Abstract
The origin of eukaryotes remains unclear1,2,3,4. 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.
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Data availability
Genomes for Ca. P. syntrophicum MK-D1, Halodesulfovibrio sp. MK-HDV and Methanogenium sp. MK-MG are available under GenBank BioProject accession numbers PRJNA557562, PRJNA557563 and PRJNA557565, respectively. The iTAG sequence data was deposited in BioProject PRJDB8518 with SRA accession numbers DRR184081–DRR184101. The 16S rRNA gene sequences of MK-D1, Halodesulfovibrio sp. MK-HDV, Methanogenium sp. MK-MG and clones obtained from primary enrichment culture were deposited in the DDBJ/EMBL/GenBank database under accession numbers LC490619–LC490624. The gene expression data of MK-D1 in BioProject PRJDB9032 with the accession number DRR199588. The cryo-electron tomograms of Ca. P. syntrophicum MK-D1 have been deposited in the EMDB with accession codes EMD-0809 and EMD-0852.
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Acknowledgements
We thank H. Ohno and T. Yamaguchi for assistance with HCR-FISH analysis; T. Terada for help with NanoSIMS sample preparation; M. Isozaki for assistance with cultivation experiments; T. Kubota for assistance with chemical analysis; K. Takishita, A. Yabuki, T. Shiratori, A. Ohashi, F. Inagaki, T. Nunoura, S. Kawagucci, T. Shibuya, S. Ishii, S. Suzuki, Y. Tsukatani, C. Chen, Y. Kuruma and R. C. Robinson for advice and discussion; A. Miyashita, Y. Yashiro, K. Aoi, M. Ehara, M. Aoki and Y. Saito for assistance with operating the bioreactor; and J. Ashi and the RV Yokosuka and RV Shinkai 6500 operation team during cruise YK06-03 (JAMSTEC) and the shipboard scientists and crews of the RV Chikyu Shakedown Cruise CK06-06 for their assistance in collecting samples. This study was partially supported by grants from the Japan Society for the Promotion of Science (JSPS) (KAKENHI grants 18687006, 21687006, 24687011, 15H02419 and 19H01005 to H.I., 18H03367 to M.K.N., 26710012, 18H02426, 18H05295 to H.T., 18H04468 and 18K18795 to M.I. and Grant-in-Aid for JSPS Fellow 16J10845 to N.N.). This work was also supported by JSPS KAKENHI grant number JP16H06280, Grant-in-Aid for Scientific Research on Innovative Areas–Platforms for Advanced Technologies and Research Resources ‘Advanced Bioimaging Support’ and the Cooperative Study Program (19-504) of National Institute for Physiological Sciences.
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Contributions
H.I. conceived the study and carried out the deep-marine sediment sampling. H.I., N.N., M.O., M.M. and S.S. conducted cultivation and culture-based experiments. M.K.N. performed metabolic reconstruction and phylogenetic analyses. M.K.N. and Y. Takaki performed genome analysis. H.I., N.N., Y. Morono, M.O., T.I., M.I., K.M., C.S. and K.U. carried out the microscopy and NanoSIMS work. M.O., Y.S. and Y.Y. performed qPCR, SSU rRNA gene analysis and DNA/RNA sequencing. Y. Takano, Y. Matsui and E.T. performed chemical analysis. H.I., M.K.N., N.N., Y. Morono, Y. Takaki, Y. Takano, K.M., C.S., T.Y., Y.K., H.T. and K.T. conducted data interpretation. H.I., M.K.N., Y. Takano, H.T., Y.K. and K.T. wrote the manuscript with input from all co-authors. All authors have read and approved the manuscript submission.
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Extended data figures and tables
Extended Data Fig. 1 Growth of MK-D1.
a, Effect of temperature on growth of MK-D1. Data are mean ± s.d. of triplicate determinations. Each data point is shown as a dot. The temperature range test was performed twice with similar results. b, c, The amino acid concentrations and growth curves of MK-D1 in pure cocultures at 20 °C. Results from cultures 1 (b) and 2 (c) are shown. Please note that the initial concentrations of amino acids were normalized to 100%. Total amino acids and several representative amino acids (Val, valine; Leu, leucine; Ile, isoleucine) are independently shown for the duplicate culture samples. Detailed iTAG-based community compositions of the cultures are shown in Supplementary Table 1.
Extended Data Fig. 2 Circular representation of MK-D1 genome.
From the outside to the centre: the distribution of the coding sequences based on the conserved (orange) or non-conserved (grey) genes in the first circle, non-coding RNAs in the second circle, GC content showing deviation from average (40.7%) in the third circle, and GC skew in the fourth circle. The GC content and GC skew were calculated using a sliding window of 2 kb in step of 10 kb. The coding sequences and RNA genes illustrate the findings for plus and minus strands.
Extended Data Fig. 3 Other representative photomicrographs of MK-D1 cultures and Methanobacterium sp. strain MO-MB1.
a, b, Fluorescence images of cells from enrichment cultures after 8 (a) and 11 (b) transfers stained with DAPI (violet) and hybridized with nucleotide probes that target MK-D1 (green) and Bacteria (red). The images are different fields of view to those shown in Fig. 1b, c, which were taken at the same time. c, A fluorescence image of cells in the enrichments after 11 transfers hybridized with nucleotide probes that target MK-D1 (green) and Archaea (but with one mismatch against MK-D1; red). Large and irregular coccoid-shaped cells stained by only ARC915 are probably Methanogenium. d, e, Dividing cells of MK-D1 with a bleb. The top-right inset image in e shows a magnification of the bleb. f, g, Cryo-EM images of MK-D1 cells and large membrane vesicles (white arrows). h, i, Ultrathin sections of MK-D1 cells with a membrane vesicle. The image i shows a magnified image of h. j, k, SEM images of MK-D1 cells with protrusions. l, Ultrathin section of a MK-D1 cell with a protrusion. m, n, Photomicrographs of pure culture of Methanobacterium sp. strain MO-MB1 cells stained with SYBR Green I. Phase-contrast (m) and fluorescence (n) images of the same field are shown. a, b, The FISH experiments were performed three times with similar results. d, e, j, k, The SEM images are representative of n = 122 recorded images that were obtained from four independent observations from four culture samples. The lipid composition experiments were repeated twice and gave similar results. f, g, The cryo-EM images are representative of n = 14 recorded images that were taken from two independent observations from two culture samples. h, i, l, The ultrathin-section images are representative of n = 131 recorded images that were obtained from six independent observations from six culture samples. m, n, The SYBR Green I staining experiment was performed once, but all 10 recorded images showed similar results. Detailed iTAG analyses of cultures are shown in Supplementary Table 1.
Extended Data Fig. 4 Ribosomal protein- and 16S rRNA gene-based phylogeny of MK-D1.
a, Phylogenomic tree of MK-D1 and select cultured archaea, eukaryotes and bacteria based on 31 ribosomal proteins conserved across the three domains (Supplementary Table 7). Ribosomal protein sequences of MK-D1, the organisms shown in the tree and MAGs of uncultured archaeal lineages (Supplementary Table 8) were aligned individually using MAFFT. MAG-derived sequences (except for Ca. Korarchaeum) were then removed for tree construction. After removing all-gap positions and concatenation, the maximum-likelihood tree was constructed using RAxML-NG. Bootstrap values around critical branching points are also shown. In total, 14,875 sites of the alignment were used for tree construction. b, A ribosomal protein-based phylogenomic tree constructed using MrBayes. Bayesian inference phylogenies were calculated using MrBayes 3.2.7a and a ribosomal protein concatenated alignment used for Fig. 4a. c, Phylogenetic tree of MK-D1 and related archaea based on 16S rRNA genes. The 16S rRNA gene sequences were aligned using SINA against the Silva v.132 alignment and the maximum-likelihood tree was calculated using RAxML.
Extended Data Fig. 5 Amino acid, cofactor and nucleotide biosynthesis capacities of MK-D1 and other Asgard archaea.
Genomes that encode proteins for the synthesis of amino acids, cofactors and nucleotides from pyruvate or acetyl-CoA (dark blue) and synthesis from other intermediates (light blue) are indicated. Those without complete pathways from pyruvate and/or acetyl-CoA are indicated in white. Halodesulfovibrio sp. strain MK-HDV and Methanogenium sp. strain MK-MG isolated in this study are also shown.
Extended Data Fig. 6 Maximum-likelihood tree of Asgard archaea urocanate hydratase.
Urocanate hydratase (HutU) homologues were obtained by BLASTp analysis of the Asgard archaea sequences against the UniProt database (release 2019_06). Of homologues with sequence similarity ≥40% and overlap ≥70%, representative sequences were selected using CD-HIT with a clustering cut-off of 70% similarity (otherwise default settings were used). Additional homologues with verified biochemical activity, sequence similarity ≥30% and overlap ≥70% were obtained by BLASTp analysis of the Asgard archaea sequences against the UniProt/SwissProt database (2019_05). Sequences were aligned using MAFFT v.7 with default settings and trimmed using trimAl v.1.2 with default settings. The maximum-likelihood tree was constructed using RAxML-NG using fixed empirical substitution matrix (LG), 4 discrete GAMMA categories, empirical amino acid frequencies from the alignment and 100 bootstrap replicates. In total, 876 sites of the alignment were used for tree construction.
Extended Data Fig. 7 Maximum-likelihood tree of Asgard archaea l-threonine/l-serine dehydratase.
a, Tree calculated for target Asgard archaea l-threonine/l-serine dehydratase (TdcB) and homologues. TdcB homologues were obtained by BLASTp analysis of the Asgard archaea sequences against the UniProt reference proteome and SwissProt database (release 2019_06). Of homologues with sequence similarity ≥40%, overlap ≥70% and predicted prosite domain PS00165 (serine/threonine dehydratases pyridoxal-phosphate attachment site), representative sequences were selected using CD-HIT with a clustering cut-off of 70% similarity (otherwise default settings were used). Additional homologues with verified biochemical activity, sequence similarity ≥30% and overlap ≥70% were obtained by BLASTp analysis of the Asgard archaea sequences against the UniProt/SwissProt database (2019_05). Sequences were aligned using MAFFT v.7 with default settings. Positions with gaps in more than 10% of the sequences were excluded from the alignment using trimAl v.1.2 (-gt 0.9; and otherwise default settings were used). The maximum-likelihood tree was constructed using PhyML using a fixed empirical substitution matrix (LG), 4 discrete GAMMA categories, empirical amino acid frequencies from the alignment and 100 bootstrap replicates (-b 100 -d aa -m LG -v e). In total, 308 sites of the alignment were used for tree construction. b, Tree calculated for a subset of sequences contained in a section of the original tree (branches that are coloured blue). Sequences were realigned and trimmed as described for a. In total, 308 sites of the alignment were used for tree construction.
Supplementary information
Supplementary Information
This file contains Supplementary Notes 1–9, Supplementary Methods, Supplementary Figures 1–18, and Supplementary References.
Supplementary Tables
This file contains Supplementary Tables 1–10
Supplementary Video 1
| Tilt-series images of a single cell of MK-D1
Supplementary Video 2
| Z-slices of the tomographic three-dimensional reconstruction from the tilt-series in Supplementary Video 1
Supplementary Video 3
| Animation of the same MK-D1 cell as in Supplementary Video 2 The cell envelope and membrane vesicles are colored in light blue and pink, respectively.
Supplementary Video 4
| Tilt-series images of MK-D1 cells
Supplementary Video 5
| Z-slices of the tomographic three-dimensional reconstruction from the tilt-series in Supplementary Video 4
Supplementary Video 6
| Animation of the same MK-D1 cells as in Supplementary Video 5 The cell envelope and membrane vesicles are colored in light blue and pink, respectively.
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Imachi, H., Nobu, M.K., Nakahara, N. et al. Isolation of an archaeon at the prokaryote–eukaryote interface. Nature 577, 519–525 (2020). https://doi.org/10.1038/s41586-019-1916-6
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DOI: https://doi.org/10.1038/s41586-019-1916-6
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