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Tlhaloso e qaqileng ea sehlahisoa
304 Ts'epe e sa hloekang e kopantsoeng ka tube /tubing
1. Tlhaloso: Coil tube / tubing ea tšepe e sa hloekang
2. Mofuta: o cheselitsoe kapa o se na seamless
3. Tekanyetso: ASTM A269, ASTM A249
4. Coil tube ea tšepe e sa hloekang OD: 6mm ho 25.4MM
5. Bolelele: 600-3500MM kapa ho ea ka tlhokahalo ea moreki.
6. Botenya ba lebota: 0.2mm ho 2.0mm.
7. Mamello: OD: +/-0.01mm;Botenya: +/-0.01%.
8. Coil ka hare ho lesoba boholo: 500MM-1500MM (e ka fetoloa ho ea ka litlhoko tsa bareki)
9. Bophahamo ba coil: 200MM-400MM (e ka fetoloa ho latela litlhoko tsa bareki)
10. Bokaholimo: E khanyang kapa e khaotsoe
11. Boitsebiso: 304, 304L, 316L, 321, 301, 201, 202, 409, 430, 410, motsoako oa 625, 825, 2205, 2507, joalo-joalo.
12. Ho paka: mekotla e lohiloeng ka mokotla oa lepolanka, pallet ea lehong, shaft ea lehong, kapa ho latela tlhoko ea moreki.
13. Teko : karolo ea lik'hemik'hale, matla a ho hlahisa, matla a thata, tekanyo ea boima
14. Tiisetso: Tlhahlobo ea motho oa boraro (mohlala :SGS TV ), joalo-joalo.
15. Kopo: Mokhabiso, thepa ea ka tlung, lipalangoang tsa oli, mocheso oa mocheso, ho etsa litšepe, ho etsa pampiri, koloi, ho lokisa lijo, bongaka, joalo-joalo.
Likarolo tsohle tsa Lik'hemik'hale le Lintho Tsa 'Mele tsa Stainless Steel joalo ka beow:
| Lintho tse bonahalang | ASTM A269 Sebopeho sa Lik'hemik'hale % Max | ||||||||||
| C | Mn | P | S | Si | Cr | Ni | Mo | NB | Nb | Ti | |
| TP304 | 0.08 | 2.00 | 0.045 | 0.030 | 1.00 | 18.0-20.0 | 8.0-11.0 | ^ | ^ | ^ . | ^ |
| TP304L | 0.035 | 2.00 | 0.045 | 0.030 | 1.00 | 18.0-20.0 | 8.0-12.0 | ^ | ^ | ^ | ^ |
| TP316 | 0.08 | 2.00 | 0.045 | 0.030 | 1.00 | 16.0-18.0 | 10.0-14.0 | 2.00-3.00 | ^ | ^ | ^ |
| TP316L | 0.035 D | 2.00 | 0.045 | 0.030 | 1.00 | 16.0-18.0 | 10.0-15.0 | 2.00-3.00 | ^ | ^ | ^ |
| TP321 | 0.08 | 2.00 | 0.045 | 0.030 | 1.00 | 17.0-19.0 | 9.0-12.0 | ^ | ^ | ^ | 5C -0,70 |
| TP347 | 0.08 | 2.00 | 0.045 | 0.030 | 1.00 | 17.0-19.0 | 9.0-12.0 | 10C -1.10 | ^ | ||
| Lintho tse bonahalang | Phekolo ea mocheso | Mocheso F (C) Min. | Ho thatafala | |
| Brinell | Rockwell | |||
| TP304 | Tharollo | 1900 (1040) | 192HBW/200HV | 90HRB |
| TP304L | Tharollo | 1900 (1040) | 192HBW/200HV | 90HRB |
| TP316 | Tharollo | 1900(1040) | 192HBW/200HV | 90HRB |
| TP316L | Tharollo | 1900(1040) | 192HBW/200HV | 90HRB |
| TP321 | Tharollo | 1900(1040) F | 192HBW/200HV | 90HRB |
| TP347 | Tharollo | 1900(1040) | 192HBW/200HV | 90HRB |
| OD, inch | OD Tolerance inch(mm) | Mamello ea WT % | Length Tolernace inch(mm) | |
| + | - | |||
| ≤ 1 / 2 | ± 0.005 ( 0.13 ) | ± 15 | 1 / 8 ( 3.2 ) | 0 |
| > 1/2 ~1 1/2 | ± 0.005(0.13) | ± 10 | 1 / 8 (3.2) | 0 |
| > 1 1 / 2 ~< 3 1 / 2 | ± 0.010(0.25) | ± 10 | 3 / 16 (4.8) | 0 |
| > 3 1 / 2 ~< 5 1 / 2 | ± 0.015(0.38) | ± 10 | 3 / 16 (4.8) | 0 |
| > 5 1 / 2 ~< 8 | ± 0.030(0.76) | ± 10 | 3 / 16 (4.8) | 0 |
| 8 ~ <12 | ± 0.040(1.01) | ± 10 | 3 / 16 (4.8) | 0 |
| 12 ~ <14 | ± 0.050(1.26) | ± 10 | 3 / 16 (4.8) | 0 |
Mekhatlo ea tlhaho ea likokoana-hloko e fapane ka phylogenetically le metabolism.Ho phaella ho lihlopha tse sa rutehang tsa lintho tse phelang1, mefuta-futa ena e boetse e na le menyetla e mengata ea ho sibolloa ha li-enzyme tsa bohlokoa tsa tikoloho le biotechnologically le metsoako ea lik'hemik'hale tsa biochemical2,3.Leha ho le joalo, ho ithuta mefuta-futa ena ho fumana litsela tsa genomic tse kopanyang metsoako e joalo le ho e tlama ho batho ba e amohelang e ntse e le phephetso.Bokhoni ba biosynthetic ba likokoana-hloko leoatleng le bulehileng bo lula bo sa tsejoe ka lebaka la mefokolo ea tlhahlobo ea lintlha tsa tharollo ea genome lefatšeng ka bophara.Mona, re hlahloba mefuta-futa le mefuta-futa ea lihlopha tsa liphatsa tsa lefutso tsa biosynthetic leoatleng ka ho kopanya liphatsa tsa lefutso tse ka bang 10,000 tse tsoang liseleng tse tsoetseng pele le lisele tse le 'ngoe tse nang le liphatsa tsa lefutso tse fetang 25,000 tse nchafalitsoeng ho tsoa lisampoleng tse fetang 1,000 tsa metsi a leoatle.Boiteko bona bo bonts'itse lihlopha tse ka bang 40,000 tsa liphatsa tsa lefutso tse ncha tsa biosynthetic, tseo tse ling tsa tsona li fumanoeng lihlopheng tsa pele tse neng li sa belaelloe tsa phylogenetic.Ho baahi bana, re khethile leloko le ruileng ka lihlopha tsa liphatsa tsa lefutso tsa biosynthetic ("Candidatus Eudormicrobiaceae") tseo e neng e le tsa phylum ea baktheria e sa lengoang 'me e kenyelletsa tse ling tsa likokoana-hloko tse fapaneng ka ho fetisisa tikolohong ena.Ho tsena, re tšoaile litsela tsa phosphatase-peptide le pytonamide, ho supa maemo a sebopeho se sa tloaelehang sa motsoako oa bioactive le enzymology, ka ho latellana.Qetellong, thuto ena e bonts'a hore na maqheka a thehiloeng ho microbiome a ka thusa joang ho hlahloba li-enzyme tse neng li sa hlalosoa pele le lijo tsa tlhaho ka microbiota le tikoloho e sa utloisisoeng hantle.
Likokoana-hloko li tsamaisa lipotoloho tsa biogeochemical lefatšeng ka bophara, li boloka marang-rang a lijo, 'me li boloka limela le liphoofolo li phela hantle5.Phapang ea tsona e kholo ea phylogenetic, metabolism le ts'ebetso e emela monyetla o motle oa ho sibolloa ha taxa1 e ncha, li-enzyme le metsoako ea lik'hemik'hale tsa biochemical, ho kenyeletsoa lihlahisoa tsa tlhaho6.Libakeng tsa tikoloho, limolek'hule tsena li fana ka likokoana-hloko tse nang le mefuta e sa tšoaneng ea mesebetsi ea 'mele le ea tikoloho, ho tloha puisanong ho ea tlhōlisanong ea 2, 7.Ho phaella mesebetsing ea bona ea pele, lihlahisoa tsena tsa tlhaho le litsela tsa tsona tsa tlhahiso ea liphatsa tsa lefutso li fana ka mehlala ea lisebelisoa tsa biotechnology le tsa phekolo2,3.Ho tsebahatsa litsela tse joalo le likhokahano ho nolofalitsoe haholo ke boithuto ba likokoana-hloko tse hlahisitsoeng.Leha ho le joalo, liphuputso tsa taxonomic tsa tikoloho ea tlhaho li bonts'itse hore bongata bo boholo ba likokoana-hloko ha lia lengoa8.Leeme lena la setso le fokotsa bokhoni ba rona ba ho sebelisa mefuta-futa e sebetsang e kentsoeng ke likokoana-hloko tse ngata4,9.
Ho hlola mefokolo ena, tsoelo-pele ea thekenoloji lilemong tse leshome tse fetileng e lumeletse bafuputsi hore ka ho toba (ke hore, ntle le setso sa pele) ba hlophise likaroloana tsa DNA tsa likokoana-hloko tse tsoang lichabeng tsohle (metagenomics) kapa lisele tse le 'ngoe.Bokhoni ba ho bokella likhechana tsena ka likhechana tse kholo tsa genome le ho aha bocha li-genome tse kopaneng tsa metagenomically (MAGs) kapa li-genomes tsa amplified single (SAGs), ka ho latellana, li bula monyetla oa bohlokoa bakeng sa lithuto tsa lekhetho tsa microbiome (ke hore, lichaba tsa likokoana-hloko le microbiome).lokisa litsela tse ncha.thepa ea liphatsa tsa lefutso tikolohong e fanoeng) 10,11,12.Ehlile, liphuputso tsa morao-rao li atolositse haholo kemelo ea phylogenetic ea mefuta-futa ea likokoana-hloko ho Earth1, 13 mme e senotse mefuta e mengata ea ts'ebetso ho sechaba sa likokoana-hloko tse sa kang tsa kengoa ke tatellano ea liphatsa tsa lefutso tsa microorganism (REFs)14.Bokhoni ba ho beha mefuta e fapaneng ea ts'ebetso e sa bonahaleng maemong a genome e amohelang (ke hore, tharollo ea genome) e bohlokoa bakeng sa ho bolela esale pele mela ea likokoana-hloko tse e-s'o be le sebopeho sa lihlahisoa tse ncha tsa tlhaho15,16 kapa bakeng sa ho sala morao metsoako e joalo ho mohlahisi oa eona oa pele17.Ka mohlala, mokhoa o kopantsoeng oa tlhahlobo ea metagenomic le ea sele e le 'ngoe ea genomic e lebisitse ho khethoeng ha Candidatus Entotheonella, sehlopha sa likokoana-hloko tse amanang le seponche tse ruileng, e le bahlahisi ba mefuta e sa tšoaneng ea lithethefatsi18.Leha ho le joalo, ho sa tsotellehe liteko tsa morao-rao tsa lipatlisiso tsa genomic tsa lichaba tse fapa-fapaneng tsa likokoana-hloko, 16,19 e fetang karolo ea bobeli ho tse tharo ea data ea lefats'e ea metagenomic bakeng sa leoatle le leholo ka ho fetisisa lefatšeng16,20 e ntse e le sieo.Kahoo, ka kakaretso, bokhoni ba biosynthetic ba microbiome ea leoatleng le bokhoni ba eona e le polokelo ea lihlahisoa tse ncha tsa enzymatic le lihlahisoa tsa tlhaho li lula li sa ithutoe haholo.
Ho hlahloba bokhoni ba biosynthetic ea likokoana-hloko tsa leoatleng lefatšeng ka bophara, re ile ra qala ka ho kopanya liphatsa tsa lefutso tsa marine tsa likokoana-hloko tse fumanoeng ka mekhoa e itšetlehileng ka setso le e seng ea setso ho theha polokelo ea boitsebiso e pharaletseng ea phylogenetics le gene function.Tlhahlobo ea bobolokelo bona ba polokelo e ile ea senola mefuta e mengata e fapaneng ea lihlopha tsa liphatsa tsa lefutso tsa biosynthetic (BGCs), tseo bongata ba tsona e leng tsa malapa a ntseng a e-s'o be le sebopeho sa gene cluster (GCF).Ho feta moo, re khethile lelapa la baktheria le sa tsejoeng le bonts'ang mefuta e mengata e tsebahalang ea BGC leoatleng le bulehileng ho fihlela joale.Re khethile litsela tse peli tsa ribosomal synthesis le post-translationally modified peptide (RiPP) bakeng sa netefatso ea liteko ho latela liphapang tsa bona tsa lefutso le litsela tse tsejoang hajoale.Sebopeho sa ts'ebetso ea litsela tsena se senotse mehlala e sa lebelloang ea enzymology hammoho le metsoako e sa tloaelehang ea sebopeho e nang le ts'ebetso ea protease inhibitory.
Qalong, re ne re rerile ho theha mohloli oa boitsebiso ba lefats'e bakeng sa tlhahlobo ea genome, re shebane le likarolo tsa eona tsa baktheria le tsa khale.Ho fihlela sena, re ile ra kopanya lintlha tsa metagenomic le disampole tse 1038 tsa metsi a leoatle ho tsoa libakeng tse 215 tse ajoang lefatšeng ka bophara (latitude range = 141.6°) le likarolo tse ngata tse tebileng (ho tloha ho 1 ho isa ho 5600 m ka botebo, tse koahelang libaka tsa pelagic, mesopelagic le mohohlo).Background21,22,23 (setšoantšo sa 1a, lintlha tse atolositsoeng, setšoantšo sa 1a le Letlapa la Tlatsetso 1).Ntle le ho fana ka sebaka se pharaletseng, lisampole tsena tse tlhotliloeng li ile tsa re lumella ho bapisa likarolo tse fapaneng tsa microbiome ea metsing, ho kenyeletsoa le kokoana-hloko e ngata (<0.2 µm), prokaryotic-rich (0.2–3 µm), e nang le likaroloana tse ngata (0.8 µm). ).-20 µm) le likolone tse se nang kokoana-hloko (>0.2 µm).
a, Kakaretso ea liphatsa tsa lefutso tse 1038 tse fumanehang phatlalatsa (metagenomics) ea lichaba tsa leoatle tse nyenyane tse bokelletsoeng libakeng tse 215 tse ajoang lefatšeng ka bophara (62°S ho isa 79°N le 179°W ho isa 179°E.).Lithaele tsa 'mapa © Esri.Mehloli: GEBCO, NOAA, CHS, OSU, UNH, CSUMB, National Geographic, DeLorme, NAVTEQ, le Esri.b, metagenomes tsena li ne li sebelisetsoa ho tsosolosa MAGs (mekhoa le boitsebiso bo eketsehileng), tse fapaneng ka bongata le boleng (mekhoa) ho li-datasets (tse tšoailoeng ka mebala).Li-MAG tse hahiloeng bocha li ile tsa tlatselletsoa ka liphatsa tsa lefutso tse fumanehang phatlalatsa (ka ntle), ho kenyeletsoa MAG26 e entsoeng ka letsoho, SAG27 le REF.27 Kopanya OMD.c, ha ho bapisoa le litlaleho tse fetileng tse thehiloeng feela ho SAG (GORG)20 kapa MAG (GEM)16, OMD e ntlafatsa sebopeho sa genomic sa metse ea likokoana-hloko tsa leoatleng (sekhahla sa ho bala 'mapa oa metagenomic; mokhoa) ka makhetlo a mabeli ho isa ho a mararo ka boemeli bo tsitsitseng haholoanyane ka botebo le latitude..<0.2, n=151, 0.2-0.8, n=67, 0.2-3, n=180, 0.8-20, n=30, >0.2, n=610, <30°, n = 132, 30–60° , n = 73, >60°, n = 42, EPI, n = 174, MES, n = 45, BAT, n = 28. d, OMD e hlopha ka mekhahlelo ea lihlopha tsa mefuta (95% e bolela nucleotide identity) e khetholla kakaretso ea mefuta e ka bang 8300, e fetang halofo ea eona e neng e e-so ka e khetholloa ho latela litlhaloso tsa taxonomic tse sebelisang GTDB (version 89) e, ho hlophisoa ha mefuta ka mofuta oa genome ho bontšitse hore MAG, SAG le REFs lia tlatsana hantle ho bonts'a mefuta e sa tšoaneng ea phylogenetic. microbiome ea metsing.Ka ho khetheha, 55%, 26% le 11% ea mefuta e ne e le e tobileng bakeng sa MAG, SAG le REF, ka ho latellana.BATS, Bermuda Atlantic Time Series;GEM, liphatsa tsa lefutso tsa microbiome ea Lefatše;GORG, genome ea litšupiso tsa leoatle la lefatše;HOT, letoto la nako la Leoatle la Hawaii.
Re sebelisa dataset ena, re ile ra tsosolosa kakaretso ea 26,293 MAGs, haholo-holo baktheria le archaeal (setšoantšo sa 1b le lintlha tse atolositsoeng, setšoantšo sa 1b).Re thehile li-MAGs tsena ho tsoa likopanong tse arohaneng ho fapana le ho kopanya lisampole tsa metagenomic ho thibela ho putlama ha phapang ea tatellano ea tlhaho lipakeng tsa lisampole tse tsoang libakeng tse fapaneng kapa linako (mekhoa).Ho feta moo, re hlophisitse likhechana tsa genomic ho ipapisitse le ho ata ha tsona ho palo e kholo ea disampole (ho tloha lisampole tse 58 ho isa ho tse 610, ho latela lipatlisiso; mokhoa).Re fumane hore ona ke mohato o nkang nako empa e le oa bohlokoa24 o ileng oa tlotsoa mesebetsing e mengata ea kaho e kholo ea MAG16, 19, 25 mme e ntlafatsa haholo bongata (ka karolelano makhetlo a 2.7) le boleng (+ 20% ka karolelano) ea genome.e tsosolositsoeng ho tloha metagenome ea metsing e ithutoang mona (lintlha tse atolositsoeng, setšoantšo sa 2a le boitsebiso bo eketsehileng).Ka kakaretso, boiteko bona bo entse hore ho be le keketseho ea 4.5 ea lik'hemik'hale tse nyenyane tsa marine MAG (6-fold haeba ho nahanoa feela li-MAG tsa boleng bo phahameng) ha li bapisoa le mohloli o pharaletseng oa MAG o fumanehang kajeno16 (Mekhoa).Sete ena e sa tsoa thehoa ea MAG e ile ea kopanngoa le 830 e nkiloeng ka letsoho MAG26s, 5969 SAG27s le 1707 REFs.Mefuta e mashome a mabeli a metso e supileng ea libaktheria tsa leoatleng le li-archaea li entse pokello e kopaneng ea liphatsa tsa lefutso tsa 34,799 (setšoantšo sa 1b).
Ka mor'a moo re ile ra hlahloba mohloli o sa tsoa thehoa ho ntlafatsa bokhoni ba oona ba ho emela lichaba tse phelang metsing le ho hlahloba phello ea ho kopanya mefuta e fapaneng ea genome.Ka karolelano, re fumane hore e akaretsa hoo e ka bang 40-60% ea data ea marine metagenomic (Setšoantšo sa 1c), makhetlo a mabeli ho isa ho a mararo ho koahela litlaleho tsa MAG-feela tse fetileng ka botebo le latitude More serial 16 kapa SAG20.Ho feta moo, ho lekanya ka mokhoa o hlophisehileng mefuta-futa ea lekhetho pokellong e hlophisitsoeng, re ile ra hlakisa liphatsa tsa lefutso tsohle re sebelisa lisebelisoa tsa Genome Taxonomy Database (GTDB) (mekhoa) 'me ra sebelisa boitsebiso bo tloaelehileng ba genome-wide nucleotide ea 95%.28 ho khetholla lihlopha tsa mefuta e 8,304 (mefuta).Likarolo tse peli ho tse tharo tsa mefuta ena (ho kenyeletsoa le lihlopha tse ncha) li ne li e-s'o hlahe ho GTDB, eo 2790 e ileng ea fumanoa ho sebelisoa MAG e tsosolositsoeng thutong ena (setšoantšo sa 1d).Ho phaella moo, re fumane hore mefuta e fapaneng ea liphatsa tsa lefutso e tlatsana haholo: 55%, 26%, le 11% ea mefuta e entsoe ka ho feletseng ka MAG, SAG, le REF, ka ho latellana (setšoantšo sa 1e).Ntle le moo, MAG e ne e akaretsa mefuta eohle e 49 e fumanehang ka har'a kholomo ea metsi, athe SAG le REF li ne li emela tse 18 le 11 feela ka ho latellana.Leha ho le joalo, SAG e molemo e emela mefuta-futa ea lihlopha tse tloaelehileng (lintlha tse atolositsoeng, setšoantšo sa 3a), tse kang Pelagic Bacteriales (SAR11), le SAG e koahelang mefuta e ka bang 1300 le MAG feela mefuta e 390.Haholo-holo, li-REF li ne li sa kopane le li-MAG kapa li-SAG maemong a mefuta mme li emetse> 95% ea liphatsa tsa lefutso tse ka bang 1000 tse sa fumanoeng libakeng tse bulehileng tsa leoatleng tse ithutoang mona, haholoholo ka lebaka la tšebelisano le mefuta e meng ea mehlala e ikemetseng ea leoatle (mohlala, li-sediments) .kapa moemedi wa moamoheli).Ho etsa hore e fumanehe haholo ho sechaba sa mahlale, sesebelisoa sena sa marine genome, se kenyelletsang le likhechana tse sa hlalosoang (mohlala, ho tsoa ho phages e boletsoeng esale pele, lihlekehlekeng tsa genomic, le likaroloana tsa genome tseo ho senang data e lekaneng bakeng sa kaho bocha ea MAG), e ka bapisoa le data ea lekhetho. .Fumana litlhaloso hammoho le ts'ebetso ea liphatsa tsa lefutso le maemo a maemo ho Database ea Ocean Microbiology (OMD; https://microbiomics.io/ocean/).
Ka mor'a moo, re ile ra ikemisetsa ho hlahloba leruo le bocha ba bokhoni ba biosynthetic ka har'a li-microbiome tsa leoatle tse bulehileng.Ho fihlela sena, re ile ra qala ka ho sebelisa antiSMASH bakeng sa li-MAG, li-SAG, le li-REF kaofela tse fumanehang ho 1038 marine metagenomes (mekhoa) ho bolela esale pele kakaretso ea 39,055 BGCs.Ka mor'a moo re arola li-GCF tse 6907 tse sa hlokeng mosebetsi le tse 151 tsa lihlopha tsa liphatsa tsa lefutso (GCCs; Supplementary Table 2 le mekhoa) ho ikarabella bakeng sa ho fokotseha ha tlhaho (ke hore, BGC e tšoanang e ka kenyelletsoa ka mefuta e mengata ea liphatsa tsa lefutso) le data ea metagenomic Ho arohana ha li-BGC tse tsepamisitsoeng.Li-BGC tse sa phethahalang ha lia ka tsa eketseha haholo, haeba li le teng (Tlhaloso ea Tlatsetso), palo ea li-GCF le li-GCC, ka ho latellana, tse nang le bonyane setho se le seng sa BGC se tsitsitseng ho 44% le 86% ea linyeoe.
Boemong ba GCC, re fumane mefuta e mengata e fapaneng ea li-RiPP tse boletsoeng esale pele le lihlahisoa tse ling tsa tlhaho (setšoantšo sa 2a).Har'a bona, mohlala, li-arylpolyenes, carotenoids, ectoines, le siderophores ke tsa GCC tse nang le kabo e pharaletseng ea phylogenetic le bongata bo bongata ba metagenome ea leoatle, e ka bonts'ang phetoho e pharaletseng ea likokoana-hloko tikolohong ea leoatle, ho kenyelletsa ho hanyetsa mefuta ea oksijene e sebetsang. khatello ea oxidative le osmotic..kapa ho monya tšepe (boitsebiso bo eketsehileng).Phapano ena e sebetsang e fapana le tlhahlobo ea morao-rao ea li-BGC tse ka bang limilione tse 1.2 har'a liphatsa tsa lefutso tse ka bang 190,000 tse bolokiloeng polokelong ea NCBI RefSeq (BiG-FAM/RefSeq, eo hamorao e bitsoang RefSeq)29, e bonts'itseng hore nonribosomal Synthetase peptides (NRPS) le polyketide synthethase. (PKS) BGCs (Tlhahlobo ya Tlatsetso).Re boetse re fumane 44 (29%) GCCs e amanang le hole feela ho leha e le efe RefSeq BGC (\(\bar{d}\)RefSeq> 0.4; Fig. 2a le mekhoa) le 53 (35%) GCCs feela ho MAG , e totobatsang bokhoni ho lemoha lik'hemik'hale tse neng li sa hlalosoa pele ho OMD.Ka lebaka la hore e 'ngoe le e 'ngoe ea li-GCC tsena e kanna ea emela mesebetsi e fapaneng haholo ea biosynthetic, re ile ra tsoela pele ho hlahlobisisa data boemong ba GCF ka boiteko ba ho fana ka sehlopha se nang le lintlha tse ngata tsa BGC tse boletsoeng esale pele ho khouta bakeng sa lihlahisoa tse tšoanang tsa tlhaho29.Kakaretso ea li-GCF tse 3861 (56%) tse khethiloeng ha lia ka tsa kopana le RefSeq, 'me> 97% ea li-GCF e ne e le sieo ho MIBiG, e leng e' ngoe ea li-database tse kholo ka ho fetisisa tsa BGC tse netefalitsoeng ka liteko (Setšoantšo sa 2b).Leha ho sa makatse ho fumana litsela tse ngata tse ncha maemong a sa emeloeng hantle ke genome ea litšupiso, mokhoa oa rona oa ho fetolela li-BGC ho li-GCF pele ho benchmarking o fapana le litlaleho tse fetileng tsa 16 mme o re lumella ho fana ka tlhahlobo e se nang leeme ea lintho tse ncha.Boholo ba mefuta-futa e mecha (3012 GCF kapa 78%) e lumellana le terpenes e boletsoeng esale pele, RiPP kapa lihlahisoa tse ling tsa tlhaho, 'me boholo (1815 GCF kapa 47%) e kentsoe ka mefuta e sa tsejoeng ka lebaka la bokhoni ba bona ba biosynthetic.Ho fapana le lihlopha tsa PKS le NRPS, li-BGC tsena tse kopanetsoeng ha li na monyetla oa hore li arohane nakong ea kopano ea 31 ea metagenomic 'me li lumella nako e ngata le lisebelisoa tse sebetsang hantle tsa lihlahisoa tsa tsona.
Kakaretso ea li-BGC tse 39,055 li ne li arotsoe ka li-GCF tse 6,907 le tse 151 tsa li-GCC.a, kemedi ya data (ka hare ho kantle).Khokahano ea maemo a holimo ea libaka tsa BGC tse ipapisitseng le GCC, tse 53 tsa tsona li hlophisitsoe ke MAG feela.GCC e na le li-BGC tse tsoang ho taxa tse fapaneng (maqhubu a liheke tse fetotsoeng) le lihlopha tse fapaneng tsa BGC (boholo ba selikalikoe bo lumellana le makhetlo a eona).Bakeng sa GCC ka 'ngoe, karolo e ka ntle e emela palo ea li-BGC, ho ata (liperesente tsa lisampole), le sebaka (bonyane BGC cosine sebaka (min(dMIBiG))) ho tloha BiG-FAM ho ea BGC.Li-GCC tse nang le li-BGC tse amanang haufi-ufi le li-BGC tse netefalitsoeng ka liteko (MIBiG) li totobatsoa ka metsu.b Ho bapisa GCF le li-BGC tse boletsoeng esale pele (BiG-FAM) le tse netefalitsoeng ka liteko (MIBiG), ho fumanoe li-GCF tse ncha tse 3861 (d–>0.2).Boholo (78%) ba khoutu ena bakeng sa RiPP, terpenes, le lihlahisoa tse ling tsa tlhaho tse behang.c, liphatsa tsa lefutso tsohle ho OMD tse fumanoang ho 1038 metagenomes ea marine li ile tsa behoa sefateng sa motheo sa GTDB ho bontša tšireletso ea phylogenetic ea OMD.Li-clade tse se nang liphatsa tsa lefutso ho OMD li bonts'oa ka bohlooho.Palo ea li-BGC e tsamaisana le palo e kholo ka ho fetisisa ea li-BGC tse boletsoeng esale pele ka genome ho clade e fanoeng.Bakeng sa ho hlaka, 15% ea ho qetela ea li-node e oa.Metsu e bontša lihlopha tse ruileng ka BGC (>15 BGC), ntle le Mycobacterium, Gordonia (ea bobeli ho Rhodococcus), le Crocosphaera (ea bobeli ho Synechococcus).d, Tse sa tsejoeng c.Eremiobacterota e bontšitse mefuta-futa e phahameng ka ho fetisisa ea biosynthetic (index ea Shannon e thehiloeng ho mofuta oa sehlahisoa sa tlhaho).Sehlopha se seng le se seng se emela genome e nang le li-BGC tse ngata ka ho fetisisa mofuteng.T1PKS, PKS mofuta oa I, T2/3PKS, PKS mofuta oa II le mofuta oa III.
Ntle le maruo le bocha, re hlahloba sebopeho sa biogeographic sa bokhoni ba biosynthetic ba microbiome ea leoatleng.Ho hlophisoa ha lisampole ka karolelano ea ho ajoa ha linomoro tsa likopi tsa metagenomic GCF (Mekhoa) ho bonts'itse hore baahi ba sebaka se tlase, ba bokaholimo, ba ruileng ba prokaryotic le ba futsanehileng ke kokoana-hloko, haholo-holo ho tloha holim'a metsi kapa metsi a tebileng a letsatsi, ba ne ba ruile RiPP le BGC terpenes.Ka lehlakoreng le leng, metse ea polar, leoatle le tebileng, kokoana-hloko le likaroloana tse ruileng li ne li amahanngoa le bongata bo phahameng ba NRPS le PKS BGC (lintlha tse atolositsoeng, setšoantšo sa 4 le boitsebiso bo eketsehileng).Qetellong, re fumane hore metse e ithutoang hantle ea tropike le pelagic ke mehloli e tšepisang ka ho fetisisa ea terpenes e ncha (Augmented Data Figure).Monyetla o phahameng ka ho fetisisa oa PKS, RiPP le lihlahisoa tse ling tsa tlhaho (Setšoantšo sa 5a se nang le lintlha tse atolositsoeng).
Ho tlatselletsa boithuto ba rona ba bokhoni ba biosynthetic ba likokoana-hloko tsa leoatleng, re ne re ikemiselitse ho etsa 'mapa oa kabo ea tsona ea phylogenetic le ho tseba likarolo tse ncha tse matlafalitsoeng ke BGC.Ho fihlela sena, re ile ra beha liphatsa tsa lefutso tsa likokoana-hloko tsa metsing ka har'a sefate sa baktheria sa GTDB13 se tloaelehileng le sa archaeal phylogenetic 'me ra koahela litsela tseo ba li kopanyang (Setšoantšo sa 2c).Re hlokometse ha bonolo lihlopha tse 'maloa tse ntlafalitsoeng tsa BGC (tse emeloang ke BGCs tse fetang 15) ka lisampole tsa metsi a leoatle (mekhoa) tse tsejoang ka bokhoni ba tsona ba biosynthetic, joalo ka cyanobacteria (Synechococcus) le libaktheria tsa Proteus, joalo ka Tistrella32,33, kapa tse sa tsoa khahla tlhokomelo ea tsona. lihlahisoa tsa tlhaho .tse kang Myxococota (Sandaracinaceae), Rhodococcus le Planctomycetota34,35,36.Hoa thahasellisa hore ebe re fumane meloko e mengata e neng e e-so ka e hlahlojoa lihlopheng tsena.Ka mohlala, mefuta eo e nang le bokhoni bo bongata ba biosynthetic ho phyla Planctomycetota le Myxococcota e ne e le ea litaelo tsa bonkgetheng tse sa hlalosoang le genera, ka ho latellana (Tlaleho ea Tlatsetso 3).Ha li kopane, sena se fana ka maikutlo a hore OMD e fana ka phihlelo ea tlhahisoleseding ea phylogenetic e neng e sa tsejoe pele, ho kenyelletsa le likokoana-hloko, tse ka 'nang tsa emela lipakane tse ncha tsa ho sibolloa ha enzyme le lihlahisoa tsa tlhaho.
Ka mor'a moo, re ile ra khetholla sehlopha se matlafalitsoeng sa BGC ka ho se bale feela palo e kholo ea BGC e kenyellelitsoeng ke litho tsa eona, empa hape le ka ho hlahloba mefuta-futa ea li-BGC tsena, tse hlalosang makhetlo a mangata a mefuta e fapaneng ea lihlahisoa tsa tlhaho (Fig. )..Re fumane hore mefuta e fapaneng ka ho fetesisa ea biosynthetically e ne e emeloa ke MAGs ea baktheria e entsoeng ka mokhoa o ikhethileng thutong ena.Libaktheria tsena ke tsa phylum Candidatus Eremiobacterota e sa lengoang, e lulang e sa hlahlojoe ntle le lithuto tse seng kae tsa genomic37,38.Hoa hlokomeleha hore "ca.Mofuta oa Eremiobacterota o hlahlobiloe feela tikolohong ea lefatše39 me ha o tsejoe hore o kenyelletsa litho tse ruisitsoeng ho BGC.Mona re tsosolositse li-MAG tse robeli tsa mefuta e tšoanang (nucleotide identity> 99%) 23. Ka hona re fana ka tlhahiso ea lebitso la mofuta "Candidatus Eudoremicrobium malaspinii", e reheletsoeng ka nereid (sea nymph), mpho e ntle ka litšōmo le maeto a Bagerike.'Ka.Ho latela tlhaloso ea phylogenetic 13, E. malaspinii ha e na beng ka eena ba kileng ba tsejoa ka tlase ho boemo ba tatellano 'me kahoo ke ea lelapa le lecha la baktheria leo re le sisinyang "Ca.E. malaspinii” joalo ka mofuta oa mofuta le “Ca.Eudormicrobiaceae” joalo ka lebitso la semmuso (Tlhahiso ea Tlatsetso).Kaho e khutšoane ea metagenomic ea 'Ca.Morero oa genome oa E. malaspinii o tiisitsoe ka ho kenya letsoho ho fokolang haholo, tatellano ea nako e telele ea metagenomic le kopano e lebisitsoeng ea sampuli e le 'ngoe (Mekhoa) e le chromosome e le 'ngoe ea 9.63 Mb e nang le 75 kb ka bobeli.e le eona feela ntho e sa hlakang e setseng.
Ho theha moelelo oa phylogenetic oa mofuta ona, re batlile mefuta e 40 e amanang haufi-ufi ho lisampole tse eketsehileng tsa eukaryotic-enriched metagenomic ho tloha leetong la Leoatle la Tara ka ho tsosolosoa ha genome.Ka bokhuts'oane, re hokahanya lipalo tsa metagenomic le likaroloana tsa genomic tse amanang le "Ca.E. malaspinii” mme ho nahanoa hore sekhahla se eketsehang sa khiro sampoleng ena se bontša boteng ba beng ka bona ba bang (mekhoa).Ka lebaka leo, re fumane 10 MAGs, motsoako oa 19 MAGs e emelang mefuta e mehlano ea lihlopha tse tharo ka har'a lelapa le sa tsoa hlalosoa (ke hore "Ca. Eudormicrobiaceae").Ka mor'a ho hlahloba ka letsoho le taolo ea boleng (lintlha tse atolositsoeng, setšoantšo sa 6 le boitsebiso bo eketsehileng), re fumane hore "Ca.Mefuta ea Eudormicrobiaceae e hlahisa liphatsa tsa lefutso tse kholoanyane (8 Mb) le bokhoni bo bongata ba biosynthetic (14 ho isa ho 22 BGC ka mofuta) ho feta litho tse ling tsa "Ca".Clade Eremiobacterota (ho fihlela ho 7 BGC) (Setšoantšo sa 3a-c).
a, Phylogenetic maemo a tse hlano 'Ca.Mefuta ea Eudormicrobiaceae e bonts'itse borui ba BGC bo ikhethang ho melapo ea leoatle e boletsoeng thutong ena.Sefate sa phylogenetic se kenyelletsa tsohle 'Ca.MAG Eremiobacterota le litho tsa phyla tse ling (linomoro tsa genome ka masakaneng) tse fanoeng ho GTDB (mofuta oa 89) li ne li sebelisoa bakeng sa semelo sa ho iphetola ha lintho (Mekhoa).Likarolo tse ka ntle ka ho fetisisa li emela lihlopha boemong ba lelapa ("Ca. Eudormicrobiaceae" le "Ca. Xenobiaceae") le boemong ba sehlopha ("Ca. Eremiobacteria").Mefuta e mehlano e hlalositsoeng thutong ena e emeloa ke likhoutu tsa alphanumeric le mabitso a binomial a sisintsoeng (Tlhahlobo ea Tlatsetso).b, ho lokile.Mefuta ea Eudormicrobiaceae e na le likarolo tse supileng tse tloaelehileng tsa BGC.Ho ba sieo ha BGC ka har'a clade ea A2 ho ne ho bakoa ke ho se phethehe ha moemeli oa MAG (Letlapa la Tlatsetso 3).Li-BGC li tobane le "Ca.Amphithomicrobium” le “Ca.Amphithomicrobium” (clades A le B) ha e bonngwe.c, Li-BGC tsohle li kentsoe joalo ka "Ca.Eudoremicrobium taraoceanii e ile ea fumanoa e hlahisoa ka metaranscripttomes tse 623 tse nkiloeng maoatleng a Tara.Masakana a tiileng a bontša mongolo o sebetsang.Li-circles tsa Orange li bolela liphetoho tse fetotsoeng tsa log2 ka tlase le ka holim'a sekhahla sa polelo ea mofuta oa tlhokomelo ea ntlo (mekhoa).d, li-curve tse ngata (mekhoa) tse bontšang 'Ca.Mefuta ea Eudormicrobiaceae e atile libakeng tse ngata tsa leoatle le kholomong eohle ea metsi (ho tloha holimo ho ea botebong ba bonyane 4000 m).Ho latela likhakanyo tsena, re fumane hore 'Ca.E. malaspinii 'e etsa karolo ea 6% ea lisele tsa prokaryotic libakeng tse tebileng tsa pelagic tse amanang le lijo-thollo.Re ile ra nka hore mofuta o teng setšeng haeba o ne o fumanoa karolong efe kapa efe ea boholo ba lera le fanoeng la botebo.IO - Indian Ocean, NAO - North Atlantic, NPO - North Pacific, RS - Red Sea, SAO - South Atlantic, SO - Southern Ocean, SPO - South Pacific.
Ho ithuta ka bongata le kabo ea Ca.Eudormicrobiaceae, eo, joalo ka ha re fumane, e hlahelletseng ka bongata maoatleng a leoatle, hammoho le kholomong eohle ea metsi (setšoantšo sa 3d).Sebakeng sa heno, ba etsa karolo ea 6% ea likokoana-hloko tsa leoatleng, e leng se etsang hore e be karolo ea bohlokoa ea likokoana-hloko tsa leoatleng lefatšeng ka bophara.Ho feta moo, re fumane litaba tse amanang le Ca.Mefuta ea Eudormicrobiaceae le maemo a bona a polelo ea BGC a ne a phahame ka ho fetisisa karolong e ntlafalitsoeng ea eukaryotic (setšoantšo sa 3c le data e atolositsoeng, setšoantšo sa 7), e bontšang hore ho ka khoneha ho sebelisana le ntho e itseng, ho akarelletsa le plankton.Maikutlo ana a tšoana le 'Ca.Li-Eudoremicrobium BGC tse hlahisang lihlahisoa tsa tlhaho tsa cytotoxic ka litsela tse tsejoang li ka 'na tsa bontša boitšoaro ba ho hlasela (Tlhahlobo ea Tlatsetso le Boitsebiso bo Atolositsoeng, Figure 8), tse tšoanang le tse ling tse jang liphoofolo tse hlahisang metabolite tse kang Myxococcus41.Ho sibolloa ha Ca.Eudormicrobiaceae e fumanehang hanyane (leoatleng le tebileng) kapa eukaryotic ho fapana le lisampole tsa prokaryotic e ka hlalosa hore na ke hobaneng ha libaktheria tsena le mefuta ea tsona e sa lebelloang ea BGC e lula e sa hlaka molemong oa lipatlisiso tsa lijo tsa tlhaho.
Qetellong, re ile ra leka ho netefatsa tšepiso ea mosebetsi oa rona o thehiloeng ho microbiome ho sibolla litsela tse ncha, li-enzyme le lihlahisoa tsa tlhaho.Har'a lihlopha tse fapaneng tsa li-BGC, tsela ea RiPP e tsejoa ho kenyelletsa mefuta e mengata ea lik'hemik'hale le ts'ebetso ka lebaka la liphetoho tse fapaneng tsa morao-rao tsa peptide ea mantlha ka li-enzyme tse hōlileng42.Kahoo re ile ra khetha 'Ca.Eudoremicrobium' RiPP BGCs (Lipalo 3b le 4a-e) li thehiloe ho tšoana le BGC efe kapa efe e tsejoang (\(\bar{d}\)MIBiG le \(\bar{d}\)RefSeq ka holimo ho 0.2) .
a–c, In vitro heterologous expression le in vitro enzymatic assays ea novel (\(\bar{d}\)RefSeq = 0.29) sehlopha sa RiPP biosynthesis e khethehileng bakeng sa mefuta e tebileng ea leoatle la Ca.E. malaspinii' e ile ea lebisa tlhahisong ea lihlahisoa tsa diphosphorylated.c, liphetoho tse khetholloang ho sebelisa high-resolution (HR) MS / MS (karohano e bontšitsoeng ke b le y ions mohahong oa lik'hemik'hale) le NMR (lintlha tse atolositsoeng, setšoantšo sa 9).d, peptide ena ea phosphorylated e bonts'a inhibition e tlase ea micromolar ea mammalian neutrophil elastase, e sa fumanoeng peptide ea taolo le peptide e senyang metsi (ho tlosoa ha lik'hemik'hale ho baka ho felloa ke metsi).Teko e ile ea phetoa ka makhetlo a mararo ka liphello tse tšoanang.Mohlala, polelo e makatsang ea buka ea bobeli \(\bar{d}\)RefSeq = 0.33) sehlopha sa protheine biosynthesis e hlakisa tšebetso ea li-enzyme tse' nè tse holileng tse fetolang 46 amino acid core peptide.Masala a silafalitsoe ho latela sebaka sa phetoho e boletsoeng esale pele ke HR-MS/MS, ho ngola isotope, le tlhahlobo ea NMR (Tlhahlobo ea Tlatsetso).Dashed coloration e bontša hore phetoho e etsahala ho e 'ngoe ea masala a mabeli.Palo ke pokello ea libopeho tse ngata tse fapaneng ho bonts'a tšebetso ea li-enzyme tsohle tse holileng khubung e le 'ngoe.h, Papiso ea lintlha tsa NMR bakeng sa mokokotlo oa amide N-methylation.Liphetho tse felletseng li bonts'oa feiga.10 e nang le data e atolositsoeng.i, Phylogenetic boemo ba FkbM e hōlileng tsebong protheine cluster enzyme har'a libaka tsohle tsa FkbM tse fumanoang polokelong ea MIBiG 2.0 e senola enzyme ea lelapa lena le mosebetsi oa N-methyltransferase (Tlhaloso ea Tlatsetso).Lits'oants'o tsa moralo oa li-BGCs (a, e), libopeho tsa peptide tse tlang pele (b, f), le likarolo tsa lik'hemik'hale tsa lihlahisoa tsa tlhaho (c, g) lia bontšoa.
Tsela ea pele ea RiPP (\(\bar{d}\)MIBiG = 0.41, \(\bar{d}\)RefSeq = 0.29) e fumanoe feela mefuteng e tebileng ea leoatle "Ca.E. malaspinii” le likhoutu bakeng sa Peptide- precursor (Setšoantšo sa 4a, b).Ho enzyme ena e hōlileng tsebong, re khethile sebaka se le seng se sebetsang sa homologous ho domain dehydration ea lantipeptide synthase eo ka tloaelo e bakang phosphorylation le ho tlosoa ha 43 (Tlhaloso ea Tlatsetso).Ka hona, re bolela esale pele hore phetoho ea peptide ea pele e kenyelletsa ho felloa ke metsi ka mehato e 'meli.Leha ho le joalo, ka ho sebelisa tandem mass spectrometry (MS/MS) le nuclear magnetic resonance spectroscopy (NMR), re fumane polyphosphorylated linear peptide (Fig. 4c).Le hoja re ne re sa lebelloa, re fumane lintlha tse 'maloa tsa bopaki bo tšehetsang hore ke sehlahisoa sa ho qetela: lihlopha tse peli tse fapaneng tsa heterologous le litlhahlobo tsa dehydration in vitro, ho khetholla masala a bohlokoa a fetotsoeng sebakeng sa catalytic dehydration sa enzyme e hōlileng tsebong.tsohle li entsoe bocha ka "Ca".Genome ea E. malaspinii (lintlha tse atolositsoeng, setšoantšo sa 9 le boitsebiso bo eketsehileng) 'me, qetellong, mosebetsi oa likokoana-hloko oa sehlahisoa sa phosphorylated, empa eseng mokhoa oa lik'hemik'hale o nang le lik'hemik'hale (setšoantšo sa 4d).Ha e le hantle, re fumane hore e bonts'a ts'ebetso e tlaase ea micromolar protease inhibitory khahlanong le neutrophil elastase, e bapisoang le lihlahisoa tse ling tse amanang le tlhaho sebakeng sa mahloriso (IC50 = 14.3 μM) 44, ho sa tsotellehe hore karolo ea tikoloho e sa ntse e lokela ho hlakisoa.Ho latela liphetho tsena, re etsa tlhahiso ea ho reha tsela "phospheptin".
Nyeoe ea bobeli ke tsela e rarahaneng ea RiPP e tobileng 'Ca.Mofuta oa Eudoremicrobium (\(\bar{d}\)MIBiG = 0.46, \(\bar{d}\)RefSeq = 0.33) e ile ea boleloa esale pele hore e tla kenyelletsa lihlahisoa tsa tlhaho tsa protheine (setšoantšo sa 4e).Litsela tsena li na le thahasello e khethehileng ea biotechnology ka lebaka la tekano e lebelletsoeng le mefuta e fapaneng ea liphetoho tse sa tloaelehang tsa lik'hemik'hale tse thehiloeng ke li-enzyme tse kenyellelitsoeng ke BGCs45 tse batlang li le khutšoaane.Re fumane hore protheine ena e fapane le liprotheine tse neng li tsejoa pele ka hore ha e na NX5N motif e kholo ea polyceramides le lanthionine loop ea landornamides 46.Ho hlola mefokolo ea lipaterone tse tloaelehileng tsa heterologous, re li sebelisitse hammoho le sistimi e tloaelehileng ea Microvirgula aerodenitrificans ho hlahisa li-enzyme tse 'ne tse holileng tsa litsela (mekhoa).Re sebelisa motsoako oa MS / MS, isotope labeling, le NMR, re ile ra lemoha li-enzyme tsena tse hōlileng tsebong karolong ea 46-amino acid ea peptide (setšoantšo sa 4f, g, data e atolositsoeng, litšoantšo tsa 10-12 le boitsebiso bo eketsehileng).Har'a li-enzyme tse hōlileng tsebong, re ile ra bontša ponahalo ea pele ea setho sa lelapa sa FkbM O-methyltransferase 47 tseleng ea RiPP 'me ka tšohanyetso re fumane hore enzyme ena e hōlileng tsebong e hlahisa mokokotlo oa N-methylation (setšoantšo sa 4h, i le boitsebiso bo eketsehileng).Le hoja phetoho ena e tsejoa ka lihlahisoa tsa tlhaho tsa NRP48, N-methylation ea enzymatic ea li-amide bonds ke karabelo e rarahaneng empa ea biotechnologically49 eo ho fihlela joale e bileng e thahasellisang ho lelapa la RiPP la borosine.Lintlha tse khethehileng 50,51.Ho tsebahatsoa ha ts'ebetso ena malapeng a mang a li-enzyme le RiPP ho ka bula lits'ebetso tse ncha le ho atolosa mefuta e fapaneng ea ts'ebetso ea liprotheine tsa 52 le mefuta ea tsona ea lik'hemik'hale.Ho latela liphetoho tse khethiloeng le bolelele bo sa tloaelehang ba sebopeho sa sehlahisoa se reriloeng, re fana ka lebitso la tsela "pythonamide".
Ho sibolloa ha enzymology e neng e sa lebelloa lelapeng le nang le ts'ebetso ea li-enzyme e bonts'a ts'episo ea genomics ea tikoloho bakeng sa litšibollo tse ncha, hape e bonts'a matla a fokolang a ts'ebetso e thehiloeng ho tatellano ea homology feela.Kahoo, hammoho le litlaleho tsa non-canonical bioactive polyphosphorylated RiPPs, liphetho tsa rona li bonts'a boleng bo tebileng ba lisebelisoa empa bo bohlokoa boitekong ba maiketsetso ba baeloji ho senola ka botlalo matlotlo a sebetsang, mefuta-futa le meaho e sa tloaelehang ea metsoako ea biochemical.
Mona re bonts'a mefuta e mengata ea bokhoni ba biosynthetic bo kentsoeng ke likokoana-hloko le moelelo oa tsona oa genomic ho microbiome ea leoatleng, ho thusa lipatlisiso tsa kamoso ka ho etsa hore mohlodi o fumanehe ho setjhaba sa saense (https://microbiomics.io/ocean/).Re fumane hore boholo ba lintho tse ncha tsa phylogenetic le tse sebetsang li ka fumanoa feela ka ho tsosolosa MAGs le SAGs, haholo-holo metseng ea likokoana-hloko tse sa sebelisoeng hantle tse ka tataisang boiteko ba nakong e tlang ba ho hlahloba likokoana-hloko.Leha re tla tsepamisa maikutlo mona ho 'Ca.Eudormicrobiaceae” joalo ka leloko haholo-holo le nang le “talente” ea biosynthetically, bongata ba li-BGC tse boletsoeng esale pele ka har'a microbiota e sa sibolloang e ka 'nang ea kenyelletsa li-enzymologies tse neng li sa hlalosoa pele tse hlahisang metsoako e nang le liketso tsa bohlokoa tsa tikoloho le/kapa tsa biotechnological.
Li-datasets tsa Metagenomic tse tsoang lithutong tse kholo tsa maoatle le letoto la nako tse nang le tatellano e tebileng e tebileng li kenyellelitsoe ho eketsa ts'ireletso ea lichaba tsa lefats'e tsa likokoana-hloko tsa maoatleng a leoatle, mekhahlelo e tebileng le ha nako e ntse e feta.Lintlha tsena (Supplementary Table 1 le Figure 1) li kenyelletsa metagenomics ho tloha lisampole tse bokeletsoeng maoatleng a Tara (viral enriched, n=190; prokaryotic enriched, n=180)12,22 le BioGEOTRACES expedition (n=480).Hawaiian Oceanic Time Series (HOT, n = 68), Bermuda-Atlantic Time Series (BATS, n = 62)21 le Malaspina Expedition (n = 58)23.Ho bala ka tatellano ho tsoa likhechanang tsohle tsa metagenomic ho ile ha tlhotloa bakeng sa boleng ho sebelisa BBMap (v.38.71) ka ho tlosa li-adapter tsa tatellano ho tsoa ho bala, ho tlosa limmapa tse hlophisitsoeng ho latela tatellano ea taolo ea boleng (PhiX genomes), le ho sebelisa trimq = 14, maq = 20 e lahla boleng bo bobe ba ho bala, maxns = 0 le minlength = 45. Litlhahlobo tse latelang li ile tsa tsamaisoa kapa tsa kopanngoa le QC e baloang haeba ho boletsoe (bbmerge.sh minoverlap=16).Ho baloa ha QC ho ne ho tloaelehile (bbnorm.sh target = 40, minddepth = 0) pele ho haha ho sebelisa metaSPAdes (v.3.11.1 kapa v.3.12 ha ho hlokahala)53.Li-scaffold contigs (tseo ka morao ho moo li bitsoang li-scaffolds) li ile tsa qetella li tlhotliloeng ka bolelele (≥1 kb).
Mehlala ea 1038 ea metagenomic e ile ea aroloa ka lihlopha, 'me bakeng sa sehlopha ka seng sa lisampole, litekanyetso tsa taolo ea boleng ba metagenomic tsa lisampole kaofela li ne li bapisoa le masakaneng a sampole ka 'ngoe, e leng se ileng sa fella ka hore palo e latelang ea lihlopha tse peli tse nang le lihlopha tse peli li balehe: Likoekoana tsa Tara Marine - E matlafalitsoe. (190 × 190), Prokaryotes e Matlafalitsoeng (180 × 180), BioGEOTRACES, HOT le BATS (610 × 610) le Malaspina (58 × 58).Ho etsa 'mapa ho ile ha etsoa ho sebelisoa Burrows-Wheeler-Aligner (BWA) (v.0.7.17-r1188)54 e lumellang hore ho baloe ho tsamaisana le libaka tsa bobeli (ho sebelisa -a folakha).Litlhophiso li ne li tlhotliloeng hore bonyane li be bolelele ba metheo ea 45, li na le ≥97% boitsebiso, le nako ea ho bala ≥80%.Lifaele tsa BAM tse hlahisitsoeng li ile tsa sebetsoa ho sebelisoa jgi_summarize_bam_contig_depths script bakeng sa MetaBAT2 (v.2.12.1)55 ho fana ka khaso ea intra- le lipakeng tsa lisampole bakeng sa sehlopha ka seng.Qetellong, li-brackets li ile tsa hlophisoa ho eketsa kutloisiso ka ho tsamaisa MetaBAT2 ka bomong ho mehlala eohle e nang le -minContig 2000 le -maxEdges 500. Re sebelisa MetaBAT2 ho e-na le ensemble boxer hobane e bontšitsoe litekong tse ikemetseng ho ba sebapali se le seng se sebetsang ka ho fetisisa.le makhetlo a 10 ho isa ho a 50 ka potlako ho feta li-boxers tse ling tse sebelisoang ka tloaelo57.Ho lekola phello ea likhokahano tsa bongata, sampole e khethiloeng ka mokhoa o sa reroang ea metagenomics (10 bakeng sa e 'ngoe le e' ngoe ea li-database tse peli tsa Tara Ocean, 10 bakeng sa BioGEOTRACES, 5 bakeng sa letoto le leng le le leng la nako, le 5 bakeng sa Malaspina) e boetse e sebelisa lisampole feela.Mehlala ea ka hare e hlophisitsoe ka lihlopha ho fumana boitsebiso ba litaba.(Tlhahisoleseling e 'Ngoe).
Liphatsa tsa lefutso tsa tlatsetso (tsa kantle) li kenyelelitsoe tlhahlobisong e latelang, e leng MAGs a 830 a khethiloeng ka letsoho ho tsoa setsing sa data sa Tara Oceans26, 5287 SAGs ho tsoa ho dataset ea GORG20, le data ho tsoa polokelong ea MAR (MarDB v. 4) ho tsoa ho 1707 REFs e ka thoko le 682 SAGs) 27. Bakeng sa datha tsa MarDB, liphatsa tsa lefutso li khethoa ho ipapisitsoe le metadata e fumanehang haeba mohlala oa mofuta o lumellana le polelo e tloaelehileng e latelang: '[S|s]ingle.?[C|c]ell|[C|c]ulture| [I|i] ka thoko'.
Boleng ba setshelo se seng le se seng sa metagenomic le liphatsa tsa lefutso tsa kantle bo ile ba hlahlojoa ho sebelisoa CheckM (v.1.0.13) le Anvi'o's Lineage Workflow (v.5.5.0)58,59.Haeba CheckM kapa Anvi'o e tlaleha ≥50% botlalo / botlalo le ≤10% tšilafalo / ho fokotseha, joale boloka lisele tsa metagenomic le liphatsa tsa lefutso tsa ka ntle bakeng sa tlhahlobo ea morao-rao.Joale lintlha tsena li ne li kopanngoa hore li be le botlalo (mcpl) le ho bolela tšilafalo (mctn) ho arola boleng ba genome ho latela mekhoa ea sechaba60 ka tsela e latelang: boleng bo phahameng: mcpl ≥ 90% le mctn ≤ 5%;boleng bo botle: mcpl ≥ 70%, mctn ≤ 10%, boleng bo bohareng: mcpl ≥ 50% le mctn ≤ 10%, boleng bo botle: mcpl ≤ 90% kapa mctn ≥ 10%.Joale liphatsa tsa lefutso tse tlhotliloeng li ile tsa amahanngoa le lintlha tsa boleng (Q le Q') ka tsela e latelang: Q = mcpl – 5 x mctn Q' = mcpl – 5 x mctn + mctn x (phapang ea mathata)/100 + 0.5 x log[N50] .(e kentsoeng ho dRep61).
Ho lumella tlhahlobo ea papiso lipakeng tsa mehloli e fapaneng ea data le mefuta ea genome (MAG, SAG le REF), liphatsa tsa lefutso tse 34,799 li ile tsa tlosoa ho ipapisitsoe le genome-wide average nucleotide identity (ANI) ho sebelisa dRep (v.2.5.4).Pheta)61 e nang le 95% thresholds28,62 (-comp 0 -con 1000 -sa 0.95 -nc 0.2) le liphatsa tsa lefutso tsa kopi e le 'ngoe tse sebelisang SpecI63 tse fanang ka lihlopha tsa genome maemong a mefuta.Ho khethiloe genome e emelang sehlopha ka seng sa dRep ho latela lintlha tsa boleng bo holimo (Q') tse hlalositsoeng ka holimo, tse neng li nkuoa li emela mofuta.
Ho lekola lebelo la 'mapa, BWA (v.0.7.17-r1188, -a) e ile ea sebelisoa ho etsa 'mapa lihlopha tsohle tse 1038 tsa lipalo tsa metagenomic ka liphatsa tsa lefutso tse 34,799 tse fumanehang ho OMD.Litemana tse laoloang ke boleng li ile tsa etsoa 'mapa ka mokhoa o le mong 'me li-alignments tse hlahisitsoeng li ile tsa hloekisoa ho boloka li-alignments ≥45 bp ka bolelele.le boitsebiso ≥95%.Karolelano ea ponts'o bakeng sa sampole e 'ngoe le e 'ngoe ke liperesente tsa lipalo tse setseng ka mor'a ho sefa li arotsoe ka palo eohle ea lipalo tsa taolo ea boleng.Ho sebelisa mokhoa o ts'oanang, e 'ngoe le e' ngoe ea metagenomes ea 1038 e ile ea fokotsoa ho kenya limilione tse 5 (lintlha tse atolositsoeng, setšoantšo sa 1c) 'me li bapisoa le GORG SAG ho OMD le ho GEM16 eohle.Palo ea li-MAG tse fumanoeng metsing a leoatle ka har'a lethathamo la GEM16 li ile tsa khethoa ke lipotso tsa bohlokoa tsa mehloli ea metagenomic, ho khetha lisampole tsa metsi a leoatle (mohlala, ho fapana le maoatle a leoatle).Ka ho khetheha, re khetha "aquatic" e le "ecosystem_category", "marine" as "ecosystem_type", le sefa "habitat" joalo ka "deep ocean", "marine", "maritime oceanic", "pelagic marine", "marine water" , "Leoatle", "Metsi a Leoatle", "Metsi a Leoatleng a ka holim'a metsi", "Metsi a Leoatleng a ka holim'a metsi".Sena se entse hore 5903 MAGs (734 boleng bo phahameng) e abuoe ho feta 1823 OTUs (maikutlo mona).
Liphatsa tsa lefutso tsa prokaryotic li ne li hlalositsoe ka mokhoa oa taxonomic ho sebelisa GTDB-Tk (v.1.0.2)64 e nang le li-parameter tsa kamehla tse lebisitsoeng ho GTDB r89 version 13. Anvi'o e ne e sebelisetsoa ho khetholla liphatsa tsa lefutso tsa eukaryotic tse thehiloeng ho bolela esale pele le ho hopola ≥50% le redundancy ≤ 10%.Tlhaloso ea taxonomic ea mofuta o hlalosoa e le e 'ngoe ea liphatsa tsa lefutso tsa eona.Ntle le li-eukaryotes (148 MAG), genome e 'ngoe le e' ngoe e ile ea qala ho hlalosoa ka mokhoa o sebetsang ho sebelisa prokka (v.1.14.5) 65, ho reha liphatsa tsa lefutso tse feletseng, ho hlalosa "archaea" kapa "bacteria" parameters ha ho hlokahala, eo hape e tlalehoang bakeng sa bao e seng ba- liphatsa tsa lefutso.le libaka tsa CRISPR, har'a likarolo tse ling tsa genomic.Hlalosa liphatsa tsa lefutso tse boletsoeng esale pele ka ho tsebahatsa liphatsa tsa lefutso tsa kopi e le 'ngoe (uscMG) u sebelisa fetchMG (v.1.2)66, abela lihlopha tsa ortholog le potso u sebelisa emapper (v.2.0.1)67 e thehiloeng ho eggNOG (v.5.0)68.Database ea KEGG (e hatisitsoeng Hlakola 10, 2020) 69. Mohato oa ho qetela o entsoe ka ho bapisa liprotheine ho database ea KEGG ho sebelisoa DIAMOND (v.0.9.30)70 e nang le potso le sehlooho sa ≥70%.Liphetho li ile tsa tlhotloa hape ho latela NCBI Prokaryotic Genome Annotation Pipeline71 ho ipapisitsoe le bitrate ≥ 50% ea boholo bo lebelletsoeng ba bitrate (link ka boeona).Tatelano ea liphatsa tsa lefutso e ile ea boela ea sebelisoa e le ho kenya letsoho ho khetholla BGC ho genome ho sebelisa antiSMASH (v.5.1.0)72 e nang le li-parameter tsa kamehla le ho phatloha ho fapaneng ha lihlopha.Liphatsa tsa lefutso tsohle le litlhaloso li hlophisitsoe ho OMD hammoho le metadata ea maemo e fumanehang webosaeteng (https://microbiomics.io/ocean/).
Ho ts'oana le mekhoa e hlalositsoeng pele12,22 re sebelisitse CD-HIT (v.4.8.1) ho kopanya > limilione tse 56.6 tsa liphatsa tsa lefutso tsa protheine tse tsoang ho likokoana-hloko tsa baktheria le tsa khale ho tloha ho OMD ho ea ho 95% ea boitsebiso le liphatsa tse khutšoanyane (90%) > Lihlopha tsa liphatsa tsa lefutso tse limilione tse 17.7.Tatelano e telele ka ho fetisisa e khethiloe e le leloko le emelang sehlopha ka seng sa lefutso.Joale li-metagenomes tse 1038 li ile tsa bapisoa le litho tsa sehlopha sa> BWA (-a) tse limilione tse 17.7 'me lifaele tsa BAM tse hlahisitsoeng li ile tsa tlhotloa ho boloka li-alignments feela ka ≥95% ea boitsebiso le ≥45 li-alignments tsa motheo.Bolelele bo tloaelehileng ba liphatsa tsa lefutso bo ile ba baloa ka ho qala ho baloa ho tloha ho tatellano e ikhethang e ntle ka ho fetisisa, 'me ka mor'a moo, bakeng sa ho kenya 'mapa o sa utloahaleng, ho eketsa lipalo-palo ho liphatsa tsa lefutso tse tsamaellanang le palo ea tsona tse kentsoeng ka mokhoa o ikhethileng.
Liphatsa tsa lefutso tse tsoang ho OMD e atolositsoeng (e nang le li-MAG tse eketsehileng tse tsoang "Ca. Eudormicrobiaceae", bona ka tlase) li kenyelelitsoe ho mOTUs74 metagenomic analysis tool database (v.2.5.1) ho theha boitsebiso bo atolositsoeng ba mOTU.Ke liphatsa tsa lefutso tse tšeletseng feela tsa kopi e le 'ngoe (li-genome tse 23,528) tse pholohileng ho li-uscMG tse leshome.Katoloso ea polokelo ea boitsebiso e ile ea fella ka lihlopha tse eketsehileng tse 4,494 boemong ba mefuta.Li-metagenomes tsa 1038 li ile tsa hlahlojoa ho sebelisoa li-parameter tsa moOTU tse sa feleng (v.2).Kakaretso ea liphatsa tsa lefutso tse 989 tse ka har'a lihlopha tse 644 tsa mOTU (95% REF, 5% SAG le 99.9% tsa MarDB) ha lia fumanoa ke profil ea moOTU.Sena se bonts'a mehloli e fapaneng ea tlatsetso ea ho itšehla thajana ea liphatsa tsa lefutso tsa MarDB (boholo ba liphatsa tsa lefutso tse sa lemohuoang li amahanngoa le lintho tse phelang tse arohaneng le sediments, mabotho a leoatle, joalo-joalo).Ho tswelapele ho tsepamisitse maikutlo tikolohong ya mawatle a bulehileng phuputsong ena, re ba qheletse ka thoko tshekatshekong ya tlase ntle le haeba ba ile ba fumanwa kapa ba kenyeletswa polokelongtshebetsong e atolositsweng ya mOTU e thehilweng phuputsong ena.
Li-BGC tsohle tse tsoang ho MAG, SAG le REF ho OMD (bona ka holimo) li ne li kopantsoe le li-BGC tse khetholloang ho li-scaffolds tsohle tsa metagenomic (antiSMASH v.5.0, li-parameter tsa kamehla) 'me li khetholloa ka ho sebelisa BiG-SLICE (v.1.1) (PFAM domain)75.Ho ipapisitsoe le likarolo tsena, re ile ra khomphutha libaka tsohle tsa cosine lipakeng tsa BGC mme ra li arola (lihokelo tse bolelang) ho GCF le GCC re sebelisa litekanyo tsa sebaka sa 0.2 le 0.8 ka ho latellana.Merero ena ke tlhophiso ea menyako e neng e sebelisoa pele ho sebelisoa Euclidean distance75 mmoho le cosine distance, e fokotsang phoso e 'ngoe ea leano la mantlha la ho kopanya la BiG-SLICE (Tlhahlobo ea Tlatsetso).
Joale li-BGC li ile tsa tlhotloa ho boloka feela ≥5 kb e kentsoeng scaffolds ho fokotsa kotsi ea ho arohana joalo ka ha ho hlalositsoe pele16 le ho qhelela ka thoko MarDB REFs le SAGs tse sa fumanoeng ho 1038 metagenomes (bona ka holimo).Sena se ile sa fella ka hore kakaretso ea li-BGC tse 39,055 li kenngoe ke genome ea OMD, ka 14,106 e eketsehileng e khetholloang ka likhechana tsa metagenomic (ke hore ha e kopane le MAGs).Li-BGC tsena tsa "metagenomic" li ne li sebelisetsoa ho hakanya karolo ea matla a maoatleng a biosynthesis a sa kang a kenngoa polokelong ea litaba (Tlhahlobo ea Tlatsetso).BGC e 'ngoe le e' ngoe e ne e khetholloa ka mokhoa o sebetsang ho latela mefuta ea lihlahisoa tse boletsoeng esale pele tse hlalosoang ke li-anti-SMASH kapa likarolo tse ngata tsa lihlahisoa tse hlalositsoeng ho BiG-SCAPE76.Ho thibela leeme la lisampole ho quantification (taxonomic le sebopeho sa ts'ebetso sa GCC/GCF, sebaka sa GCF le GCC ho li-database tsa litšupiso, le bongata ba metagenomic ea GCF), ka ho boloka BGC e telele ka ho fetesisa ka GCF bakeng sa mofuta o mong le o mong, 39,055 BGCs e ile ea etsoa hape, ka ho fella ka kakaretso ea 17,689 BGC.
Bocha ba GCC le GCF bo ile ba hlahlobjoa ho ipapisitsoe le sebaka se pakeng tsa database e baloang (RefSeq database ho BiG-FAM)29 le tse netefalitsoeng ka liteko (MIBIG 2.0)30 BGC.Bakeng sa e 'ngoe le e 'ngoe ea baemeli ba 17,689 ba BGC, re khethile sebaka se senyenyane sa cosine ho ea sebakeng sa polokelo ea boitsebiso.Mehole ena e fokolang e ntan'o lekanngoa (e bolelang) ho latela GCF kapa GCC, ka moo ho loketseng.GCF e nkoa e le ncha haeba sebaka sa polokelo ea boitsebiso se le seholo ho feta 0.2, e lumellanang le karohano e ntle pakeng tsa (karolelano) ea GCF le referense.Bakeng sa GCC, re khetha 0.4, e leng habeli moeli o hlalosoang ke GCF, ho koala likamano tsa nako e telele le lihokelo.
Bongata ba metagenomic ea BGC bo hakantsoe e le bongata bo boholo ba liphatsa tsa lefutso tsa eona tsa biosynthetic (joalo ka ha ho laetsoe ke anti-SMASH) tse fumanehang ho tsoa ho liprofaele tsa boemo ba lefutso.Bongata ba metagenomic ba GCF e 'ngoe le e 'ngoe kapa GCC bo ile ba baloa e le kakaretso ea li-BGC tsa baemeli (ho tse 17,689).Limmapa tsena tse ngata ka mor'a moo li ile tsa tloaeleha bakeng sa ho hlophisoa ha cellular ho sebelisa palo ea sampuli ea mOTU, e neng e boetse e ikarabella bakeng sa boiteko ba ho latellana (data e atolositsoeng, setšoantšo sa 1d).Ho ata ha GCF kapa GCC ho ile ha baloa e le peresente ea lisampole tse nang le bongata> 0.
Sebaka sa Euclidean lipakeng tsa lisampole se baloa ho tsoa ho profil e tloaelehileng ea GCF.Libaka tsena li ile tsa fokotsoa ka boholo ho sebelisoa UMAP77, 'me sephetho se ile sa sebelisoa bakeng sa lihlopha tse sa laoleheng tse thehiloeng ho tšubuhlellano ho sebelisa HDBSCAN78.Palo e nepahetseng ea bonyane ba lintlha bakeng sa sehlopha (ka hona palo ea lihlopha) e sebelisoang ke HDBSCAN e khethoa ka ho eketsa monyetla oa ho ba setho sa sehlopha.Lihlopha tse khethiloeng (le sampole e leka-lekaneng ea lihlopha tsena ho ikarabella bakeng sa leeme tlhahlobong ea permutational multivariate of variance (PERMANOVA)) li ile tsa lekoa bakeng sa bohlokoa khahlanong le libaka tse sa fokotseheng tsa Euclidean tse sebelisang PERMANOVA.Kakaretso ea boholo ba liphatsa tsa lefutso tsa lisampole bo ile ba baloa ho ipapisitsoe le bongata bo lekanyelitsoeng ba mOTU le boholo bo hakantsoeng ba genome ea litho tsa genome.Haholo-holo, karolelano ea boholo ba genome ea moTU ka 'ngoe e ne e hakanngoa e le karolelano ea boholo ba genome ea litho tsa eona e lokisoang bakeng sa ho phethahala (ka mor'a ho sefa) (mohlala, 75% genome e feletseng e nang le bolelele ba 3 Mb e na le boholo bo fetotsoeng ba 4 Mb).bakeng sa liphatsa tsa lefutso tse mahareng tse nang le botšepehi ≥70%.Kakaretso ea boholo ba genome bakeng sa sampole ka 'ngoe e ile ea baloa e le kakaretso ea boholo ba genome ea mOTU e lekantsoeng ka bongata bo itseng.
Sehlopha se tlhotliloeng sa li-BGC tse kentsoeng liphatsa tsa lefutso ho OMD li bontšoa lifateng tsa GTDB tsa baktheria le tsa khale (ka ≥5 kb meralo, ho sa kenyeletsoe REF le SAG MarDB tse sa fumanoeng ho 1038 metagenomes, bona ka holimo) le likarolo tsa tsona tsa lihlahisoa tse boletsoeng esale pele ho latela phylogenetic. boemo ba genome (sheba ka holimo).Re ile ra qala ka ho fokotsa lintlha ka mefuta, re sebelisa genome e nang le li-BGC tse ngata ka ho fetisisa mefuteng eo e le moemeli.Bakeng sa pono, baemeli ba ile ba aroloa hape ka lihlopha tsa lifate, 'me hape, bakeng sa clade e' ngoe le e 'ngoe ea lisele, genome e nang le palo e kholo ka ho fetisisa ea BGC e khethiloe e le moemeli.Mefuta e matlafalitsoeng ea BGC (bonyane genome e le 'ngoe e nang le > 15 BGCs) e ile ea hlahlobisisoa hape ka ho bala Shannon Diversity Index bakeng sa mefuta ea lihlahisoa tse kentsoeng ho li-BGC tseo.Haeba mefuta eohle ea lihlahisoa tse boletsoeng esale pele e tšoana, mefuta e nyalisitsoeng ea lik'hemik'hale le li-BGC tse ling tse rarahaneng (joalokaha ho boletsoe esale pele ke anti-SMAH) li nkuoa e le tsa mofuta o le mong oa sehlahisoa, ho sa tsotelehe tatellano ea tsona sehlopheng (mohlala, protheine-bacteriocin le bacteriocin-proteoprotein fusion. mmele).lebasetere).
DNA e setseng (e hakantsoeng ho ba 6 ng) ho tsoa ho sampole ea Malaspina MP1648, e tsamaellanang le sampole ea biological SAMN05421555 mme e tsamaellana le Illumina SRR3962772 metagenomic read set for short read, processed ho latela PacBio sequencing protocol with ultra-low input to use DNA samplication GSMBio kit (100-980-000) le SMRTbell Express 2.0 tlhophiso ea thempleite kit (100-938-900).Ka bokhutšoanyane, DNA e setseng e ile ea khaoloa, ea lokisoa le ho hloekisoa (ProNex beads) ho sebelisoa Covaris (g-TUBE, 52104).DNA e hloekisitsoeng joale e tlas'a tokisetso ea laebrari, ho hōlisa, ho hloekisa (ProNex beads) le khetho ea boholo (> 6 kb, Blue Pippin) pele ho mohato oa ho qetela oa tlhoekiso (ProNex beads) le ho latellana sethaleng sa Sequel II.
Ho tsosolosoa ha tse peli tsa pele ca.Bakeng sa MAG Eremiobacterota, re khethile li-ANI tse ling tse tšeletseng> 99% (tsena li kenyelelitsoe ho Setšoantšo sa 3), tseo qalong li ileng tsa tlhotloa ho latela lintlha tse silafatsang (hamorao li ile tsa tsejoa e le liphatsa tsa lefutso, bona ka tlase).Hape re fumane terei e ngotsoeng "Ca".Eremiobacterota” ho tsoa lithutong tse fapaneng23 mme a li sebelisa hammoho le li-MAG tse robeli ho tsoa thutong ea rona e le sesupo sa lipalo tsa metagenomic ho tsoa ho lisampole tse 633 tsa eukaryotic tse ntlafalitsoeng (> 0.8 µm) tse sebelisang BWA (v.0.7.17) Ref -r1188, - folakha) bakeng sa mehlala e theohileng 'mapa (e baloa limilione tse 5).Ho ipapisitsoe le limmapa tse ikhethileng tsa ntlafatso (e tlhotliloeng ka boitsebahatso ba 95% le 80% ea ho bala), li-metagenomes tse 10 (karolo e lebelletsoeng ≥5 ×) li khethiloe bakeng sa kopano le metagenome e eketsehileng ea 49 (kabelo e lebelletsoeng ≥1 ×) bakeng sa khokahano ea litaba.Ho sebelisoa liparamente tse tšoanang le tse kaholimo, lisampole tsena li ile tsa tlanngoe 'me tsa eketsoa tse 10 tsa 'Ca'.MAG Eremiobacterota e tsosolositsoe.Li-MAGs tsena tsa 16 (ho sa bale tse peli tse seng li ntse li le ka har'a database) li tlisa palo eohle ea liphatsa tsa lefutso ho OMD e atolositsoeng ho 34,815.Li-MAG li abeloa maemo a taxonomic ho ipapisitse le ho tšoana ha bona le boemo ba bona ho GTDB.Li-MAG tsa 18 li ile tsa hlakoloa ka ho sebelisa dRep ho mefuta ea 5 (intraspecific ANI> 99%) le 3 genera (intrageneric ANI 85% ho 94%) ka har'a lelapa le le leng79.Baemeli ba mefuta ba ile ba khethoa ka letsoho ho latela botšepehi, tšilafalo, le N50.Lenane la mabitso le sisintsweng le fanoe ho Boitsebiso ba Tlatsetso.
Lekola botšepehi le tšilafalo ea 'Ca.MAG Eremiobacterota, re ile ra lekola boteng ba uscMG, hammoho le liphatsa tsa lefutso tse sebelisoang ke CheckM le Anvi'o tsa leloko le li-domain-specific single-copy marker marker sets.Ho tsebahatsoa ha likopi tse peli ho tse 40 tsa uscMG ho netefalitsoe ke kaho bocha ea phylogenetic (sheba ka tlase) ho thibela tšilafalo efe kapa efe e ka bang teng (sena se tsamaisana le 5% ho ipapisitse le liphatsa tsa lefutso tse 40).Thuto e eketsehileng ea baemeli ba bahlano ba MAGs 'Ca.Boemo bo tlaase ba litšila ho liphatsa tsa lefutso tsena tse tsosolositsoeng bo ile ba tiisoa bakeng sa mefuta ea Eremiobacterota e sebelisang sebopeho se sebetsanang sa Anvi'o se thehiloeng ho bongata le ho latellana ha lihlopha tse amanang le tsona (Tlhaloso ea Tlatsetso)59.
Bakeng sa tlhahlobo ea phylogenomic, re khethile li-MAG tse hlano tsa baemeli "Ca".Eudormicrobiaceae", mefuta eohle ea "Ca.Genome ea Eremiobacterota le litho tsa phyla tse ling (ho akarelletsa le UBP13, Armatimonadota, Patescibacteria, Dormibacterota, Chloroflexota, Cyanobacteria, Actinobacteria le Planctomycetota) e fumaneha ho tloha GTDB (r89)13.Liphatsa tsa lefutso tsena kaofela li hlalositsoe joalo ka ha ho hlalositsoe pele bakeng sa ho ntšoa ha mofuta oa kopi e le 'ngoe le tlhaloso ea BGC.Liphatsa tsa lefutso tsa GTDB li ile tsa bolokoa ho latela maemo a ka holimo a botšepehi le tšilafalo.Tlhahlobo ea Phylogenetic e entsoe ka mokhoa oa ho sebetsa oa Anvi'o Phylogenetics59.Sefate se hahiloe ho sebelisoa IQTREE (v.2.0.3) (likhetho tsa kamehla le -bb 1000)80 ka ho tsamaisana ha liprotheine tsa 39 tandem ribosomal tse khetholloang ke Anvi'o (MUSCLE, v.3.8.1551)81.Maemo a hae a fokotsehile.ho koahela bonyane 50% ea genome82 le Planctomycecota e ile ea sebelisoa e le sehlopha se ka thōko se ipapisitseng le topology ea lifate tsa GTDB.Sefate se le seng sa li-uscMG tse 40 se hahiloe ho sebelisoa lisebelisoa le li-parameter tse tšoanang.
Re sebelisitse Traitar (v.1.1.2) ka li-parameter tsa kamehla (phenotype, ho tloha li-nucleotides)83 ho bolela esale pele litšobotsi tse tloaelehileng tsa likokoana-hloko.Re ile ra lekola mokhoa oa bophelo oa bosholu o ka bang teng o ipapisitse le index84 e neng e entsoe pele e ipapisitseng le litaba tsa mofuta oa protheine-coding genome.Ka ho khetheha, re sebelisa DIAMOND ho bapisa liprotheine tsa genome khahlanong le database ea OrthoMCL (v.4) 85 re sebelisa likhetho -more-sensive -id 25 -query-cover 70 -subject-cover 70 -top 20 LE bala liphatsa tsa lefutso tse tsamaellanang le liphatsa tsa lefutso tsa libatana le tse sa jeng.Lenane ke phapang lipakeng tsa palo ea matshwao a tlatlapuoeng le ao e seng ahlatsi.E le taolo e eketsehileng, re boetse re hlahlobile "Ca" genome.Ntho ea Entotheonella TSY118 e ipapisitse le kamano ea eona le Ca.Eudoremicrobium (boholo bo boholo ba genome le bokhoni ba biosynthetic).Ka mor'a moo, re ile ra leka likhokahano tse ka bang teng lipakeng tsa liphatsa tsa lefutso tsa liphoofolo tse jang liphoofolo tse jang liphoofolo le tse sa jeng le matla a biosynthetic a Ca.Eudormicrobiaceae” mme e fumane hore ha ho na mofuta o fetang o le mong (ho tsoa mofuteng ofe kapa ofe oa mofuta oa lets'oao, ke hore mofuta oa predator/non-predator) o kopanang le BGC, ho fana ka maikutlo a hore BGC ha e ferekanye mats'oao a tlhaselo.Tlhaloso e eketsehileng ea genomic ea replicons scrambled e ile ea etsoa ho sebelisoa TXSSCAN (v.1.0.2) ho hlahloba ka ho khetheha tsamaiso ea secretion, pili, le flagella86.
Baemeli ba bahlano ba 'Ca' ba ile ba etsoa 'mapa ka ho etsa limmapa tse 623 tsa metatranscriptomes tse tsoang likarolong tse matlafatsang tsa prokaryotic le eukaryotic tsa Tara oceans22,40,87 (ho sebelisa BWA, v.0.7.17-r1188, -a folakha).Eudormicrobiaceae genome.Lifaele tsa BAM li ile tsa sebetsoa ka FeatureCounts (v.2.0.1)88 ka mor'a hore 80% e baloe le 95% ea ho sefa boitsebiso (e nang le likarolo tsa likhethoCounts -primary -O -fraction -t CDS,tRNA -F GTF -g ID -p ) E baloa palo ea tse kentsoeng ka jini.Limmapa tse hlahisitsoeng li ne li tloaeleha bakeng sa bolelele ba liphatsa tsa lefutso le bongata ba liphatsa tsa lefutso tsa mOTU (bolelele bo tloaelehileng bo tloaelehileng ba palo ea ho kenya liphatsa tsa lefutso tse nang le palo> ho fapana ho tloha ho sampuli ho ea ho sampuli nakong ea tatellano.Likarolo tse joalo li lumella tlhahlobo ea papiso, ho fokotsa mathata a sebopeho ha ho sebelisoa lintlha tse ngata tse amanang.Ke mehlala feela e nang le > 5 ea liphatsa tsa lefutso tsa 10 tsa mOTU tse ileng tsa nkoa bakeng sa tlhahlobo e eketsehileng ho lumella karolo e kholo e lekaneng ea genome ho fumanoa.
Boemo bo tloaelehileng ba transcriptome ea 'Ca.E. taraoceanii e ile ea fokotsoa ho sebelisoa UMAP 'me sephetho se ile sa sebelisoa bakeng sa lihlopha tse sa laoleheng ho sebelisoa HDBSCAN (bona ka holimo) ho fumana boemo ba polelo.PERMANOVA e leka bohlokoa ba liphapang pakeng tsa lihlopha tse khethiloeng sebakeng sa pele (se sa fokotsoang) sa sebaka.Polelo e fapaneng pakeng tsa maemo ana e ile ea lekoa ho pholletsa le genome (bona ka holimo) le litsela tsa 201 KEGG li ile tsa khetholloa ka lihlopha tse 6 tse sebetsang, e leng: BGC, tsamaiso ea secretion le liphatsa tsa lefutso tse tsoang ho TXSSCAN, li-enzyme tse senyang (protease le peptidase), le libatana le tse seng liphatsa tsa lefutso.li-index tsa liphoofolo tse jang liphoofolo.Bakeng sa sampole e 'ngoe le e 'ngoe, re balile polelo e tloaelehileng e tloaelehileng bakeng sa sehlopha ka seng (hlokomela hore polelo ea BGC ka boeona e baloa e le polelo ea bohareng ea liphatsa tsa lefutso tsa biosynthetic bakeng sa BGC eo) 'me e lekoa hore na e bohlokoa hakae ho pholletsa le linaha (teko ea Kruskal-Wallis e lokiselitsoe FDR).
Liphatsa tsa lefutso tsa maiketsetso li ile tsa rekoa ho GenScript le li-primer tsa PCR li ile tsa rekoa ho Microsynth.Phusion polymerase e tsoang ho Thermo Fisher Scientific e ne e sebelisetsoa ho holisa DNA.Li-plasmids tsa NucleoSpin, gel ea NucleoSpin le lisebelisoa tsa tlhoekiso ea PCR tse tsoang Macherey-Nagel li ne li sebelisetsoa tlhoekiso ea DNA.Li-enzyme tsa thibelo le T4 DNA ligase li ile tsa rekoa ho tsoa New England Biolabs.Lik'hemik'hale tse ling ntle le isopropyl-β-d-1-thiogalactopyranoside (IPTG) (Biosynth) le 1,4-dithiothreitol (DTT, AppliChem) li ile tsa rekoa ho Sigma-Aldrich 'me tsa sebelisoa ntle le ho hloekisoa ho eketsehileng.Lithibela-mafu chloramphenicol (Cm), spectinomycin dihydrochloride (Sm), ampicillin (Amp), gentamicin (Gt), le carbenicillin (Cbn) li ile tsa rekoa ho AppliChem.Likarolo tsa mecha ea litaba ea Bacto Tryptone le Bacto Yeast Extract li ile tsa rekoa ho BD Biosciences.Trypsin bakeng sa tatellano e ile ea rekoa ho Promega.
Tatelano ea liphatsa tsa lefutso e ntšitsoe ho anti-SMASH e boletsoeng esale pele ea BGC 75.1.E. malaspinii (Boitsebiso ba tlatsetso).
Liphatsa tsa lefutso embA (locus, MALA_SAMN05422137_METAG-framework_127-gene_5), embM (locus, MALA_SAMN05422137_METAG-framework_127-gene_4), le embAM (ho kenyeletsoa libaka tsa intergene) li hahiloe ka tatellano ea 5 le li-complex55 e etselitsoe polelo ho E neng.Mofuta oa embA o ile oa kenngoa sebakeng sa pele sa cloning se ngata (MCS1) sa pACYCDuet-1(CmR) le pCDFDuet-1(SmR) e nang le libaka tsa BamHI le tsa HindIII tsa cleavage.Liphatsa tsa lefutso tsa embM le embMopt (codon-optimized) li ile tsa kenngoa ho MCS1 pCDFDuet-1(SmR) le BamHI le HindIII mme tsa behoa sebakeng sa bobeli sa cloning sa pCDFDuet-1(SmR) le pRSFDuet-1(KanR) (MCS2) le NdeI/ChoI.Khasete ea embAM e ile ea kengoa ho pCDFDuet1(SmR) ka libaka tsa BamHI le tsa HindIII.Mofuta oa orf3/embI (locus, MALA_SAMN05422137_METAG-scaffold_127-gene_3) e hahiloe ka ho fetellana ka katoloso ea PCR ho sebelisoa li-primers EmbI_OE_F_NdeI le EmbI_OE_R_XhoI, e kolobisitsoeng ka NdeI/Xeted-XhoICTM1MCDM e le 'ngoe ea li-pCD le li-XhoI tse tšoanang. li-enzyme (tlatsetso tafole).6).Thibelo ea tšilo ea enzyme le ligation e entsoe ho latela protocol ea moetsi (New England Biolabs).
Nako ea poso: Mar-14-2023