Isolation of an archaeon at the prokaryote eukaryote interface Author Imachi, Hiroyuki Author Nobu, Masaru K. Author Nakahara, Nozomi Author Morono, Yuki Author Ogawara, Miyuki Author Takaki, Yoshihiro Author Takano, Yoshinori Author Uematsu, Katsuyuki Author Ikuta, Tetsuro Author Ito, Motoo Author Matsui, Yohei Author Miyazaki, Masayuki Author Murata, Kazuyoshi Author Saito, Yumi Author Sakai, Sanae Author Song, Chihong Author Tasumi, Eiji Author Yamanaka, Yuko Author Yamaguchi, Takashi Author Kamagata, Yoichi Author Tamaki, Hideyuki Author Takai, Ken text Nature 2020 2020-01-15 41586 1 23 journal article 10.1038/s41586-019-1916-6 2332fa6a-fa7e-46a6-aaa9-db2473b654e1 PMC7015854 31942073 3609900 ‘ 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). Fig.2| Syntrophic amino acid utilization of MK-D1.a ,Genome-based metabolic reconstruction of MK-D1.Metabolic pathways identified (coloured or black) and not identified (grey)are shown.For identified pathways,each step (solid line) or process (dotted) is marked by whether it is oxidative (red), reductive (blue),ATP-yielding (orange)or ATP-consuming (purple).Wavy arrows indicate exchange of compounds:formate,H 2 , amino acids,vitamin B 12 , biotin,lipoate and thiamine pyrophosphate(TPP),which are predicted to be metabolized or synthesized by the partnering Halodesulfovibrio and/or Methanogenium .Biosynthetic pathways are indicated with a yellow background.Metatranscriptomics-detected amino-acid-catabolizing pathways are indicated (black dots above amino acids).DHDH,4,5-dihydroxy- 2,6-dioxohexanoate;DHDG,2-dehydro-3-deoxy-d-gluconate;DHDG6P, 3-dehydro-3-deoxy-d-gluconate 6-phosphate;Ac-CoA,acetyl-CoA;uro, urocanate;Fo-Glu,formyl glutamate;CH 3 =H 4 F,methylene-tetrahydrofolate; CH≡H 4 F,methenyl-tetrahydrofolate;Fo-H 4 F,formyl-tetrahydrofolate;2OB, 2-oxobutyrate;Prop-CoA,propionyl-CoA;ACAC,acetoacetate;GB-CoA, γ-amino-butyryl-CoA;But-CoA,butyryl-CoA;Fd,ferredoxin;XSH/X-S-S-X, thiol/disulfide pair;TCA,tricarboxylic acid cycle;PPP,pentose-phosphate pathway. b–e , NanoSIMS analysis of a highly purified MK-D1 culture incubated with a mixture of 13 C- and 15 N-labelled amino acids. b , Green fluorescent micrograph of SYBR Green I-stained cells.Aggregates are MK-D1,and filamentous cells are Methanobacterium sp.strain MO-MB1 (fluorescence can be weak owing to the high rigidity and low permeability of the cell membrane (Extended Data Fig.2m,n ;see also ref. 49 ). c , NanoSIMS ion image of 12 C(cyan). d ,NanoSIMS ion image of 12 C 15 N/ 12 C 14 N (magenta). e , Overlay image of b–d . d ,The colour bar indicates the relative abundance of 15 N expressed as 15 N/ 14 N. Scale bars 5µm.The NanoSIMS analysis was performed without replicates due to its slow growth rate and low cell density.However,to ensure the reproducibility,we used two different types of highly purified cultures of MK-D1 (see Methods).Representative of n = 8 recorded images.The iTAG analysis of the imaged culture is shown in Supplementary Table 1. 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). Fig.3 | Microscopy characterization and lipid composition of MK-D1. a–c , SEM images of MK-D1.Single cell ( a ), aggregated cells covered with EPS-like materials ( b ) and a dividing cell with polar chains of blebs ( c ). d , Cryo-electron tomography image of MK-D1.The top-right inset image shows a magnification of the boxed area to show the cell envelope structure. e , Cryo-EM image of large membrane vesicles attached to and surrounding MK-D1 cells. f , Ultrathin section of an MK-D1 cell and a membrane vesicle.The bottom-right inset image shows a magnified view of the membrane vesicle. g , h ,SEM images of MK-D1 cells producing long branching ( g )and straight ( h ) membrane protrusions. i ,Ultrathin section of a MK-D1 cell with protrusions. j , A total ion chromatogram of gas chromatography–mass spectrometry (GC–MS) for lipids extracted from a highly purified MK-D1 culture.The chemical structures of isoprenoids and their relative compositions are also shown (Supplementary Fig.2 ).Scale bars, 1 µm ( b , c , g , h ),500 nm ( a , d , e , i ) and200 nm ( f ). a–c , g , h , SEM images are representative of n = 122 recorded images that were obtained from four independent observations from four culture samples. d , e , Cryo-EM images are representative of n =14 recorded images that were taken from two independent observations from two culture samples. f , i , The ultrathin section images are representative of n = 131 recorded images that were obtained from six independent observations from six culture samples.White arrows in the images indicate large membrane vesicles.The lipid composition experiments were repeated twice and gave similar results.Detailed iTAG-based community compositions of the cultures are shown in Supplementary Table 1. 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.