Illuminating the coevolution of photosynthesis and Bacteria.

Life harnessing light energy transformed the relationship between biology and Earth-bringing a massive flux of organic carbon and oxidants to Earth's surface that gave way to today's organotrophy- and respiration-dominated biosphere. However, our understanding of how life drove this transition has largely relied on the geological record; much remains unresolved due to the complexity and paucity of the genetic record tied to photosynthesis. Here, through holistic phylogenetic comparison of the bacterial domain and all photosynthetic machinery (totally spanning >10,000 genomes), we identify evolutionary congruence between three independent biological systems-bacteria, (bacterio)chlorophyll-mediated light metabolism (chlorophototrophy), and carbon fixation-and uncover their intertwined history. Our analyses uniformly mapped progenitors of extant light-metabolizing machinery (reaction centers, [bacterio]chlorophyll synthases, and magnesium-chelatases) and enzymes facilitating the Calvin-Benson-Bassham cycle (form I RuBisCO and phosphoribulokinase) to the same ancient Terrabacteria organism near the base of the bacterial domain. These phylogenies consistently showed that extant phototrophs ultimately derived light metabolism from this bacterium, the last phototroph common ancestor (LPCA). LPCA was a non-oxygen-generating (anoxygenic) phototroph that already possessed carbon fixation and two reaction centers, a type I analogous to extant forms and a primitive type II. Analyses also indicate chlorophototrophy originated before LPCA. We further reconstructed evolution of chlorophototrophs/chlorophototrophy post-LPCA, including vertical inheritance in Terrabacteria, the rise of oxygen-generating chlorophototrophy in one descendant branch near the Great Oxidation Event, and subsequent emergence of Cyanobacteria. These collectively unveil a detailed view of the coevolution of light metabolism and Bacteria having clear congruence with the geological record.

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.

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.

The nexus of syntrophy-associated microbiota in anaerobic digestion revealed by long-term enrichment and community survey.

Anaerobic digestion (AD) processes are known to effectively convert organic waste to CO2 and CH4 , but much of the microbial ecology remains unclear. Specifically, we have limited insights into symbiotic syntroph and methanogen ('syntrophy') acid degradation, although they are essential for preventing process deterioration. Also, we often observed many uncharacterized or uncultivated organisms, but poorly understood their role(s) in relation to syntrophy. To define syntrophy-associated populations, this study enriched methanogenic communities with propionate, butyrate, benzoate, acetate, formate and H2 from two different inocula over 3 years. 16S pyrotag analysis revealed core populations of known syntrophs (six clades) and methanogens (nine clades) associated with acid degradation, and evidence for substrate- and/or inoculum-dependent specificity in syntrophic partnerships. Based on comprehensive re-evaluation of publically available microbial community data for AD, the known syntrophs and methanogens identified were clearly representatives of the AD-associated syntrophs and methanogens. In addition, uncultivated clades related to Bacteroidetes, Firmicutes, Actinobacteria and Chloroflexi were ubiquitously found in AD and enrichments. These organisms may be universally involved in AD syntrophic degradation, but only represented <23% of the yet-to-be-cultivated organisms (89 of 390 clades). Thus, the contribution of these uncultured organisms in AD remains unclear and warrants further investigation.

The genome of Syntrophorhabdus aromaticivorans strain UI provides new insights for syntrophic aromatic compound metabolism and electron flow.

How aromatic compounds are degraded in various anaerobic ecosystems (e.g. groundwater, sediments, soils and wastewater) is currently poorly understood. Under methanogenic conditions (i.e. groundwater and wastewater treatment), syntrophic metabolizers are known to play an important role. This study explored the draft genome of Syntrophorhabdus aromaticivorans strain UI and identified the first syntrophic phenol-degrading phenylphosphate synthase (PpsAB) and phenylphosphate carboxylase (PpcABCD) and syntrophic terephthalate-degrading decarboxylase complexes. The strain UI genome also encodes benzoate degradation through hydration of the dienoyl-coenzyme A intermediate as observed in Geobacter metallireducens and Syntrophus aciditrophicus. Strain UI possesses electron transfer flavoproteins, hydrogenases and formate dehydrogenases essential for syntrophic metabolism. However, the biochemical mechanisms for electron transport between these H2 /formate-generating proteins and syntrophic substrate degradation remain unknown for many syntrophic metabolizers, including strain UI. Analysis of the strain UI genome revealed that heterodisulfide reductases (HdrABC), which are poorly understood electron transfer genes, may contribute to syntrophic H2 and formate generation. The genome analysis further identified a putative ion-translocating ferredoxin : NADH oxidoreductase (IfoAB) that may interact with HdrABC and dissimilatory sulfite reductase gamma subunit (DsrC) to perform novel electron transfer mechanisms associated with syntrophic metabolism.

Metagenomic characterization of 'Candidatus†Defluviicoccus tetraformis strain TFO71', a tetrad-forming organism, predominant in an anaerobic-aerobic membrane bioreactor with deteriorated biological phosphorus removal.

In an acetate-fed anaerobic-aerobic membrane bioreactor with deteriorated enhanced biological phosphorus removal (EBPR), Defluviicoccus-related tetrad-forming organisms (DTFO) were observed to predominate in the microbial community. Using metagenomics, a partial genome of the predominant DTFO, 'Candidatus Defluviicoccus tetraformis strain TFO71', was successfully constructed and characterized. Examining the genome confirmed the presence of genes related to the synthesis and degradation of glycogen and polyhydroxyalkanoate (PHA), which function as energy and carbon storage compounds. TFO71 and 'Candidatus Accumulibacter phosphatis' (CAP) UW-1 and CAP UW-2, representative polyphosphate-accumulating organisms (PAO), have PHA metabolism-related genes with high homology, but TFO71 has unique genes for PHA synthesis, gene regulation and granule management. We further discovered genes encoding DTFO polyphosphate (polyP) synthesis, suggesting that TFO71 may synthesize polyP under untested conditions. However, TFO71 may not activate these genes under EBPR conditions because the retrieved genome does not contain all inorganic phosphate transporters that are characteristic of PAOs (CAP UW-1, CAP UW-2, Microlunatus phosphovorus NM-1 and Tetrasphaera species). As a first step in characterizing EBPR-associated DTFO metabolism, this study identifies important differences between DTFO and PAO that may contribute to EBPR community competition and deterioration.

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.

Metatranscriptomics-guided genome-scale metabolic reconstruction reveals the carbon flux and trophic interaction in methanogenic communities.

BACKGROUND: Despite rapid advances in genomic-resolved metagenomics and remarkable explosion of metagenome-assembled genomes (MAGs), the function of uncultivated anaerobic lineages and their interactions in carbon mineralization remain largely uncertain, which has profound implications in biotechnology and biogeochemistry. RESULTS: In this study, we combined long-read sequencing and metatranscriptomics-guided metabolic reconstruction to provide a genome-wide perspective of carbon mineralization flow from polymers to methane in an anaerobic bioreactor. Our results showed that incorporating long reads resulted in a substantial improvement in the quality of metagenomic assemblies, enabling the effective recovery of 132 high-quality genomes meeting stringent criteria of minimum information about a metagenome-assembled genome (MIMAG). In addition, hybrid assembly obtained 51% more prokaryotic genes in comparison to the short-read-only assembly. Metatranscriptomics-guided metabolic reconstruction unveiled the remarkable metabolic flexibility of several novel Bacteroidales-affiliated bacteria and populations from Mesotoga sp. in scavenging amino acids and sugars. In addition to recovering two circular genomes of previously known but fragmented syntrophic bacteria, two newly identified bacteria within Syntrophales were found to be highly engaged in fatty acid oxidation through syntrophic relationships with dominant methanogens Methanoregulaceae bin.74 and Methanothrix sp. bin.206. The activity of bin.206 preferring acetate as substrate exceeded that of bin.74 with increasing loading, reinforcing the substrate determinantal role. CONCLUSION: Overall, our study uncovered some key active anaerobic lineages and their metabolic functions in this complex anaerobic ecosystem, offering a framework for understanding carbon transformations in anaerobic digestion. These findings advance the understanding of metabolic activities and trophic interactions between anaerobic guilds, providing foundational insights into carbon flux within both engineered and natural ecosystems. Video Abstract.

Microscopic and metatranscriptomic analyses revealed unique cross-domain parasitism between phylum Candidatus Patescibacteria/candidate phyla radiation and methanogenic archaea in anaerobic ecosystems.

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 (r2 ≥ 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.IMPORTANCECulture-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.

Microbiological insights into anaerobic phenol degradation mechanisms and bulking phenomenon in a mesophilic upflow anaerobic sludge blanket reactor in long-term operation.

In this study, a long-term operation of 2,747 days was conducted to evaluate the performance of the upflow anaerobic sludge blanket (UASB) reactor and investigated the degradation mechanisms of high-organic loading phenol wastewater. During the reactor operation, the maximum chemical oxygen demand (COD) removal rate of 6.1 ± 0.6 kg/m3/day under 1,680 mg/L phenol concentration was achieved in the mesophilic UASB reactor. After a significant change in the operating temperature from 24.0 ± 4.1 °C to 35.9 ± 0.6 °C, frequent observations of floating and washout of the bloated granular sludge (novel types of the bulking phenomenon) were made in the UASB reactor, suggesting that the change in operating temperature could be a trigger for the bulking phenomenon. Through the metagenomic analysis, phenol degradation mechanisms were predicted that phenol was converted to 4-hydroxybenzoate via two possible routes by Syntrophorhabdaceae and Pelotomaculaceae bacteria. Furthermore, the degradation of 4-hydroxybenzoate to benzoyl-CoA was carried out by members of Syntrophorhabdaceae and Smithellaceae. In the bulking sludge, a predominant presence of Nanobdellota, belonging to DPANN archaea, was detected. The metagenome-assembled genome of the Nanobdellota lacks many biosynthetic pathways and has several genes for the symbiotic lifestyle such as trimeric autotransporter adhesin-related protein. Furthermore, the Nanobdellota have significant correlations with several methanogenic archaea that are predominantly present in the UASB reactor. Considering the results of this study, the predominant Nanobdellota may negatively affect the growth of the methanogens through the parasitic lifestyle and change the balance of microbial interactions in the granular sludge ecosystem.

Development of an internal two-stage upflow anaerobic reactor integrating biostimulation strategies to enhance the degradation of aromatic compounds in wastewater from purified terephthalic acid and dimethyl terephthalate manufacturing processes.

In this study, we aimed to establish high-rate biological treatment of purified terephthalic acid (PTA) and dimethyl terephthalate (DMT) wastewater that minimizes the inhibitory effects of high concentration benzoate and acetate. To achieve this, we developed a novel bioreactor system and biostimulation strategy. An internal two-stage upflow anaerobic (ITUA) reactor was operated with (i) a packed bed containing green tuff medium underlying (ii) a compartment seeded with anaerobic granular sludge. Ethylene glycol was amended to stimulate syntrophic interactions. Continuous operation of the system for 1,026 days achieve an organic removal rate of 11.0 ± 0.6 kg COD/m3/d. The abundance of aromatic degraders significantly increased during operation. Thus, we successfully developed a high-rate treatment system to treat wastewater from the PTA/DMT manufacturing processes by activating syntrophs in an ITUA reactor.

The origin and evolution of methanogenesis and Archaea are intertwined.

Methanogenesis has been widely accepted as an ancient metabolism, but the precise evolutionary trajectory remains hotly debated. Disparate theories exist regarding its emergence time, ancestral form, and relationship with homologous metabolisms. Here, we report the phylogenies of anabolism-involved proteins responsible for cofactor biosynthesis, providing new evidence for the antiquity of methanogenesis. Revisiting the phylogenies of key catabolism-involved proteins further suggests that the last Archaea common ancestor (LACA) was capable of versatile H2-, CO2-, and methanol-utilizing methanogenesis. Based on phylogenetic analyses of the methyl/alkyl-S-CoM reductase family, we propose that, in contrast to current paradigms, substrate-specific functions emerged through parallel evolution traced back to a nonspecific ancestor, which likely originated from protein-free reactions as predicted from autocatalytic experiments using cofactor F430. After LACA, inheritance/loss/innovation centered around methanogenic lithoautotrophy coincided with ancient lifestyle divergence, which is clearly reflected by genomically predicted physiologies of extant archaea. Thus, methanogenesis is not only a hallmark metabolism of Archaea, but the key to resolve the enigmatic lifestyle that ancestral archaea took and the transition that led to physiologies prominent today.

Identification and cultivation of anaerobic bacterial scavengers of dead cells.

The cycle of life and death and Earth's carbon cycle(s) are intimately linked, yet how bacterial cells, one of the largest pools of biomass on Earth, are recycled back into the carbon cycle remains enigmatic. In particular, no bacteria capable of scavenging dead cells in oxygen-depleted environments have been reported thus far. In this study, we discover the first anaerobes that scavenge dead cells and the two isolated strains use distinct strategies. Based on live-cell imaging, transmission electron microscopy, and hydrolytic enzyme assays, one strain (designated CYCD) relied on cell-to-cell contact and cell invagination for degrading dead food bacteria where as the other strain (MGCD) degraded dead food bacteria via excretion of lytic extracellular enzymes. Both strains could degrade dead cells of differing taxonomy (bacteria and archaea) and differing extents of cell damage, including those without artificially inflicted physical damage. In addition, both depended on symbiotic metabolic interactions for maximizing cell degradation, representing the first cultured syntrophic Bacteroidota. We collectively revealed multiple symbiotic bacterial decomposition routes of dead prokaryotic cells, providing novel insight into the last step of the carbon cycle.

Draft Genome Sequence of the Extremely Haloalkaliphilic and Homoacetic Bacterium Natroniella acetigena Z-7937T.

Natroniella acetigena Z-7937T (= DSM 9952T) is a heterotrophic homoacetogenic natronophile. The draft genome sequence is 2.6 Mb in 116 contigs, with a G+C content of 34.1%.

Draft Genome Sequence of the Alkaliphilic, Lithoautotrophic Homoacetogen Fuchsiella alkaliacetigena Z-7100T.

Fuchsiella alkaliacetigena is a spore-forming, alkaliphilic hydrogentrophic homoacetogen that was isolated from the soda lake Lake Tanatar III in Russia. The genome of the type strain Z-7100 (= DSM 24880) is 2.9 Mb, with a G+C content of 36.2%.

Symbiosis between Candidatus Patescibacteria and Archaea Discovered in Wastewater-Treating Bioreactors.

Each prokaryotic domain, Bacteria and Archaea, contains a large and diverse group of organisms characterized by their ultrasmall cell size and symbiotic lifestyles (potentially commensal, mutualistic, and parasitic relationships), namely, Candidatus Patescibacteria (also known as the Candidate Phyla Radiation/CPR superphylum) and DPANN archaea, respectively. Cultivation-based approaches have revealed that Ca. Patescibacteria and DPANN symbiotically interact with bacterial and archaeal partners and hosts, respectively, but that cross-domain symbiosis and parasitism have never been observed. By amending wastewater treatment sludge samples with methanogenic archaea, we observed increased abundances of Ca. Patescibacteria (Ca. Yanofskybacteria/UBA5738) and, using fluorescence in situ hybridization (FISH), discovered that nearly all of the Ca. Yanofskybacteria/UBA5738 cells were attached to Methanothrix (95.7 ± 2.1%) and that none of the cells were attached to other lineages, implying high host dependency and specificity. Methanothrix filaments (multicellular) with Ca. Yanofskybacteria/UBA5738 attached had significantly more cells with no or low detectable ribosomal activity (based on FISH fluorescence) and often showed deformations at the sites of attachment (based on transmission electron microscopy), suggesting that the interaction is parasitic. Metagenome-assisted metabolic reconstruction showed that Ca. Yanofskybacteria/UBA5738 lacks most of the biosynthetic pathways necessary for cell growth and universally conserves three unique gene arrays that contain multiple genes with signal peptides in the metagenome-assembled genomes of the Ca. Yanofskybacteria/UBA5738 lineage. The results shed light on a novel cross-domain symbiosis and inspire potential strategies for culturing CPR and DPANN. IMPORTANCE 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. We explored the possibility of symbiotic interactions with Archaea by amending wastewater treatment sludge samples that were rich in Ca. Patescibacteria and Archaea with an isolate archaeon that is closely related to a methanogen population abundant in situ (Methanothrix). This strategic cultivation successfully established enrichment cultures that were mainly comprised of Ca. Patescibacteria (family level lineage Ca. Yanofskybacteria/UBA5738) and Methanothrix, in which we found highly specific physical interactions between the two organisms. Microscopic observations based on transmission electron microscopy, target-specific fluorescence in situ hybridization, and metagenomic analyses showed evidence that the interaction is likely parasitic. The results show a novel cross-domain parasitism between Bacteria and Archaea and suggest that the amendment of host Archaea may be an effective approach in culturing novel Ca. Patescibacteria.

Complete Genome Sequence of "Candidatus Hydrogeosomobacter endosymbioticus," an Intracellular Bacterial Symbiont of the Anaerobic Ciliate Scuticociliate GW7.

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.

Novel Cross-domain Symbiosis between Candidatus Patescibacteria and Hydrogenotrophic Methanogenic Archaea Methanospirillum Discovered in a Methanogenic Ecosystem.

To identify novel cross-domain symbiosis between Candidatus Patescibacteria and Archaea, we performed fluorescence in situ hybridization (FISH) on enrichment cultures derived from methanogenic bioreactor sludge with the newly designed 32-520-1066 probe targeting the family-level uncultured clade 32-520/UBA5633 lineage in the class Ca. Paceibacteria. All FISH-detectable 32-520/UBA5633 cells were attached to Methanospirillum, indicating high host specificity. Transmission electron microscopy observations revealed 32-520/UBA5633-like cells that were specifically adherent to the plug structure of Methanospirillum-like rod-shaped cells. The metagenome-assembled genomes of 32-520/UBA5633 encoded unique gene clusters comprising pilin signal peptides and type IV pilins. These results provide novel insights into unseen symbiosis between Ca. Patescibacteria and Archaea.