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The microbial communities inhabiting the fronds of duckweeds have not been investigated in as much detail as those on the roots. We herein examined the microbial communities in three duckweed species using 16S rRNA amplicon sequencing and compared them to those on the roots. The microbial compositions of the fronds were distinct from those of the roots in the three species. Various types of taxonomic bacteria, including rarely cultivated phyla, Acidobacteria, Armatimonadetes, and Verrucomicrobia, were also isolated from the fronds, but at a slightly lower abundance than those from the roots. These results suggest that duckweed fronds are an alternative source for isolating rare and novel microbes, which may otherwise be recalcitrant to cultivation using conventional strategies.

Imachi, Hiroyuki, Nobu, Masaru K., Nakahara, Nozomi, Morono, Yuki, Ogawara, Miyuki, Takaki, Yoshihiro, Takano, Yoshinori, Uematsu, Katsuyuki, Ikuta, Tetsuro, Ito, Motoo, Matsui, Yohei, Miyazaki, Masayuki, Murata, Kazuyoshi, Saito, Yumi, Sakai, Sanae, Song, Chihong, Tasumi, Eiji, Yamanaka, Yuko, Yamaguchi, Takashi, Kamagata, Yoichi, Tamaki, Hideyuki, Takai, Ken (2020): Isolation of an archaeon at the prokaryote eukaryote interface. Nature 41586: 1-23, DOI: 10.1038/s41586-019-1916-6

‘ Candidatus Prometheoarchaeum syntrophicum ’ strain MK-D1 for the isolated archaeon (see Supplementary Note 3 for reasons why the provisional Candidatus status is necessary despite isolation). Cell biology, physiology and metabolism We further characterized MK-D1 using the pure co-cultures and highly purified cultures.Microscopy analyses showed that the cells were small cocci (approximately300–750nm in diameter (average,550 nm)),and generally formed aggregates surrounded by extracellular polymer substances (EPS) (Fig.3a,b and Extended Data Fig.3),consistent with previous observations using FISH 15, 17. MK-D1 cells were easily identifiable given the morphological difference from their co-culture partner Methanogenium (highly irregular coccoid cells of ≥2 µm; Fig. 1d, e). Dividing cells had less EPS and a ring-like structure around the cells (Fig. 3c). Cryo-electron microscopy (cryo-EM) and transmission electron microscopy (TEM) analyses revealed that the cells contain no visible organelle-like inclusions (Fig. 3 d–f and Supplementary Videos 1–6), in contrast to previous suggestions 6. For cryo-EM, cells were differentiated from vesicles on the basis of the presence of cytosolic material (although DNA and ribosomes could not be differentiated),EPS on the cell surface and cell sizes that were consistent with observations by SEM and TEM analyses (Supplementary Videos 4–6). The cells produce membrane vesicles (50–280 nm in diameter) (Fig. 3 b–f) and chains of blebs (Fig. 3c). MK-D1 cells also form membrane-based cytosol-connected protrusions of various lengths that have diameters of 80–100 nm,and display branching with a homogeneous appearance unlike those of other archaea (Fig.3 g–i; confirmed using both SEM and TEM).These protrusions neither form elaborate networks (as in Pyrodictium 18) nor intercellular connections (Pyrodictium, Thermococcus and Haloferax 18 – 20), suggesting differences in physiological functions.The MK-D1 cell envelope may be composed of a membrane and a surrounding S-layer, given the presence of four genes that encode putative S-layer proteins (Supplementary Fig. 1), stalk-like structures on the surface of the vesicles (Fig. 3e and Extended Data Fig.3f, g) and the even distance between the inner and outer layers of the cell envelope (Fig.3d). Lipid composition analysis of the MK-D1 and Methanogenium co-culture revealed typical archaeal isoprenoid signatures—C 20 -phytane and C 40 -biphytanes with 0–2cyclopentane rings were obtained after ether-cleavage treatment(Fig.3j). Considering the lipid data obtained from a reference Methanogenium isolate (99.3% 16S rRNA gene identity; Supplementary Fig. 2), MK-D1 probably contains C 20 -phytane and C 40 -biphytanes with 0–2 rings. The MK-D1 genome encoded most of the genes necessary to synthesize ether-type lipids—although geranylgeranylglyceryl phosphate synthase was missing—and lacked genes for ester-type lipid synthesis (Supplementary Tables 3, 4). MK-D1 can degrade amino acids anaerobically, as confirmed by monitoring the depletion of amino acids during the growth of pure co-cultures (Extended Data Fig. 1b, c). We further verify the utilization of amino acids by quantifying the uptake of a mixture of 13 C- and 15 N-labelled amino acids through nanometre-scale secondary ion mass spectrometry (NanoSIMS) (Fig. 2 b–e). Cell aggregates of MK-D1 incorporated amino-acid-derived nitrogen, demonstrating the capacity of MK-D1 to utilize amino acids for growth.Notably,the 13 C-labelling of methane and CO 2 varied depending on the methanogenic partner, indicating that MK-D1 produces both hydrogen and formate from amino acids for interspecies electron transfer(Extended Data Table 2). Indeed, addition of high concentrations of hydrogen or formate completely suppressed growth of MK-D1 (Extended Data Table 3). The syntrophic partner was replaceable—MK-D1 could also grow syntrophically with Methanobacterium sp. strain MO-MB1 21 instead of Methanogenium (Fig.2 b–e). Although 14 different culture conditions were applied,none enhanced the cell yield,which indicates specialization of the degradation of amino acids and/or peptides (Extended Data Table 3). To further characterize the physiology of the archaeon,we analysed the complete MK-D1 genome (Extended Data Fig.2 and Supplementary Tables 2–6). The genome only encodes one hydrogenase (NiFe hydrogenase MvhADG–HdrABC) and formate dehydrogenase (molybdopterin-dependent FdhA),suggesting that these enzymes mediate reductive H 2 and formate generation,respectively. MK-D1 represents, to our knowledge, the first cultured archaeon that can produce and syntrophically transfer H 2 and formate using the above enzymes.We also found genes encoding proteins for the degradation of ten amino acids.Most of the identified amino-acid-catabolizing pathways only recover energy through the degradation of a 2-oxoacid intermediate (that is,pyruvate or 2-oxobutyrate; Fig.2a and Supplementary Table 4). MK-D1 can degrade 2-oxoacids hydrolytically (through 2-oxoacid-formate lyases) or oxidatively (through 2-oxoacid:ferredoxin oxidoreductases) to yield acyl-CoA intermediates that can be further degraded for ATP generation. In the hydrolytic path, the carboxylate group of the amino acid is released as formate that can be directly handed off to partnering methanogenic archaea or SRB. In the oxidative path, 2-oxoacid oxidation is coupled with release of amino acid carboxylate as CO 2 and reduction of ferredoxin,which can be re-oxidized through H + and/or CO 2 reduction to H 2 and formate,respectively (through the electron-confurcating NiFe hydrogenaseMvhADG–HdrABC or formate dehydrogenase FdhA). On the basis of 13 C-amino-acid-based experiments (Supplementary Note 4), MK-D1 can probably switch between syntrophic interaction through 2-oxoacid hydrolysis and oxidation depending on the partner(s). Etymology. Prometheoarchaeum, Prometheus (Greek):a Greek god who shaped humans out of mud and gave them the ability to create fire; archaeum from archaea (Greek):an ancient life.The genus name is an analogy between the evolutionary relationship this organism and the origin of eukaryotes,and the involvement of Prometheus in the origin of humans from sediments and the acquisition of an unprecedented oxygen-driven energy-harnessing ability.The species name, syntrophicum, syn (Greek):together with; trephein (Greek) nourish; icus (Latin) pertaining to. The species name refers to the syntrophic substrate utilization property of this strain. Locality. Isolated from deep-sea methane-seep sediment of the Nankai Trough at 2,533 m water depth,off the Kumano area,Japan. Diagnosis. Anaerobic,amino-acid-oxidizing archaeon,small coccus, around 550 nm in diameter,syntrophically grows with hydrogen- and formate-using microorganisms.It produces membrane vesicles,chains of blebs and membrane-based protrusions., Published as part of Imachi, Hiroyuki, Nobu, Masaru K., Nakahara, Nozomi, Morono, Yuki, Ogawara, Miyuki, Takaki, Yoshihiro, Takano, Yoshinori, Uematsu, Katsuyuki, Ikuta, Tetsuro, Ito, Motoo, Matsui, Yohei, Miyazaki, Masayuki, Murata, Kazuyoshi, Saito, Yumi, Sakai, Sanae, Song, Chihong, Tasumi, Eiji, Yamanaka, Yuko, Yamaguchi, Takashi, Kamagata, Yoichi, Tamaki, Hideyuki & Takai, Ken, 2020, Isolation of an archaeon at the prokaryote eukaryote interface, pp. 1-23 in Nature 41586 on pages 2-4, DOI: 10.1038/s41586-019-1916-6, http://zenodo.org/record/3609900, {"references":["15. Aoki, M. et al. A long-term cultivation of an anaerobic methane-oxidizing microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor. PLoS ONE 9, e 105356 (2014).","17. Knittel, K., Losekann, T., Boetius, A., Kort, R. & Amann, R. Diversity and distribution of methanotrophic archaea at cold seeps. Appl. Environ. Microbiol. 71, 467 - 479 (2005).","6. Zaremba-Niedzwiedzka, K. et al. Asgard archaea illuminate the origin of eukaryotic cellular complexity. Nature 541, 353 - 358 (2017).","18. Albers, S. - V. & Meyer, B. H. The archaeal cell envelope. Nat. Rev. Microbiol. 9, 414 - 426 (2011).","20. Rosenshine, I., Tchelet, R. & Mevarech, M. The mechanism of DNA transfer in the mating system of an archaebacterium. Science 245, 1387 - 1389 (1989).","13. Pushkarev, A. et al. A distinct abundant group of microbial rhodopsins discovered using functional metagenomics. Nature 558, 595 - 599 (2018).","49. Nakamura, K. et al. Application of pseudomurein endoisopeptidase to fluorescence in situ hybridization of methanogens within the family Methanobacteriaceae. Appl. Environ. Microbiol. 72, 6907 - 6913 (2006).","21. Imachi, H. et al. Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor. ISME J. 5, 1913 - 1925 (2011)."]}

We investigated the in situ gene expression profile of sulfur-turf microbial mats dominated by an uncultured large sausage-shaped Aquificae bacterium, a key metabolic player in sulfur-turfs in sulfidic hot springs. A reverse transcription-PCR analysis revealed that the genes responsible for sulfide, sulfite, and thiosulfate oxidation and carbon fixation via the reductive TCA cycle were continuously expressed in sulfur-turf mats taken at different sampling points, seasons, and years. These results suggest that the uncultured large sausage-shaped bacterium has the ability to grow chemolithoautotrophically and plays key roles as a primary producer in the sulfidic hot spring ecosystem in situ.

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 H-2/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 H-2 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.

The family Methanomicrobiaceae comprises hydrogen- and formate-utilizing methanogens. Genome sequencing of nine species of Methanomicrobiaceae has been conducted so far. Here, we report three additional draft genome sequences of Methanomicrobiaceae, those of Methanoculleus horonobensis JCM 15517 (=T10T), Methanoculleus thermophilus DSM 2373 (=CR-1T), and Methanofollis ethanolicus JCM 15103 (=HASUT).

International audience; High-affinity H2 -oxidizing bacteria possessing group 5 [NiFe]-hydrogenase genes are important contributors to atmospheric hydrogen (H2 ) uptake in soil environments. Although previous studies reported the occurrence of a significant H2 uptake activity in vegetation, there has been no report on the identification and diversity of the responsible microorganisms. Here, we show the existence of plant-associated bacteria with the ability to consume atmospheric H2 that may be a potential energy source required for their persistence in plants. Detection of the gene hhyL - encoding the large subunit of group 5 [NiFe]-hydrogenase - in plant tissues showed that plant-associated high-affinity H2 -oxidizing bacteria are widely distributed in herbaceous plants. Among a collection of 145 endophytic isolates, 7 Streptomyces strains were shown to possess hhyL gene and exhibit high- or intermediate-affinity H2 uptake activity. Inoculation of Arabidopsis thaliana (thale cress) and Oryza sativa (rice) seedlings with selected isolates resulted in an internalization of the bacteria in plant tissues. H2 uptake activity per bacterial cells was comparable between plant and soil, demonstrating that both environments are favorable for the H2 uptake activity of streptomycetes. This study first demonstrated the occurrence of plant-associated high-affinity H2 -oxidizing bacteria and proposed their potential contribution as a sink for atmospheric H2 .

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.

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Additional file 3: Figure S1. Comparison of the taxonomic composition (at the phylum level) of reads generated using long-read (this study) and short-read [38] platforms. Data from nine Japanese lakes sampled in 2015 were averaged for both water layers. Note that the same DNA extracts were used for both studies. Figure S2. The relative abundance of ASVs in each sample for each lineage. Rows and columns are clustered based on the Bray-Curtis dissimilarity among samples and ASVs, respectively (see Materials and Methods for detail). Abbreviations for sample names follow those in Fig. 5. Legends are shown at the bottom. Figure S3. Clustering of samples based on Bray–Curtis dissimilarity of amplicon sequence variant composition for each lineage. Abbreviations for sample names follow those in Fig. 5. Figure S4. Comparison of the five most abundant amplicon sequence variants (ASVs) between temporal replicates (2010 and 2015) collected in Lake Biwa for each water layer. Bars indicate the relative abundances of ASVs within each lineage and are ordered by abundance rank for each sample. Gray lines indicate succession of ranks between two time points; N.D., not detected. Supplementary Text (R script).

Additional file 3: Figure S1. Comparison of the taxonomic composition (at the phylum level) of reads generated using long-read (this study) and short-read [38] platforms. Data from nine Japanese lakes sampled in 2015 were averaged for both water layers. Note that the same DNA extracts were used for both studies. Figure S2. The relative abundance of ASVs in each sample for each lineage. Rows and columns are clustered based on the Bray-Curtis dissimilarity among samples and ASVs, respectively (see Materials and Methods for detail). Abbreviations for sample names follow those in Fig. 5. Legends are shown at the bottom. Figure S3. Clustering of samples based on Bray–Curtis dissimilarity of amplicon sequence variant composition for each lineage. Abbreviations for sample names follow those in Fig. 5. Figure S4. Comparison of the five most abundant amplicon sequence variants (ASVs) between temporal replicates (2010 and 2015) collected in Lake Biwa for each water layer. Bars indicate the relative abundances of ASVs within each lineage and are ordered by abundance rank for each sample. Gray lines indicate succession of ranks between two time points; N.D., not detected. Supplementary Text (R script).

Additional file 2: Supplementary Table S2. Comparison of the 23Sr (original) and 23Sr-mod (modified for the present study) primers, showing their coverage for each phylum. Coverage was determined using the TestProbe 3.0 tool with reference to the SILVA LSU 132 Parc database (Quast et al., [55] allowing no mismatches.

Additional file 2: Supplementary Table S2. Comparison of the 23Sr (original) and 23Sr-mod (modified for the present study) primers, showing their coverage for each phylum. Coverage was determined using the TestProbe 3.0 tool with reference to the SILVA LSU 132 Parc database (Quast et al., [55] allowing no mismatches.

Viruses are diverse and play significant ecological roles in marine ecosystems. However, our knowledge of genome-level diversity in viruses is biased toward those isolated from few culturable hosts. Here, we determined 1,352 nonredundant complete viral genomes from marine environments. Lifting the uncertainty that clouds short incomplete sequences, whole-genome-wide analysis suggests that these environmental genomes represent hundreds of putative novel viral genera. Predicted hosts include dominant groups of marine bacteria and archaea with no isolated viruses to date. Some of the viral genomes encode many functionally related enzymes, suggesting a strong selection pressure on these marine viruses to control cellular metabolisms by accumulating genes., Metagenomics has revealed the existence of numerous uncharacterized viral lineages, which are referred to as viral “dark matter.” However, our knowledge regarding viral genomes is biased toward culturable viruses. In this study, we analyzed 1,600 (1,352 nonredundant) complete double-stranded DNA viral genomes (10 to 211 kb) assembled from 52 marine viromes. Together with 244 previously reported uncultured viral genomes, a genome-wide comparison delineated 617 genus-level operational taxonomic units (OTUs) for these environmental viral genomes (EVGs). Of these, 600 OTUs contained no representatives from known viruses, thus putatively corresponding to novel viral genera. Predicted hosts of the EVGs included major groups of marine prokaryotes, such as marine group II Euryarchaeota and SAR86, from which no viruses have been isolated to date, as well as Flavobacteriaceae and SAR116. Our analysis indicates that marine cyanophages are already well represented in genome databases and that one of the EVGs likely represents a new cyanophage lineage. Several EVGs encode many enzymes that appear to function for an efficient utilization of iron-sulfur clusters or to enhance host survival. This suggests that there is a selection pressure on these marine viruses to accumulate genes for specific viral propagation strategies. Finally, we revealed that EVGs contribute to a 4-fold increase in the recruitment of photic-zone viromes compared with the use of current reference viral genomes. IMPORTANCE Viruses are diverse and play significant ecological roles in marine ecosystems. However, our knowledge of genome-level diversity in viruses is biased toward those isolated from few culturable hosts. Here, we determined 1,352 nonredundant complete viral genomes from marine environments. Lifting the uncertainty that clouds short incomplete sequences, whole-genome-wide analysis suggests that these environmental genomes represent hundreds of putative novel viral genera. Predicted hosts include dominant groups of marine bacteria and archaea with no isolated viruses to date. Some of the viral genomes encode many functionally related enzymes, suggesting a strong selection pressure on these marine viruses to control cellular metabolisms by accumulating genes.