Prometheoarchaeum syntrophicum

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, pp. 1-23 : 2-4

‘ 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.

Figures

Fig.3 | Microscopy characterizationand lipid composition ofMK-D1. a–c, SEMimages ofMK-D1.Singlecell (a), aggregated cellscovered with EPS-like materials (b) and adividing cell with polar chainsof blebs (c). d, Cryo-electron tomographyimage of MK-D1.The top-right insetimage showsa magnification of the boxed area to show the cell envelopestructure.e, Cryo-EM imageof large membranevesicles attachedto and surrounding MK-D1 cells.f, Ultrathin section of anMK-D1 cell and a membranevesicle.The bottom-right insetimage showsamagnified viewof the membranevesicle.g, h,SEM imagesof MK-D1 cells producinglong branching (g)and straight (h) membraneprotrusions. i,Ultrathin section of aMK-D1 cell with protrusions.j, A total ionchromatogram of gas chromatography–massspectrometry (GC–MS) forlipids extractedfrom ahighly purified MK-D1 culture.The chemical structures ofisoprenoids and

Fig.1 | Growthcurves and photomicrographs ofthe culturedLokiarchaeota strainMK-D1.a, Growth curves of MK-D1 inanaerobic medium supplemented withcasamino acids (CA)alone;casaminoacids with20 amino acids(AAs)and powderedmilk (PM);or peptone withpowdered milk.Resultsare alsoshown forcultures fed with10- and 100-fold dilutionof casamino acids,20 amino acidsand powderedmilk.b,c, Fluorescence images of cellsfrom enrichment cultures after8 (b)and 11 (c)transfers stained withDAPI (violet) and hybridized withnucleotide probesthat targetMK-D1 (green) andBacteria (red).Piecharts show the relative abundance of microbialpopulations based onSSU rRNA gene-tag sequencing(iTAG)analysis.d, A fluorescenceimage of cellsfrom enrichment cultures after11 transfers hybridized withnucleotide probesthat target MK-D1 (green) andMethanogenium(red).TheFISH experiments were performedthree times withsimilar results.e, SEM image of a highlypurified co-culture of MK-D1 andMethanogenium. White arrows indicate Methanogeniumcells.We observed four different co-cultureswith Methanogenium.Representative of n = 40 recordedimages.Thedetailed iTAG-based communitycompositions of cultures correspondingto each of theimages areshown inSupplementary Table 1.Scale bars,10 Μm (b, c) and 5Μm (d, e).

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 ahighly purified MK-D1 culture incubated with amixture of 13C- and15N-labelled amino acids.b, Green fluorescent micrograph of SYBR Green I-stained cells.Aggregates are MK-D1,and filamentous cells areMethanobacterium sp.strainMO-MB1 (fluorescence can be weak owing tothe high rigidity andlow permeability of the cellmembrane (Extended Data Fig.2m,n;see also ref.49).c, NanoSIMS ion image of 12C(cyan). d,NanoSIMS ion image of 12C15N/12C14N (magenta).e, Overlay image of b–d. d,The colour bar indicates the relative abundance of 15Nexpressed as 15N/14N. Scale bars 5Μm.TheNanoSIMS analysis was performedwithout replicates due to itsslow growth rate andlow cell density.However,to ensurethe reproducibility,we usedtwo different types of highly purifiedcultures of MK-D1 (see Methods).Representative of n = 8 recordedimages.TheiTAG analysis of theimaged culture is shown inSupplementary Table 1.

Fig.2| Syntrophic amino acidutilizationofMK-D1.a,Genome-based metabolicreconstruction of MK-D1.Metabolicpathways identified (coloured or black) andnot identified (grey)are shown.For identified pathways,each step (solid line) or process (dotted) is markedby whether itis oxidative (red), reductive (blue),ATP-yielding (orange)or ATP-consuming (purple).Wavy arrowsindicate exchange of compounds:formate,H2, amino acids,vitamin B12, biotin,lipoateand thiamine pyrophosphate(TPP),which are predicted tobe metabolizedor synthesized bythe partnering Halodesulfovibrioand/or Methanogenium.Biosynthetic pathways areindicated with ayellow background.Metatranscriptomics-detected amino-acid-catabolizing pathwaysare indicated (black dots aboveamino 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,formylglutamate;CH3=H4F,methylene-tetrahydrofolate; CH≡H4F,methenyl-tetrahydrofolate;Fo-H4F,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 ahighly purified MK-D1 culture incubated with amixture of 13C- and15N-labelled amino acids.b, Green fluorescent micrograph of SYBR Green I-stained cells.Aggregates are MK-D1,and filamentous cells areMethanobacterium sp.strainMO-MB1 (fluorescence can be weak owing tothe high rigidity andlow permeability of the cellmembrane (Extended Data Fig.2m,n;see also ref.49).c, NanoSIMS ion image of 12C(cyan). d,NanoSIMS ion image of 12C15N/12C14N (magenta).e, Overlay image of b–d. d,The colour bar indicates the relative abundance of 15Nexpressed as 15N/14N. Scale bars 5Μm.TheNanoSIMS analysis was performedwithout replicates due to itsslow growth rate andlow cell density.However,to ensurethe reproducibility,we usedtwo different types of highly purifiedcultures of MK-D1 (see Methods).Representative of n = 8 recordedimages.TheiTAG analysis of theimaged culture is shown inSupplementary Table 1.