558 lines
88 KiB
XML
558 lines
88 KiB
XML
<document ID-DOI="10.1038/s41586-019-1916-6" ID-GBIF-Dataset="2332fa6a-fa7e-46a6-aaa9-db2473b654e1" ID-PMC="PMC7015854" ID-PubMed="31942073" ID-Zenodo-Dep="3609900" checkinTime="1579171683687" checkinUser="plazi" docAuthor="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" docDate="2020" docId="03F887ABFFFDFFE0761FFDF68FF0FA12" docLanguage="en" docName="nature.s41586-019-1916-6.pdf" docOrigin="Nature 41586" docStyle="DocumentStyle{}" docTitle="Prometheoarchaeum syntrophicum Imachi, Nobu, Nakahara, Morono, Ogawara, Takaki, Takano, Uematsu, Ikuta, Ito, Matsui, Miyazaki, Murata, Saito, Sakai, Song, Tasumi, Yamanaka, Yamaguchi, Kamagata, Tamaki & Takai, 2020, Candidatus" docType="treatment" docVersion="9" lastPageNumber="4" masterDocId="FFC1FFD3FFFCFFE3737FFF9B8B2FFFB6" masterDocTitle="Isolation of an archaeon at the prokaryote eukaryote interface" masterLastPageNumber="23" masterPageNumber="1" pageNumber="2" updateTime="1668137217727" updateUser="ExternalLinkService">
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<mods:titleInfo>
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<mods:title>Isolation of an archaeon at the prokaryote eukaryote interface</mods:title>
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<mods:namePart>Imachi, Hiroyuki</mods:namePart>
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<mods:namePart>Nobu, Masaru K.</mods:namePart>
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<mods:namePart>Nakahara, Nozomi</mods:namePart>
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<mods:namePart>Morono, Yuki</mods:namePart>
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<mods:namePart>Ogawara, Miyuki</mods:namePart>
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<mods:namePart>Takaki, Yoshihiro</mods:namePart>
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<mods:namePart>Takano, Yoshinori</mods:namePart>
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<mods:namePart>Uematsu, Katsuyuki</mods:namePart>
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<mods:namePart>Ikuta, Tetsuro</mods:namePart>
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<mods:namePart>Ito, Motoo</mods:namePart>
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<mods:namePart>Matsui, Yohei</mods:namePart>
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<mods:namePart>Miyazaki, Masayuki</mods:namePart>
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<mods:namePart>Murata, Kazuyoshi</mods:namePart>
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<mods:namePart>Saito, Yumi</mods:namePart>
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<mods:namePart>Sakai, Sanae</mods:namePart>
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<mods:namePart>Song, Chihong</mods:namePart>
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<mods:namePart>Yamanaka, Yuko</mods:namePart>
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<mods:namePart>Yamaguchi, Takashi</mods:namePart>
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<mods:namePart>Kamagata, Yoichi</mods:namePart>
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<mods:namePart>Tamaki, Hideyuki</mods:namePart>
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<mods:namePart>Takai, Ken</mods:namePart>
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<mods:title>Nature</mods:title>
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<mods:date>2020</mods:date>
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<mods:number>2020-01-15</mods:number>
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<mods:number>41586</mods:number>
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<treatment ID-DOI="http://doi.org/10.5281/zenodo.5920940" ID-GBIF-Taxon="161756097" ID-Zenodo-Dep="5920940" LSID="urn:lsid:plazi:treatment:03F887ABFFFDFFE0761FFDF68FF0FA12" httpUri="http://treatment.plazi.org/id/03F887ABFFFDFFE0761FFDF68FF0FA12" lastPageId="3" lastPageNumber="4" pageId="1" pageNumber="2">
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‘
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<taxonomicNameLabel box="[1382,1497,622,641]" pageId="1" pageNumber="2">Candidatus</taxonomicNameLabel>
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</emphasis>
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<taxonomicName box="[816,1192,650,670]" class="Methanomicrobia" family="Methanomicrobiaceae" kingdom="Archaea" order="Methanomicrobiales" pageId="1" pageNumber="2" phylum="Euryarchaeota" rank="species" species="syntrophicum" status="candidatus">Prometheoarchaeum syntrophicum</taxonomicName>
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’ strain
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<figureCitation box="[1265,1333,650,670]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="1" pageNumber="2">MK-D1</figureCitation>
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for the isolated archaeon (see Supplementary Note 3 for reasons why the provisional
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<taxonomicName box="[816,931,708,727]" class="Mollicutes" family="Acholeplasmataceae" genus="Candidatus" higherTaxonomySource="GBIF" kingdom="Bacteria" order="Acholeplasmatales" pageId="1" pageNumber="2" rank="genus">
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<emphasis box="[816,931,708,727]" italics="true" pageId="1" pageNumber="2">Candidatus</emphasis>
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status is necessary despite isolation).
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</paragraph>
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<subSubSection lastPageId="3" lastPageNumber="4" pageId="1" pageNumber="2" type="description">
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<paragraph blockId="1.[816,1498,783,1988]" box="[816,1315,783,809]" pageId="1" pageNumber="2">
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<emphasis bold="true" box="[816,1315,783,809]" pageId="1" pageNumber="2">Cell biology, physiology and metabolism</emphasis>
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</heading>
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</paragraph>
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<paragraph blockId="1.[816,1498,783,1988]" pageId="1" pageNumber="2">
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We further characterized
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<figureCitation box="[1066,1131,822,842]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="1" pageNumber="2">MK-D1</figureCitation>
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||
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) (
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<figureCitation box="[994,1082,936,956]" captionStart="Fig" captionStartId="3.[106,137,924,942]" captionTargetId="figure@3.[757,1004,383,630]" captionTargetPageId="3" captionText="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" figureDoi="http://doi.org/10.5281/zenodo.3610575" httpUri="https://zenodo.org/record/3610575/files/figure.png" pageId="1" pageNumber="2">Fig.3a,b</figureCitation>
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and Extended Data
|
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<figureCitation box="[1275,1331,936,956]" captionStart="Fig" captionStartId="3.[106,137,924,942]" captionTargetId="figure@3.[757,1004,383,630]" captionTargetPageId="3" captionText="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" figureDoi="http://doi.org/10.5281/zenodo.3610575" httpUri="https://zenodo.org/record/3610575/files/figure.png" pageId="1" pageNumber="2">Fig.3</figureCitation>
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),consistent with previous observations using FISH
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<superScript attach="left" box="[1150,1176,960,973]" fontSize="5" pageId="1" pageNumber="2">
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<bibRefCitation author="Aoki, M." box="[1150,1162,960,973]" journalOrPublisher="PLoS ONE" pageId="1" pageNumber="2" pagination="105356" part="9" refId="ref6670" refString="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)." title="A long-term cultivation of an anaerobic methane-oxidizing microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor" type="journal article" year="2014">15</bibRefCitation>
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,
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<bibRefCitation author="Knittel, K. & Losekann, T. & Boetius, A. & Kort, R. & Amann, R." box="[1165,1176,960,973]" journalOrPublisher="Appl. Environ. Microbiol." pageId="1" pageNumber="2" pagination="467 - 479" part="71" refId="ref6747" refString="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)." title="Diversity and distribution of methanotrophic archaea at cold seeps" type="journal article" year="2005">17</bibRefCitation>
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</superScript>
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.
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<figureCitation box="[1186,1251,965,985]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="1" pageNumber="2">MK-D1</figureCitation>
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cells were easily identifiable given the morphological difference from their co-culture partner
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||
<taxonomicName box="[816,982,1023,1042]" class="Methanomicrobia" family="Methanomicrobiaceae" genus="Methanogenium" kingdom="Archaea" order="Methanomicrobiales" pageId="1" pageNumber="2" phylum="Euryarchaeota" rank="genus">
|
||
<emphasis box="[816,982,1023,1042]" italics="true" pageId="1" pageNumber="2">Methanogenium</emphasis>
|
||
</taxonomicName>
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||
(highly irregular coccoid cells of ≥2 µm;
|
||
<figureCitation box="[1395,1482,1022,1042]" captionStart="Fig" captionStartId="1.[106,137,1156,1174]" captionTargetBox="[113,779,133,1131]" captionTargetPageId="1" captionText="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)." figureDoi="http://doi.org/10.5281/zenodo.3609902" httpUri="https://zenodo.org/record/3609902/files/figure.png" pageId="1" pageNumber="2">Fig. 1d, e</figureCitation>
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||
). Dividing cells had less EPS and a ring-like structure around the cells (
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||
<figureCitation box="[823,895,1080,1100]" captionStart="Fig" captionStartId="3.[106,137,924,942]" captionTargetId="figure@3.[757,1004,383,630]" captionTargetPageId="3" captionText="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" figureDoi="http://doi.org/10.5281/zenodo.3610575" httpUri="https://zenodo.org/record/3610575/files/figure.png" pageId="1" pageNumber="2">Fig. 3c</figureCitation>
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). Cryo-electron microscopy (cryo-EM) and transmission electron microscopy (TEM) analyses revealed that the cells contain no visible organelle-like inclusions (
|
||
<figureCitation box="[1191,1249,1137,1157]" captionStart="Fig" captionStartId="3.[106,137,924,942]" captionTargetId="figure@3.[757,1004,383,630]" captionTargetPageId="3" captionText="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" figureDoi="http://doi.org/10.5281/zenodo.3610575" httpUri="https://zenodo.org/record/3610575/files/figure.png" pageId="1" pageNumber="2">Fig. 3</figureCitation>
|
||
d–f and Supplementary Videos 1–6), in contrast to previous suggestions
|
||
<bibRefCitation author="Zaremba-Niedzwiedzka, K." box="[1299,1307,1161,1174]" journalOrPublisher="Nature" pageId="1" pageNumber="2" pagination="353 - 358" part="541" refId="ref6310" refString="6. Zaremba-Niedzwiedzka, K. et al. Asgard archaea illuminate the origin of eukaryotic cellular complexity. Nature 541, 353 - 358 (2017)." title="Asgard archaea illuminate the origin of eukaryotic cellular complexity" type="journal article" year="2017">
|
||
<superScript attach="left" box="[1299,1307,1161,1174]" fontSize="5" pageId="1" pageNumber="2">6</superScript>
|
||
</bibRefCitation>
|
||
. 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) (
|
||
<figureCitation box="[930,987,1338,1358]" captionStart="Fig" captionStartId="3.[106,137,924,942]" captionTargetId="figure@3.[757,1004,383,630]" captionTargetPageId="3" captionText="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" figureDoi="http://doi.org/10.5281/zenodo.3610575" httpUri="https://zenodo.org/record/3610575/files/figure.png" pageId="1" pageNumber="2">Fig. 3</figureCitation>
|
||
b–f) and chains of blebs (
|
||
<figureCitation box="[1244,1313,1338,1358]" captionStart="Fig" captionStartId="3.[106,137,924,942]" captionTargetId="figure@3.[757,1004,383,630]" captionTargetPageId="3" captionText="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" figureDoi="http://doi.org/10.5281/zenodo.3610575" httpUri="https://zenodo.org/record/3610575/files/figure.png" pageId="1" pageNumber="2">Fig. 3c</figureCitation>
|
||
).
|
||
<figureCitation box="[1332,1399,1338,1358]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="1" pageNumber="2">MK-D1</figureCitation>
|
||
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 (
|
||
<figureCitation box="[1404,1459,1424,1444]" captionStart="Fig" captionStartId="3.[106,137,924,942]" captionTargetId="figure@3.[757,1004,383,630]" captionTargetPageId="3" captionText="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" figureDoi="http://doi.org/10.5281/zenodo.3610575" httpUri="https://zenodo.org/record/3610575/files/figure.png" pageId="1" pageNumber="2">Fig.3</figureCitation>
|
||
g–i; confirmed using both SEM and TEM).These protrusions neither
|
||
<taxonomicName form="elaborate" pageId="1" pageNumber="2" rank="form">form elaborate</taxonomicName>
|
||
networks (as in
|
||
<taxonomicName box="[1065,1186,1482,1501]" class="Thermoprotei" family="Pyrodictiaceae" genus="Pyrodictium" kingdom="Archaea" order="Desulfurococcales" pageId="1" pageNumber="2" phylum="Crenarchaeota" rank="genus">
|
||
<emphasis box="[1065,1186,1482,1501]" italics="true" pageId="1" pageNumber="2">Pyrodictium</emphasis>
|
||
</taxonomicName>
|
||
<bibRefCitation author="Albers, S. - V. & Meyer, B. H." box="[1185,1198,1476,1489]" journalOrPublisher="Nat. Rev. Microbiol." pageId="1" pageNumber="2" pagination="414 - 426" part="9" refId="ref6798" refString="18. Albers, S. - V. & Meyer, B. H. The archaeal cell envelope. Nat. Rev. Microbiol. 9, 414 - 426 (2011)." title="The archaeal cell envelope" type="journal article" year="2011">
|
||
<superScript attach="left" box="[1185,1198,1476,1489]" fontSize="5" pageId="1" pageNumber="2">18</superScript>
|
||
</bibRefCitation>
|
||
) nor intercellular connections (
|
||
<taxonomicName box="[825,951,1511,1530]" class="Thermoprotei" family="Pyrodictiaceae" genus="Pyrodictium" kingdom="Archaea" order="Desulfurococcales" pageId="1" pageNumber="2" phylum="Crenarchaeota" rank="genus">
|
||
<emphasis box="[825,951,1511,1530]" italics="true" pageId="1" pageNumber="2">Pyrodictium</emphasis>
|
||
</taxonomicName>
|
||
,
|
||
<taxonomicName box="[961,1108,1511,1530]" class="Thermococci" family="Thermococcaceae" genus="Thermococcus" kingdom="Archaea" order="Thermococcales" pageId="1" pageNumber="2" phylum="Euryarchaeota" rank="genus">
|
||
<emphasis box="[961,1108,1511,1530]" italics="true" pageId="1" pageNumber="2">Thermococcus</emphasis>
|
||
</taxonomicName>
|
||
and
|
||
<taxonomicName box="[1155,1258,1511,1530]" class="Halobacteria" family="Halobacteriaceae" genus="Haloferax" kingdom="Archaea" order="Halobacteriales" pageId="1" pageNumber="2" phylum="Euryarchaeota" rank="genus">
|
||
<emphasis box="[1155,1258,1511,1530]" italics="true" pageId="1" pageNumber="2">Haloferax</emphasis>
|
||
</taxonomicName>
|
||
<superScript attach="left" box="[1258,1295,1505,1518]" fontSize="5" pageId="1" pageNumber="2">
|
||
<bibRefCitation author="Albers, S. - V. & Meyer, B. H." box="[1258,1272,1505,1518]" journalOrPublisher="Nat. Rev. Microbiol." pageId="1" pageNumber="2" pagination="414 - 426" part="9" refId="ref6798" refString="18. Albers, S. - V. & Meyer, B. H. The archaeal cell envelope. Nat. Rev. Microbiol. 9, 414 - 426 (2011)." title="The archaeal cell envelope" type="journal article" year="2011">18</bibRefCitation>
|
||
–
|
||
<bibRefCitation author="Rosenshine, I. & Tchelet, R. & Mevarech, M." box="[1279,1295,1505,1518]" journalOrPublisher="Science" pageId="1" pageNumber="2" pagination="1387 - 1389" part="245" refId="ref6875" refString="20. Rosenshine, I., Tchelet, R. & Mevarech, M. The mechanism of DNA transfer in the mating system of an archaebacterium. Science 245, 1387 - 1389 (1989)." title="The mechanism of DNA transfer in the mating system of an archaebacterium" type="journal article" year="1989">20</bibRefCitation>
|
||
</superScript>
|
||
), suggesting differences in physiological functions.The
|
||
<figureCitation box="[1177,1241,1538,1558]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="1" pageNumber="2">MK-D1</figureCitation>
|
||
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
|
||
<figureCitation box="[816,868,1624,1644]" captionStart="Fig" captionStartId="1.[106,137,1156,1174]" captionTargetBox="[113,779,133,1131]" captionTargetPageId="1" captionText="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)." figureDoi="http://doi.org/10.5281/zenodo.3609902" httpUri="https://zenodo.org/record/3609902/files/figure.png" pageId="1" pageNumber="2">Fig. 1</figureCitation>
|
||
), stalk-like structures on the surface of the vesicles (
|
||
<figureCitation box="[1388,1455,1624,1644]" captionStart="Fig" captionStartId="3.[106,137,924,942]" captionTargetId="figure@3.[757,1004,383,630]" captionTargetPageId="3" captionText="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" figureDoi="http://doi.org/10.5281/zenodo.3610575" httpUri="https://zenodo.org/record/3610575/files/figure.png" pageId="1" pageNumber="2">Fig. 3e</figureCitation>
|
||
and Extended Data
|
||
<figureCitation box="[966,1049,1653,1673]" captionStart="Fig" captionStartId="3.[106,137,924,942]" captionTargetId="figure@3.[757,1004,383,630]" captionTargetPageId="3" captionText="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" figureDoi="http://doi.org/10.5281/zenodo.3610575" httpUri="https://zenodo.org/record/3610575/files/figure.png" pageId="1" pageNumber="2">Fig.3f, g</figureCitation>
|
||
) and the even distance between the inner and outer layers of the cell envelope (
|
||
<figureCitation box="[1142,1210,1682,1702]" captionStart="Fig" captionStartId="3.[106,137,924,942]" captionTargetId="figure@3.[757,1004,383,630]" captionTargetPageId="3" captionText="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" figureDoi="http://doi.org/10.5281/zenodo.3610575" httpUri="https://zenodo.org/record/3610575/files/figure.png" pageId="1" pageNumber="2">Fig.3d</figureCitation>
|
||
). Lipid composition analysis of the
|
||
<figureCitation box="[876,940,1710,1730]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="1" pageNumber="2">MK-D1</figureCitation>
|
||
and
|
||
<taxonomicName box="[984,1145,1711,1730]" class="Methanomicrobia" family="Methanomicrobiaceae" genus="Methanogenium" kingdom="Archaea" order="Methanomicrobiales" pageId="1" pageNumber="2" phylum="Euryarchaeota" rank="genus">
|
||
<emphasis box="[984,1145,1711,1730]" italics="true" pageId="1" pageNumber="2">Methanogenium</emphasis>
|
||
</taxonomicName>
|
||
co-culture revealed typical archaeal isoprenoid signatures—C
|
||
<bibRefCitation author="Rosenshine, I. & Tchelet, R. & Mevarech, M." box="[1059,1074,1748,1761]" journalOrPublisher="Science" pageId="1" pageNumber="2" pagination="1387 - 1389" part="245" refId="ref6875" refString="20. Rosenshine, I., Tchelet, R. & Mevarech, M. The mechanism of DNA transfer in the mating system of an archaebacterium. Science 245, 1387 - 1389 (1989)." title="The mechanism of DNA transfer in the mating system of an archaebacterium" type="journal article" year="1989">
|
||
<subScript attach="left" box="[1059,1074,1748,1761]" fontSize="5" pageId="1" pageNumber="2">20</subScript>
|
||
</bibRefCitation>
|
||
-phytane and C
|
||
<subScript attach="left" box="[1218,1234,1748,1761]" fontSize="5" pageId="1" pageNumber="2">40</subScript>
|
||
-biphytanes with 0–2cyclopentane rings were obtained after ether-cleavage treatment(
|
||
<figureCitation box="[1422,1483,1768,1788]" captionStart="Fig" captionStartId="3.[106,137,924,942]" captionTargetId="figure@3.[757,1004,383,630]" captionTargetPageId="3" captionText="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" figureDoi="http://doi.org/10.5281/zenodo.3610575" httpUri="https://zenodo.org/record/3610575/files/figure.png" pageId="1" pageNumber="2">Fig.3j</figureCitation>
|
||
). Considering the lipid data obtained from a reference
|
||
<taxonomicName box="[1335,1497,1797,1816]" class="Methanomicrobia" family="Methanomicrobiaceae" genus="Methanogenium" kingdom="Archaea" order="Methanomicrobiales" pageId="1" pageNumber="2" phylum="Euryarchaeota" rank="genus">
|
||
<emphasis box="[1335,1497,1797,1816]" italics="true" pageId="1" pageNumber="2">Methanogenium</emphasis>
|
||
</taxonomicName>
|
||
isolate (99.3% 16S rRNA gene identity; Supplementary
|
||
<figureCitation box="[1359,1417,1825,1845]" captionStart="γ-amino-butyryl-CoA" captionStartId="2.[816,1004,1019,1038]" captionTargetBox="[157,1445,134,995]" captionTargetId="graphics@2.[245,1171,203,978]" captionText="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." figureDoi="http://doi.org/10.5281/zenodo.3609906" httpUri="https://zenodo.org/record/3609906/files/figure.png" pageId="1" pageNumber="2">Fig. 2</figureCitation>
|
||
),
|
||
<figureCitation box="[1431,1497,1825,1845]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="1" pageNumber="2">MK-D1</figureCitation>
|
||
probably contains C
|
||
<subScript attach="left" box="[1025,1041,1863,1876]" fontSize="5" pageId="1" pageNumber="2">20</subScript>
|
||
-phytane and C
|
||
<subScript attach="left" box="[1198,1215,1863,1876]" fontSize="5" pageId="1" pageNumber="2">40</subScript>
|
||
-biphytanes with 0–2 rings. The
|
||
<figureCitation box="[859,925,1882,1902]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="1" pageNumber="2">MK-D1</figureCitation>
|
||
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).
|
||
</paragraph>
|
||
<caption ID-DOI="http://doi.org/10.5281/zenodo.3609904" ID-Zenodo-Dep="3609904" httpUri="https://zenodo.org/record/3609904/files/figure.png" pageId="2" pageNumber="3" startId="2.[106,137,1020,1038]" subCaptionStartIDs="2.[954,984,1206,1224]" subCaptionStarts="Fig.2m" targetBox="[157,1445,134,995]" targetPageId="2">
|
||
<paragraph blockId="2.[106,781,1019,1438]" pageId="2" pageNumber="3">
|
||
<emphasis bold="true" box="[106,568,1019,1038]" pageId="2" pageNumber="3">Fig.2| Syntrophic amino acid utilization of MK-D1.a</emphasis>
|
||
,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
|
||
<subScript attach="left" box="[556,562,1162,1172]" fontSize="4" pageId="2" pageNumber="3">2</subScript>
|
||
, amino acids,vitamin B
|
||
<subScript attach="left" box="[764,776,1162,1172]" fontSize="4" pageId="2" pageNumber="3">12</subScript>
|
||
, biotin,lipoate and thiamine pyrophosphate(TPP),which are predicted to be metabolized or synthesized by the partnering
|
||
<taxonomicName box="[503,658,1207,1224]" class="Deltaproteobacteria" family="Desulfovibrionaceae" genus="Halodesulfovibrio" higherTaxonomySource="GBIF" kingdom="Bacteria" order="Desulfovibrionales" pageId="2" pageNumber="3" phylum="Proteobacteria" rank="genus">
|
||
<emphasis box="[503,658,1207,1224]" italics="true" pageId="2" pageNumber="3">Halodesulfovibrio</emphasis>
|
||
</taxonomicName>
|
||
and/or
|
||
<taxonomicName box="[106,248,1234,1251]" class="Methanomicrobia" family="Methanomicrobiaceae" genus="Methanogenium" kingdom="Archaea" order="Methanomicrobiales" pageId="2" pageNumber="3" phylum="Euryarchaeota" rank="genus">
|
||
<emphasis box="[106,248,1234,1251]" italics="true" pageId="2" pageNumber="3">Methanogenium</emphasis>
|
||
</taxonomicName>
|
||
.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
|
||
<subScript attach="left" box="[456,462,1376,1386]" fontSize="4" pageId="2" pageNumber="3">3</subScript>
|
||
=H
|
||
<subScript attach="both" box="[488,495,1376,1386]" fontSize="4" pageId="2" pageNumber="3">4</subScript>
|
||
F,methylene-tetrahydrofolate; CH≡H
|
||
<subScript attach="both" box="[159,166,1402,1412]" fontSize="4" pageId="2" pageNumber="3">4</subScript>
|
||
F,methenyl-tetrahydrofolate;Fo-H
|
||
<subScript attach="both" box="[466,473,1402,1412]" fontSize="4" pageId="2" pageNumber="3">4</subScript>
|
||
F,formyl-tetrahydrofolate;2OB, 2-oxobutyrate;Prop-CoA,propionyl-CoA;ACAC,acetoacetate;GB-CoA,
|
||
</paragraph>
|
||
<paragraph blockId="2.[816,1484,1019,1411]" pageId="2" pageNumber="3">
|
||
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.
|
||
<emphasis bold="true" box="[897,931,1073,1091]" pageId="2" pageNumber="3">b–e</emphasis>
|
||
, NanoSIMS analysis of a highly purified MK-D1 culture incubated with a mixture of
|
||
<bibRefCitation author="Pushkarev, A." box="[964,975,1097,1107]" journalOrPublisher="Nature" pageId="2" pageNumber="3" pagination="595 - 599" part="558" refId="ref6603" refString="13. Pushkarev, A. et al. A distinct abundant group of microbial rhodopsins discovered using functional metagenomics. Nature 558, 595 - 599 (2018)." title="A distinct abundant group of microbial rhodopsins discovered using functional metagenomics" type="journal article" year="2018">
|
||
<superScript attach="right" box="[964,975,1097,1107]" fontSize="4" pageId="2" pageNumber="3">13</superScript>
|
||
</bibRefCitation>
|
||
C- and
|
||
<bibRefCitation author="Aoki, M." box="[1032,1043,1097,1107]" journalOrPublisher="PLoS ONE" pageId="2" pageNumber="3" pagination="105356" part="9" refId="ref6670" refString="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)." title="A long-term cultivation of an anaerobic methane-oxidizing microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor" type="journal article" year="2014">
|
||
<superScript attach="right" box="[1032,1043,1097,1107]" fontSize="4" pageId="2" pageNumber="3">15</superScript>
|
||
</bibRefCitation>
|
||
N-labelled amino acids.
|
||
<emphasis bold="true" box="[1248,1260,1100,1118]" pageId="2" pageNumber="3">b</emphasis>
|
||
, Green fluorescent micrograph of SYBR Green I-stained cells.Aggregates are MK-D1,and filamentous cells are
|
||
<taxonomicName authorityName="Kluyver & van Niel" authorityYear="1936" box="[998,1164,1154,1171]" class="Methanobacteria" family="Methanobacteriaceae" genus="Methanobacterium" kingdom="Archaea" order="Methanobacteriales" pageId="2" pageNumber="3" phylum="Euryarchaeota" rank="genus">
|
||
<emphasis box="[998,1164,1154,1171]" italics="true" pageId="2" pageNumber="3">Methanobacterium</emphasis>
|
||
</taxonomicName>
|
||
sp.strain MO-MB1 (fluorescence can be weak owing to the high rigidity and low permeability of the cell membrane (Extended Data
|
||
<figureCitation box="[954,1037,1206,1224]" captionStart="Fig" captionStartId="2.[106,137,1020,1038]" captionTargetBox="[157,1445,134,995]" captionTargetId="graphics@2.[245,1171,203,978]" captionText="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." figureDoi="http://doi.org/10.5281/zenodo.3609904" httpUri="https://zenodo.org/record/3609904/files/figure.png" pageId="2" pageNumber="3">Fig.2m,n</figureCitation>
|
||
;see also ref.
|
||
<bibRefCitation author="Nakamura, K." box="[1146,1159,1204,1214]" journalOrPublisher="Appl. Environ. Microbiol." pageId="2" pageNumber="3" pagination="6907 - 6913" part="72" refId="ref8055" refString="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)." title="Application of pseudomurein endoisopeptidase to fluorescence in situ hybridization of methanogens within the family Methanobacteriaceae" type="journal article" year="2006">
|
||
<superScript attach="right" box="[1146,1159,1204,1214]" fontSize="4" pageId="2" pageNumber="3">49</superScript>
|
||
</bibRefCitation>
|
||
).
|
||
<emphasis bold="true" box="[1173,1183,1206,1224]" pageId="2" pageNumber="3">c</emphasis>
|
||
, NanoSIMS ion image of
|
||
<superScript attach="right" box="[1393,1404,1204,1214]" fontSize="4" pageId="2" pageNumber="3">12</superScript>
|
||
C(cyan).
|
||
<emphasis bold="true" box="[816,828,1233,1251]" pageId="2" pageNumber="3">d</emphasis>
|
||
,NanoSIMS ion image of
|
||
<superScript attach="right" box="[1039,1050,1231,1241]" fontSize="4" pageId="2" pageNumber="3">12</superScript>
|
||
C
|
||
<superScript attach="both" box="[1063,1074,1231,1241]" fontSize="4" pageId="2" pageNumber="3">15</superScript>
|
||
N/
|
||
<superScript attach="right" box="[1096,1107,1231,1241]" fontSize="4" pageId="2" pageNumber="3">12</superScript>
|
||
C
|
||
<superScript attach="both" box="[1120,1131,1231,1241]" fontSize="4" pageId="2" pageNumber="3">14</superScript>
|
||
N (magenta).
|
||
<emphasis bold="true" box="[1245,1255,1233,1251]" pageId="2" pageNumber="3">e</emphasis>
|
||
, Overlay image of
|
||
<emphasis bold="true" box="[1411,1446,1233,1251]" pageId="2" pageNumber="3">b–d</emphasis>
|
||
.
|
||
<emphasis bold="true" box="[816,828,1260,1278]" pageId="2" pageNumber="3">d</emphasis>
|
||
,The colour bar indicates the relative abundance of
|
||
<superScript attach="right" box="[1270,1281,1257,1267]" fontSize="4" pageId="2" pageNumber="3">15</superScript>
|
||
N expressed as
|
||
<superScript attach="right" box="[1411,1422,1257,1267]" fontSize="4" pageId="2" pageNumber="3">15</superScript>
|
||
N/
|
||
<superScript attach="right" box="[1444,1455,1257,1267]" fontSize="4" pageId="2" pageNumber="3">14</superScript>
|
||
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
|
||
<emphasis box="[1162,1173,1368,1384]" italics="true" pageId="2" pageNumber="3">n</emphasis>
|
||
= 8 recorded images.The iTAG analysis of the imaged culture is shown in Supplementary Table 1.
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph blockId="2.[106,787,1510,1988]" pageId="2" pageNumber="3">
|
||
<figureCitation box="[129,197,1510,1530]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="2" pageNumber="3">MK-D1</figureCitation>
|
||
can degrade amino acids anaerobically, as confirmed by monitoring the depletion of amino acids during the growth of pure co-cultures (Extended Data
|
||
<figureCitation box="[391,482,1567,1587]" captionStart="Fig" captionStartId="1.[106,137,1156,1174]" captionTargetBox="[113,779,133,1131]" captionTargetPageId="1" captionText="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)." figureDoi="http://doi.org/10.5281/zenodo.3609902" httpUri="https://zenodo.org/record/3609902/files/figure.png" pageId="2" pageNumber="3">Fig. 1b, c</figureCitation>
|
||
). We further verify the utilization of amino acids by quantifying the uptake of a mixture of
|
||
<bibRefCitation author="Pushkarev, A." box="[750,764,1591,1604]" journalOrPublisher="Nature" pageId="2" pageNumber="3" pagination="595 - 599" part="558" refId="ref6603" refString="13. Pushkarev, A. et al. A distinct abundant group of microbial rhodopsins discovered using functional metagenomics. Nature 558, 595 - 599 (2018)." title="A distinct abundant group of microbial rhodopsins discovered using functional metagenomics" type="journal article" year="2018">
|
||
<superScript attach="right" box="[750,764,1591,1604]" fontSize="5" pageId="2" pageNumber="3">13</superScript>
|
||
</bibRefCitation>
|
||
C- and
|
||
<bibRefCitation author="Aoki, M." box="[150,163,1620,1633]" journalOrPublisher="PLoS ONE" pageId="2" pageNumber="3" pagination="105356" part="9" refId="ref6670" refString="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)." title="A long-term cultivation of an anaerobic methane-oxidizing microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor" type="journal article" year="2014">
|
||
<superScript attach="right" box="[150,163,1620,1633]" fontSize="5" pageId="2" pageNumber="3">15</superScript>
|
||
</bibRefCitation>
|
||
N-labelled amino acids through nanometre-scale secondary ion mass spectrometry (NanoSIMS) (
|
||
<figureCitation box="[485,541,1653,1673]" captionStart="γ-amino-butyryl-CoA" captionStartId="2.[816,1004,1019,1038]" captionTargetBox="[157,1445,134,995]" captionTargetId="graphics@2.[245,1171,203,978]" captionText="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." figureDoi="http://doi.org/10.5281/zenodo.3609906" httpUri="https://zenodo.org/record/3609906/files/figure.png" pageId="2" pageNumber="3">Fig. 2</figureCitation>
|
||
b–e). Cell aggregates of
|
||
<figureCitation box="[106,173,1682,1702]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="2" pageNumber="3">MK-D1</figureCitation>
|
||
incorporated amino-acid-derived nitrogen, demonstrating the capacity of
|
||
<figureCitation box="[257,324,1710,1730]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="2" pageNumber="3">MK-D1</figureCitation>
|
||
to utilize amino acids for growth.Notably,the
|
||
<bibRefCitation author="Pushkarev, A." box="[106,119,1734,1747]" journalOrPublisher="Nature" pageId="2" pageNumber="3" pagination="595 - 599" part="558" refId="ref6603" refString="13. Pushkarev, A. et al. A distinct abundant group of microbial rhodopsins discovered using functional metagenomics. Nature 558, 595 - 599 (2018)." title="A distinct abundant group of microbial rhodopsins discovered using functional metagenomics" type="journal article" year="2018">
|
||
<superScript attach="right" box="[106,119,1734,1747]" fontSize="5" pageId="2" pageNumber="3">13</superScript>
|
||
</bibRefCitation>
|
||
C-labelling of methane and CO
|
||
<subScript attach="none" box="[427,434,1748,1761]" fontSize="5" pageId="2" pageNumber="3">2</subScript>
|
||
varied depending on the methanogenic partner, indicating that
|
||
<figureCitation box="[414,481,1768,1788]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="2" pageNumber="3">MK-D1</figureCitation>
|
||
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
|
||
<figureCitation box="[560,626,1854,1874]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="2" pageNumber="3">MK-D1</figureCitation>
|
||
(Extended Data Table 3). The syntrophic partner was replaceable—MK-D1 could also grow syntrophically with
|
||
<taxonomicName authorityName="Kluyver & van Niel" authorityYear="1936" box="[374,576,1912,1931]" class="Methanobacteria" family="Methanobacteriaceae" genus="Methanobacterium" kingdom="Archaea" order="Methanobacteriales" pageId="2" pageNumber="3" phylum="Euryarchaeota" rank="genus">
|
||
<emphasis box="[374,576,1912,1931]" italics="true" pageId="2" pageNumber="3">Methanobacterium</emphasis>
|
||
</taxonomicName>
|
||
sp. strain MO-MB1
|
||
<bibRefCitation author="Imachi, H." box="[773,786,1906,1919]" journalOrPublisher="ISME J." pageId="2" pageNumber="3" pagination="1913 - 1925" part="5" refId="ref6914" refString="21. Imachi, H. et al. Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor. ISME J. 5, 1913 - 1925 (2011)." title="Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor" type="journal article" year="2011">
|
||
<superScript attach="none" box="[773,786,1906,1919]" fontSize="5" pageId="2" pageNumber="3">21</superScript>
|
||
</bibRefCitation>
|
||
instead of
|
||
<taxonomicName box="[209,374,1941,1960]" class="Methanomicrobia" family="Methanomicrobiaceae" genus="Methanogenium" kingdom="Archaea" order="Methanomicrobiales" pageId="2" pageNumber="3" phylum="Euryarchaeota" rank="genus">
|
||
<emphasis box="[209,374,1941,1960]" italics="true" pageId="2" pageNumber="3">Methanogenium</emphasis>
|
||
</taxonomicName>
|
||
(
|
||
<figureCitation box="[384,438,1940,1960]" captionStart="γ-amino-butyryl-CoA" captionStartId="2.[816,1004,1019,1038]" captionTargetBox="[157,1445,134,995]" captionTargetId="graphics@2.[245,1171,203,978]" captionText="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." figureDoi="http://doi.org/10.5281/zenodo.3609906" httpUri="https://zenodo.org/record/3609906/files/figure.png" pageId="2" pageNumber="3">Fig.2</figureCitation>
|
||
b–e). Although 14 different culture conditions were applied,none enhanced the cell yield,which indicates
|
||
</paragraph>
|
||
<paragraph blockId="2.[816,1499,1510,1988]" pageId="2" pageNumber="3">specialization of the degradation of amino acids and/or peptides (Extended Data Table 3).</paragraph>
|
||
<paragraph blockId="2.[816,1499,1510,1988]" lastBlockId="3.[106,787,1338,1988]" lastPageId="3" lastPageNumber="4" pageId="2" pageNumber="3">
|
||
To further characterize the physiology of the archaeon,we analysed the complete
|
||
<figureCitation box="[955,1021,1596,1616]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="2" pageNumber="3">MK-D1</figureCitation>
|
||
genome (Extended Data
|
||
<figureCitation box="[1273,1326,1596,1616]" captionStart="γ-amino-butyryl-CoA" captionStartId="2.[816,1004,1019,1038]" captionTargetBox="[157,1445,134,995]" captionTargetId="graphics@2.[245,1171,203,978]" captionText="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." figureDoi="http://doi.org/10.5281/zenodo.3609906" httpUri="https://zenodo.org/record/3609906/files/figure.png" pageId="2" pageNumber="3">Fig.2</figureCitation>
|
||
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
|
||
<subScript attach="left" box="[931,938,1720,1733]" fontSize="5" pageId="2" pageNumber="3">2</subScript>
|
||
and formate generation,respectively.
|
||
<figureCitation box="[1316,1381,1710,1730]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="2" pageNumber="3">MK-D1</figureCitation>
|
||
represents, to our knowledge, the first cultured archaeon that can produce and syntrophically transfer H
|
||
<subScript attach="left" box="[1066,1073,1777,1790]" fontSize="5" pageId="2" pageNumber="3">2</subScript>
|
||
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;
|
||
<figureCitation box="[1155,1217,1882,1902]" captionStart="γ-amino-butyryl-CoA" captionStartId="2.[816,1004,1019,1038]" captionTargetBox="[157,1445,134,995]" captionTargetId="graphics@2.[245,1171,203,978]" captionText="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." figureDoi="http://doi.org/10.5281/zenodo.3609906" httpUri="https://zenodo.org/record/3609906/files/figure.png" pageId="2" pageNumber="3">Fig.2a</figureCitation>
|
||
and Supplementary Table 4).
|
||
<figureCitation box="[816,881,1911,1931]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="2" pageNumber="3">MK-D1</figureCitation>
|
||
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
|
||
<subScript attach="left" box="[162,169,1462,1475]" fontSize="5" pageId="3" pageNumber="4">2</subScript>
|
||
and reduction of ferredoxin,which can be re-oxidized through H
|
||
<superScript attach="left" box="[122,130,1476,1489]" fontSize="5" pageId="3" pageNumber="4">+</superScript>
|
||
and/or CO
|
||
<subScript attach="left" box="[236,243,1490,1503]" fontSize="5" pageId="3" pageNumber="4">2</subScript>
|
||
reduction to H
|
||
<subScript attach="left" box="[390,397,1490,1503]" fontSize="5" pageId="3" pageNumber="4">2</subScript>
|
||
and formate,respectively (through the electron-confurcating NiFe hydrogenaseMvhADG–HdrABC or formate dehydrogenase FdhA). On the basis of
|
||
<bibRefCitation author="Pushkarev, A." box="[492,505,1534,1547]" journalOrPublisher="Nature" pageId="3" pageNumber="4" pagination="595 - 599" part="558" refId="ref6603" refString="13. Pushkarev, A. et al. A distinct abundant group of microbial rhodopsins discovered using functional metagenomics. Nature 558, 595 - 599 (2018)." title="A distinct abundant group of microbial rhodopsins discovered using functional metagenomics" type="journal article" year="2018">
|
||
<superScript attach="right" box="[492,505,1534,1547]" fontSize="5" pageId="3" pageNumber="4">13</superScript>
|
||
</bibRefCitation>
|
||
C-amino-acid-based experiments (Supplementary Note 4),
|
||
<figureCitation box="[425,491,1567,1587]" captionStart="MK-D1" captionStartId="13.[817,868,1625,1643]" captionText="MK-D1 cell witha protrusion.m, n, Photomicrographs of pureculture of Methanobacteriumsp.strain MO-MB1 cells stainedwith SYBR Green I. Phasecontrast (m)and fluorescence(n) images of the same field areshown.a, b, The FISH experiments were performedthree timeswith similarresults.d, e, j, k,The SEMimages arerepresentative of n =122 recorded images that were obtained from four independent observations from four culture samples.The lipid compositionexperiments wererepeated twiceand gavesimilar results. f, g, Thecryo-EM images are representativeof n = 14recorded images that were taken from two independentobservations from two culture samples.h, i, l, The ultrathin-sectionimages are representative of n= 131 recorded images that were obtained from sixindependent observations from sixculture samples. m, n, The SYBRGreen I staining experiment wasperformed once,but all10 recordedimages showed similarresults.Detailed iTAGanalyses of cultures are shown inSupplementary Table 1." pageId="3" pageNumber="4">MK-D1</figureCitation>
|
||
can probably switch between syntrophic interaction through 2-oxoacid hydrolysis and oxidation depending on the partner(s).
|
||
</paragraph>
|
||
</subSubSection>
|
||
<caption ID-DOI="http://doi.org/10.5281/zenodo.3610575" ID-Zenodo-Dep="3610575" httpUri="https://zenodo.org/record/3610575/files/figure.png" pageId="3" pageNumber="4" subCaptionStartIDs="3.[1328,1358,924,942]" subCaptionStarts="Fig.2" targetBox="[351,1254,133,899]" targetPageId="3">
|
||
<paragraph blockId="3.[106,780,924,1235]" pageId="3" pageNumber="4">
|
||
Fig.3 | Microscopy characterization and lipid composition of MK-D1.
|
||
<emphasis bold="true" box="[106,137,950,968]" pageId="3" pageNumber="4">a–c</emphasis>
|
||
, SEM images of MK-D1.Single cell (
|
||
<emphasis bold="true" box="[429,439,950,968]" pageId="3" pageNumber="4">a</emphasis>
|
||
), aggregated cells covered with EPS-like materials (
|
||
<emphasis bold="true" box="[197,208,977,995]" pageId="3" pageNumber="4">b</emphasis>
|
||
) and a dividing cell with polar chains of blebs (
|
||
<emphasis bold="true" box="[598,607,977,995]" pageId="3" pageNumber="4">c</emphasis>
|
||
).
|
||
<emphasis bold="true" box="[622,634,977,995]" pageId="3" pageNumber="4">d</emphasis>
|
||
, 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.
|
||
<emphasis bold="true" box="[560,570,1030,1048]" pageId="3" pageNumber="4">e</emphasis>
|
||
, Cryo-EM image of large membrane vesicles attached to and surrounding MK-D1 cells.
|
||
<emphasis bold="true" box="[627,633,1057,1075]" pageId="3" pageNumber="4">f</emphasis>
|
||
, Ultrathin section of an MK-D1 cell and a membrane vesicle.The bottom-right inset image shows a magnified view of the membrane vesicle.
|
||
<emphasis bold="true" box="[525,536,1110,1128]" pageId="3" pageNumber="4">g</emphasis>
|
||
,
|
||
<emphasis bold="true" box="[545,557,1110,1128]" pageId="3" pageNumber="4">h</emphasis>
|
||
,SEM images of MK-D1 cells producing long branching (
|
||
<emphasis bold="true" box="[382,393,1137,1155]" pageId="3" pageNumber="4">g</emphasis>
|
||
)and straight (
|
||
<emphasis bold="true" box="[517,528,1137,1155]" pageId="3" pageNumber="4">h</emphasis>
|
||
) membrane protrusions.
|
||
<emphasis bold="true" box="[106,112,1164,1182]" pageId="3" pageNumber="4">i</emphasis>
|
||
,Ultrathin section of a MK-D1 cell with protrusions.
|
||
<emphasis bold="true" box="[544,550,1164,1182]" pageId="3" pageNumber="4">j</emphasis>
|
||
, 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
|
||
</paragraph>
|
||
<paragraph blockId="3.[816,1497,923,1209]" pageId="3" pageNumber="4">
|
||
their relative compositions are also shown (Supplementary
|
||
<figureCitation box="[1328,1376,924,942]" captionStart="γ-amino-butyryl-CoA" captionStartId="2.[816,1004,1019,1038]" captionTargetBox="[157,1445,134,995]" captionTargetId="graphics@2.[245,1171,203,978]" captionText="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." figureDoi="http://doi.org/10.5281/zenodo.3609906" httpUri="https://zenodo.org/record/3609906/files/figure.png" pageId="3" pageNumber="4">Fig.2</figureCitation>
|
||
).Scale bars, 1 µm (
|
||
<emphasis bold="true" box="[866,878,950,968]" pageId="3" pageNumber="4">b</emphasis>
|
||
,
|
||
<emphasis bold="true" box="[886,896,950,968]" pageId="3" pageNumber="4">c</emphasis>
|
||
,
|
||
<emphasis bold="true" box="[904,915,950,968]" pageId="3" pageNumber="4">g</emphasis>
|
||
,
|
||
<emphasis bold="true" box="[923,935,950,968]" pageId="3" pageNumber="4">h</emphasis>
|
||
),500 nm (
|
||
<emphasis bold="true" box="[1025,1035,950,968]" pageId="3" pageNumber="4">a</emphasis>
|
||
,
|
||
<emphasis bold="true" box="[1043,1055,950,968]" pageId="3" pageNumber="4">d</emphasis>
|
||
,
|
||
<emphasis bold="true" box="[1063,1073,950,968]" pageId="3" pageNumber="4">e</emphasis>
|
||
,
|
||
<emphasis bold="true" box="[1081,1087,950,968]" pageId="3" pageNumber="4">i</emphasis>
|
||
) and200 nm (
|
||
<emphasis bold="true" box="[1209,1216,950,968]" pageId="3" pageNumber="4">f</emphasis>
|
||
).
|
||
<emphasis bold="true" box="[1231,1263,950,968]" pageId="3" pageNumber="4">a–c</emphasis>
|
||
,
|
||
<emphasis bold="true" box="[1271,1282,950,968]" pageId="3" pageNumber="4">g</emphasis>
|
||
,
|
||
<emphasis bold="true" box="[1290,1302,950,968]" pageId="3" pageNumber="4">h</emphasis>
|
||
, SEM images are representative of
|
||
<emphasis box="[969,980,978,994]" italics="true" pageId="3" pageNumber="4">n</emphasis>
|
||
= 122 recorded images that were obtained from four independent observations from four culture samples.
|
||
<emphasis bold="true" box="[1283,1295,1004,1022]" pageId="3" pageNumber="4">d</emphasis>
|
||
,
|
||
<emphasis bold="true" box="[1303,1313,1004,1022]" pageId="3" pageNumber="4">e</emphasis>
|
||
, Cryo-EM images are representative of
|
||
<emphasis box="[969,980,1032,1048]" italics="true" pageId="3" pageNumber="4">n</emphasis>
|
||
=14 recorded images that were taken from two independent observations from two culture samples.
|
||
<emphasis bold="true" box="[1163,1170,1057,1075]" pageId="3" pageNumber="4">f</emphasis>
|
||
,
|
||
<emphasis bold="true" box="[1177,1183,1057,1075]" pageId="3" pageNumber="4">i</emphasis>
|
||
, The ultrathin section images are representative of
|
||
<emphasis box="[969,980,1085,1101]" italics="true" pageId="3" pageNumber="4">n</emphasis>
|
||
= 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.
|
||
</paragraph>
|
||
</caption>
|
||
<subSubSection pageId="3" pageNumber="4" type="etymology">
|
||
<paragraph blockId="3.[106,787,1338,1988]" pageId="3" pageNumber="4">
|
||
<emphasis bold="true" box="[129,243,1653,1673]" pageId="3" pageNumber="4">Etymology</emphasis>
|
||
.
|
||
<emphasis box="[251,458,1654,1673]" italics="true" pageId="3" pageNumber="4">Prometheoarchaeum</emphasis>
|
||
,
|
||
<taxonomicName authority="(Greek)" authorityName="Greek" baseAuthorityName="Greek" box="[467,665,1653,1673]" class="Insecta" family="Castniidae" genus="Prometheus" kingdom="Animalia" order="Lepidoptera" pageId="3" pageNumber="4" phylum="Arthropoda" rank="genus">
|
||
<emphasis box="[467,584,1654,1673]" italics="true" pageId="3" pageNumber="4">Prometheus</emphasis>
|
||
(Greek)
|
||
</taxonomicName>
|
||
:a Greek god who shaped humans out of mud and gave them the ability to create fire;
|
||
<emphasis box="[106,207,1711,1730]" italics="true" pageId="3" pageNumber="4">archaeum</emphasis>
|
||
from
|
||
<emphasis box="[263,345,1711,1730]" italics="true" pageId="3" pageNumber="4">archaea</emphasis>
|
||
(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
|
||
<taxonomicName box="[542,664,1768,1788]" class="Insecta" family="Castniidae" genus="Prometheus" kingdom="Animalia" order="Lepidoptera" pageId="3" pageNumber="4" phylum="Arthropoda" rank="genus">Prometheus</taxonomicName>
|
||
in the origin of humans from sediments and the acquisition of an unprecedented oxygen-driven energy-harnessing ability.The species name,
|
||
<emphasis italics="true" pageId="3" pageNumber="4">syntrophicum</emphasis>
|
||
,
|
||
<emphasis box="[157,191,1855,1873]" italics="true" pageId="3" pageNumber="4">syn</emphasis>
|
||
(Greek):together with;
|
||
<emphasis box="[424,508,1855,1874]" italics="true" pageId="3" pageNumber="4">trephein</emphasis>
|
||
(Greek) nourish;
|
||
<emphasis box="[677,716,1855,1873]" italics="true" pageId="3" pageNumber="4">icus</emphasis>
|
||
(Latin) pertaining to. The species name refers to the syntrophic substrate utilization property of this strain.
|
||
</paragraph>
|
||
<paragraph blockId="3.[106,787,1338,1988]" pageId="3" pageNumber="4">
|
||
<emphasis bold="true" box="[129,218,1940,1960]" pageId="3" pageNumber="4">Locality</emphasis>
|
||
. Isolated from deep-sea methane-seep sediment of the Nankai Trough at 2,533 m water depth,off the Kumano area,Japan.
|
||
</paragraph>
|
||
</subSubSection>
|
||
<subSubSection pageId="3" pageNumber="4" type="diagnosis">
|
||
<paragraph blockId="3.[816,1498,1338,1444]" pageId="3" pageNumber="4">
|
||
<emphasis bold="true" box="[839,939,1338,1358]" pageId="3" pageNumber="4">Diagnosis</emphasis>
|
||
. 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.
|
||
</paragraph>
|
||
</subSubSection>
|
||
</treatment>
|
||
</document> |