Nobu, Masaru K.

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.

ABSTRACT To verify whether members of the phylum Candidatus Patescibacteria parasitize archaea, we applied cultivation, microscopy, metatranscriptomic, and protein structure prediction analyses on the Patescibacteria-enriched cultures derived from a methanogenic bioreactor. Amendment of cultures with exogenous methanogenic archaea, acetate, amino acids, and nucleoside monophosphates increased the relative abundance of Ca . Patescibacteria. The predominant Ca . Patescibacteria were families Ca . Yanofskyibacteriaceae and Ca . Minisyncoccaceae, and the former showed positive linear relationships ( r 2 ≥ 0.70) Methanothrix in their relative abundances, suggesting related growth patterns. Methanothrix and Methanospirillum cells with attached Ca . Yanofskyibacteriaceae and Ca . Minisyncoccaceae, respectively, had significantly lower cellular activity than those of the methanogens without Ca . Patescibacteria, as extrapolated from fluorescence in situ hybridization-based fluorescence. We also observed that parasitized methanogens often had cell surface deformations. Some Methanothrix -like filamentous cells were dented where the submicron cells were attached. Ca . Yanofskyibacteriaceae and Ca . Minisyncoccaceae highly expressed extracellular enzymes, and based on structural predictions, some contained peptidoglycan-binding domains with potential involvement in host cell attachment. Collectively, we propose that the interactions of Ca . Yanofskyibacteriaceae and Ca . Minisyncoccaceae with methanogenic archaea are parasitisms. IMPORTANCE Culture-independent DNA sequencing approaches have explored diverse yet-to-be-cultured microorganisms and have significantly expanded the tree of life in recent years. One major lineage of the domain Bacteria, Ca . Patescibacteria (also known as candidate phyla radiation), is widely distributed in natural and engineered ecosystems and has been thought to be dependent on host bacteria due to the lack of several biosynthetic pathways and small cell/genome size. Although bacteria-parasitizing or bacteria-preying Ca . Patescibacteria have been described, our recent studies revealed that some lineages can specifically interact with archaea. In this study, we provide strong evidence that the relationship is parasitic, shedding light on overlooked roles of Ca . Patescibacteria in anaerobic habitats.

AbstractTo verify the parasitic lifestyle ofCandidatusPatescibacteria in the enrichment cultures derived from a methanogenic bioreactor, we applied multifaceted approaches combining cultivation, microscopy, metatranscriptomic, and protein structure prediction analyses. Cultivation experiments with the addition of exogenous methanogenic archaea with acetate, amino acids, and nucleoside monophosphates and 16S rRNA gene sequencing confirmed the increase in the relative abundance ofCa. Patescibacteria and methanogens. The predominantCa. Patescibacteria wereCa. Yanofskybacteria and 32-520 lineages (to which belongs to classCa. Paceibacteria) and positive linear relationships (r2≥ 0.70) between the relative abundance ofCa. Yanofskybacteria andMethanothrix, suggesting that the tendency of the growth rate is similar to that of the host. By fluorescencein situhybridization (FISH) observations, the FISH signals ofMethanothrixandMethanospirillumcells withCa. Yanofskybacteria and with 32-520 lineages, respectively, were significantly lower than those of the methanogens withoutCa. Patescibacteria, suggesting their parasitic interaction. The TEM and SEM observations also support parasitism in that the cell walls and plugs of these methanogens associated with submicron cells were often deformed. In particular, someMethanothrix-like filamentous cells were dented where the submicron cells were attached. Metatranscriptomic and protein structure prediction analyses identified highly expressed secreted genes from the genomes ofCa. Yanofskybacteria and 32-520, and these genes contain adhesion-related domains to the host cells. Considering the results through the combination of microscopic observations, gene expression, and computational protein modeling, we propose that the interactions betweenCa. Yanofskybacteria and 32-520 belonging to classCa. Paceibacteria and methanogenic archaea are parasitism.

One highly diverse phylogenetic group of Bacteria, Ca . Patescibacteria, remains poorly understood, but, from the few cultured representatives and metagenomic investigations, they are thought to live symbiotically or parasitically with other bacteria or even with eukarya.

The bacterium “ Candidatus Hydrogenosomobacter endosymbioticus” is an intracellular symbiont of anaerobic scuticociliate GW7, which is associated with hydrogenosome together with methanogenic archaea. Here, we report a complete genome sequence of the symbiont consisting of 827 kbp. Knowing this sequence would contribute to the understanding of the metabolic interactions and evolution of the tripartite symbiosis.

AbstractA key feature that differentiates prokaryotic cells from eukaryotes is the absence of an intracellular membrane surrounding the chromosomal DNA. Here, we isolate a member of the ubiquitous, yet-to-be-cultivated phylum ‘Candidatus Atribacteria’ (also known as OP9) that has an intracytoplasmic membrane apparently surrounding the nucleoid. The isolate, RT761, is a subsurface-derived anaerobic bacterium that appears to have three lipid membrane-like layers, as shown by cryo-electron tomography. Our observations are consistent with a classical gram-negative structure with an additional intracytoplasmic membrane. However, further studies are needed to provide conclusive evidence for this unique intracellular structure. The RT761 genome encodes proteins with features that might be related to the complex cellular structure, including: N-terminal extensions in proteins involved in important processes (such as cell-division protein FtsZ); one of the highest percentages of transmembrane proteins among gram-negative bacteria; and predicted Sec-secreted proteins with unique signal peptides. Physiologically, RT761 primarily produces hydrogen for electron disposal during sugar degradation, and co-cultivation with a hydrogen-scavenging methanogen improves growth. We propose RT761 as a new species, Atribacter laminatus gen. nov. sp. nov. and a new phylum, Atribacterota phy. nov.