the first isolate of the lineage 'Asgard' archaea, and proposal of the new archaeal phylum Promethearchaeota phyl. nov. and kingdom Promethearchaeati regn. nov.

An anaerobic, mesophilic, syntrophic, archaeon strain MK-D1T, was isolated as a pure co-culture with Methanogenium sp. strain MK-MG from deep-sea methane seep sediment. This organism is, to our knowledge, the first cultured representative of 'Asgard' archaea, an archaeal group closely related to eukaryotes. Here, we describe the detailed physiology and phylogeny of MK-D1T and propose Promethearchaeum syntrophicum gen. nov., sp. nov. to accommodate this strain. Cells were non-motile, small cocci, approximately 300-750 nm in diameter and produced membrane vesicles, chains of blebs and membrane-based protrusions. MK-D1T grew at 4-30 °C with optimum growth at 20 °C. The strain grew chemoorganotrophically with amino acids, peptides and yeast extract with obligate dependence on syntrophy with H2-/formate-utilizing organisms. MK-D1T showed the fastest growth and highest maximum cell yield when grown with yeast extract as the substrate: approximately 3 months to full growth, reaching up to 6.7×106 16S rRNA gene copies ml-1. MK-D1T had a circular 4.32 Mb chromosome with a DNA G+C content of 31.1 mol%. The results of phylogenetic analyses of the 16S rRNA gene and conserved marker proteins indicated that the strain is affiliated with 'Asgard' archaea and more specifically DHVC1/DSAG/MBG-B and 'Lokiarchaeota'/'Lokiarchaeia'. On the basis of the results of 16S rRNA gene sequence analysis, the most closely related isolated relatives were Infirmifilum lucidum 3507LTT (76.09 %) and Methanothermobacter tenebrarum RMAST (77.45 %) and the closest relative in enrichment culture was Candidatus 'Lokiarchaeum ossiferum' (95.39 %). The type strain of the type species is MK-D1T (JCM 39240T and JAMSTEC no. 115508). We propose the associated family, order, class, phylum, and kingdom as Promethearchaeaceae fam. nov., Promethearchaeales ord. nov., Promethearchaeia class. nov., Promethearchaeota phyl. nov., and Promethearchaeati regn. nov., respectively. These are in accordance with ICNP Rules 8 and 22 for nomenclature, Rule 30(3)(b) for validation and maintenance of the type strain, and Rule 31a for description as a member of an unambiguous syntrophic association.

Proposed minimal standards for description of methanogenic archaea.

Methanogenic archaea are a diverse, polyphyletic group of strictly anaerobic prokaryotes capable of producing methane as their primary metabolic product. It has been over three decades since minimal standards for their taxonomic description have been proposed. In light of advancements in technology and amendments in systematic microbiology, revision of the older criteria for taxonomic description is essential. Most of the previously recommended minimum standards regarding phenotypic characterization of pure cultures are maintained. Electron microscopy and chemotaxonomic methods like whole-cell protein and lipid analysis are desirable but not required. Because of advancements in DNA sequencing technologies, obtaining a complete or draft whole genome sequence for type strains and its deposition in a public database are now mandatory. Genomic data should be used for rigorous comparison to close relatives using overall genome related indices such as average nucleotide identity and digital DNA-DNA hybridization. Phylogenetic analysis of the 16S rRNA gene is also required and can be supplemented by phylogenies of the mcrA gene and phylogenomic analysis using multiple conserved, single-copy marker genes. Additionally, it is now established that culture purity is not essential for studying prokaryotes, and description of Candidatus methanogenic taxa using single-cell or metagenomics along with other appropriate criteria is a viable alternative. The revisions to the minimal criteria proposed here by the members of the Subcommittee on the Taxonomy of Methanogenic Archaea of the International Committee on Systematics of Prokaryotes should allow for rigorous yet practical taxonomic description of these important and diverse microbes.

Multispecies Populations of Methanotrophic Methyloprofundus and Cultivation of a Likely Dominant Species from the Iheya North Deep-Sea Hydrothermal Field.

The Methyloprofundus clade is represented by uncultivated methanotrophic bacterial endosymbionts of deep-sea bathymodiolin mussels, but only a single free-living species has been cultivated to date. This study reveals the existence of free-living Methyloprofundus variants in the Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough. A clade-targeted amplicon analysis of the particulate methane monooxygenase gene (pmoA) detected 647 amplicon sequence variants (ASVs) of the Methyloprofundus clade in microbial communities newly formed in in situ colonization systems. Such systems were deployed at colonies of bathymodiolin mussels and a galatheoid crab in diffuse-flow areas. These ASVs were classified into 161 species-like groups. The proportion of the species-like groups representing endosymbionts of mussels was unexpectedly low. A methanotrophic bacterium designated INp10, a likely dominant species in the Methyloprofundus population in this field, was enriched in a biofilm formed in a methane-fed cultivation system operated at 10°C. Genomic characterization with the gene transcription data set of INp10 from the biofilm suggested traits advantageous to niche competition in environments, such as mobility, chemotaxis, biofilm formation, offensive and defensive systems, and hypoxia tolerance. The notable metabolic traits that INp10 shares with some Methyloprofundus members are the use of lanthanide-dependent XoxF as the sole methanol dehydrogenase due to the absence of the canonical MxaFI, the glycolytic pathway using fructose-6-phosphate aldolase instead of fructose-1,6-bisphosphate aldolase, and the potential to perform partial denitrification from nitrate under oxygen-limited conditions. These findings help us better understand the ecological strategies of this possibly widespread marine-specific methanotrophic clade. IMPORTANCE The Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough is characterized by abundant methane derived from organic-rich sediments and diverse chemosynthetic animal species, including those harboring methanotrophic bacterial symbionts, such as bathymodiolin mussels Bathymodiolus japonicus and "Bathymodiolus" platifrons and a galatheoid crab, Shinkaia crosnieri. Symbiotic methanotrophs have attracted significant attention, and yet free-living methanotrophs in this environment have not been studied in detail. We focused on the free-living Methyloprofundus spp. that thrive in this hydrothermal field and identified an unexpectedly large number of species-like groups in this clade. Moreover, we enriched and characterized a methanotroph whose genome sequence indicated that it corresponds to a new species in the genus Methyloprofundus. This species might be a dominant member of the indigenous Methyloprofundus population. New information on free-living Methyloprofundus populations suggests that the hydrothermal field is a promising locale at which to investigate the adaptive capacity and associated genetic diversity of Methyloprofundus spp.

Rectinema subterraneum sp. nov, a chemotrophic spirochaete isolated from the deep terrestrial subsurface.

A novel, obligately anaerobic bacterium (strain SURF-ANA1T) was isolated from deep continental subsurface fluids at a depth of 1500 m below surface in the former Homestake Gold Mine (now Sanford Underground Research Facility, in Lead, South Dakota, USA). Cells of strain SURF-ANA1T were Gram-negative, helical, non-spore-forming and were 0.25-0.55×5.0-75.0 µm with a wavelength of 0.5-0.62 µm. Strain SURF-ANA1T grew at 15-50 °C (optimally at 40 °C), at pH 4.8-9.0 (pH 7.2) and in 1.0-40.0 g l-1 NaCl (10 g l-1 NaCl). The strain grew chemoheterotrophically with hydrogen or mono-, di- and polysaccharides as electron donors. The major cellular fatty acids in order of decreasing abundance (comprising >5% of total) were 10-methyl C16:0, iso-C15:0, C18:2 and C18:0 dimethyl acetal (DMA) and C20:0 methylene-nonadecanoic acid. Phylogenetic analysis based on the 16S rRNA gene sequence of strain SURF-ANA1T indicated a closest relationship with the recently characterized Rectinema cohabitans (99%). Despite high sequence identity, because of its distinct physiology, morphology and fatty acid profile, strain SURF-ANA1T is considered to represent a novel species within the genus Rectinema, for which the name Rectinema subterraneum sp. nov. is proposed. To our knowledge, this is the first report of an isolate within the phylum Spirochaetes from the deep (>100 m) terrestrial subsurface. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and genomic sequences of strain SURF-ANA1T are KU359248 and GCF 009768935.1, respectively. The type strain of Rectinema subterraneum is SURF-ANA1T (=ATCC TSD-67=JCM 32656).

Multiple organic substrates support Mn(II) removal with enrichment of Mn(II)-oxidizing bacteria.

Three different organic substrates, K-medium, sterilized activated sludge (SAS), and methanol, were examined for utility as substrates for enriching manganese-oxidizing bacteria (MnOB) in an open bioreactor. The differences in Mn(II) oxidation performance between the substrates were investigated using three down-flow hanging sponge (DHS) reactors continuously treating artificial Mn(II)-containing water over 131 days. The results revealed that all three substrates were useful for enriching MnOB. Surprisingly, we observed only slight differences in Mn(II) removal between the substrates. The highest Mn(II) removal rate for the SAS-supplied reactor was 0.41 kg Mn⋅m-3⋅d-1, which was greater than that of K-medium, although the SAS performance was unstable. In contrast, the methanol-supplied reactor had more stable performance and the highest Mn(II) removal rate. We conclude that multiple genera of Comamonas, Pseudomonas, Mycobacterium, Nocardia and Hyphomicrobium play a role in Mn(II) oxidation and that their relative predominance was dependent on the substrate. Moreover, the initial inclusion of abiotic-MnO2 in the reactors promoted early MnOB enrichment.

Aggregatilinea lenta gen. nov., sp. nov., a slow-growing, facultatively anaerobic bacterium isolated from subseafloor sediment, and proposal of the new order Aggregatilineales ord. nov. within the class Anaerolineae of the phylum Chloroflexi.

A novel slow-growing, facultatively anaerobic, filamentous bacterium, strain MO-CFX2T, was isolated from a methanogenic microbial community in a continuous-flow bioreactor that was established from subseafloor sediment collected off the Shimokita Peninsula of Japan. Cells were multicellular filamentous, non-motile and Gram-stain-negative. The filaments were generally more than 20 µm (up to approximately 200 µm) long and 0.5-0.6 µm wide. Cells possessed pili-like structures on the cell surface and a multilayer structure in the cytoplasm. Growth of the strain was observed at 20-37 °C (optimum, 30 °C), pH 5.5-8.0 (pH 6.5-7.0), and 0-30 g l-1 NaCl (5 g l-1 NaCl). Under optimum growth conditions, doubling time and maximum cell density were estimated to be approximately 19 days and ~105 cells ml-1, respectively. Strain MO-CFX2T grew chemoorganotrophically on a limited range of organic substrates in anaerobic conditions. The major cellular fatty acids were saturated C16 : 0 (47.9 %) and C18 : 0 (36.9 %), and unsaturated C18 : 1ω9c (6.0 %) and C16 : 1ω7 (5.1 %). The G+C content of genomic DNA was 63.2 mol%. 16S rRNA gene-based phylogenetic analysis showed that strain MO-CFX2T shares a notably low sequence identity with its closest relatives, which were Thermanaerothrix daxensis GNS-1T and Thermomarinilinea lacunifontana SW7T (both 85.8 % sequence identity). Based on these phenotypic and genomic properties, we propose the name Aggregatilinea lenta gen. nov., sp. nov. for strain MO-CFX2T (=KCTC 15625T, =JCM 32065T). In addition, we also propose the associated family and order as Aggregatilineaceae fam. nov. and Aggregatilineales ord. nov., respectively.

Novel energy conservation strategies and behaviour of Pelotomaculum schinkii driving syntrophic propionate catabolism.

Under methanogenic conditions, short-chain fatty acids are common byproducts from degradation of organic compounds and conversion of these acids is an important component of the global carbon cycle. Due to the thermodynamic difficulty of propionate degradation, this process requires syntrophic interaction between a bacterium and partner methanogen; however, the metabolic strategies and behaviour involved are not fully understood. In this study, the first genome analysis of obligately syntrophic propionate degraders (Pelotomaculum schinkii HH and P. propionicicum MGP) and comparison with other syntrophic propionate degrader genomes elucidated novel components of energy metabolism behind Pelotomaculum propionate oxidation. Combined with transcriptomic examination of P. schinkii behaviour in co-culture with Methanospirillum hungatei, we found that formate may be the preferred electron carrier for P. schinkii syntrophy. Propionate-derived menaquinol may be primarily re-oxidized to formate, and energy was conserved during formate generation through newly proposed proton-pumping formate extrusion. P. schinkii did not overexpress conventional energy metabolism associated with a model syntrophic propionate degrader Syntrophobacter fumaroxidans MPOB (i.e., CoA transferase, Fix and Rnf). We also found that P. schinkii and the partner methanogen may also interact through flagellar contact and amino acid and fructose exchange. These findings provide new understanding of syntrophic energy acquisition and interactions.

Biotic manganese oxidation coupled with methane oxidation using a continuous-flow bioreactor system under marine conditions.

Biogenic manganese oxides (BioMnOx) can be applied for the effective removal and recovery of trace metals from wastewater because of their high adsorption capacity. Although a freshwater continuous-flow system for a nitrifier-based Mn-oxidizing microbial community for producing BioMnOx has been developed so far, a seawater continuous-flow bioreactor system for BioMnOx production has not been established. Here, we report BioMnOx production by a methanotroph-based microbial community by using a continuous-flow bioreactor system. The bioreactor system was operated using a deep-sea sediment sample as the inoculum with methane as the energy source for over 2 years. The BioMnOx production became evident after 370 days of reactor operation. The maximum Mn oxidation rate was 11.4 mg L-1 day-1. An X-ray diffraction analysis showed that the accumulated BioMnOx was birnessite. 16S rRNA gene-based clone analyses indicated that methanotrophic bacterial members were relatively abundant in the system; however, none of the known Mn-oxidizing bacteria were detected. A continuous-flow bioreactor system coupled with nitrification was also run in parallel for 636 days, but no BioMnOx production was observed in this bioreactor system. The comparative experiments indicated that the methanotroph-based microbial community, rather than the nitrifier-based community, was effective for BioMnOx production under the marine environmental conditions.

Variance and potential niche separation of microbial communities in subseafloor sediments off Shimokita Peninsula, Japan.

Subseafloor pelagic sediments with high concentrations of organic matter form habitats for diverse microorganisms. Here, we determined depth profiles of genes for SSU rRNA, mcrA, dsrA and amoA from just beneath the seafloor to 363.3 m below the seafloor (mbsf) using core samples obtained from the forearc basin off the Shimokita Peninsula. The molecular profiles were combined with data on lithostratigraphy, depositional age, sedimentation rate and pore-water chemistry. The SSU rRNA gene tag structure and diversity changed at around the sulfate-methane transition zone (SMTZ), whereas the profiles varied further with depth below the SMTZ, probably in connection with the variation in pore-water chemistry. The depth profiles of diversity and abundance of dsrA, a key gene for sulfate reduction, suggested the possible niche separations of sulfate-reducing populations, even below the SMTZ. The diversity and abundance patterns of mcrA, a key gene for methanogenesis/anaerobic methanotrophy, suggested a stratified distribution and separation of anaerobic methanotrophy and hydrogenotrophic or methylotrophic methanogensis below the SMTZ. This study provides novel insights into the relationships between the composition and function of microbial communities and the chemical environment in the nutrient-rich continental margin subseafloor sediments, which may result in niche separation and variability in subseafloor microbial populations.

Nitrogen and Oxygen Isotope Effects of Ammonia Oxidation by Thermophilic Thaumarchaeota from a Geothermal Water Stream.

UNLABELLED: Ammonia oxidation regulates the balance of reduced and oxidized nitrogen pools in nature. Although ammonia-oxidizing archaea have been recently recognized to often outnumber ammonia-oxidizing bacteria in various environments, the contribution of ammonia-oxidizing archaea is still uncertain due to difficulties in the in situ quantification of ammonia oxidation activity. Nitrogen and oxygen isotope ratios of nitrite (δ(15)NNO2- and δ(18)ONO2-, respectively) are geochemical tracers for evaluating the sources and the in situ rate of nitrite turnover determined from the activities of nitrification and denitrification; however, the isotope ratios of nitrite from archaeal ammonia oxidation have been characterized only for a few marine species. We first report the isotope effects of ammonia oxidation at 70°C by thermophilic Thaumarchaeota populations composed almost entirely of "Candidatus Nitrosocaldus." The nitrogen isotope effect of ammonia oxidation varied with ambient pH (25‰ to 32‰) and strongly suggests the oxidation of ammonia, not ammonium. The δ(18)O value of nitrite produced from ammonia oxidation varied with the δ(18)O value of water in the medium but was lower than the isotopic equilibrium value in water. Because experiments have shown that the half-life of abiotic oxygen isotope exchange between nitrite and water is longer than 33 h at 70°C and pH ≥6.6, the rate of ammonia oxidation by thermophilic Thaumarchaeota could be estimated using δ(18)ONO2- in geothermal environments, where the biological nitrite turnover is likely faster than 33 h. This study extended the range of application of nitrite isotopes as a geochemical clock of the ammonia oxidation activity to high-temperature environments. IMPORTANCE: Because ammonia oxidation is generally the rate-limiting step in nitrification that regulates the balance of reduced and oxidized nitrogen pools in nature, it is important to understand the biological and environmental factors underlying the regulation of the rate of ammonia oxidation. The discovery of ammonia-oxidizing archaea (AOA) in marine and terrestrial environments has transformed the concept that ammonia oxidation is operated only by bacterial species, suggesting that AOA play a significant role in the global nitrogen cycle. However, the archaeal contribution to ammonia oxidation in the global biosphere is not yet completely understood. This study successfully identified key factors controlling nitrogen and oxygen isotopic ratios of nitrite produced from thermophilic Thaumarchaeota and elucidated the applicability and its limit of nitrite isotopes as a geochemical clock of ammonia oxidation rate in nature. Oxygen isotope analysis in this study also provided new biochemical information on archaeal ammonia oxidation.

Sedimentibacter acidaminivorans sp. nov., an anaerobic, amino-acid-utilizing bacterium isolated from marine subsurface sediment.

A novel, anaerobic bacterium, strain MO-SEDIT, was isolated from a methanogenic microbial community, which was originally obtained from marine subsurface sediments collected from off the Shimokita Peninsula of Japan. Cells were Gram-stain-negative, non-motile, non-spore-forming rods, 0.4-1.4 µm long by 0.4-0.6 µm wide. The cells also formed long filaments of up to about 11 µm. The strain grew on amino acids (i.e. valine, leucine, isoleucine, methionine, glycine, phenylalanine, tryptophan, lysine and arginine), pyruvate and melezitose in the presence of yeast extract. Growth was observed at 4-37 °C (optimally at 30 °C), at pH 6.0 and 8.5 (optimally at 7.0-7.5) and in 0-60 g l- 1 NaCl (optimally 20 g NaCl l- 1). The G+C content of the DNA was 32.0 mol%. The polar lipids of strain MO-SEDIT were phosphatidylglycerol, phosphatidyl lipids and unknown lipids. The major cellular fatty acids (>10 % of the total) were C14 : 0, C16 : 1ω9 and C16 : 0 dimethyl aldehyde. Comparative sequence analysis of the 16S rRNA gene showed that strain MO-SEDIT was affiliated with the genus Sedimentibacter within the phylum Firmicutes. It was related most closely to the type strain of Sedimentibacter saalensis (94 % sequence similarity). Based on the phenotypic and genetic characteristics, strain MO-SEDIT is considered to represent a novel species of the genus Sedimentibacter, for which the name Sedimentibacter acidaminivorans sp. nov. is proposed. The type strain is MO-SEDIT ( = JCM 17293T = DSM 24004T).

Methanomicrobium antiquum sp. nov., a hydrogenotrophic methanogen isolated from deep sedimentary aquifers in a natural gas field.

A mesophilic, hydrogenotrophic methanogen, designated strain MobHT, was isolated from sediments derived from deep sedimentary, natural-gas-bearing aquifers in Japan. Strain MobHT utilized H2/CO2 or formate, but not ethanol, 1-propanol, 2-propanol, 2-butanol or cyclopentanol, for growth and methane production. In addition, acetate and tungsten were required for growth. Yeast extract stimulated the growth, but was not required. The cells were weakly motile with multiple flagella, presented as a curved-rod-shaped (0.8×2.0 µm) and occurred singly or in pairs. Strain MobHT grew at 15-40 °C (optimum 35 °C) and at pH 5.9-7.9 (optimum pH 7.0-7.5). The sodium chloride range for growth was 0-5.8 % (optimum 2 %). The G+C content of the genomic DNA was 37.6 mol%. In the phylogenetic tree based on the 16S rRNA gene sequences, strain MobHT clustered together with Methanomicrobium mobile (95.4 % in sequence similarity), and formed a distinct clade from Methanolacinia petrolearia SEBR 4847T (95.6 %) and Methanolacinia paynteri G-2000T (95.4 %). The two species of the genus Methanolacinia utilized 2-propanol, whereas strain MobHT and Methanomicrobium mobile, the sole species of the genus Methanomicrobium, do not. Based on phenotypic and phylogenetic features, we propose a novel species for the isolate with the name, Methanomicrobiumantiquum sp. nov. The type strain is MobHT (=DSM 21220T=NBRC 104160T).

In situ DNA-hybridization chain reaction (HCR): a facilitated in situ HCR system for the detection of environmental microorganisms.

In situ detection of microorganisms by fluorescence in situ hybridization (FISH) is a powerful tool for environmental microbiology, but analyses can be hampered by low rRNA content in target organisms, especially in oligotrophic environments. Here, we present a non-enzymatic, hybridization chain reaction (HCR)-based signal amplified in situ whole-cell detection technique (in situ DNA-HCR). The components of the amplification buffer were optimized to polymerize DNA amplifier probes for in situ DNA-HCR. In situ hybridization of initiator probes followed by signal amplification via HCR produced bright signals with high specificity and probe permeation into cells. The detection rates for Bacteria in a seawater sample and Archaea in anaerobic sludge samples were comparable with or greater than those obtained by catalyzed reporter deposition (CARD)-FISH or standard FISH. Detection of multiple organisms (Bacteria, Archaea and Methanosaetaceae) in an anaerobic sludge sample was achieved by simultaneous in situ DNA-HCR. In summary, in situ DNA-HCR is a simple and easy technique for detecting single microbial cells and enhancing understanding of the ecology and behaviour of environmental microorganisms in situ.

Spirochaeta psychrophila sp. nov., a psychrophilic spirochaete isolated from subseafloor sediment, and emended description of the genus Spirochaeta.

An obligately anaerobic, psychrophilic spirochaete, strain MO-SPC1(T), was isolated from a methanogenic microbial community grown in a continuous-flow bioreactor. Originally, this community was obtained from subseafloor sediments off the Shimokita Peninsula of Japan in the north-western Pacific Ocean. The cells were motile, Gram-stain-negative, helical, 0.25-0.55×3.6-15 µm, with a wavelength of approximately 0.5-0.6 µm. Strain MO-SPC1(T) grew at 0-18 °C (optimally at 15 °C), at pH 6.0-7.5 (optimally at pH 6.8-7.0) and in 20-70 g NaCl l(-1) (optimally at 30-40 NaCl l(-1)). The strain grew chemo-organotrophically with mono-, di- and polysaccharides. The major end products of glucose fermentation were acetate, ethanol, hydrogen and carbon dioxide. The abundant polar lipids of strain MO-SPC1(T) were diphosphatidylglycerol, phosphatidylglycerol, unknown phospholipids and an unknown lipid. The major cellular fatty acids (>5% of the total) were C(14 : 0), C(16 : 0), iso-C(13 : 0), iso-C(14 : 0), iso-C(15 : 0), anteiso-C(13 : 0) and anteiso-C(15 : 0). To the best of our knowledge, this is the first report of the fatty acids iso-C(13 : 0) and anteiso-C(13 : 0) from a species of the genus Spirochaeta. Isoprenoid quinones were not found. The G+C content of the genomic DNA was 39.8 mol%. 16S rRNA gene sequence-based phylogenetic analysis showed that strain MO-SPC1(T) was affiliated with the genus Spirochaeta, and its closest relatives were Spirochaeta isovalerica MA-2(T) (95.6% sequence identity) and Spirochaeta litoralis R1(T) (89.4%). Based on its phenotypic characteristics and phylogenetic traits, strain MO-SPC1(T) is placed in a separate taxon at the level of a novel species within the genus Spirochaeta, for which the name Spirochaeta psychrophila sp. nov. is proposed, reflecting its true psychrophilic physiology. The type strain is MO-SPC1(T) ( = JCM 17280(T) = DSM 23951(T)). To our knowledge, this is the first report of an isolate of the phylum Spirochaetes from a deep-sea sedimentary environment, and of an obligately psychrophilic nature.

Pelolinea submarina gen. nov., sp. nov., an anaerobic, filamentous bacterium of the phylum Chloroflexi isolated from subseafloor sediment.

A novel, anaerobic filamentous bacterium, strain MO-CFX1(T), was isolated from a methanogenic community, which was originally established from subseafloor sediments collected from off the Shimokita Peninsula, Japan. Cells were non-spore-forming, non-motile, Gram-stain-negative and filamentous. The filaments were longer than 10 µm and 130-150 nm in width. Growth of the strain was observed at 10-37 °C (optimum 25-30 °C), at pH 5.5-8.5 (optimum pH 7.0) and in 0-50 g NaCl l(-1) (optimum 15 g NaCl l(-1)). The strain was able to grow with a number of carbohydrates in the presence of yeast extract. The major cellular fatty acids were monounsaturated C18 : 1ω9, C16 : 1ω7 and saturated C18 : 0 and C16 : 0. The intact polar lipids of the strain were dominated by diacylglyceride and sphingolipid core lipid structures with monoglycosidic, mixed phosphomonoglycosidic and fatty-acid-modified monoglycosidic polar head groups. The G+C content of the genomic DNA was 52.4 mol%. Based on the comparative 16S rRNA gene sequence analysis, strain MO-CFX1(T) was affiliated with the class Anaerolineae within the phylum Chloroflexi and was most closely related to Leptolinea tardivitalis YMTK-2(T) (sequence identity of 91.0 %). Based on phenotypic and genetic properties of the novel isolate, we propose a novel species representing a new genus Pelolinea submarina gen. nov., sp. nov., for strain MO-CFX1(T) ( = JCM 17238(T), = KCTC 5975(T)). This is the first formal description, to our knowledge, of an isolate of the phylum Chloroflexi from the deep-sea sedimentary environment.

Sphaerochaeta multiformis sp. nov., an anaerobic, psychrophilic bacterium isolated from subseafloor sediment, and emended description of the genus Sphaerochaeta.

An anaerobic, psychrophilic bacterium, strain MO-SPC2(T), was isolated from a methanogenic microbial community in a continuous-flow bioreactor that was established from subseafloor sediments collected from off the Shimokita Peninsula of Japan in the north-western Pacific Ocean. Cells were pleomorphic: spherical, annular, curved rod, helical and coccoid cell morphologies were observed. Motility only occurred in helical cells. Strain MO-SPC2(T) grew at 0-17 °C (optimally at 9 °C), at pH 6.0-8.0 (optimally at pH 6.8-7.2) and in 20-40 g NaCl l(-1) (optimally at 20-30 NaCl l(-1)). The strain grew chemo-organotrophically with mono-, di- and polysaccharides. The major end products of glucose fermentation were acetate, ethanol, hydrogen and carbon dioxide. The abundant polar lipids of strain MO-SPC2(T) were phosphatidylglycolipids, phospholipids and glycolipids. The major cellular fatty acids were C14 : 0, C16 : 0 and C16 : 1ω9. Isoprenoid quinones were not detected. The G+C content of the DNA was 32.3 mol%. 16S rRNA gene-based phylogenetic analysis showed that strain MO-SPC2(T) was affiliated with the genus Sphaerochaeta within the phylum Spirochaetes, and its closest relatives were Sphaerochaeta pleomorpha Grapes(T) (88.4 % sequence identity), Sphaerochaeta globosa Buddy(T) (86.7 %) and Sphaerochaeta coccoides SPN1(T) (85.4 %). Based on phenotypic characteristics and phylogenetic traits, strain MO-SPC2(T) is considered to represent a novel species of the genus Sphaerochaeta, for which the name Sphaerochaeta multiformis sp. nov. is proposed. The type strain is MO-SPC2(T) ( = JCM 17281(T) = DSM 23952(T)). An emended description of the genus Sphaerochaeta is also proposed.

Characterization of protein glycosylation in an Asgard archaeon.

Archaeal cells are typically enveloped by glycosylated S-layer proteins. Archaeal protein glycosylation provides valuable insights not only into their adaptation to their niches but also into their evolutionary trajectory. Notably, thermophilic Thermoproteota modify proteins with N-glycans that include two GlcNAc units at the reducing end, resembling the "core structure" preserved across eukaryotes. Recently, Asgard archaea, now classified as members of the phylum Promethearchaeota, have offered unprecedented opportunities for understanding the role of archaea in eukaryogenesis. Despite the presence of genes indicative of protein N-glycosylation in this archaeal group, these have not been experimentally investigated. Here we performed a glycoproteome analysis of the firstly isolated Asgard archaeon Promethearchaeum syntrophicum. Over 700 different proteins were identified through high-resolution LC-MS/MS analysis, however, there was no evidence of either the presence or glycosylation of putative S-layer proteins. Instead, N-glycosylation in this archaeon was primarily observed in an extracellular solute-binding protein, possibly related to chemoreception or transmembrane transport of oligopeptides. The glycan modification occurred on an asparagine residue located within the conserved N-X-S/T sequon, consistent with the pattern found in other archaea, bacteria, and eukaryotes. Unexpectedly, three structurally different N-glycans lacking the conventional core structure were identified in this archaeon, presenting unique compositions that included atypical sugars. Notably, one of these sugars was likely HexNAc modified with a threonine residue, similar to modifications previously observed in mesophilic methanogens within the Methanobacteriati. Our findings advance our understanding of Asgard archaea physiology and evolutionary dynamics.

A Marine Group A isolate relies on other growing bacteria for cell wall formation.

Most of Earth's prokaryotes live under energy limitation, yet the full breadth of strategies that enable survival under such conditions remain poorly understood. Here we report the isolation of a bacterial strain, IA91, belonging to the candidate phylum Marine Group A (SAR406 or 'Candidatus Marinimicrobia') that is unable to synthesize the central cell wall compound peptidoglycan itself. Using cultivation experiments and microscopy, we show that IA91 growth and cell shape depend on other bacteria, deriving peptidoglycan, energy and carbon from exogenous muropeptide cell wall fragments released from growing bacteria. Reliance on exogenous muropeptides is traceable to the phylum's ancestor, with evidence of vertical inheritance across several classes. This dependency may be widespread across bacteria (16 phyla) based on the absence of key peptidoglycan synthesis genes. These results suggest that uptake of exogenous cell wall components could be a relevant and potentially common survival strategy in energy-limited habitats like the deep biosphere.