304 Insimbi engenasici edityanisiweyo ityhubhu edityanisiweyo / ityhubhu ye-zhemical zomponent, Amandla e-Biosynthetic yeGlobal Marine Microbiome

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Inkcazo yemveliso eneenkcukacha

304 Intsimbi engatyiwayo edityanisiweyo ityhubhu /ityhubhu
1. Inkcazo: ityhubhu ye-coil yensimbi engenasici / i-tubing
2. Uhlobo: i-welded okanye ingenamthungo
3. Umgangatho: ASTM A269, ASTM A249
4. I-tube ye-coil yensimbi engenasici ye-OD: 6mm ukuya kwi-25.4MM
5. Ubude: 600-3500MM okanye ngokwemfuno yomthengi ngamnye.
6. Ubunzima bodonga: 0.2mm ukuya ku-2.0mm.

7. Ukunyamezela: OD: +/-0.01mm;Ukutyeba: +/-0.01%.

8. Ubungakanani bomngxuma wekhoyili wangaphakathi: 500MM-1500MM (inokulungelelaniswa ngokweemfuno zabathengi)

9. Ubude bekhoyili: 200MM-400MM (inokulungelelaniswa ngokweemfuno zabathengi)

10. Umphezulu: Uqaqambile okanye uvaliwe
11. Izinto eziphathekayo: 304, 304L, 316L, 321, 301, 201, 202, 409, 430, 410, i-alloy 625, 825, 2205, 2507, njl.
12. Ukupakisha: iingxowa ezilukiweyo ezikwicala leplanga, ipallet yomthi, shaft yomthi, okanye ngokwemfuno yomthengi
13. Uvavanyo : icandelo lekhemikhali, amandla esivuno, amandla okuqina, ukulinganisa ubunzima
14. Isiqinisekiso: Uhlolo lweqela lesithathu (umzekelo :SGS TV ), njl.
15. Isicelo: Umhlobiso, ifenitshala, ukuthuthwa kweoli, ukutshintshwa kobushushu, ukwenza izithuko, ukwenza iphepha, imoto, ukutya, unyango, njl.

Uluntu lwendalo lwe-microbial luhluke ngokwe-phylogenetically kunye ne-metabolically.Ukongeza kumaqela angafundiweyo ezinto eziphilayo1, olu yantlukwano lukwabambe amandla atyebileyo okufunyanwa kwe-ecology kunye ne-biotechnologically ebalulekileyo enzymes kunye ne-biochemical compounds2,3.Nangona kunjalo, ukufunda le yantlukwano ukumisela iindlela ze-genomic ezenza iikhompawundi ezinjalo kwaye zibophelele kwiinginginya zazo ezahlukeneyo kuhlala kungumngeni.Amandla e-biosynthetic we-microorganisms kulwandle oluvulekileyo ahlala engaziwa kakhulu ngenxa yothintelo kuhlalutyo lwedatha yesisombululo se-genome kwisikali sehlabathi.Apha, siphonononga iyantlukwano kunye neyantlukwano yamaqela emfuza ebhayoloji elwandle ngokudibanisa malunga ne-10,000 yeejenomes zemicrobial ezisuka kwiiseli ezikhuliswayo kunye neeseli enye ezineedrafti ezingaphezulu kwama-25,000 ezisandula ukwakhiwa ngokutsha kwiisampulu zamanzi olwandle angaphezu kwe-1,000.Ezi nzame zichonge malunga ne-40,000 yokubeka uninzi lwamaqela amatsha emfuza ye-biosynthetic, amanye awo afunyenwe kumaqela e-phylogenetic awayengacingelwa ngaphambili.Kwaba bantu, sichonge umnombo otyetyiswe kumaqela emfuza e-biosynthetic (“Candidatus Eudormicrobiaceae”) eyayiyeye-bacterial phylum engalinywanga kwaye ibandakanya ezinye zezona microorganisms ezahlukeneyo zebhayosynthetically kule ndawo.Kwezi, siye sabonakalisa iindlela ze-phosphatase-peptide kunye ne-pytonamide, ukuchonga iimeko zesakhiwo esingaqhelekanga se-bioactive compound kunye ne-enzymology, ngokulandelelanayo.Ukuqukumbela, olu phononongo lubonisa indlela izicwangciso ezisekelwe kwi-microbiome ezinokwenza ukuba kuhlolwe ii-enzymes ezingachazwanga ngaphambili kunye nokutya kwendalo kwi-microbiota engaqondakaliyo kunye nokusingqongileyo.
Ii-Microbes ziqhuba imijikelo ye-biogeochemical yehlabathi, igcina i-webs yokutya, kwaye igcina izityalo nezilwanyana zisempilweni5.I-phylogenetic yabo enkulu, i-metabolic and functional diversity imele amandla atyebileyo ekufumaneni i-taxa1 entsha, i-enzymes kunye neekhompawundi ze-biochemical, kubandakanywa neemveliso zendalo6.Kwiindawo zokuhlala, ezi molekyuli zibonelela ngee-microorganisms ngeendlela ezahlukeneyo ze-physiological and ecological, ukusuka kunxibelelwano ukuya kukhuphiswano lwe-2, 7.Ukongeza kwimisebenzi yabo yangaphambili, ezi mveliso zendalo kunye neendlela zabo zokuvelisa i-genetically coded zibonelela ngemizekelo ye-biotechnological and therapeutic applications2,3.Ukuchongwa kwezo ndlela kunye noqhagamshelo kuququzelelwe kakhulu luphononongo lwee-microbes ezikhuliswayo.Nangona kunjalo, izifundo zetaxonomic zokusingqongileyo kwendalo zibonise ukuba uninzi lweemicroorganisms azilinywanga8.Olu thatho cala lwenkcubeko lunciphisa amandla ethu okusebenzisa iyantlukwano esebenzayo efakwe kwii-microbes ezininzi4,9.
Ukoyisa le mida, inkqubela phambili yetekhnoloji kule minyaka ilishumi idlulileyo ivumele abaphandi ukuba ngokuthe ngqo (okt, ngaphandle kwenkcubeko yangaphambili) balandelelanise amaqhekeza e-microbial DNA asuka kuluntu lonke (metagenomics) okanye iiseli enye.Ukukwazi ukudibanisa ezi ziqwenga zibe ngamaqhekeza amakhulu e-genome kunye nokwakha kwakhona iigenomes ezidityanisiweyo ezininzi ze-metagenomically (MAGs) okanye i-single amplified genomes (SAGs), ngokulandelelanayo, kuvula ithuba elibalulekileyo lezifundo zetaxocentric ye-microbiome (okt, uluntu lwe-microbial kunye ne-microbiome).vula iindlela ezintsha.imathiriyeli yemfuzo yakhe kwindawo ethile) 10,11,12.Ngokwenene, uphando lwakutsha nje luye lwandisa kakhulu ukubonakaliswa kwe-phylogenetic yokwahlukana kwe-microbial ku-Earth1, i-13 kwaye ibonise ubuninzi beentlobo ezahlukeneyo zokusebenza kuluntu olulodwa lwe-microbial olungagqunywanga ngaphambili lulandelelwano lwe-microorganism reference genome (REFs)14.Ukukwazi ukubeka ulwahlulo olusebenzayo olungafunyaniswanga kumxholo wejenome yomkhosi (oko kukuthi, isisombululo se-genome) sibaluleke kakhulu ekuqikeleleni imigca yemicrobial engekacaciswa ekucingelwa ukuba ifakele iimveliso zendalo ezintsha15,16 okanye ukulandelela ezo khompawundi zibuyela kumvelisi wazo wokuqala17.Ngokomzekelo, uhlalutyo oludibeneyo lwe-metagenomic kunye ne-single-cell genomic analysis luye lwakhokelela ekuchongeni i-Candidatus Entotheonella, iqela leebhaktheriya ezinxulumene ne-sponge ezityebileyo, njengabavelisi beentlobo ezahlukeneyo zeziyobisi18.Nangona kunjalo, ngaphandle kweenzame zakutsha nje zophononongo lwe-genomic lweendawo ezahlukeneyo ze-microbial, i-16,19 ngaphezulu kwesibini kwisithathu sedatha yemetagenomic yehlabathi yolona lwandlekazi lukhulu lwe-ecosystems16,20 lusalahlekile.Ke, ngokubanzi, amandla e-biosynthetic e-microbiome yolwandle kunye nokubanakho kwayo njengendawo yokugcina yenoveli ye-enzymatic kunye neemveliso zendalo zihlala zingafundiswanga.
Ukuphonononga ukubanakho kwe-biosynthetic yee-microbiomes zaselwandle kumlinganiselo wehlabathi, siqale sadibanisa ii-microbial genomes zaselwandle ezifunyenwe kusetyenziswa iindlela ezixhomekeke kwinkcubeko kunye nezingezizo ezenkcubeko ukwenza uvimba weenkcukacha obanzi we-phylogenetics kunye nomsebenzi wemfuza.Ukuhlolwa kwesi siseko sogcino-lwazi kubonakalise iindidi ngeendidi ezininzi zamaqela emfuza yebhayosynthetic (BGCs), uninzi lwazo ziphantsi kweentsapho ezingekabinacharacterized gene gene cluster (GCF).Ukongeza, sichonge usapho lwebhaktiriya olungaziwayo olubonisa ukohluka okwaziwayo kwe-BGC kulwandle oluvulekileyo ukuza kuthi ga ngoku.Sikhethe iindlela ezimbini ze-ribosomal synthesis kunye ne-post-translationally modified peptide (RiPP) iindlela zokuqinisekiswa kovavanyo ngokusekelwe kwiiyantlukwano zofuzo kwiindlela ezaziwayo ngoku.Ukubonakaliswa kokusebenza kwezi ndlela kuveze imizekelo engalindelekanga ye-enzymology kunye neekhompawundi zesakhiwo ezingaqhelekanga ezinomsebenzi we-protease inhibitory.
Ekuqaleni, sasijolise ekudaleni i-data yedatha yehlabathi jikelele yohlalutyo lwe-genome, igxininise kumacandelo ayo ebhaktheriya kunye ne-archaeal.Ukuza kuthi ga ngoku, sihlanganise idatha ye-metagenomic kunye neesampulu zamanzi olwandle angama-1038 ukusuka kwiziza zesampulu ezisasazwe kwihlabathi jikelele ezingama-215 (uluhlu lobubanzi = 141.6 °) kunye neeleya ezininzi ezinzulu (ukusuka ku-1 ukuya kwi-5600 m ubunzulu, ezigquma ipelagic, i-mesopelagic kunye nemimandla enzonzobileyo).Imvelaphi21,22,23 (Umfanekiso 1a, idatha eyandisiweyo, umzobo 1a kunye neTheyibhile eyoNgezelelweyo 1).Ukongeza ekuboneleleni ngejografi ebanzi, ezi sampuli zihluzwayo ngokukhethiweyo zisivumele ukuba sithelekise amacandelo ahlukeneyo e-microbiome yaselwandle, kuquka i-virus-rich (<0.2 µm), i-prokaryotic-rich (0.2–3 µm), i-particle-rich (0.8 µm) ).-20 µm) kunye neekoloni eziphelelwe yintsholongwane (>0.2 µm).
a, Iyonke ngama-1038 eegenomes afumanekayo eluntwini (metagenomics) yoluntu lwemicrobial yaselwandle eqokelelwe kwiindawo ezingama-215 ezisasazwe kwihlabathi (62°S ukuya kuma-79°N kunye ne-179°W ukuya kwi-179°E.).Iithayile zemephu © Esri.Imithombo: GEBCO, NOAA, CHS, OSU, UNH, CSUMB, National Geographic, DeLorme, NAVTEQ, kunye noEsri.b, ezi metagenomes zisetyenziselwa ukuvuselela ii-MAG (iindlela kunye nolwazi olongezelelweyo), olwahlukileyo ngobuninzi kunye nomgangatho (iindlela) kwiidatha (eziphawulwe ngombala).Ii-MAGs ezihlaziyiweyo zongezwa ngeegenomes ezifumaneka esidlangalaleni (zangaphandle), kubandakanywa i-MAG26 eyenziwe ngesandla, i-SAG27 kunye ne-REF.27 Qokelela i-OMD.c, xa kuthelekiswa neengxelo zangaphambili ezisekelwe kuphela kwi-SAG (GORG)20 okanye i-MAG (GEM)16, i-OMD iphucula ukubonakaliswa kwe-genomic ye-marine microbial community (ireyithi yokufunda imephu ye-metagenomic; indlela) ngokuphindwe kabini ukuya kathathu kunye nokumelwa okuhambelana ngakumbi ngobunzulu kunye isibanzi..<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, ukuhlelwa kwe-OMD ngokwenqanaba leqela leentlobo (ama-95% athetha i-nucleotide identity) ichonga iyonke malunga neentlobo ezingama-8300, ezingaphezu kwesiqingatha zazo ezingazange zibonakaliswe ngaphambili ngokwenkcazo ye-taxonomic kusetyenziswa i-GTDB (uguqulelo 89) e, ukuhlelwa kweentlobo ngohlobo lwe-genome kubonise ukuba i-MAG, i-SAG kunye ne-REFs ziyancedisana kakuhle ekuboniseni iyantlukwano ye-phylogenetic i-microbiome yolwandle.Ngokukodwa, i-55%, i-26% kunye ne-11% yeentlobo zazithe ngqo kwi-MAG, i-SAG kunye ne-REF, ngokulandelanayo.BATS, Bermuda Atlantic Time Series;I-GEM, i-genomes ye-microbiome yoMhlaba;I-GORG, ijenome yesalathiso solwandle jikelele;HOT, uthotho lwexesha loLwandlekazi lwaseHawaii.
Ukusebenzisa le datha, sakha kwakhona i-26,293 MAGs iyonke, ininzi ibhaktheriya kunye ne-archaeal (Umfanekiso we-1b kunye nedatha eyandisiweyo, i-Fig. 1b).Senze la ma-MAGs kwiindibano ezisuka ngokwahlukeneyo kunokuba zidibanise iisampulu ze-metagenomic ukuthintela ukuwa kokwahluka kolandelelwano lwendalo phakathi kweesampulu ezivela kwiindawo ezahlukeneyo okanye amaxesha ngamaxesha (iindlela).Ukongeza, sahlulahlula amaqhekeza e-genomic ngokusekwe kulungelelwaniso lwazo lokuxhaphaka kwinani elikhulu leisampulu (ukusuka kwi-58 ukuya kwi-610 iisampulu, kuxhomekeke kuphando; indlela).Sifumanise ukuba eli linyathelo elithatha ixesha kodwa elibalulekileyo24 elathi latsitywa kwimisebenzi emininzi yolwakhiwo lwe-MAG16, 19, 25 kwaye iphucula kakhulu ubungakanani (i-2.7-fold on avareji) kunye nomgangatho (+20% ngokomndilili) igenome.yakhiwe ngokutsha kwi-metagenome yolwandle efundwa apha (idatha eyandisiweyo, i-Fig. 2a kunye nolwazi olongezelelweyo).Ngokubanzi, ezi nzame zibangele ukwanda kwe-4.5-fold in marine microbial MAGs (i-6-fold if only high-quality MAGS ziqwalaselwa) xa kuthelekiswa neyona nto ibanzi i-MAG resource ekhoyo namhlanje16 (Iindlela).Le seti ye-MA entsha iye yadityaniswa ne-830 ye-MAG26s ekhethwe ngesandla, 5969 SAG27s kunye ne-1707 REFs.Iintlobo ezingamashumi amabini anesixhenxe zebhaktiriya zaselwandle kunye ne-archaea zenze iqoqo elidibeneyo le-34,799 genomes (Umfanekiso 1b).
Emva koko siye savavanya isibonelelo esitsha sokuphucula ukukwazi ukumela uluntu lwe-microbial yaselwandle kunye nokuvavanya impembelelo yokudibanisa iintlobo ezahlukeneyo ze-genome.Ngokomyinge, sifumene ukuba igubungela malunga ne-40-60% yedatha ye-metagenomic yolwandle (Umfanekiso 1c), kabini ukuya kathathu ukugubungela iingxelo ze-MAG kuphela kubunzulu kunye nobubanzi I-serial 16 okanye i-SAG20 yangaphambili.Ukongeza, ukulinganisa ngokucwangcisekileyo iyantlukwano yetaxonomic kwingqokelela esekiweyo, siye sachaza zonke iigenomes sisebenzisa i-Genome Taxonomy Database (GTDB) toolkit (iindlela) kwaye sasebenzisa isazisi somndilili we-genome-wide nucleotide yama-95%.28 ukuchonga i-8,304 iintlobo zeentlobo (iintlobo).Isibini esithathwini solu didi (kubandakanywa nemihlathi emitsha) aluzange luvele ngaphambili kwi-GTDB, apho i-2790 yafunyanwa kusetyenziswa i-MAG eyakhiwe ngokutsha kolu phononongo (Fig. 1d).Ukongezelela, sifumene ukuba iintlobo ezahlukeneyo ze-genomes zihambelana kakhulu: i-55%, i-26%, kunye ne-11% yeentlobo zezilwanyana ziqulunqwe ngokupheleleyo kwi-MAG, SAG, kunye ne-REF, ngokulandelanayo (Umfanekiso 1e).Ukongeza, i-MAG igubungele zonke iintlobo ze-49 ezifunyenwe kwikholamu yamanzi, ngelixa i-SAG kunye ne-REF imele kuphela i-18 kunye ne-11 yazo, ngokulandelanayo.Nangona kunjalo, i-SAG imele ngcono ukuhlukahluka kweentlobo eziqhelekileyo (idatha eyandisiweyo, i-Fig. 3a), njenge-Pelagic Bacteriales (SAR11), kunye ne-SAG egubungela phantse iintlobo ze-1300 kunye ne-MAG kuphela iindidi ezingama-390.Ngokucacileyo, ii-REFs azifane zinxibelelene nee-MAGs okanye ii-SAGs kwinqanaba leentlobo kwaye zimele > i-95% ye-genomes emalunga ne-1000 engafumanekiyo kwiiseti zemetagenomic yolwandle oluvulekileyo olufundiweyo apha, ikakhulu ngenxa yokunxibelelana nezinye iintlobo zemizekelo yokuzimela yaselwandle ezimeleyo (umz. intlenga) .okanye umamkeli-umncedisi).Ukwenza ukuba ifumaneke ngokubanzi kuluntu lwezenzululwazi, lo mthombo we-genome waselwandle, oquka iziqwenga ezingachazwanga (umzekelo, ukusuka kwi-phages eqikelelweyo, iziqithi ze-genomic, kunye namaqhekeza e-genome apho kungekho datha eyaneleyo yokwakhiwa kwakhona kwe-MAG), inokuthelekiswa nedatha ye-taxonomic. .Ukufikelela amanqakwana kunye nomsebenzi wemfuza kunye neeparamitha zomxholo kwi-Ocean Microbiology Database (OMD; https://microbiomics.io/ocean/).
Emva koko siye sajongana nobutyebi kunye nobutsha bezinto eziphilayo ezinokubakho kwii-microbiomes zolwandle oluvulekileyo.Ukuza kuthi ga ngoku, siqale sasebenzisa i-antiSMASH yazo zonke ii-MAG, ii-SAG, kunye nee-REF ezifunyenwe kwii-metagenomes zaselwandle ezili-1038 (iindlela) ukuqikelela i-39,055 BGCs iyonke.Emva koko sidibanise ezi kwii-GCFs ezingafunekiyo ze-6907 kunye ne-151 ye-gene cluster populations (GCCs; iTheyibhile eyoNgezelelweyo yesi-2 kunye neendlela) ukuhlawula ukungabikho kwezinto eziphathekayo (okt, i-BGC efanayo inokufakwa ngekhowudi kwii-genomes ezininzi) kunye nedatha ye-metagenomic Ukuhlukana kwee-BGC ezigxininisiweyo.I-BGCs engaphelelanga ayizange inyuke kakhulu, ukuba kukho (uLwazi oloNgezelelweyo), inani le-GCFs kunye ne-GCCs, ngokulandelanayo, eliqulethe ubuncinane ilungu le-BGC elingenasiphelo kwi-44% kunye ne-86% yamatyala.
Kwinqanaba le-GCC, sifumene iindidi ezininzi ze-RiPP eziqikelelweyo kunye nezinye iimveliso zendalo (umzobo 2a).Phakathi kwazo, umzekelo, i-arylpolyenes, i-carotenoids, i-ectoines, kunye ne-siderophores ye-GCC kunye nokusabalalisa okubanzi kwe-phylogenetic kunye nobuninzi obuphezulu kwi-metagenomes ye-oceanic, enokuthi ibonise ukulungelelaniswa okubanzi kwee-microorganisms kwindawo yolwandle, kuquka ukuchasana neentlobo ze-oksijini ezisebenzayo, uxinzelelo lwe-oxidative kunye ne-osmotic..okanye ukufunxwa kwentsimbi (ulwazi olongezelelekileyo).Le yantlukwano isebenzayo iyachasana nohlalutyo lwakutsha nje malunga ne-1.2 yezigidi ze-BGCs phakathi malunga ne-190,000 ye-genomes egcinwe kwi-NCBI RefSeq database (BiG-FAM/RefSeq, emva koku kubhekiselwa kuyo njenge-RefSeq)29, ebonise ukuba i-nonribosomal Synthetase peptides (NRPS) kunye ne-polyketide synthetase (PKS) ii-BGCs (uLwazi oloNgezelelweyo).Siphinde safumana i-44 (29%) ye-GCC kuphela ehambelana kude kuyo nayiphi na i-RefSeq BGC (\(\bar{d}\)RefSeq> 0.4; Umzobo 2a kunye neendlela) kunye ne-53 (35%) ye-GCC kuphela kwi-MAG, egxininisa amandla ukufumanisa iikhemikhali ezingachazwanga ngaphambili kwi-OMD.Ngenxa yokuba nganye kwezi GCCs kusenokwenzeka ukuba imele imisebenzi eyahluka-hlukeneyo yebhayoloji, siye sahlalutya ngakumbi idatha kwinqanaba le-GCF kwinzame zokubonelela ngeqela elineenkcukacha ezithe kratya zee-BGC eziqikelelwe ukuba zifake ikhowudi yeemveliso zendalo ezifanayo29.Iyonke i-3861 (56%) echongiweyo ye-GCFs ayizange idibane ne-RefSeq, kwaye > i-97% ye-GCFs yayingekho kwi-MIBiG, enye yogcino-lwazi olukhulu lwee-BGCs eziqinisekisiweyo (Figure 2b).Nangona ingothusi ukufumanisa iindlela ezininzi zenoveli ezinokuthi zibekho kwisethingi ezingabonakaliswa kakuhle yi-reference genome, indlela yethu yokuphindaphinda ii-BGCs kwii-GCFs phambi kokuba ibhentshimarking ihluke kwiingxelo zangaphambili ze-16 kwaye isivumela ukuba sinikeze uhlolo olungakhethi cala lobutsha.Uninzi lweyantlukwano entsha (3012 GCF okanye 78%) ihambelana neeterpenes eziqikelelweyo, iRiPP okanye ezinye iimveliso zendalo, kwaye uninzi (1815 GCF okanye 47%) lufakwe kwiikhowudi kwiintlobo ezingaziwayo ngenxa yesakhono sazo se-biosynthetic.Ngokungafaniyo namaqela e-PKS kunye ne-NRPS, ezi BGCs ezihlangeneyo azinakwenzeka ukuba ziqhekeke ngexesha lendibano ye-metagenomic 31 kwaye zivumele ixesha elininzi kunye nezixhobo zokusebenza ezisebenza ngamandla kwiimveliso zazo.
Zizonke ii-BGCs ezingama-39,055 zadityaniswa zaba ngama-6,907 eGCFs kunye ne-151 GCCs.a, ukumelwa kwedatha (yangaphakathi yangaphandle).Ukuhlanganiswa kwe-Hierarchical ye-BGC imigama esekelwe kwi-GCC, i-53 yayo ilungiswe ngu-MAG kuphela.I-GCC iqulethe ii-BGCs ezivela kwi-taxa eyahlukeneyo (i-ln-transformed gate frequency) kunye neeklasi ezahlukeneyo ze-BGC (ubungakanani besangqa buhambelana nokuphindaphinda kwayo).Kwi-GCC nganye, umaleko wangaphandle umele inani le-BGCs, ukuxhaphaka (ipesenti yeesampulu), kunye nomgama (ubuncinci be-BGC cosine umgama (min(dMIBiG))) ukusuka kwi-BiG-FAM ukuya kwi-BGC.Ii-GCC ezine-BGC ezisondeleleneyo nee-BGC ezingqinisiswe ngomfuniselo (MIBiG) ziphawulwe ngeentolo.b Ukuthelekisa i-GCF kunye nee-GCF eziqikelelweyo (BiG-FAM) kunye nee-BGC eziqinisekisiweyo (MIBiG), ezingama-3861 (d–>0.2) ezintsha ze-GCF zafunyanwa.Uninzi (78%) yale khowudi ye-RiPP, i-terpenes, kunye nezinye iimveliso zendalo ezibekayo.c, zonke iigenomes kwi-OMD ezifunyenwe kwi-1038 i-metagenomes yolwandle yafakwa kwisiseko somthi we-GTDB ukubonisa ukugubungela i-phylogenetic ye-OMD.Iiklasi ezingenazo naziphi na iigenomes kwi-OMD ziboniswa ngwevu.Inani le-BGCs lihambelana nelona nani likhulu le-BGCs eqikelelweyo ngokwe-genome nganye kwi-clade enikiweyo.Ukucaca, i-15% yokugqibela ye-nodes idilikile.Iintolo zibonisa izigaba ezityebileyo kwi-BGC (>15 BGC), ngaphandle kweMycobacterium, Gordonia (yesibini kuphela ukuya kwiRhodococcus), kunye neCrocosphaera (yesibini kuphela kwiSynechococcus).d, akwaziwa c.I-Eremiobacterota ibonise ukuhlukana okuphezulu kwe-biosynthetic (i-Shannon index esekelwe kuhlobo lwemveliso yendalo).Iqela ngalinye limele i-genome ene-BGCs ezininzi kwiintlobo.T1PKS, PKS uhlobo I, T2/3PKS, PKS uhlobo II kunye nohlobo III.
Ukongeza kubutyebi kunye nobutsha, siphonononga ubume be-biogeographic yesakhono se-biosynthetic ye-microbiome yolwandle.Ukwahlulahlulwa kweesampulu ngokwe-avareji ye-metagenomic yekopi ye-GCF yokuhanjiswa kwenani (IiNdlela) kubonise ukuba isibanzi esisezantsi, umphezulu, uluntu olutyebileyo lweprokaryotic kunye noluntu oluhlwempuzekileyo lwentsholongwane, ubukhulu becala lusuka kumphezulu okanye kumanzi anzulu elanga, lwalutyebile kwi-RiPP kunye ne-BGC terpenes.Ngokwahlukileyo, i-polar, i-deep-sea, i-virus- kunye ne-particle-rich-rich community zadibaniswa nobuninzi obuphezulu be-NRPS kunye ne-PKS BGC (idatha eyandisiweyo, i-Fig 4 kunye nolwazi olongezelelweyo).Okokugqibela, siye safumanisa ukuba uluntu olufundelwe kakuhle lwetropikhi kunye ne-pelagic yeyona mithombo ithembisayo ye-terpenes emitsha (uMzobo weDatha owandisiweyo).Amandla aphezulu kwi-PKS, i-RiPP kunye nezinye iimveliso zendalo (Umfanekiso 5a kunye nedatha eyandisiweyo).
Ukuncedisa uphononongo lwethu lwesakhono se-biosynthetic yee-microbiomes zaselwandle, sijonge ukwenza imephu yokusasazwa kwe-phylogenetic kunye nokuchonga amaqhosha amatsha e-BGC-etyetyisiweyo.Ukuza kuthi ga ngoku, sibeke iigenomes zemicrobes zaselwandle kumthi oqhelekileyo we-GTDB13 webhaktiriya kunye ne-archaeal phylogenetic kwaye sogqume iindlela ezifaka i-biosynthetic ezizifakelayo (Fig. 2c).Sibhaqe ngokulula amaqhosha e-BGC-etyetyisiweyo (emelwe ngaphezulu kwe-15 BGCs) kwiisampulu zamanzi olwandle (iindlela) ezaziwa ngesakhono sazo se-biosynthetic, njenge-cyanobacteria (i-Synechococcus) kunye neentsholongwane ze-Proteus, ezifana ne-Tistrella32,33, okanye kutshanje zitsale ingqalelo yazo. iimveliso zendalo .njenge-Myxococota (i-Sandaracinaceae), i-Rhodococcus kunye ne-Planctomycetota34,35,36.Okubangel 'umdla kukuba, sifumene imigca emininzi engazange ibonwe ngaphambili kula maqela.Ngokomzekelo, ezo ntlobo ze-biosynthetic ezityebileyo kwi-phyla Planctomycetota kunye ne-Myxococcota yayiyeyama-odolo abaviwa angabonakaliyo kunye negenera, ngokulandelanayo (Itheyibhile eyoNgezelelweyo 3).Kuthatyathwe kunye, oku kubonisa ukuba i-OMD ibonelela ngokufikelela kulwazi olungaziwa ngaphambili lwe-phylogenetic, kubandakanywa ne-microorganisms, enokuthi imele iithagethi ezintsha ze-enzyme kunye nokufunyanwa kwemveliso yendalo.
Okulandelayo, sibonakalise i-clade etyetyisiweyo ye-BGC ngokubala kuphela inani eliphezulu le-BGCs ekhowudiweyo ngamalungu ayo, kodwa kwakhona ngokuvavanya iyantlukwano yezi BGC, echaza ukuphindaphinda kweentlobo ezahlukeneyo zeemveliso zomgqatswa wendalo (Fig. )..Sifumene ukuba ezona ntlobo zeentlobo ngeentlobo zebhayoloji zimelwe zii-MAG zebhaktiriya ezikhethekileyo kolu phononongo.Ezi bhaktheriya ziyingxenye ye-phylum Candidatus Eremiobacterota engahlanjululwayo, ehlala ingahlolisiswanga ngaphandle kwezifundo ezimbalwa ze-genomic37,38.Kuyaphawuleka ukuba “ca.I-genus Eremiobacterota ihlalutywe kuphela kwindawo yomhlaba39 kwaye ayaziwa ukuba ibandakanya nawaphi na amalungu atyetyiswe kwi-BGC.Apha siye sakha kwakhona ii-MAG ezisibhozo zohlobo olufanayo (i-nucleotide identity> 99%) 23. Ngoko ke siphakamisa igama lezilwanyana elithi "Candidatus Eudoremicrobium malaspinii", elibizwa ngokuba yi-nereid (i-nymph yolwandle), isipho esihle kwi-mythology yamaGrike kunye nohambo.'Ka.Ngokutsho kwe-phylogenetic annotation 13, i-E. malaspinii ayinazo izalamane ezaziwa ngaphambili ngaphantsi kwenqanaba lokulandelelana kwaye ngoko ke ngowentsapho entsha yebhaktiriya esiyiphakamisayo "Ca.E. malaspinii” njengohlobo lweentlobo kunye ne “Ca.Eudormicrobiaceae” njengegama elisemthethweni (uLwazi oloNgezelelweyo).Ulwakhiwo olufutshane lwemetagenomic ye'Ca.Iprojekthi ye-E. malaspinii genome yaqinisekiswa ngegalelo eliphantsi kakhulu, ukulandelelana kwemetagenomic efundwa ixesha elide kunye nendibano ekujoliswe kuyo yesampulu enye (Iindlela) njengekhromozomi enye ye-9.63 Mb enokuphindaphindwa kwe-75 kb.njengokungacaci kakuhle okuseleyo.
Ukuseka umxholo we-phylogenetic wolu hlobo, siye safuna iintlobo ze-40 ezinxulumene ngokusondeleyo kwiisampuli ezongezelelweyo ze-eukaryotic-enriched metagenomic ukusuka kwi-expedition ye-Tara Ocean ngokusebenzisa ukwakhiwa ngokutsha kwe-genome ekujoliswe kuyo.Ngokufutshane, siye sanxulumanisa ufundo lwemetagenomic kumaqhekeza e-genomic anxulumene “noCa.E. malaspinii "kwaye kuqikelelwa ukuba ukunyuka kwesantya sokuqesha kule sampuli kubonisa ubukho bezinye izalamane (iindlela).Ngenxa yoko, sifumene i-MAG ye-10, inhlanganisela ye-MAG ye-19 emele iintlobo ezintlanu kwiintlobo ezintathu kwintsapho esanda kuchazwa (okt "Ca. Eudormicrobiaceae").Emva kokuhlolwa ngesandla kunye nolawulo lomgangatho (idatha eyandisiweyo, i-Fig. 6 kunye nolwazi olongezelelweyo), sifumene ukuba "Ca.Iintlobo ze-Eudormicrobiaceae zibonakalisa i-genomes ezinkulu (8 Mb) kunye ne-biosynthetic etyebileyo enokubakho (i-14 ukuya kwi-22 ye-BGC ngohlobo ngalunye) kunamanye amalungu e-"Ca".Clade Eremiobacterota (ukuya kwi-7 BGC) (Umfanekiso 3a-c).
a, izikhundla zePhylogenetic zesihlanu 'Ca.Iintlobo ze-Eudormicrobiaceae zibonise ubutyebi be-BGC obukhethekileyo kwimizila yolwandle echongiweyo kolu phononongo.Umthi wephylogenetic ubandakanya yonke i-'Ca.I-MAG Eremiobacterota kunye namalungu enye i-phyla (amanani e-genome kwizibiyeli) anikezelwe kwi-GTDB (uhlobo lwama-89) asetyenziswe kwimvelaphi ye-evolution (Iindlela).Iingqimba zangaphandle zimelela ukuhlelwa kwinqanaba lentsapho ("Ca. Eudormicrobiaceae" kunye ne "Ca. Xenobiaceae") kunye nenqanaba leklasi ("Ca. Eremiobacteria").Iindidi ezintlanu ezichazwe kolu phononongo zimelwe ziikhowudi zealphanumeric kunye namagama acetywayo abinimial (uLwazi oloNgezelelweyo).b, kulungile.Iintlobo ze-Eudormicrobiaceae zabelana ngeenuclei ezisixhenxe eziqhelekileyo ze-BGC.Ukungabikho kwe-BGC kwi-clade ye-A2 kwakungenxa yokungaphelelanga kommeli we-MAG (iThebhile eyoNgezelelweyo 3).Ii-BGCs zithe ngqo kwi “Ca.Amphithomicrobium” kunye ne “Ca.I-Amphithomicrobium” (i-clades A kunye no-B) ayiboniswa.c, Zonke ii-BGCs zifakwe ngekhowudi njenge “Ca.I-Eudoremicrobium taraoceanii yafunyanwa ibonakaliswe kwii-623 ze-metaranscripttomes ezithathwe kwiilwandle zaseTara.Izangqa eziqinileyo zibonisa ushicilelo olusebenzayo.Izangqa eziorenji zibonisa utshintsho oluguquliweyo lwelog2 ngezantsi nangaphezulu komyinge wokubonakaliswa kofuzo (iindlela).d, iigophe zobuninzi ezizalanayo (iindlela) ezibonisa 'Ca.Iintlobo ze-Eudormicrobiaceae zixhaphake kwiindawo ezininzi zolwandle kunye nakuluhlu lwamanzi lonke (ukusuka kumphezulu ukuya kubunzulu bama-4000 m ubuncinane).Ngokusekelwe kolu qikelelo, sifumanise ukuba 'Ca.I-E. malaspinii 'ibalelwa ukuya kuthi ga kwi-6% yeeseli zeprokaryotic kwindawo enzulu yolwandle yepelagic enxulumene nokutya okuziinkozo.Sithathele ingqalelo uhlobo olukhoyo kwisiza ukuba lufunyenwe kulo naliphi na iqhezu lobungakanani bomaleko obunzulu obunikiweyo.IO - i-Indian Ocean, i-NAO - i-North Atlantic, i-NPO - i-North Pacific, i-RS - uLwandle oluBomvu, i-SAO - i-South Atlantic, i-SO - i-Southern Ocean, i-SPO - i-South Pacific.
Ukufunda ngobuninzi kunye nokusasazwa kweCa.I-Eudormicrobiaceae, leyo, njengoko sifumene, ilawula kwiindawo ezininzi zolwandle, kunye nakwikholomu yonke yamanzi (Umfanekiso 3d).Ekuhlaleni, benza i-6% ye-microbial community yaselwandle, ibenza babe yinxalenye ebalulekileyo ye-microbiome yolwandle yehlabathi.Ukongeza, sifumene umxholo ohambelana neCa.Iintlobo ze-Eudormicrobiaceae kunye nemigangatho yazo yokubonakalisa i-BGC yayiphezulu kwi-fraction ye-eukaryotic ecebileyo (Umfanekiso we-3c kunye nedatha eyandisiweyo, i-Fig. 7), ebonisa ukusebenzisana okunokwenzeka kunye ne-particulate matter, kubandakanywa neplankton.Olu qwalaselo lufana no-'Ca.I-Eudoremicrobium BGCs ezivelisa iimveliso zendalo ze-cytotoxic ngeendlela ezaziwayo zinokubonisa ukuziphatha okuphangayo (uLwazi oloNgezelelweyo kunye neNkcukacha eYandisiweyo, uMfanekiso 8), ngokufanayo nezinye izilwanyana ezivelisa ngokukodwa i-metabolites ezifana ne-Myxococcus41.Ukufunyanwa kweCa.I-Eudormicrobiaceae ekufumanekeni kancinci (ulwandle olunzulu) okanye i-eukaryotic kuneesampuli zeprokaryotic inokuchaza ukuba kutheni ezi bhaktheriya kunye neentlobo zazo ze-BGC ezingalindelekanga zihlala zingacacanga kumxholo wophando lokutya kwendalo.
Ekugqibeleni, siye sazama ukuvavanya isithembiso somsebenzi wethu osekwe kwi-microbiome ekufumaneni iindlela ezintsha, ii-enzymes, kunye neemveliso zendalo.Phakathi kweeklasi ezahlukeneyo ze-BGCs, indlela ye-RiPP iyaziwa ngokuba yi-encode yekhemikhali ecebileyo kunye nokwahlukana okusebenzayo ngenxa yokuguqulwa okuhlukeneyo emva kokuguqulwa kwe-peptide engundoqo ngama-enzymes avuthiwe42.Ngoko ke sakhetha ezimbini 'Ca.I-Eudoremicrobium 'RiPP BGCs (Amanani 3b kunye ne-4a-e) asekelwe kufana nayo nayiphi na i-BGC eyaziwayo (\(\bar{d}\)MIBiG kunye \(\bar{d}\)RefSeq ngasentla kwe-0.2) .
a–c, In vitro heterologous expression kunye ne-vitro enzymatic assays yenoveli (\(\bar{d}\)RefSeq = 0.29) cluster of RiPP biosynthesis specific for deep sea species Ca.E. malaspinii' ikhokelele ekuvelisweni kweemveliso zediphosphorylated.c, ukuguqulwa okuchongiweyo kusetyenziswa i-high-resolution (HR) i-MS / MS (i-fragmentation eboniswe yi-b kunye ne-y ion kwi-chemical structure) kunye ne-NMR (idatha eyandisiweyo, i-Fig. 9).d, le peptide ye-phosphorylated ibonisa inhibition ephantsi ye-micromolar ye-mammalian neutrophil elastase, engafumanekiyo kwi-peptide yokulawula kunye ne-peptide ye-dehydrating (ukususwa kweekhemikhali kubangelwa ukuchithwa kwamanzi).Uvavanyo luphindwe kathathu ngeziphumo ezifanayo.Ngokomzekelo, ukubonakaliswa kwe-heterologous yenoveli yesibini \ (\ bar{d} \) RefSeq = 0.33) iqoqo le-protein biosynthesis licacisa umsebenzi wee-enzyme ezine ezivuthiweyo eziguqula i-46 ye-amino acid core peptide.Iintsalela zingcoliswe ngokwendawo yohlengahlengiso oluqikelelweyo yi-HR-MS/MS, ukuleyibhelishwa kwe-isotope, kunye nohlalutyo lwe-NMR (uLwazi oloNgezelelweyo).Umbala oqhawukileyo ubonisa ukuba uhlengahlengiso lwenzeka nakweyiphi na kwezi ntsalela zimbini.Umzobo yingqokelela yezinto ezininzi ezakhayo ze-heterologous ukubonisa umsebenzi wazo zonke ii-enzyme ezivuthiweyo kwinucleus efanayo.h, Umzobo wedatha ye-NMR ye-backbone amide N-methylation.Iziphumo ezipheleleyo ziboniswa kwifig.10 kunye nedatha eyandisiweyo.I, indawo ye-Phylogenetic ye-enzyme ye-protein ye-FkbM ekhulileyo phakathi kwazo zonke iindawo ze-FkbM ezifumaneka kwi-database ye-MIBiG 2.0 ibonisa i-enzyme yale ntsapho kunye nomsebenzi we-N-methyltransferase (uLwazi olongezelelweyo).Imizobo ecwangcisiweyo ye-BGCs (a, e), i-precursor peptide structures (b, f), kunye ne- putative chemical structures yeemveliso zendalo (c, g) zibonisiwe.
Indlela yokuqala ye-RiPP (\(\bar{d}\)MIBiG = 0.41, \(\bar{d}\)RefSeq = 0.29) ifunyenwe kuphela kwiintlobo ze-deep-sea "Ca.E. malaspinii” kunye neekhowudi zePeptide- precursor (Fig. 4a, b).Kule enzyme evuthiweyo, siye safumanisa i-domain eyodwa esebenzayo ye-homologous kwi-domain ye-dehydration ye-lantipeptide synthase eyenza i-phosphorylation kunye nokususwa okulandelayo kwe-43 (uLwazi olongezelelweyo).Ke ngoko, siqikelela ukuba ukuguqulwa kwe-peptide yangaphambili kubandakanya ukuphelelwa ngamanzi emzimbeni ngamanyathelo amabini.Nangona kunjalo, sisebenzisa i-tandem mass spectrometry (MS/MS) kunye ne-nuclear magnetic resonance spectroscopy (NMR), sichonge i-polyphosphorylated linear peptide (Fig. 4c).Nangona kungalindelekanga, sifumene imigca emininzi yobungqina bokuxhasa ukuba yimveliso yokugqibela: imikhosi emibini eyahlukeneyo ye-heterologous kwaye akukho dehydration in vitro assays, ukuchongwa kweentsalela eziphambili eziguqulwe kwindawo ye-catalytic dehydration ye-enzyme evuthiweyo.zonke zakhiwe ngokutsha ngu-“Ca”.I-E. malaspinii genome (idatha eyandisiweyo, i-9 kunye nolwazi olongezelelweyo) kwaye, ekugqibeleni, umsebenzi we-biological wemveliso ye-phosphorylated, kodwa kungekhona ifom ye-chemically synthesized dehydrated (Fig. 4d).Enyanisweni, sifumene ukuba ibonisa umsebenzi ophantsi we-micromolar protease inhibitory ngokuchasene ne-neutrophil elastase, xa kuthelekiswa nezinye iimveliso zendalo ezihambelanayo kwi-concentration range (IC50 = 14.3 μM) 44, nangona indima ye-ecological ihlala icaciswa.Ngokusekelwe kwezi ziphumo, sicebisa ukubiza indlela "phospheptin".
Imeko yesibini yindlela yeRiPP entsonkothileyo ethe ngqo kwi'Ca.I-genus Eudoremicrobium (\(\ bar{d}\) MIBiG = 0.46, \(\ bar{d}\) RefSeq = 0.33) yaxelwa kwangaphambili ukuba ifake imveliso yeprotheni yendalo (Umfanekiso 4e).Ezi ndlela zinomdla othile we-biotechnology ngenxa yoxinaniso olulindelekileyo kunye neentlobo ezahlukeneyo zohlengahlengiso lweekhemikhali ezingaqhelekanga ezisekwe zii-enzymes ezikhowudwe zii-BGCs45 ezimfutshane.Sifumene ukuba le proteni ihluke kwiiprotheni ezibonakaliswe ngaphambili kuba ayinayo i-NX5N ephambili ye-polyceramides kunye ne-lanthionine loop ye-landornamides 46.Ukoyisa imida yeepateni zokuthetha eziqhelekileyo ze-heterologous, siye sazisebenzisa kunye nenkqubo ye-Microvirgula aerodenitrificans yesiko ukubonisa ii-enzymes zeendlela ezine ezivuthiweyo (iindlela).Ukusebenzisa udibaniso lwe-MS / MS, ukubhalwa kwe-isotope, kunye ne-NMR, sifumene ezi enzymes ezivuthiweyo kwi-46-amino acid core ye-peptide (umzobo we-4f, g, idatha eyandisiweyo, i-Figs. 10-12 kunye nolwazi olongezelelweyo).Phakathi kwama-enzymes aqolileyo, sibonakalise ukubonakala kokuqala kwelungu lentsapho ye-FkbM O-methyltransferase ye-47 kwindlela ye-RiPP kwaye ngokungalindelekanga safumanisa ukuba le enzyme evuthiweyo ingenisa umqolo we-N-methylation (umzobo 4h, i kunye nolwazi olongezelelweyo).Nangona olu hlengahlengiso luyaziwa kwiimveliso ze-NRP48 zendalo, i-N-methylation ye-enzymatic ye-amide bond iyinkimbinkimbi kodwa i-biotechnologically ebalulekileyo ye-reaction49 esele inomdla kwintsapho ye-RiPP ye-borosines.Iinkcukacha 50,51.Ukuchongwa kwalo msebenzi kwezinye iintsapho ze-enzymes kunye ne-RiPP inokuvula izicelo ezintsha kunye nokwandisa ukuhlukahluka kokusebenza kweeprotheni ze-52 kunye nokuhlukahluka kweekhemikhali.Ngokusekelwe kwiinguqu ezichongiweyo kunye nobude obungaqhelekanga besakhiwo semveliso ecetywayo, siphakamisa igama lendlela "pythonamide".
Ukufunyanwa kwe-enzymology engalindelekanga kusapho oluphawulwe ngokusebenzayo lwe-enzymes lubonisa isithembiso se-genomics yendalo esingqongileyo kwizinto ezintsha ezifunyenweyo, kwaye ikwabonisa amandla alinganiselweyo okusebenza okusekwe kulandelelwano lwe-homology yodwa.Ke, kunye neengxelo ze-non-canonical bioactive polyphosphorylated RiPPs, iziphumo zethu zibonisa ukubaluleka kobutyebi kodwa okubalulekileyo kwiinzame zebhayoloji yokwenziwa ukutyhila ngokupheleleyo ubutyebi obusebenzayo, ukwahlukana, kunye nezakhiwo ezingaqhelekanga zeekhompawundi ze-biochemical.
Apha sibonisa uluhlu lwezinto ezinokwenzeka ze-biosynthetic ezifakwe kwi-microbes kunye nomxholo wazo we-genomic kwi-microbiome yolwandle lwehlabathi, ukuququzelela uphando lwexesha elizayo ngokwenza ubutyebi obuvelayo bufumaneke kuluntu lwezenzululwazi (https://microbiomics.io/ocean/).Sifumene ukuba ininzi ye-phylogenetic kunye ne-novelty esebenzayo inokufumaneka kuphela ngokuphinda kuhlaziywe ii-MAG kunye ne-SAGs, ngokukodwa kwiindawo ezincinci ezincinci ezinokukhokela iinzame ze-bioprospecting ezizayo.Nangona siza kugxila apha ku-'Ca.I-Eudormicrobiaceae” njengomnombo ingakumbi “obanesiphiwo” sebhayoloji, uninzi lwee-BGCs ezixelwe kwangaphambili kwi-microbiota engafunyaniswanga ekunokwenzeka ukuba ifakele ii-enzymologies ezingachazwanga ngaphambili ezivelisa iikhompawundi kunye nezenzo ezibalulekileyo zokusingqongileyo kunye/okanye zebhayoloji.
Iiseti zedatha ye-Metagenomic evela kwizifundo eziphambili ze-oceanographic kunye nothotho lwexesha ezinobunzulu obaneleyo bolandelelwano zibandakanyiwe ekwandiseni ukugutyungelwa kweentsholongwane zaselwandle zehlabathi kwiilwandle zolwandle, iileya ezinzulu kunye nokuhamba kwexesha.Ezi datha (iTheyibhile eyoNgezelelweyo 1 kunye noMzobo 1) zibandakanya i-metagenomics evela kwiisampuli eziqokelelwe kwiilwandle zaseTara (i-viral enriched, n = 190; iprokaryotic etyetyisiweyo, n = 180) 12,22 kunye ne-BioGEOTRACES expedition (n=480).I-Hawaiian Oceanic Time Series (HOT, n = 68), i-Bermuda-Atlantic Time Series (BATS, n = 62) 21 kunye ne-Malaspina Expedition (n = 58)23.Ukulandelelanisa ukufundwa kuzo zonke iziqwenga ze-metagenomic zahluzwa umgangatho usebenzisa i-BBMap (v.38.71) ngokususa i-adapters zolandelelwano ukusuka ekufundeni, ukususa ukufundwa kwemephu kulandelelwano lokulawula umgangatho (i-PhiX genomes), kunye nokusebenzisa i-trimq = 14, maq = 20 ilahla umgangatho ophantsi wokufunda, maxns = 0 kunye nobude bemizuzu = 45. Uhlalutyo olulandelayo lwaqhutywa okanye lwadityaniswa ne-QC efundwayo ukuba icacisiwe (bbmerge.sh minoverlap=16).Ukufundwa kwe-QC kwakuqhelekileyo (i-bbnorm.sh target = 40, i-minddepth = 0) ngaphambi kokwakha usebenzisa i-metaSPAdes (v.3.11.1 okanye i-v.3.12 ukuba iyadingeka)53.Iziphumo ze-scaffold contigs (emva koku zibhekiselwa kuzo njenge-scaffolds) ekugqibeleni zahluzwa ngobude (≥1 kb).
Iisampulu ze-1038 ze-metagenomic zahlulwa zibe ngamaqela, kwaye kwiqela ngalinye leesampuli, ukulawulwa komgangatho we-metagenomic ukufundwa kwazo zonke iisampulu zadityaniswa kwizibiyeli zesampulu nganye ngokwahlukileyo, okubangele inani elilandelayo leqela elifakwe kwizibiyeli lifundeka ngolu hlobo: Iintsholongwane zaseTara zaseMarine - Ziphuculwe. (190 × 190), Prokaryotes Enriched (180 × 180), BioGEOTRACES, HOT kunye BATS (610 × 610) kunye Malaspina (58 × 58).Imephu yenziwa ngokusebenzisa i-Burrows-Wheeler-Aligner (BWA) (v.0.7.17-r1188)54 evumela ukufundwa ukuba kuhambelane neziza zesibini (usebenzisa i-flag).Ulungelelwaniso lwahluzwa ukuba lube ubuncinane iziseko ze-45 ubude, zine-≥97% yesazisi, kunye ne-span ≥80% efundwayo.Iifayili ze-BAM ezibe neziphumo zasingathwa kusetyenziswa i-jgi_summarize_bam_contig_depths script ye-MetaBAT2 (v.2.12.1)55 ukunika i-intra- and inter-sample coverage kwiqela ngalinye.Ekugqibeleni, izibiyeli zadityaniswa ukuze kwandiswe ubuntununtunu ngokuqhuba ngamnye i-MetaBAT2 kuzo zonke iisampulu ezine-minContig 2000 kunye ne-maxEdges 500. Sisebenzisa i-MetaBAT2 endaweni ye-ensemble boxer kuba iboniswe kwiimvavanyo ezizimeleyo ukuba ibe yeyona bhokisi isebenzayo enye.kunye ne-10 ukuya ku-50 amaxesha ngokukhawuleza kunezinye iimbethi manqindi ezidla ngokusetyenziswa57.Ukuvavanya umphumo wokulungelelaniswa kobuninzi, i-subsample ekhethiweyo ye-metagenomics (i-10 nganye yeedatha ezimbini ze-Tara Ocean, i-10 ye-BioGEOTRACES, i-5 ngexesha lexesha ngalinye, kunye ne-5 ye-Malaspina) eyongezelelweyo isetyenziswe iisampuli kuphela.Iisampulu zangaphakathi zibekwe ngokwamaqela ukufumana ulwazi lokhuseleko.(Iinkcukacha ezongezelelweyo).
Iigenomes ezongezelelweyo (zangaphandle) zifakwe kuhlalutyo olulandelayo, ezizezi i-830 ze-MAGs ezikhethwe ngesandla kwi-subset yedatha ye-Tara Oceans26, i-5287 SAGs esuka kwi-dataset ye-GORG20, kunye nedatha evela kwi-database ye-MR (MarDB v. 4) ukusuka kwi-1707 i-REFs eyodwa kunye 682 SAGs) 27. Kuluhlu lwedatha ye-MarDB, iigenomes zikhethwa ngokusekwe kwimetadata ekhoyo ukuba udidi lwesampulu ludibana nala mabinzana aqhelekileyo alandelayo: '[S|s]ingle.?[C|c]ell|[C|c]ulture| [I|i] ndedwa'.
Umgangatho wesitya ngasinye se-metagenomic kunye ne-genomes yangaphandle yavavanywa kusetyenziswa i-CheckM (v.1.0.13) kunye ne-Anvi'o's Lineage Workflow (v.5.5.0) 58,59.Ukuba i-CheckM okanye i-Anvi'o ibika ≥50% ukugqibeka / ukugqiba kunye ≤10% ukungcola / ukungafuneki, ke gcina iiseli ze-metagenomic kunye ne-genomes yangaphandle ukuze uhlalutye kamva.La manqaku aye adityaniswa abe yintsingiselo epheleleyo (mcpl) kunye nokungcoliseka kwentsingiselo (mctn) ukuhlela umgangatho we-genome ngokwemiqathango yoluntu60 ngolu hlobo lulandelayo: umgangatho ophezulu: mcpl ≥ 90% kunye ne-mctn ≤ 5%;umgangatho omhle: mcpl ≥ 70%, mctn ≤ 10%, umgangatho ophakathi: mcpl ≥ 50% kunye ne-mctn ≤ 10%, umgangatho ofanelekileyo: mcpl ≤ 90% okanye mctn ≥ 10%.Iigenomes ezihluziweyo zaye zadityaniswa namanqaku asemgangathweni (Q kunye ne-Q') ngolu hlobo lulandelayo: Q = mcpl – 5 x mctn Q' = mcpl – 5 x mctn + mctn x (ukuhluka kobunzima)/100 + 0.5 x log[N50].(isetyenziswe kwi-dRep61).
Ukuvumela uhlalutyo lokuthelekisa phakathi kwemithombo yedatha eyahlukeneyo kunye neentlobo ze-genome (i-MAG, i-SAG kunye ne-REF), i-34,799 ye-genomes yachithwa ngokusekelwe kwi-genome-wide average nucleotide identity (ANI) usebenzisa i-dRep (v.2.5.4).Ukuphindaphinda)61 kunye ne-95% ye-ANI thresholds28,62 (-comp 0 -con 1000 -sa 0.95 -nc 0.2) kunye nejene zophawu lwekopi enye usebenzisa i-SpecI63 ebonelela ngokuhlanganiswa kwe-genome kwinqanaba leentlobo.I-genome emele ikhethwe kwiqela ngalinye le-dRep ngokowona mgangatho uphezulu wamanqaku (Q') achazwe ngasentla, athathwa njengomele uhlobo.
Ukuvavanya isantya semephu, i-BWA (v.0.7.17-r1188, -a) yayisetyenziselwa ukwenza imephu zonke iiseti ze-1038 ze-metagenomic ezifundwayo kunye ne-34,799 genomes equlethwe kwi-OMD.Ukufundwa okulawulwa ngumgangatho kufakwe kwimowudi yokuphela kokuphela kwaye ulungelelwaniso olubangelwayo lwahluzwa ukuze kugcinwe ulungelelwaniso kuphela ≥45 bp ubude.kunye nesazisi ≥95%.Umlinganiselo wokuboniswa kwisampulu nganye yipesenti yokufunda okuseleyo emva kohluzo olwahlulwe ngenani elipheleleyo lofundo lolawulo lomgangatho.Ukusebenzisa indlela efanayo, nganye ye-metagenomes ye-1038 yancitshiswa kwi-5 yezigidi zokufakwa (idatha eyandisiweyo, i-Fig. 1c) kwaye ihambelana ne-GORG SAG kwi-OMD nakuyo yonke i-GEM16.Isixa see-MAGs ezifunyenwe kumanzi olwandle kwikhathalogu ye-GEM16 yagqitywa yimibuzo yegama elingundoqo lemithombo ye-metagenomic, ukukhetha iisampulu zamanzi olwandle (umzekelo, ngokuchasene ne-sediments yolwandle).Ngokukodwa, sikhetha "aquatic" njenge "ecosystem_category", "marine" as "ecosystem_type", kwaye sihluze "indawo yokuhlala" njenge "deep ocean", "marine", "maritime oceanic", "pelagic marine", "amanzi olwandle" , "Ulwandle", "Amanzi oLwandle", "Amanzi oLwandle angaphezulu", "Amanzi oLwandle angaphezulu".Oku kubangele i-5903 MAGs (i-734 umgangatho ophezulu) isasazwe ngaphezu kwe-1823 OTUs (imibono apha).
Iigenomes zeProkaryotic zachazwa ngokwetaxonomically kusetyenziswa i-GTDB-Tk (v.1.0.2)64 enemilinganiselo engagqibekanga ejolise kwi-GTDB r89 uguqulelo 13. I-Anvi'o yasetyenziselwa ukuchonga i-eukaryotic genomes ngokusekelwe kuqikelelo lwesizinda kunye nokukhumbula ≥50% kunye nokungafuneki ≤ 10%.Inkcazo ye-taxonomic yohlobo oluthile ichazwa njengenye yeegenomes ezimele.Ngaphandle kwe-eukaryotes (i-148 MAG), i-genome nganye yaqala ukuchazwa ngokusebenzayo kusetyenziswa i-prokka (v.1.14.5) 65, ibiza i-genes epheleleyo, ichaza "i-archaea" okanye "iibhaktheriya" iparameters njengoko kufuneka, ekwabikwe ngokungeyo- iikhowudi zofuzo.kunye nemimandla ye-CRISPR, phakathi kwezinye iimpawu ze-genomic.Chaza izakhi zofuzo eziqikelelweyo ngokuchonga iijini ze-universal-copy marker (uscMG) usebenzisa i-fetchMG (v.1.2)66, yabela amaqela e-ortholog kunye nombuzo usebenzisa i-emapper (v.2.0.1)67 esekelwe kwi-eggNOG (v.5.0)68.I-database ye-KEGG (epapashwe ngoFebruwari 10, 2020) 69. Inyathelo lokugqibela lenziwe ngokuthelekisa iiprotheni kwi-database ye-KEGG usebenzisa i-DIAMOND (v.0.9.30) 70 kunye nombuzo kunye nesihloko somxholo we-≥70%.Iziphumo zahluzwa ngakumbi ngokwe-NCBI Prokaryotic Genome Annotation Pipeline71 ngokusekelwe kwi-bitrate ≥ 50% yobuninzi obulindelekileyo bebitrate (ikhonkco ngokwayo).Ukulandelelana kweGene kwasetyenziswa njengegalelo ukuchonga i-BGCs kwi-genome usebenzisa i-antiSMASH (v.5.1.0)72 eneparameters engagqibekanga kunye nokuqhuma kweqela elihlukeneyo.Zonke iigenomes kunye neenkcazo zihlanganiswe kwi-OMD kunye nemetadata yomxholo ekhoyo kwiwebhu (https://microbiomics.io/ocean/).
Ngokufanayo neendlela ezichazwe ngaphambili12,22 sasebenzisa i-CD-HIT (v.4.8.1) ukudibanisa> i-56.6 yezigidi ze-protein-coding genomes ezivela kwi-bacterial and archaeal genomes ukusuka kwi-OMD ukuya kwi-95% yesazisi kunye nemfuza emfutshane (i-90% i-coverage)73 ukuya kuthi ga > 17.7 yezigidi gene klass.Olona landelelwano lude lukhethwe njenge gene yommeli kwiqela ngalinye lemfuza.I-metagenomes ye-1038 yaza yadityaniswa> 17.7 yezigidi ze-BWA (-a) amalungu e-cluster kunye neefayile ze-BAM ezibangelwayo zahluzwa ukuze kugcinwe ulungelelwaniso kuphela nge-≥95% yesazisi sepesenti kunye ≥45 ulungelelwaniso lwesiseko.Ubuninzi bemfuza obuqhelekisiweyo bobude bubalwe ngokubala kuqala ukufakwa kolona lungelelwaniso lubalaseleyo kwaye emva koko, kufakelo olukwimaphu entsokothileyo, ukongeza amanani amaqhezu kuhlobo lwemfuza oluhambelanayo ngokulinganayo nenani labo lofakelo olulodwa.
Iigenomes ezivela kwi-OMD eyandisiweyo (kunye nee-MAGs ezongezelelweyo ezivela kwi-"Ca. Eudormicrobiaceae", bona ngezantsi) zongezwa kwi-moTUs74 yedatha yesixhobo sokuhlalutya i-metagenomic (v.2.5.1) ukudala i-database ye-reference ye-moTU eyandisiweyo.Kuphela ziigenomes ezinekopi enye (23,528 genomes) ezisindileyo kwishumi le-uscMGs.Ukwandiswa kwedathabheyisi kubangele i-4,494 yamaqela awongezelelweyo kwinqanaba leentlobo.Ii-metagenome ze-1038 zahlalutywa kusetyenziswa iiparamitha ze-moTU ezingagqibekanga (v.2).Iyonke ngama-989 eegenomes eziqulethwe kumaqela e-MOTU angama-644 (95% REF, 5% SAG kunye ne-99.9% ye-MarDB) ayifunyaniswanga yiprofayili ye-moTU.Oku kubonakalisa imithombo eyahlukeneyo eyongezelelweyo yokwahlulwa elwandle kwi-MarDB genomes (uninzi lweegenomes ezingabhaqwanga zinxulunyaniswa nezinto eziphilayo ezibekwe zodwa kwintlenga, iinginginya zaselwandle, njl. njl.).Ukuze siqhubeke sigxininise kwindawo evulekileyo yolwandle kolu phononongo, asibandakanyi kuhlalutyo olusezantsi ngaphandle kokuba zifunyenwe okanye zibandakanyiwe kwisiseko sedatha ye-moTU eyandisiweyo eyenziwe kolu phononongo.
Zonke ii-BGC ezivela kwi-MAG, i-SAG kunye ne-REF kwi-OMD (jonga ngasentla) zidibene ne-BGC ezichongiweyo kuzo zonke i-scaffolds ze-metagenomic (i-antiSMASH v.5.0, i-parameters engagqibekanga) kwaye ibonakaliswe ngokusebenzisa i-BiG-SLICE (v.1.1) (i-PFAM domain)75.Ngokusekwe kwezi mpawu, sibale yonke imigama ye-cosine phakathi kwe-BGC saza sayenza ngokwamaqela (amakhonkco aqhelekileyo) kwi-GCF kunye ne-GCC sisebenzisa imida yomgama we-0.2 kunye ne-0.8 ngokulandelelanayo.Le mida luhlengahlengiso olusetyenziswa ngaphambili kusetyenziswa umgama we-Euclidean75 kunye nomgama we-cosine, onciphisa enye yempazamo kwisicwangciso sokuqala sokuhlanganisa i-BiG-SLICE (IiNkcukacha ezoNgeziweyo).
Ii-BGCs ke zahluzwa ukuze zigcine kuphela i-≥5 kb ekhowudiweyo kwi-scaffolds ukunciphisa umngcipheko wokuqhekeka njengoko kuchaziwe ngaphambili16 kunye nokungabandakanyi ii-MarDB REFs kunye nee-SAGs ezingafunyanwanga kwi-1038 metagenomes (jonga ngasentla).Oku kubangele ukuba i-39,055 ye-BGCs iyonke ifakwe kwikhowudi ye-OMD genome, kunye ne-14,106 eyongezelelweyo echongiweyo kwiiqhekeza ze-metagenomic (okt engadityaniswanga kwii-MAGs).Ezi BGCs “zemetagenomic” zasetyenziselwa ukuqikelela umlinganiselo we-marine microbiome biosynthesis enokubakho engafakwanga kuvimba weenkcukacha (uLwazi oloNgezelelweyo).I-BGC nganye yayibonakaliswe ngokufanelekileyo ngokweentlobo zemveliso eqikelelweyo echazwe yi-anti-SMASH okanye iindidi zeemveliso ezinqabileyo ezichazwe kwi-BiG-SCAPE76.Ukuthintela ukuthathwa kwesampulu kucalucalulo (taxonomic and functional composition of GCC/GCF, the GCF and GCC to reference databases, and metagenomic abundance of GCF), ngokugcina kuphela eyona BGC inde kwiGCF nganye kuhlobo ngalunye, 39,055 BGCs ziye zadityaniswa ngakumbi, okukhokelela kwi-17,689 BGC iyonke.
Ubutsha be-GCC kunye ne-GCF bavavanywa ngokusekwe kumgama phakathi kwesiseko sedatha esibaliweyo (i-RefSeq database kwi-BiG-FAM) 29 kunye novavanyo oluqinisekisiweyo (MIBIG 2.0)30 BGC.Kwi-17,689 yabameli be-BGCs nganye, sikhethe owona mgama umncinci we-cosine kwisiseko sedatha.Le migama isezantsi ke ngoko i-avareji (ephakathi) ngokwe-GCF okanye i-GCC, ngokufanelekileyo.I-GCF ithathwa njengentsha ukuba umgama wesiseko sedatha mkhulu kune-0.2, ehambelana nokwahlula okufanelekileyo phakathi kwe-GCF (umyinge) kunye nesalathiso.Kwi-GCC, sikhetha i-0.4, ephindwe kabini umqobo ochazwe yi-GCF, ukuvala ubudlelwane bexesha elide kunye namakhonkco.
Ubuninzi be-metagenomic ye-BGC yaqikelelwa njengobuninzi bobuninzi be-biosynthetic genes yayo (njengoko igqitywe yi-anti-SMASH) ekhoyo kwiiprofayili zodidi lwemfuza.Ubuninzi bemetagenomic ye-GCF nganye okanye i-GCC iye yabalwa njengesambuku sabameli be-BGCs (ngaphandle kwe-17,689).Ezi mephu zininzi zaye zenziwa njengesiqhelo ukubunjwa kweselula kusetyenziswa i-moTU yesampulu nganye, ekwachaza iinzame zolandelelwano (idatha eyandisiweyo, iFig. 1d).Ukuxhaphaka kwe-GCF okanye i-GCC kubalwa njengepesenti yeesampulu ezinobuninzi> 0.
Umgama we-Euclidean phakathi kweisampulu ubaliwe ukusuka kwiprofayile yeGCF eqhelekileyo.Le migama yancitshiswa ngokobukhulu kusetyenziswa i-UMAP77 kwaye iziphumo zokuzinzisa ziye zasetyenziselwa ukuhlanganisana okusekwe kuxinaniso olungagadwanga kusetyenziswa i-HDBSCAN78.Elona nani lincinci lincinci lamanqaku eqela (kwaye kungoko inani lamaqela) asetyenziswa yi-HDBSCAN limiselwa ngokwandiswa kwamathuba okwanda kobulungu beqela.Amaqela achongiweyo (kunye nesampulu elungeleleneyo engacwangciswanga yala maqela ukuba aphendule ngokuthatha icala kuhlalutyo lwe-permutational multivariate of variance (PERMANOVA)) zavavanywa ngokubaluleka ngokuchasene nemigama ye-Euclidean engancitshiswanga kusetyenziswa iPERMANOVA.Umyinge wesayizi ye-genome yeesampulu zibalwe ngokusekelwe kubuninzi obunxulumene ne-moTU kunye nobungakanani obuqikelelweyo be-genome yamalungu e-genomes.Ngokukodwa, ubungakanani be-genome ye-moTU nganye buqikelelwa njengomyinge wesayizi ye-genome yamalungu ayo alungiswe ukugqibezela (emva kokucoca) (umzekelo, i-75% ye-genome epheleleyo kunye nobude be-3 Mb inobungakanani obuhlengahlengisiweyo be-4 Mb).kwiigenomes eziphakathi kunye nokunyaniseka ≥70%.Umndilili wesayizi yegenome kwisampulu nganye emva koko yabalwa njengesambuku semoOTU yobungakanani begenome obulinganiswa ngobuninzi obunxulumeneyo.
Iseti ehluziweyo ye-genome-encoded BGCs kwi-OMD iboniswa kwi-bacterial and archaeal GTDB imithi (kwi-≥5 kb frameworks, ngaphandle kwe-REF kunye ne-SAG MarDB ayifumanekanga kwi-1038 metagenomes, bona ngasentla) kunye neendidi zazo zemveliso eqikelelweyo ngokusekelwe kwi-phylogenetic. indawo yegenome (jonga ngasentla).Siqale sanciphisa idatha ngeentlobo, sisebenzisa i-genome ene-BGCs ezininzi kwezo ntlobo njengommeli.Ukujonga, abameli baye bahlulwa kwakhona bangamaqela emithi, kwaye kwakhona, kwi-clade nganye eneseli, i-genome equlethe inani elikhulu le-BGCs yakhethwa njengommeli.Iintlobo ezityetyisiweyo ze-BGC (ubuncinci i-genome enye ene>15 BGCs) zaphinda zahlalutywa ngokubala i-Shannon Diversity Index yeentlobo zemveliso ezifakwe kwii-BGCs.Ukuba zonke iintlobo zeemveliso eziqikelelweyo ziyafana, imixube yeekhemikhali kunye nezinye ii-BGC ezintsonkothileyo (njengoko ziqikelelwe yi-anti-SMAH) zithathwa njengezizezohlobo olunye lwemveliso, kungakhathaliseki ukuba zilandelelanise njani kwiqela (umz. iprotein-bacteriocin kunye ne-bacteriocin-proteoprotein fusion umzimba).umxube).
I-DNA eseleyo (kuqikelelwa ukuba yi-6 ng) ukusuka kwisampulu ye-Malaspina MP1648, ehambelana nesampulu yebhayoloji SAMN05421555 kwaye ifaniswe ne-Illumina SRR3962772 iseti yemetagenomic yokufunda ukufunda okufutshane, iqhutywe ngokulandelelana kwe-PacBio yolandelelwano lweprotocol kunye ne-ultra-low input to use DNA isampuli ye-GSMBio ikhithi (100-980-000) kunye ne-SMRTbell Express 2.0 ikhithi yokulungiselela itemplate (100-938-900).Ngokufutshane, i-DNA eseleyo yasikwa, yalungiswa kwaye yahlanjululwa (i-ProNex beads) isebenzisa i-Covaris (g-TUBE, 52104).I-DNA ehlanjululweyo emva koko ixhomekeke kumalungiselelo elayibrari, ukukhulisa, ukuhlanjululwa (i-ProNex beads) kunye nokukhethwa kobukhulu (> 6 kb, i-Blue Pippin) phambi kwesinyathelo sokugqibela sokucoca (i-ProNex beads) kunye nokulandelelana kwi-platform ye-Sequel II.
Ukwakhiwa ngokutsha ezimbini zokuqala ca.Kwi-MAG Eremiobacterota, sichonge ii-ANI ezintandathu ezongezelelweyo> 99% (ezi zifakwe kwi-Figure 3), eziye zahluzwa ekuqaleni ngokusekelwe kumanqaku okungcola (kamva zichongiwe njenge-gene duplications, jonga ngezantsi).Siphinde safumana itreyi ebhalwe "Ca".I-Eremiobacterota” evela kwizifundo ezahlukeneyo23 kwaye yazisebenzisa kunye nee-MAG ezisibhozo kuphononongo lwethu njengesalathiso sokufundwa kwe-metagenomic ukusuka kwi-633 eukaryotic etyetyisiweyo (>0.8 µm) iisampulu kusetyenziswa i-BWA (v.0.7.17) Ref -r1188, - iflegi) yokuthoba isampled ukwenza imephu (izigidi ezi-5 ziyafundwa).Ngokusekelwe kwiimephu ezikhethekileyo zokutyebisa (ihluzwe nge-95% yokulungelelaniswa kwesazisi kunye ne-80% yokufunda), i-metagenomes ye-10 (i-coverage elindelekileyo ≥5 ×) ikhethwe kwindibano kunye ne-49 metagenomes eyongezelelweyo (i-coverage elindelekileyo ≥1 ×) yokulungelelaniswa komxholo.Kusetyenziswa iparameters ezifanayo nezingasentla, ezi sampuli zaye zafakwa kwaye ii-'Ca's ezili-10 zongezwa.I-MAG Eremiobacterota ibuyiselwe.Ezi 16 MAGs (ngaphandle kokubala ezimbini esele zise-database) zizisa inani elipheleleyo le-genomes kwi-OMD eyandisiweyo ukuya kwi-34,815.Ii-MAGs zabelwa iirenki zetaxonomic ngokusekwe kukufaniswa kwazo kwe-genomic kunye nesikhundla kwi-GTDB.I-18 MAGs yachithwa ngokusebenzisa i-dRep kwiintlobo ze-5 (intraspecific ANI> 99%) kunye ne-3 genera (intrageneric ANI 85% ukuya kwi-94%) kwintsapho efanayo79.Abameli beentlobo bakhethwa ngesandla ngokusekelwe kwimfezeko, ungcoliseko, kunye ne-N50.Uluhlu lwamagama olucetyiswayo lunikiwe kuLwazi oloNgezelelweyo.
Vavanya imfezeko kunye nokungcoliseka kwe-'Ca.I-MAG Eremiobacterota, siye savavanya ubukho be-uscMG, kunye nomgca- kunye ne-domain-specific single-copy single-marker gene sets esetyenziswa yi-CheckM kunye ne-Anvi'o.Ukuchongwa kweekopi ezi-2 kwezingama-40 ze-uscMG zaqinisekiswa yi-phylogenetic reconstruction (jonga ngezantsi) ukuze kuthintelwe nakuphi na ungcoliseko olunokubakho (oku kuhambelana ne-5% ngokusekelwe kula ma-40 marker genes).Uphononongo olongezelelweyo lwabameli abahlanu be-MAGAs 'Ca.Inqanaba eliphantsi lokungcola kwezi genomes zakhiwe ngokutsha zaqinisekiswa kwiintlobo ze-Eremiobacterota zisebenzisa i-interface ye-Anvi'o esebenzayo esekelwe kubuninzi kunye nokulandelelana kokuqulunqwa kolandelelwano (IiNkcukacha ezongezelelweyo)59.
Uhlalutyo lwe-phylogenomic, sikhethe ii-MAG ezimele ezintlanu "Ca".Eudormicrobiaceae”, zonke iintlobo “Ca.I-genome ye-Eremiobacterota kunye namalungu enye i-phyla (kuquka i-UBP13, i-Armatimonadota, i-Patescibacteria, i-Dormibacterota, i-Chloroflexota, i-Cyanobacteria, i-Actinobacteria kunye ne-Planctomycetota) ifumaneka kwi-GTDB (r89)13.Zonke ezi genomes ziye zachazwa njengoko kuchaziwe ngaphambili kwikopi enye yokukhutshwa kohlobo lofuzo kunye ne-BGC yenkcazo.Iigenome zeGTDB ziye zalondolozwa ngokwale milinganiselo ingasentla yemfezeko kunye nongcoliseko.Uhlalutyo lwe-Phylogenetic lwenziwa kusetyenziswa i-Anvi'o Phylogenetics59 workflow.Umthi wakhiwa kusetyenziswa i-IQTREE (v.2.0.3) (izinketho ezingagqibekanga kunye -bb 1000)80 ngolungelelwaniso lwe-39 tandem ribosomal proteins ezichongwe ngu-Anvi'o (MUSCLE, v.3.8.1551)81.Izikhundla zakhe zancitshiswa.ukugubungela ubuncinane i-50% ye-genome82 kunye ne-Planctomycecota yasetyenziswa njengeqela langaphandle elisekelwe kwi-topology yemithi ye-GTDB.Umthi omnye wee-uscMG ezingama-40 wakhiwa kusetyenziswa izixhobo ezifanayo kunye neeparamitha.
Sasebenzisa i-Traitar (v.1.1.2) kunye neeparitha ezingagqibekanga (phenotype, ukusuka kwi-nucleotides) i-83 ukuqikelela iimpawu eziqhelekileyo ze-microbial.Siphonononge indlela yokuphila enokubakho ngokusekwe kwisalathiso se-predatory index84 esixhomekeke kumxholo we-protein-coding gene kwi-genome.Ngokukodwa, sisebenzisa i-DIAMOND ukuthelekisa iiprotheyini kwi-genome ngokuchasene nedatha ye-OrthoMCL (v.4) 85 usebenzisa iinketho-more-sensive -id 25 -query-cover 70 -subject-cover 70 -top 20 AND ubale iigene ezihambelanayo. iijini ezimakishayo zamarhamncwa kunye nezingezizo.Isalathiso ngumahluko phakathi kwenani lokumakishwa kwamaxhoba kunye nokungaphangi.Njengolawulo olongezelelweyo, siphinde sahlalutya i-"Ca" genome.I-Entotheonella TSY118 factor isekwe kunxulumano neCa.I-Eudoremicrobium (ubungakanani begenome enkulu kunye namandla e-biosynthetic).Okulandelayo, siye savavanya amakhonkco anokubakho phakathi kwezilwanyana ezizingelayo kunye nohlobo lwe-non-predator marker kunye namandla e-biosynthetic eCa.Eudormicrobiaceae” kwaye yafumanisa ukuba akukho jini ngaphezulu kwesinye (kusuka kulo naluphi na uhlobo lwejini yokumakisha, oko kukuthi ijini yepredator/non-predator) ewelana ne-BGC, icebisa ukuba i-BGC ayibhidanisi imiqondiso yezilwanyana.Inkcazo eyongezelelweyo ye-genomic yee-replicons ezikhutshiweyo zenziwa kusetyenziswa i-TXSSCAN (v.1.0.2) ukujonga ngokukodwa inkqubo yokufihla, i-pili, kunye ne-flagella86.
Abahlanu abamele i-'Ca's benziwe imephu ye-623 metatranscriptoms ukusuka kwiprokaryotic kunye ne-eukaryotic enrichment fractions of the Tara oceans22,40,87 (usebenzisa i-BWA, v.0.7.17-r1188, -iflegi).Eudormicrobiaceae genome.Iifayile ze-BAM zalungiswa nge-FeatureCounts (v.2.0.1)88 emva kwe-80% efundiweyo kunye ne-95% yokuhluza isazisi (kunye neempawu zokhethoAbalwa -engundoqo -O -fraction -t CDS,tRNA -F GTF -g ID -p ) Ibala inani lofakelo kwijeni nganye.Iimephu ezenziweyo zaziqhelekile kubude bemfuza kunye nesiphawuli sobuninzi bemfuza ye-moTU (ubude obuqhelekileyo bokufakela ubalo lwemfuza ngesibalo sofakelo >0) kunye nelogi-iguqulelwe ukuya kuma-22.74 ukufumana imbonakalo ezalanayo ngeseli nganye kwinqanaba lejeni ngalinye, ekwacacisa kwakhona ukwahluka ukusuka kwisampulu ukuya kwisampulu ngexesha lokulandelelana.Ulungelelwaniso olunjalo luvumela uhlalutyo lokuthelekisa, ukunciphisa iingxaki zokuqulunqa xa usebenzisa idatha yobuninzi obunxulumene.Kuphela iisampulu ezine->5 ye-10 ye-moTU ye-moTU ye-marker gene zaye zaqwalaselwa ukuze kuhlalutyelwe ngakumbi ukuvumela inxalenye enkulu eyaneleyo ye-genome ukuba ibonwe.
Iprofayile yombhalo oqhelekileyo we 'Ca.I-E. taraoceanii yayiphantsi konciphiso lwe-dimensionality isebenzisa i-UMAP kwaye umboniso ophumayo wasetyenziselwa ukuhlanganisana okungagadwanga kusetyenziswa i-HDBSCAN (jonga ngasentla) ukumisela ubume bentetho.I-PERMANOVA ivavanya ukubaluleka kweyantlukwano phakathi kwamaqela achongiweyo kwindawo yokuqala (engancitshiswanga) umgama.Ukubonakaliswa okwahlukileyo phakathi kwezi meko kwavavanywa kuyo yonke i-genome (jonga ngasentla) kunye neendlela ze-201 KEGG zichongiwe kumaqela asebenzayo e-6, angala: i-BGC, inkqubo ye-secretion kunye ne-flagellar genes evela kwi-TXSSCAN, i-enzymes yokunciphisa (i-protease kunye ne-peptidases), kunye ne-predatory and non- imizila yemfuza.izalathisi zesalathisi.Kwisampulu nganye, sibale i-median yesiqhelo yokuvakaliswa kudidi ngalunye (qaphela ukuba imbonakalo ye-BGC ngokwayo ibalwa njenge-median ye-biosynthetic genes yaloo BGC) kwaye ivavanyelwe ukubaluleka kumazwe onke (uvavanyo lwe-Kruskal-Wallis lulungelelaniselwe i-FDR).
Iijini zokwenziwa zathengwa kwiGenScript kunye neeprimer zePCR zathengwa kwiMicrosynth.I-Phusion polymerase evela kwi-Thermo Fisher Scientific yayisetyenziselwa ukukhulisa i-DNA.I-NucleoSpin plasmids, ijeli ye-NucleoSpin kunye nekiti yokucoca i-PCR evela e-Macherey-Nagel yayisetyenziselwa ukuhlanjululwa kwe-DNA.Uthintelo lwe-enzymes kunye ne-T4 DNA ligase zathengwa eNew England Biolabs.Iikhemikhali ngaphandle kwe-isopropyl-β-d-1-thiogalactopyranoside (IPTG) (Biosynth) kunye ne-1,4-dithiothreitol (DTT, AppliChem) zathengwa kwi-Sigma-Aldrich kwaye zisetyenziswe ngaphandle kokucoca.Amayeza okubulala iintsholongwane chloramphenicol (Cm), spectinomycin dihydrochloride (Sm), ampicillin (Amp), gentamicin (Gt), kunye carbenicillin (Cbn) zathengwa AppliChem.I-Bacto Tryptone kunye ne-Bacto Yeast Extract amacandelo eendaba zathengwa kwi-BD Biosciences.I-Trypsin yokulandelelana yathengwa kwi-Promega.
Ulandelelwano lweGene lukhutshwe kwi-anti-SMASH eqikelelweyo ye-BGC 75.1.E. malaspinii (Ulwazi olongezelelweyo).
I-genenes embA (locus, MALA_SAMN05422137_METAG-framework_127-gene_5), embM (locus, MALA_SAMN05422137_METAG-framework_127-gene_4), kunye ne-embAM (kuquka imimandla ye-intergene) yacwangciswa ngaphandle kolungelelwaniso lwe-5C kunye ne-p. ilungiselelwe ukubonakaliswa kwi-E nini.I-embA gene yafakwa kwisiza sokuqala se-cloning (MCS1) ye-pACYCDuet-1(CmR) kunye ne-pCDFDuet-1(SmR) eneendawo zokucoca i-BamHI kunye ne-HindIII.I-embM kunye ne-embMopt genes (i-codon-optimized) yafakwa kwi-MCS1 pCDFDuet-1 (SmR) kunye ne-BamHI kunye ne-HindIII kwaye yabekwa kwindawo yesibini ye-cloning site ye-pCDFDuet-1 (SmR) kunye ne-pRSFDuet-1 (KanR) (MCS2) kunye NdeI/ChoI.Ikhasethi ye-embAM yafakwa kwi-pCDFDuet1(SmR) kunye ne-BamHI kunye ne-HindIII iziza zokucanda.I-orf3/embI gene (locus, MALA_SAMN05422137_METAG-scaffold_127-gene_3) yakhiwa ngolwandiso oludityanisiweyo lwe-PCR kusetyenziswa iiprimers EmbI_OE_F_NdeI kunye ne-EmbI_OE_R_XhoI, yetyiswe nge-NDEI/Xhot-XhoImx kwi-pCDEM1 efanayo ne-ligag ii-enzymes (Ezongezelelweyo itafile).6).Unyino lwe-enzyme yokuthintela kunye ne-ligation lwenziwa ngokweprotocol yomenzi (i-New England Biolabs).