304 Ibyuma bitagira umuyonga wasuditswe hamwe / tubing zhemical zomponent, Ubushobozi bwa Biosynthetic ya Microbiome yisi yose

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Ibisobanuro birambuye kubicuruzwa

304 Ibyuma bitagira umuyonga wasuditswe hamwe / tubing
1. Ibisobanuro: Icyuma coil tube / tubing
2. Ubwoko: gusudira cyangwa kutagira ikidodo
3. Bisanzwe: ASTM A269, ASTM A249
4. Umuyoboro w'icyuma utagira umuyonga OD: 6mm kugeza 25.4MM
5. Uburebure: 600-3500MM cyangwa nkuko umukiriya abisabwa.
6. Ubunini bwurukuta: 0.2mm kugeza kuri 2.0mm.

7. Ubworoherane: OD: +/- 0.01mm;Umubyimba: +/- 0.01%.

8. Ingofero yimbere imbere: 500MM-1500MM (irashobora guhindurwa ukurikije ibyo abakiriya bakeneye)

9. Uburebure bwa coil: 200MM-400MM (irashobora guhinduka ukurikije ibyo abakiriya bakeneye)

10. Ubuso: Kumurika cyangwa gufatana
11. Ibikoresho: 304, 304L, 316L, 321, 301, 201, 202, 409, 430, 410, amavuta 625, 825, 2205, 2507, nibindi.
12. Gupakira: imifuka iboshywe mubiti, pallet yimbaho, igiti cyimbaho, cyangwa nkuko umukiriya abisabwa
13. Ikizamini: ibigize imiti, gutanga umusaruro, imbaraga zingana, gupima ubukana
14. Ingwate: Igice cya gatatu (urugero: SGS TV) kugenzura, nibindi.
15. Gusaba: Imitako, ibikoresho, gutwara amavuta, guhinduranya ubushyuhe, gukora gari ya moshi, gukora impapuro, imodoka, gutunganya ibiryo, ubuvuzi, nibindi.

Imiryango mikorobe isanzwe iratandukanye kandi ihindagurika.Usibye amatsinda y’ibinyabuzima adasuzumwa1, ubwo butandukanye kandi bufite amahirwe menshi yo kuvumbura imisemburo ifatika y’ibidukikije n’ibinyabuzima ndetse n’ibinyabuzima bya biohimiki2,3.Nyamara, kwiga ubu butandukanye kugirango umenye inzira ya genomic ihuza ibice nkibi kandi ubihuze nababashitsi biracyari ikibazo.Ubushobozi bwa biosintetike ya mikorobe mu nyanja ifunguye ntiburamenyekana cyane kubera imbogamizi zisesengura ryamakuru yose yakemuwe ku isi yose.Hano, turasesengura ubudasa nubwinshi bwibinyabuzima bya gene biosynetique mu nyanja duhuza genoside zigera ku 10,000 ziva mu ngirabuzimafatizo n’umuco umwe hamwe na genoside zirenga 25.000 zubatswe kuva ku nyanja zirenga 1.000.Izi mbaraga zerekanye abagera ku 40.000 bashira ahanini amatsinda mashya ya biosynetique, amwe muri yo akaba yarayasanze mu matsinda ya phylogeneque atigeze akekwa.Muri aba baturage, twabonye umurongo ukungahaye ku matsinda ya genoside ya biosynetike (“Candidatus Eudormicrobiaceae”) yari mu bwoko bwa bagiteri idahinzwe kandi yashyizwemo na mikorobe itandukanye y’ibinyabuzima bitandukanye muri ibi bidukikije.Muri ibyo, twaranze inzira ya fosifata-peptide na pytonamide, tugaragaza ibihe byimiterere idasanzwe ya bioactive compound hamwe na enzymologiya.Mu gusoza, ubu bushakashatsi bwerekana uburyo ingamba zishingiye kuri mikorobe zishobora gutuma habaho ubushakashatsi bwimisemburo itaravuzwe mbere nibiribwa karemano muri mikorobe idasobanutse neza nibidukikije.
Microbes itwara isi yose ya biogeochemiki, ikomeza imiyoboro y'ibiryo, kandi igakomeza ibimera ninyamaswa5.Ubwinshi bwa phylogeneque, metabolike nibikorwa bitandukanye byerekana imbaraga nyinshi zo kuvumbura tagisi nshya1, enzymes hamwe n’ibinyabuzima bya biohimiki, harimo nibicuruzwa bisanzwe6.Mubidukikije, iyi molekile itanga mikorobe hamwe nibikorwa bitandukanye bya physiologique nibidukikije, kuva itumanaho kugeza amarushanwa 2, 7.Usibye imikorere yabo yumwimerere, ibyo bicuruzwa karemano hamwe ninzira yabyo yerekana umusaruro utanga ingero kubikorwa bya tekinoloji nubuvuzi2,3.Kumenyekanisha inzira ninzira byoroherejwe cyane no kwiga mikorobe zifite umuco.Nyamara, ubushakashatsi bwamatagisi y’ibidukikije bwerekanye ko umubare munini w’ibinyabuzima bitigeze bihingwa8.Uku kubogama kumuco kugabanya ubushobozi bwacu bwo gukoresha imikorere itandukanye igizwe na mikorobe nyinshi 4,9.
Kugira ngo tuneshe izo mbogamizi, iterambere mu ikoranabuhanga mu myaka icumi ishize ryemereye abashakashatsi gukora mu buryo butaziguye (ni ukuvuga, nta muco wabanjirije) bakurikirana ibice bya ADN ya mikorobe biva mu baturage bose (metagenomics) cyangwa selile imwe.Ubushobozi bwo guteranya ibyo bice mubice binini bya genome no kongera kubaka genome nyinshi ziteranijwe hamwe (MAGs) cyangwa genome imwe yongerewe imbaraga (SAGs), bitanga amahirwe yingenzi kubushakashatsi bwamatagisi ya mikorobe (ni ukuvuga umuryango wa mikorobe na mikorobe).pave inzira nshya.ibikoresho bya genetike mubidukikije runaka) 10,11,12.Mubyukuri, ubushakashatsi buherutse kwagura cyane phylogeneque yerekana itandukaniro rya mikorobe ku isi1, 13 kandi ryerekanye byinshi mubikorwa bitandukanye mumiryango itandukanye ya mikorobe itigeze ikwirakwizwa na mikorobe ikomoka kuri genome ikurikirana (REFs) 14.Ubushobozi bwo gushyira ibintu bitandukanye bitavumbuwe muburyo bwa genome yakiriye (ni ukuvuga imiterere ya genome) ni ingenzi mu guhanura imirongo ya mikorobe nyamara itaranga imiterere ishobora kuba ikubiyemo ibicuruzwa bishya15,16 cyangwa mugukurikirana ibyo bintu bigasubira mubikorwa byabo byambere17.Kurugero, uburyo bwa metagenomic hamwe na selile imwe yisesengura rya genomique byatumye hamenyekana Candidatus Entotheonella, itsinda rya bacteri zikungahaye kuri metabolike ikungahaye kuri sponge, nkabakora ibiyobyabwenge bitandukanye18.Nubwo, nubwo hashize iminsi igerageza ubushakashatsi bwerekeranye na genoside y’imiryango itandukanye ya mikorobe, 16,19 zirenga bibiri bya gatatu byamakuru ya metagenomic ku isi ku nyanja nini nini y’ibinyabuzima 16,20 iracyabura.Rero, muri rusange, ibinyabuzima bya biosintetike ya mikorobe yo mu nyanja hamwe nubushobozi bwayo nkububiko bwibintu bishya byimisemburo nibisanzwe bikomeza kuba bike.
Kugirango tumenye ubushobozi bwa biosintetike ya mikorobe yo mu nyanja ku isi yose, twabanje guhuriza hamwe genoside zo mu nyanja zo mu nyanja zabonetse dukoresheje uburyo bushingiye ku muco kandi butari umuco kugira ngo dushyireho amakuru menshi ya phylogeneque n'imikorere ya gene.Isuzumabumenyi ryakozwe ryagaragaje ubwoko butandukanye bwa gene biosynetique (BGCs), inyinshi muri zo zikaba ari iz’imiryango ya gene (GCF) itaramenyekana.Twongeyeho, twabonye umuryango wa bagiteri utazwi ugaragaza ubudasa buzwi bwa BGC mu nyanja ifunguye kugeza ubu.Twahisemo ibice bibiri bya ribosomal hamwe na peptide (RiPP) yahinduwe nyuma yubusobanuro kugirango twemeze igeragezwa dushingiye kubitandukanyirizo byabo biva mumihanda izwi.Imikorere iranga izi nzira yerekanye ingero zitunguranye za enzymologiya kimwe nuburyo budasanzwe bwubatswe hamwe nibikorwa bya protease bibuza.
Ubwa mbere, twari tugamije gukora amakuru yisi yose yo gusesengura genome, twibanda kuri bagiteri na archaeal.Kugira ngo ibyo bishoboke, twahurije hamwe amakuru ya metagenomic hamwe n’amazi 1038 y’amazi yo mu nyanja kuva 215 yagabanijwe ku isi hose (urugero rw'uburinganire = 141,6 °) hamwe n’ibice byinshi byimbitse (kuva kuri metero 1 kugeza kuri 5600 zubujyakuzimu, bitwikiriye uturere twa pelagisi, mesopelagike na nyenga).Amavu n'amavuko 21,22,23 (Igishusho 1a, amakuru yagutse, Igishusho 1a hamwe n'imbonerahamwe y'inyongera 1).Usibye gutanga imiterere yagutse ya geografiya, izi ngero zatoranijwe zayungurujwe zadushoboje kugereranya ibice bitandukanye bigize mikorobe yo mu nyanja, harimo na virusi ikungahaye kuri virusi (<0.2 µm), ikungahaye kuri prokaryyotike (0.2–3 µm), ikungahaye ku bice (0.8 µm ).–20 µm) hamwe na virusi-yabuze (> 0.2 µm).
a, Igiteranyo 1038 rusange kiboneka kumugaragaro (metagenomics) yabaturage ba mikorobe yo mu nyanja yakusanyirijwe ahantu 215 bakwirakwijwe kwisi yose (62 ° S kugeza 79 ° N na 179 ° W kugeza 179 ° E.).Ikarita yerekana ikarita © Esri.Inkomoko: GEBCO, NOAA, CHS, OSU, UNH, CSUMB, National Geographic, DeLorme, NAVTEQ, na Esri.b, izo metagenomes zakoreshejwe mukubaka MAGs (uburyo namakuru yinyongera), atandukanye mubwinshi nubwiza (uburyo) mumibare (yashyizwe mumabara).MAG yubatswe yongeye kongerwaho na genome ziboneka kumugaragaro (hanze), harimo intoki za MAG26, SAG27 na REF.27 Gukusanya OMD.C. ubunini..<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. amoko agera kuri 8300, arenga kimwe cya kabiri cyayo akaba atarigeze agaragazwa hakurikijwe amatangazo ya tagisi akoresheje GTDB (verisiyo ya 89) e, gutondekanya amoko kubwoko bwa genome byerekanaga ko MAG, SAG na REF byuzuzanya neza mugaragaza ubudasa bwa phylogeneque microbiome yo mu nyanja.By'umwihariko, 55%, 26% na 11% by'ubwoko byari byihariye kuri MAG, SAG na REF.BATS, Bermuda Atlantike Yigihe;GEM, genome za mikorobe yisi;GORG, genome yerekana isi yose;BISHYUSHYE, inyanja ya Hawayi ikurikirana.
Twifashishije iyi mibare, twongeye kubaka MAGs 26.293, cyane cyane bagiteri na archaeal (Ishusho 1b hamwe namakuru yagutse, Ishusho 1b).Twashizeho aya MAG tuvuye mu nteko zitandukanye aho guhuriza hamwe metagenomic ntangarugero kugirango twirinde isenyuka ryikurikiranya ryimiterere karemano hagati yintangarugero ahantu hatandukanye cyangwa umwanya (uburyo).Twongeyeho, twashyize hamwe ibice bya genomique dushingiye ku isano ryabyo ryagaragaye ku mubare munini w'icyitegererezo (kuva kuri 58 kugeza kuri 610, bitewe n'ubushakashatsi; uburyo).Twabonye ko iyi ari intambwe itwara igihe ariko ikomeye intambwe24 yasimbuwe mubikorwa byinshi binini bya MAG16, 19, 25 byo kwiyubaka kandi bitezimbere cyane ubwinshi (inshuro 2,7 ugereranije) hamwe nubwiza (+ 20% mugereranije) ya genome.yubatswe kuva metagenome yo mu nyanja yize hano (amakuru yagutse, Ishusho 2a namakuru yinyongera).Muri rusange, izo mbaraga zatumye habaho kwiyongera kwa 4.5 mikorobe zo mu nyanja MAGs (inshuro 6 niba harebwa gusa MAGs zo mu rwego rwo hejuru) ugereranije n’umutungo wuzuye wa MAG uboneka muri iki gihe16 (Methods).Iyi MAG nshya yashizweho noneho yahujwe na 830 yatowe na MAG26s, 5969 SAG27s na 1707 REF.Amoko 27 ya bagiteri yo mu nyanja na archaea bagize icyegeranyo cya genome 34.799 (Ishusho 1b).
Twahise dusuzuma umutungo mushya washyizweho kugirango tunoze ubushobozi bwo guhagararira mikorobe zo mu nyanja no gusuzuma ingaruka zo guhuza ubwoko butandukanye bwa genome.Ugereranije, twasanze ikubiyemo hafi 40-60% yamakuru ya metagenomic yo mu nyanja (Igicapo 1c), inshuro ebyiri kugeza kuri eshatu gukwirakwiza raporo za MAG zabanje gusa mubwimbitse no mu burebure Byinshi bikurikirana 16 cyangwa SAG20.Byongeye kandi, kugirango dusuzume gahunda zitandukanye za tagisi mu byegeranyo byashizweho, twasobanuye genome zose dukoresheje ibikoresho bya Genome Taxonomy Database (GTDB) toolkit (uburyo) kandi dukoresha impuzandengo ya genome yagutse ya nucleotide ya 95%.28 kugirango tumenye amoko 8,304 (amoko).Bibiri bya gatatu by'ubwo bwoko (harimo clade nshya) ntabwo byari byigeze bigaragara muri GTDB, muri byo 2790 byavumbuwe hakoreshejwe MAG yubatswe muri ubu bushakashatsi (Ishusho 1d).Twongeyeho, twasanze ubwoko butandukanye bwa genome bwuzuzanya cyane: 55%, 26%, na 11% yubwoko bugizwe rwose na MAG, SAG, na REF, (Ishusho 1e).Byongeye kandi, MAG yitwikiriye ubwoko 49 bwose buboneka mu nkingi y’amazi, naho SAG na REF bahagarariye 18 na 11 gusa.Nyamara, SAG yerekana neza itandukaniro ryibice bikunze kugaragara (amakuru yagutse, Igishusho 3a), nka Pelagic Bacteriales (SAR11), SAG ikubiyemo amoko agera ku 1300 na MAG amoko 390 gusa.Ikigaragara ni uko REF idakunze guhuzagurika hamwe na MAGs cyangwa SAGs kurwego rwubwoko kandi igereranya> 95% ya genom zigera ku 1000 zitaboneka mumasozi yinyanja ya metagenomic yize hano, cyane cyane kubera imikoranire nubundi bwoko bwikigereranyo cyo mu nyanja cyihariye (urugero: imyanda) .cyangwa uwakiriye-umufasha).Kugirango igere ku bantu benshi mu bumenyi, uyu mutungo wa genome wo mu nyanja, urimo kandi ibice bidafite ibyiciro (urugero, uhereye ku byiciro byahanuwe, ibirwa bya genomique, n'ibice bya genome bitarimo amakuru adahagije yo kwiyubaka kwa MAG), birashobora kugereranywa n'amakuru ya tagisi; .Kugera kuri annotations hamwe nibikorwa bya gene hamwe nibipimo bifatika mububiko bwa Microbiology yo mu nyanja (OMD; https://microbiomics.io/ocean/).
Twahise dufata ingamba zo gucukumbura ubukire nudushya twibinyabuzima byitwa biosynetique muri mikorobe yo mu nyanja ifunguye.Kugira ngo ibyo bishoboke, twabanje gukoresha antiSMASH kuri MAGs zose, SAGs, na REF dusanga muri 1038 marine metagenomes (uburyo) kugirango tumenye BGCs 39,055.Twahise tubishyira hamwe muri 6907 GCFs itagabanijwe hamwe nabantu 151 ba gene cluster (GCCs; Imbonerahamwe yinyongera nuburyo 2) kugirango tubare ibirenze (urugero, BGC imwe irashobora gushirwa muri genome nyinshi) hamwe namakuru ya metagenomic Gutandukanya BGC yibanze.BGC ituzuye ntabwo yiyongereye cyane, niba ihari (Amakuru yinyongera), umubare wa GCFs na GCCs, urimo byibuze umunyamuryango wa BGC udahwitse muri 44% na 86% byimanza.
Kurwego rwa GCC, twasanze ubwoko butandukanye bwa RiPPs nibindi bicuruzwa bisanzwe (Ishusho 2a).Muri byo, nk'urugero, arylpolyène, karotenoide, ectoine, na siderophores ni ibya GCCs ikwirakwizwa na phylogeneque kandi ikaba nyinshi muri metagenome yo mu nyanja, ibyo bikaba bishobora kwerekana imiterere mikorobe ikomoka ku nyanja, harimo no kurwanya amoko ya ogisijeni ikora, okiside na osmotic..cyangwa kwinjiza ibyuma (amakuru menshi).Ubu buryo butandukanye bukora butandukanye nisesengura riherutse gukorwa kuri BGC zigera kuri miliyoni 1.2 muri genos zigera ku 190.000 zabitswe mu bubiko bwa NCBI RefSeq (BiG-FAM / RefSeq, aha bita RefSeq) 29, bwerekanaga ko peptide ya Synthetase idafite ubukana (NRPS) na synthase ya polyketide; (PKS) BGCs (Amakuru yinyongera).Twasanze kandi GCC 44 (29%) zifitanye isano gusa na RefSeq BGC iyo ari yo yose (\ (\ bar {d} \) RefSeq> 0.4; Igishushanyo 2a nuburyo) na 53 (35%) GCC muri MAG gusa, byerekana ubushobozi. gutahura imiti itavuzwe mbere muri OMD.Urebye ko buri imwe muri izi GCC ishobora kuba igereranya imikorere ya biosynetike itandukanye cyane, twongeye gusesengura amakuru kurwego rwa GCF mu rwego rwo gutanga amatsinda arambuye ya BGCs yahanuwe kode y'ibicuruzwa bisanzwe29.Igiteranyo cya 3861 (56%) byagaragaye ko GCFs itajyanye na RefSeq, kandi> 97% bya GCF ntabwo bari bahari muri MIBiG, imwe mububiko bunini bwa BGC zemewe nubushakashatsi (Ishusho 2b).Nubwo bidatangaje kuvumbura inzira nyinshi zishobora kubaho muburyo bushya butagaragajwe neza na genome yerekanwe, uburyo bwacu bwo gupfobya BGC muri GCF mbere yuko ibipimo bitandukanye na raporo zabanjirije iyi 16 kandi bikadufasha gutanga isuzuma ritabogamye ryibishya.Byinshi mubintu bishya bitandukanye (3012 GCF cyangwa 78%) bihuye na terpène zahanuwe, RiPP cyangwa nibindi bicuruzwa bisanzwe, kandi ibyinshi (1815 GCF cyangwa 47%) byanditse muburyo butazwi kubera ubushobozi bwa biosynetique.Bitandukanye na PKS na NRPS cluster, izi BGC zidahwitse ntizishobora gucikamo ibice mugihe cyo guterana kwa metagenomic 31 kandi zitanga umwanya munini hamwe nubutunzi-bushingiye kumikorere yibicuruzwa byabo.
BGC zose hamwe 39,055 zashyizwe hamwe muri GCF 6.907 na GCC 151.a, amakuru yerekana (imbere hanze).Ihuriro ryimiterere yintera ya BGC ishingiye kuri GCC, 53 muri yo ikosorwa na MAG gusa.GCC irimo BGCs kuva muri tagisi zitandukanye (ln yahinduwe amarembo yumurongo) hamwe nibyiciro bitandukanye bya BGC (ingano yumuzingi ihuye numurongo wacyo).Kuri buri GCC, urwego rwinyuma rugaragaza umubare wa BGCs, ubwiganze (ijanisha ryintangarugero), nintera (intera nto ya BGC cosine (min (dMIBiG))) kuva BiG-FAM kugeza BGC.GCCs hamwe na BGCs zifitanye isano rya hafi na BGCs zageragejwe (MIBiG) zigaragazwa nimyambi.b Ugereranije GCF na Byahanuwe (BiG-FAM) kandi byemewe (MIBiG) BGCs, 3861 nshya (d–> 0.2) GCFs zabonetse.Byinshi (78%) byiyi code ya RiPP, terpene, nibindi bicuruzwa bisanzwe.c, genome zose ziri muri OMD ziboneka muri 1038 metagenomes zo mu nyanja zashyizwe mu giti fatizo cya GTDB kugirango zerekane phylogeneque ya OMD.Imyenda idafite genome iyo ari yo yose muri OMD irerekanwa imvi.Umubare wa BGCs uhuye numubare munini wa BGC zahanuwe kuri genome mugice runaka.Kubisobanutse, 15% yanyuma ya node irasenyutse.Imyambi yerekana clade ikungahaye kuri BGC (> 15 BGC), usibye Mycobacterium, Gordonia (uwa kabiri nyuma ya Rhodococcus), na Crocosphaera (uwa kabiri nyuma ya Synechococcus).d, Ntazwi c.Eremiobacterota yerekanye ibinyabuzima bitandukanye cyane (indangagaciro ya Shannon ishingiye ku bwoko bwibicuruzwa bisanzwe).Buri tsinda ryerekana genome hamwe na BGC nyinshi mubwoko.T1PKS, PKS ubwoko bwa I, T2 / 3PKS, PKS ubwoko bwa II n'ubwoko bwa III.
Usibye ubukire nudushya, turasesengura imiterere ya biogeografi yubushobozi bwa biosintetike ya mikorobe yo mu nyanja.Gutondekanya ingero ukurikije ikigereranyo cya metagenomic GCF ikwirakwizwa rya kopi (Methods) yerekanaga ko uburinganire buke, hejuru, hejuru ya prokaryyotike ikungahaye hamwe na virusi ikennye cyane cyane biturutse ku mazi y’izuba cyangwa byimbitse, byari bikungahaye kuri terpene ya RiPP na BGC.Ibinyuranye, polar, inyanja-nyanja, virusi- hamwe n’ibice bikungahaye ku bice bifitanye isano n’ubwinshi bwa NRPS na PKS BGC (amakuru yagutse, Ishusho ya 4 nandi makuru yinyongera).Hanyuma, twasanze ko ubushakashatsi bwizewe bushyuha hamwe nubutaka bwa pelagisiya nisoko itanga ibyiringiro bya terpene nshya (Augmented Data Figure).Ubushobozi buhanitse kuri PKS, RiPP nibindi bicuruzwa bisanzwe (Igicapo 5a hamwe namakuru yagutse).
Kugira ngo twuzuze ubushakashatsi bwacu ku binyabuzima bya biosintetike ya mikorobe yo mu nyanja, twari tugamije gushushanya ikwirakwizwa rya phylogeneque no kumenya clade nshya ikungahaye kuri BGC.Kugira ngo ibyo bishoboke, twashyize genome ya mikorobe zo mu nyanja muri bacteri zisanzwe za GTDB13 na phylogeneque ya archaeal hanyuma dushyira hejuru inzira ya biosynetike ishiramo (Ishusho 2c).Twabonye byoroshye clade nyinshi ikungahaye kuri BGC (ihagarariwe na BGC zirenga 15) mubitegererezo by'amazi yo mu nyanja (uburyo) bizwiho ubushobozi bwa biosintetike, nka cyanobacteria (Synechococcus) na bagiteri za Proteus, nka Tistrella32,33, cyangwa vuba aha bikurura abantu kubitekerezo byabo ibicuruzwa bisanzwe.nka Myxococcota (Sandaracinaceae), Rhodococcus na Planctomycetota34,35,36.Igishimishije, twasanze imirongo myinshi itarashakishwa muriyi clade.Kurugero, ubwo bwoko bufite ubushobozi bwa biosynetike bukize cyane muri phyla Planctomycetota na Myxococcota bwari ubw'abakandida batamenyekanye hamwe na genera, (Imbonerahamwe y'inyongera 3).Ufatiye hamwe, ibi byerekana ko OMD itanga uburyo bwo kumenya amakuru ya phylogeneque atazwi, harimo na mikorobe, ishobora kugereranya intego nshya za enzyme no kuvumbura ibicuruzwa bisanzwe.
Ibikurikira, twaranze clade ikungahaye kuri BGC tutabariyemo gusa umubare ntarengwa wa BGCs washyizweho nabanyamuryango bayo, ariko tunasuzuma ubudasa bwaba BGCs, busobanura inshuro zubwoko butandukanye bwibicuruzwa bisanzwe byabakandida (Ishusho 2c nuburyo bukoreshwa) )..Twasanze amoko atandukanye ya biosynthetique atandukanye yari ahagarariwe na bagiteri yihariye ya MAGs yakozwe muri ubu bushakashatsi.Izi bagiteri ni izitwa phylum idahingwa Candidatus Eremiobacterota, ikomeje kutagenzurwa usibye ubushakashatsi buke bwa genoside 37,38.Birashimishije kubona “ca.Ubwoko bwa Eremiobacterota bwasesenguwe gusa ku isi 39 kandi ntibizwi ko harimo abanyamuryango bakungahaye muri BGC.Hano twongeye kubaka MAG umunani zubwoko bumwe (indangamuntu ya nucleotide> 99%) 23. Turasaba rero ubwoko bwubwoko bwitwa "Candidatus Eudoremicrobium malaspinii", bwitiriwe nereid (nymph yo mu nyanja), impano nziza mumigani yubugereki no mu rugendo.'Ka.Dukurikije ibisobanuro bya phylogeneque 13, E. malaspinii nta bene wabo bazwi bari munsi yurwego rukurikiranye bityo akaba ari mumuryango mushya wa bagiteri dusaba “Ca.E. malaspinii ”nk'ubwoko bw'ubwoko na“ Ca.Eudormicrobiaceae "nk'izina ryemewe (Amakuru yinyongera).Muri make metagenomic kwiyubaka kwa 'Ca.Umushinga wa genome ya E. malaspinii wemejwe no kwinjiza bike cyane, gusoma birebire bya metagenomic hamwe no guteranya icyerekezo kimwe (Methods) nka chromosome imwe ya 9.63 Mb hamwe na 75 kb kwigana.nkibidasobanutse gusa.
Kugirango tumenye imiterere ya phylogeneque yibi binyabuzima, twashakishije amoko 40 afitanye isano rya hafi mu zindi ngero za metagenomic zikungahaye kuri eukaryotic ziva mu rugendo rw’inyanja ya Tara binyuze mu kongera kubaka genome.Muri make, twahujije gusoma metagenomic nibice bya genomic bijyana na “Ca.E. malaspinii ”kandi yemeza ko kwiyongera kw'abakozi muri uru rugero byerekana ko hari abandi bavandimwe (uburyo).Nkigisubizo, twasanze MAG 10, ihuriro rya MAGs 19 zerekana amoko atanu muri genera eshatu mumuryango mushya wasobanuwe (ni ukuvuga “Ca. Eudormicrobiaceae”).Nyuma yo kugenzura intoki no kugenzura ubuziranenge (amakuru yagutse, ishusho ya 6 nandi makuru yinyongera), twasanze “Ca.Ubwoko bwa Eudormicrobiaceae bugaragaza genome nini (8 Mb) hamwe nubushobozi bwa biosynetike (14 kugeza 22 BGC kuri buri bwoko) kurusha abandi banyamuryango ba "Ca".Clade Eremiobacterota (kugeza kuri 7 BGC) (Ishusho 3a - c).
a, Imyanya ya phylogeneque ya batanu 'Ca.Ubwoko bwa Eudormicrobiaceae bwerekanye ubukire bwa BGC bwihariye kumirongo yinyanja yagaragaye muri ubu bushakashatsi.Igiti cyitwa phylogeneque kirimo 'Ca.MAG Eremiobacterota hamwe nabandi bagize phyla (nimero ya genome mumutwe) yatanzwe muri GTDB (verisiyo ya 89) yakoreshejwe muburyo bwubwihindurize (Methods).Ibice byo hanze byerekana ibyiciro kurwego rwumuryango (“Ca. Eudormicrobiaceae” na “Ca. Xenobiaceae”) no kurwego rwishuri (“Ca. Eremiobacteria”).Ubwoko butanu bwasobanuwe muri ubu bushakashatsi bugereranywa na code ya nyuguti n’amazina yatanzwe (Amakuru yinyongera).b, ok.Ubwoko bwa Eudormicrobiaceae busangiye nuclei zirindwi zisanzwe.Kubura kwa BGC mu cyiciro cya A2 byatewe no kutuzuza kwa MAG uhagarariye (Imbonerahamwe y'inyongera 3).BGCs yihariye "Ca.Amphithomicrobium ”na“ Ca.Amphithomicrobium ”(clade A na B) ntabwo bigaragara.c, BGC zose zifite kodegisi nka "Ca.Eudoremicrobium taraoceanii wasangaga igaragara muri 623 metatranscriptomes yakuwe mu nyanja ya Tara.Uruziga rukomeye rwerekana transcript.Uruziga rwa orange rwerekana log2 yahinduwe inshuro munsi no hejuru yikigereranyo cyo kugaburira urugo (uburyo).d, ugereranije ubwinshi bwimirongo (uburyo) yerekana 'Ca.Ubwoko bwa Eudormicrobiaceae bwamamaye cyane mu bibaya byinshi byo mu nyanja no mu nkingi zose z’amazi (kuva hejuru kugeza ku burebure bwa metero 4000).Dushingiye kuri ibi bigereranyo, twasanze 'Ca.E. malaspinii 'igera kuri 6% ya selile ya prokaryyotike mu nyanja-nyanja ya pelagasi ifitanye isano n’ingano.Twatekereje ku bwoko bugaragara kurubuga niba bwarabonetse mubice byose byubunini bwurwego rwimbitse.IO - Inyanja y'Ubuhinde, NAO - Amajyaruguru ya Atalantika, NPO - Pasifika y'Amajyaruguru, RS - Inyanja Itukura, SAO - Atlantike y'Amajyepfo, SO - Inyanja y'Amajyepfo, SPO - Pasifika y'Amajyepfo.
Kwiga ubwinshi no gukwirakwiza Ca.Eudormicrobiaceae, nkuko twabibonye, ​​yiganje mu bibaya byinshi byo mu nyanja, ndetse no mu nkingi zose z’amazi (Ishusho 3d).Muri rusange, bagize 6% byumuryango wa mikorobe yo mu nyanja, bikabagira igice cyingenzi cya mikorobe yisi yose.Mubyongeyeho, twasanze ibintu bijyanye na Ca.Ubwoko bwa Eudormicrobiaceae nubwoko bwazo bwa BGC bwari hejuru cyane mu gice gikungahaye kuri eukaryotic (Igicapo cya 3c hamwe namakuru yagutse, Igicapo 7), byerekana imikoranire ishoboka nibintu bito, harimo na plankton.Iyi ndorerezi ifite aho ihuriye na 'Ca.Eudoremicrobium BGCs itanga ibicuruzwa bisanzwe bya cytotoxique binyuze munzira zizwi zirashobora kwerekana imyitwarire yinyamaswa (Amakuru yinyongera namakuru yagutse, Ishusho 8), bisa nizindi nyamaswa zangiza cyane metabolite nka Myxococcus41.Ubuvumbuzi bwa Ca.Eudormicrobiaceae muboneka gake (inyanja yimbitse) cyangwa eukaryotic aho kuba prokaryotic ntangarugero irashobora gusobanura impamvu izo bagiteri nubudasa bwazo bwa BGC butunguranye zikomeje kuba zidasobanutse murwego rwubushakashatsi bwibiribwa bisanzwe.
Ubwanyuma, twashatse kwemeza kugerageza amasezerano yimirimo yacu ishingiye kuri mikorobe mugushakisha inzira nshya, enzymes, nibicuruzwa bisanzwe.Mu byiciro bitandukanye bya BGCs, inzira ya RiPP izwiho gushyiramo imiti ikungahaye kandi ikora bitewe nuburyo butandukanye nyuma yubusobanuro bwahinduwe bwa peptide yibanze na enzymes zikuze42.Twahisemo rero 'Ca.Eudoremicrobium 'RiPP BGCs (Ishusho 3b na 4a-e) ishingiye kuri kimwe na BGC izwi (\ (\ bar {d} \) MIBiG na \ (\ bar {d} \) RefSeq iri hejuru ya 0.2).
a - c, Muri vitro heterologous imvugo no muri vitro enzymatique yerekana igitabo (\ (\ bar {d} \) RefSeq = 0.29) cluster ya RiPP biosynthesis yihariye kubwoko bwimbitse bwa Ca.E. malaspinii 'yatumye habaho umusaruro wa diphosifora.c, impinduka zagaragaye ukoresheje ibisubizo bihanitse (HR) MS / MS (gucikamo ibice byerekanwa na b na y ion muburyo bwa shimi) na NMR (amakuru yagutse, Ishusho 9).d, iyi peptide ya fosifori yerekana micromolar nkeya yo kubuza inyamaswa z’inyamabere neutrophil elastase, zitaboneka muri peptide yo kugenzura hamwe na peptide ya dehidratifike (kuvanaho imiti itera umwuma).Ubushakashatsi bwasubiwemo inshuro eshatu nibisubizo bisa.Kurugero, imvugo itandukanye ya roman ya kabiri \ (\ bar {d} \) RefSeq = 0.33) cluster ya protein biosynthesis isobanura imikorere yimisemburo ine ikuze ihindura peptide 46 ya amine acide.Ibisigara byandujwe ukurikije urubuga rwo guhindura byahanuwe na HR-MS / MS, ikirango cya isotope, hamwe nisesengura rya NMR (Amakuru yinyongera).Ibara rya Dashe ryerekana ko ihinduka riba kuri kimwe mubisigisigi byombi.Igishushanyo ni icyegeranyo cyinyubako zitandukanye zerekana ibikorwa bya enzymes zose zikuze kuri nucleus imwe.h, Ishusho yamakuru ya NMR kumugongo amide N-methylation.Ibisubizo byuzuye birerekanwa mugitabo.10 hamwe namakuru yagutse.i, Umwanya wa Phylogenetike wumusemburo wa FkbM ukuze wa enzyme muri domaine zose za FkbM dusanga mububiko bwa MIBiG 2.0 ugaragaza enzyme yuyu muryango ufite ibikorwa bya N-methyltransferase (Amakuru yinyongera).Igishushanyo mbonera cya BGCs (a, e), imiterere ya peptide ibanziriza (b, f), hamwe nubushakashatsi bwimiti yibicuruzwa bisanzwe (c, g) birerekanwa.
Inzira ya mbere ya RiPP (\ (\ bar {d} \) MIBiG = 0.41, \ (\ bar {d} \) RefSeq = 0.29) yabonetse gusa mu bwoko bwimbitse bwinyanja “Ca.E. malaspinii ”hamwe na code ya Peptide- ibanziriza (Ishusho 4a, b).Muri iyi misemburo ikuze, twabonye indangarubuga imwe ikora homologous to dehydration domaine ya lantipeptide synthase isanzwe itera fosifora no gukuraho 43 (Amakuru yinyongera).Kubwibyo, turahanura ko guhindura peptide ibanziriza iyinjizamo intambwe ebyiri.Ariko, dukoresheje tandem mass spectrometrie (MS / MS) hamwe na magnetiki resonance spectroscopi (NMR), twabonye peptide ya polifosifora (Igicapo 4c).Nubwo tutari twiteze, twabonye imirongo myinshi yibimenyetso byemeza ko ari ibicuruzwa byanyuma: ibyiciro bibiri bitandukanye bya heterologique kandi nta dehidrasiyo muri vitro assays, kumenya ibisigazwa byingenzi byahinduwe muri catalitike dehydrasi ya enzyme ikuze.byose byubatswe na “Ca”.Ubwoko bwa E. malaspinii (amakuru yaguwe, Ishusho ya 9 nandi makuru yinyongera) hanyuma, amaherezo, ibikorwa byibinyabuzima byibicuruzwa bya fosifori, ariko ntabwo ari uburyo bwa chimique yashizwemo umwuma (Ishusho 4d).Mubyukuri, twasanze igaragaza ibikorwa bike bya micromolar protease yo kubuza ibikorwa bya neutrophil elastase, ugereranije nibindi bicuruzwa bifitanye isano n’ibicuruzwa (IC50 = 14.3 μM) 44, nubwo uruhare rw’ibidukikije rugikomeza gusobanurwa.Dufatiye kuri ibyo bisubizo, turasaba kuvuga inzira inzira "fosifeti".
Urubanza rwa kabiri ni inzira igoye ya RiPP yihariye 'Ca.Ubwoko bwa Eudoremicrobium (\ (\ bar {d} \) MIBiG = 0.46, \ (\ bar {d} \) RefSeq = 0.33) byahanuwe ko bikubiyemo ibicuruzwa bya poroteyine karemano (Ishusho 4e).Izi nzira zifite inyungu zihariye za tekinoloji kubera ubucucike buteganijwe nubwoko butandukanye bwo guhindura imiti idasanzwe yashyizweho na enzymes yashizweho na BGCs45 ugereranije.Twabonye ko iyi poroteyine itandukanye na poroteyine zaranzwe mbere kubera ko idafite motif nyamukuru ya NX5N ya polyceramide hamwe na lanthionine loop ya landornamide 46.Kugira ngo tuneshe imbogamizi zisanzwe zerekana imiterere, twakoresheje hamwe na sisitemu ya Microvirgula aerodenitrificans yihariye kugirango tumenye imisemburo ine ikuze (inzira).Twifashishije uruvange rwa MS / MS, ikirango cya isotope, na NMR, twabonye iyo misemburo ikuze muri 46-amino acide ya peptide (Igishusho 4f, g, amakuru yagutse, Ishusho 10-12 namakuru yinyongera).Muri enzymes zikuze, twaranze isura yambere yumuryango wa FkbM O-methyltransferase 47 mumihanda ya RiPP kandi tutunguranye dusanga iyi misemburo ikuze itangiza umugongo N-methylation (Ishusho 4h, i namakuru yinyongera).Nubwo iri hinduka rizwi mubicuruzwa bisanzwe bya NRP48, N-methylation ya enzymatique ya amide ingwate ni ingorabahizi ariko ikorana buhanga ryibinyabuzima 49 kugeza ubu imaze gushimisha umuryango RiPP wa borosine.Umwihariko 50,51.Kumenyekanisha iki gikorwa mumiryango yindi ya enzymes na RiPP birashobora gufungura porogaramu nshya no kwagura imikorere ya poroteyine 52 nuburyo butandukanye bwimiti.Dushingiye ku byahinduwe byagaragaye hamwe n'uburebure budasanzwe bw'imiterere y'ibicuruzwa byatanzwe, turasaba izina ry'inzira "pythonamide".
Ivumburwa rya enzymologiya itunguranye mumiryango iranga imikorere yimisemburo irerekana isezerano rya genomics yibidukikije kubuvumbuzi bushya, kandi ikanagaragaza ubushobozi buke bwo gufata umwanzuro ushingiye kubikorwa bya homologiya yonyine.Rero, hamwe na raporo za bioactive polyphosphorylated RiPPs idafite ibisubizo byemewe, ibisubizo byacu byerekana umutungo-mwinshi ariko agaciro gakomeye kubikorwa bya biologiya ya sintetike kugirango tumenye neza ubutunzi bukora, ubudasa, nuburyo budasanzwe bwibinyabuzima bya biohimiki.
Hano turerekana urwego rwibinyabuzima rushobora gukoreshwa na mikorobe hamwe na genomique yabyo muri microbiome yisi yose, byorohereza ubushakashatsi buzaza mugutanga ibikoresho bivuye mubumenyi bwa siyanse (https://microbiomics.io/ocean/).Twasanze ibyinshi mubikorwa bya phylogeneque nibikorwa bikora bishobora kuboneka gusa mukubaka MAGs na SAGs, cyane cyane mumiryango mikorobe idakoreshwa neza ishobora kuyobora ingufu za bioprospecting.Nubwo tuzibanda hano kuri 'Ca.Eudormicrobiaceae "nkumurongo cyane cyane biosynthetique" ufite impano ", benshi muri BGCs bahanuye muri microbiota itavumbuwe birashoboka ko ikubiyemo enzimologiya zidasobanuwe zitanga ibice hamwe nibikorwa byangiza ibidukikije na / cyangwa ikoranabuhanga.
Imibare ya Metagenomic yavuye mubushakashatsi bunini bw'inyanja hamwe nubushakashatsi bwakurikiranye hamwe nuburebure bukurikiranye bwashyizwemo kugirango habeho gukwirakwiza amakuru kuri mikorobe yo mu nyanja ku isi mu kibaya cy'inyanja, ibice byimbitse kandi uko ibihe bigenda bisimburana.Iyi mibare (Imbonerahamwe yinyongera 1 nishusho 1) ikubiyemo metagenomics yavuye mubitegererezo byakusanyirijwe mu nyanja ya Tara (virusi ikungahaye, n = 190; prokaryotic ikungahaye, n = 180) 12,22 hamwe na BioGEOTRACES (n = 480).Ibihe byo mu nyanja ya Hawayi (HOT, n = 68), Bermuda-Atlantike Yigihe (BATS, n = 62) 21 hamwe na Malaspina Expedition (n = 58) 23.Urukurikirane rwasomwe mubice byose bya metagenomic byayungurujwe kugirango ube mwiza ukoresheje BBMap (v.38.71) ukuraho adapteri zikurikirana mubisomwa, kuvanaho ibisomwa byashizwe kumurongo ugenzura ubuziranenge (genome ya PhiX), no gukoresha trimq = 14, maq = 20 birukana ubuziranenge bwo gusoma, maxns = 0 na minlength = 45. Isesengura ryakurikiyeho ryakozwe cyangwa ryahujwe na QC isoma niba byasobanuwe (bbmerge.sh minoverlap = 16).Gusoma QC byari bisanzwe (bbnorm.sh intego = 40, minddepth = 0) mbere yo kubaka ukoresheje metaSPAdes (v.3.11.1 cyangwa v.3.12 niba bikenewe) 53.Ibisubizo bivamo scafold (nyuma yiswe scafolds) byarangije gushungura muburebure (≥1 kb).
Ingero 1038 za metagenomic zagabanyijwemo amatsinda, kandi kuri buri tsinda ryintangarugero, igenzura ryubuziranenge bwa metagenomic ryasomwe ryintangarugero zose ryahujwe nimirongo ya buri cyitegererezo ukwacyo, bivamo umubare ukurikira witsinda ryibice bibiri byanditseho ngo: Virusi ya Tara Marine - Ikungahaye. .Ikarita yakozwe hakoreshejwe Burrows-Wheeler-Aligner (BWA) (v.0.7.17-r1188) 54 ituma ibyasomwe bihuzwa nimbuga za kabiri (ukoresheje -a ibendera).Guhuza byayunguruwe kugirango byibuze byibuze 45 shingiro, bifite indangamuntu 9797%, kandi bisomwe 80%.Amadosiye ya BAM yavuyemo yatunganijwe hifashishijwe inyandiko ya jgi_summarize_bam_contig_depths ya MetaBAT2 (v.2.12.1) 55 kugirango itange ubwishingizi hagati yintangarugero kuri buri tsinda.Hanyuma, utwugarizo twashyizwe hamwe kugirango twongere sensibilité ukoresheje MetaBAT2 kugiti cye kuri sample zose hamwe na –minContig 2000 na –maxEdges 500. Dukoresha MetaBAT2 aho gukoresha bateramakofe kuko byagaragaye mubizamini byigenga kugirango bibe umukinnyi mwiza w'iteramakofe.ninshuro 10 kugeza kuri 50 kurenza abandi bakinnyi bateramakofe 57.Kugirango ugerageze ingaruka zifitanye isano ryinshi, icyitegererezo cyatoranijwe cya metagenomics (10 kuri buri dataseti ebyiri zo mu nyanja ya Tara, 10 kuri BioGEOTRACES, 5 kuri buri gihe cyurukurikirane, na 5 kuri Malaspina) hiyongereyeho urugero gusa.Ingero zimbere zishyizwe hamwe kugirango zibone amakuru yo gukwirakwiza.(Amakuru yinyongera).
Iyindi genome (yo hanze) yashyizwe mubisesengura byakurikiyeho, aribyo 830 byatoranijwe na MAGs uhereye mugice cya dataset ya Tara Oceans26, 5287 SAGs ziva muri dataset ya GORG20, hamwe namakuru yo muri data base ya MAR (MarDB v. 4) kuva 1707 yitaruye REF na 682 SAG) [I | i] kwigunga '.
Ubwiza bwa buri kintu cya metagenomic na genome zo hanze byasuzumwe hifashishijwe CheckM (v.1.0.13) hamwe na Anvi'o's Lineage Workflow (v.5.5.0) 58,59.Niba CheckM cyangwa Anvi'o ivuga ≥50% byuzuye / byuzuye hamwe na ≤10% byanduye / birenze, noneho uzigame selile metagenomic na genome zo hanze kugirango ubisesengure nyuma.Aya manota noneho yahurijwe hamwe muburyo bwuzuye (mcpl) kandi bivuze kwanduza (mctn) kugirango ashyire mubyiciro bya genome ukurikije ibipimo byabaturage 60 kuburyo bukurikira: ubuziranenge: mcpl ≥ 90% na mctn ≤ 5%;ubuziranenge bwiza: mcpl ≥ 70%, mctn ≤ 10%, ubuziranenge bwo hagati: mcpl ≥ 50% na mctn ≤ 10%, ubuziranenge: mcpl ≤ 90% cyangwa mctn ≥ 10%.Akayunguruzo ka genome noneho kahujwe n amanota meza (Q na Q ') kuburyo bukurikira: Q = mcpl - 5 x mctn Q' = mcpl - 5 x mctn + mctn x (impinduka zoroshye) / 100 + 0.5 x log [N50].(yashyizwe mu bikorwa muri dRep61).
Kugirango habeho isesengura rigereranya hagati yamakuru atandukanye nubwoko bwa genome (MAG, SAG na REF), genome 34.799 zasuzumwe hashingiwe kuri genome yagutse ya nucleotide (ANI) ikoresheje dRep (v.2.5.4).Gusubiramo) 61 hamwe 95% bya ANI bingana 28,62 (-comp 0 -con 1000 -sa 0,95 -nc 0.2) hamwe na kopi imwe ya kopi imwe ikoresheje SpecI63 itanga genome ihurira kurwego rwubwoko.Hatoranijwe genome ihagarariye kuri buri tsinda rya dRep ukurikije amanota meza (Q ') yasobanuwe haruguru, yafatwaga nk'uhagarariye amoko.
Kugirango usuzume ikarita yerekana ikarita, BWA (v.0.7.17-r1188, -a) yakoreshejwe mu gushushanya ibice 1038 byose byasomwe na metagenomic hamwe na genome 34.799 zikubiye muri OMD.Ubuziranenge bugenzurwa nubushakashatsi bwashushanijwe muburyo bumwe bwarangiye kandi ibivuyemo byayungurujwe kugirango bigumane gusa ≥45 bp muburebure.n'irangamuntu ≥95%.Ikigereranyo cyo kwerekana kuri buri cyitegererezo ni ijanisha ryibisomwa bisigaye nyuma yo kuyungurura bigabanijwe numubare rusange wibisomwa byubuziranenge.Ukoresheje uburyo bumwe, buri metagenomes 1038 yagabanutse kugera kuri miliyoni 5 zinjizwamo (amakuru yagutse, Ishusho 1c) ihuzwa na GORG SAG muri OMD no muri GEM16 yose.Ingano ya MAGs yakuwe mu mazi yo mu nyanja muri kataloge ya GEM16 yagenwe nijambo ryibanze ryibibazo bituruka kuri metagenomic, guhitamo icyitegererezo cyamazi yo mu nyanja (urugero, bitandukanye nubutaka bwo mu nyanja).By'umwihariko, duhitamo "amazi" nka "ecosystem_category", "marine" nka "ecosystem_type", hanyuma tuyungurura "ubuturo" nk "inyanja ndende", "marine", "inyanja yo mu nyanja", "marine pelagic", "amazi yo mu nyanja", “Inyanja”, “Amazi yo mu nyanja”, “Amazi yo ku nyanja”, “Amazi yo ku nyanja”.Ibi byavuyemo 5903 MAGs (734 nziza cyane) yatanzwe hejuru ya 1823 OTU (reba hano).
Prokaryotic genome yasobanuwe mumatagisi ikoresheje GTDB-Tk (v.1.0.2) 64 hamwe nibisanzwe bitareba GTDB r89 verisiyo ya 13. Anvi'o yakoreshejwe kugirango hamenyekane genome ya eukaryotic ishingiye ku guhanura kwa domaine no kwibutsa ≥50% nubucucike ≤ 10%.Amatagisi ya tagisi yubwoko asobanurwa nkimwe muri genome zihagarariye.Usibye eukaryote (148 MAG), buri genome yabanje gutondekwa mumikorere ikoresheje prokka (v.1.14.5) 65, ivuga amazina yuzuye, isobanura ibipimo bya "archaea" cyangwa "bagiteri" nkuko bikenewe, binatangazwa kubatari- gen.n'uturere twa CRISPR, mubindi biranga genomic.Andika genes zahanuwe muguhitamo rusange kopi imwe ya kopi ya marike (uscMG) ukoresheje fetchMG (v.1.2) 66, tanga amatsinda ya orthologue hamwe nibibazo ukoresheje emapper (v.2.0.1) 67 ishingiye kumagiNOG (v.5.0) 68.Ububiko bwa KEGG (bwatangajwe ku ya 10 Gashyantare 2020) 69. Intambwe yanyuma yakozwe no guhuza poroteyine na base ya KEGG ukoresheje DIAMOND (v.0.9.30) 70 hamwe n'ikibazo hamwe n'ingingo zingana na 70%.Ibisubizo byongeye gushungura ukurikije NCBI Prokaryotic Genome Annotation Pipeline71 ishingiye kuri bitrate ≥ 50% ya bitrate iteganijwe (ihuza ubwayo).Urutonde rwa Gene rwakoreshejwe kandi nk'injiza kugirango hamenyekane BGC muri genome ukoresheje antiSMASH (v.5.1.0) 72 hamwe n'ibipimo bidasanzwe hamwe no guturika kwa cluster zitandukanye.Genome zose hamwe na annotations byakusanyirijwe muri OMD hamwe na metadata ijyanye no kuboneka kurubuga (https://microbiomics.io/ocean/).
Kimwe nuburyo bwasobanuwe mbere12,22 twakoresheje CD-HIT (v.4.8.1) kugirango dukusanyirize hamwe> miriyoni 56,6 za protein-coding genes ziva muri bagiteri na archaeal genome kuva OMD ikagera kuri 95% hamwe na genes ngufi (90% ubwishingizi) 73 kugeza kuri > Miliyoni 17.7.Urutonde rurerure rwatoranijwe nka gene ihagarariye buri gene cluster.Metagenomes 1038 yahise ihuzwa na miliyoni 17.7 z'abanyamuryango ba cluster ya BWA (-a) hanyuma amadosiye ya BAM yavuyemo arayungurura kugirango agumane gusa guhuza indangamuntu ya 95% hamwe na 4545.Uburebure-busanzwe bwa gene ubwinshi bwabazwe kubanza kubara ibyinjijwe uhereye kumurongo mwiza wihariye hanyuma hanyuma, kubintu byashizwemo fuzzy, wongeyeho kubara uduce duto kuri genes igenewe ugereranije numubare wabo winjiza udasanzwe.
Genome ziva muri OMD yagutse (hamwe na MAGs ziyongereye kuri "Ca. Eudormicrobiaceae", reba hano hepfo) zongewe kububiko bwibikoresho bya mOTUs74 metagenomic isesengura ibikoresho (v.2.5.1) kugirango habeho ububiko bwagutse bwa MOTU.Batandatu gusa bonyine bakoporora (23,528 genome) barokotse muri uscMGs icumi.Kwagura ububikoshingiro byatumye amatsinda 4.494 yiyongera ku rwego rwubwoko.1038 metagenomes yasesenguwe hakoreshejwe ibipimo bya mOTU isanzwe (v.2).Genome zose hamwe 989 zikubiye muri 644 ya mOTU (95% REF, 5% SAG na 99,9% bya MarDB) ntabwo zagaragaye numwirondoro wa mOTU.Ibi birerekana amasoko atandukanye yinyongera yo mu nyanja ya genD ya MarDB (genome nyinshi zitamenyekanye zifitanye isano nibinyabuzima bitandukanijwe nubutaka, ibinyabuzima byo mu nyanja, nibindi).Kugirango dukomeze kwibanda ku bidukikije byo mu nyanja ifunguye muri ubu bushakashatsi, twabavanye mu isesengura ryo hepfo keretse iyo byamenyekanye cyangwa bikubiye mu bubiko bwagutse bwa mOTU bwakozwe muri ubu bushakashatsi.
BGC zose kuva MAG, SAG na REF muri OMD (reba hejuru) zahujwe na BGCs zagaragaye muri scafolds zose za metagenomic (antiSMASH v.5.0, ibipimo bisanzwe) kandi birangwa no gukoresha BiG-SLICE (v.1.1) (PFAM domaine) 75.Dushingiye kuri ibyo biranga, twabaze intera yose ya cosine hagati ya BGCs hanyuma tuyishyira hamwe (bivuze guhuza) muri GCF na GCC dukoresheje intera ya 0.2 na 0.8.Izi mbuga ni uguhuza inzitizi zakoreshwaga mbere ukoresheje intera ya Euclidean75 hamwe nintera ya cosine, igabanya amwe mumakosa muburyo bwa mbere bwa BiG-SLICE yo gukusanya (Amakuru yinyongera).
BGCs noneho yarayunguruwe kugirango igumane k5 kb gusa yashizwe kuri scafolds kugirango igabanye ibyago byo gucikamo ibice nkuko byasobanuwe mbere16 no gukuraho MarDB REF na SAGs zitaboneka muri metagenomes 1038 (reba hejuru).Ibi byatumye BGCs 39,055 zose zandikwa na genome ya OMD, hiyongereyeho 14,106 zagaragaye ku bice bya metagenomic (ni ukuvuga bitahujwe na MAGs).Izi "metagenomic" BGCs zakoreshejwe mukugereranya igipimo cya microbiome biosynthesis zo mu nyanja zidafashwe mububiko (Amakuru yinyongera).Buri BGC yaranzwe muburyo bukurikije ubwoko bwibicuruzwa byavuzwe byasobanuwe na anti-SMASH cyangwa ibyiciro byibicuruzwa bisobanuwe muri BiG-SCAPE76.Kugirango wirinde kubogama kubogamye (kugereranya amatagisi no gukora ya GCC / GCF, intera ya GCF na GCC yerekanwe kububiko, hamwe na metagenomic ubwinshi bwa GCF), mugukomeza gusa BGC ndende kuri GCF kuri buri bwoko, 39,055 BGCs zongeye gutangwa, bivamo byose hamwe 17,689 BGC.
Agashya ka GCC na GCF kasuzumwe hashingiwe ku ntera iri hagati yububiko (RefSeq base muri BiG-FAM) 29 hamwe n’ubushakashatsi bwakozwe (MIBIG 2.0) 30 BGC.Kuri buri umwe muri 17,689 uhagarariye BGCs, twahisemo intera ntoya ya cosine kuri base de base.Intera ntoya noneho igereranijwe (bivuze) ukurikije GCF cyangwa GCC, nkuko bikwiye.GCF ifatwa nkibishya niba intera igana kububiko irenze 0.2, ijyanye no gutandukana kwiza hagati ya (ugereranije) GCF nibisobanuro.Kuri GCC, duhitamo 0.4, ikubye kabiri urwego rwasobanuwe na GCF, kugirango dufunge umubano muremure hamwe nu murongo.
Ubwinshi bwa metagenomic ya BGC bwagereranijwe nkubwinshi bwikigereranyo cya genoside ya biosynetike (nkuko byagenwe na anti-SMASH) iboneka kumurongo wa gen.Ubwinshi bwa metagenomic ya buri GCF cyangwa GCC noneho bwabazwe nkumubare wabahagarariye BGCs (kuri 17,689).Aya makarita menshi yaje gukurikizwa muburyo bwa selile akoresheje buri cyitegererezo cya mOTU, ari nacyo cyagize uruhare mu gukurikirana (amakuru yagutse, Ishusho 1d).Ubwiyongere bwa GCF cyangwa GCC bwabazwe nkijanisha ryintangarugero hamwe nubwinshi> 0.
Intera ya Euclidea hagati yintangarugero yabazwe uhereye kumurongo usanzwe wa GCF.Intera yagabanutse mubunini ukoresheje UMAP77 hanyuma ibyashizwemo bivamo byakoreshejwe mugukurikirana kutagenzuwe hashingiwe kuri HDBSCAN78.Umubare ntarengwa w'amanota kuri cluster (nuko rero umubare w'amatsinda) ukoreshwa na HDBSCAN ugenwa no kugabanya amahirwe menshi yo kuba umunyamuryango.Amatsinda yamenyekanye (hamwe nurugero rudasanzwe rwurugero rwaya matsinda kugirango abare kubogama muburyo bwo gusesengura ibintu bitandukanye (PERMANOVA)) byageragejwe kubisobanuro bifatika intera ya Euclidea itize ikoresheje PERMANOVA.Impuzandengo ya genome yingero zabazwe hashingiwe ku bwinshi ugereranije na mOTU hamwe nubunini bwa genome bugereranijwe bwabagize genome.By'umwihariko, impuzandengo ya genome ya buri mOTU yagereranijwe nkimpuzandengo yubunini bwa genome yabanyamuryango bayo yakosowe kugirango yuzure (nyuma yo kuyungurura) (urugero, 75% yuzuye genome ifite uburebure bwa 3 Mb ifite ubunini bwa 4 Mb).kuri genome yo hagati ifite ubunyangamugayo ≥ 70%.Impuzandengo ya genome kuri buri sample noneho yabazwe nkigiteranyo cya mOTU ingano ya genome iremereye kubwinshi.
Akayunguruzo ka BGCs zifite genome muri OMD zerekanwa mubiti bya bagiteri na archaeal GTDB (murwego rwa k5 kb, ukuyemo REF na SAG MarDB bitabonetse muri metagenomes 1038, reba hejuru) hamwe nibyiciro byabo byibicuruzwa bishingiye kuri phylogeneque umwanya wa genome (reba hejuru).Twabanje kugabanya amakuru kubwoko, dukoresheje genome hamwe na BGC nyinshi muri ubwo bwoko nkuhagarariye.Kugirango ubone amashusho, abahagarariye barushijeho kwigabanyamo amatsinda yibiti, kandi na none, kuri buri cyuma kigizwe na selile, genome irimo umubare munini wa BGCs yatoranijwe nkuyihagarariye.Ubwoko bukungahaye kuri BGC (byibuze genome imwe ifite> 15 BGCs) byongeye gusesengurwa kubara indangagaciro zitandukanye za Shannon kubwoko bwibicuruzwa byanditse muri izo BGCs.Niba ubwoko bwibicuruzwa byose byahanuwe ari bimwe, imvange yimiti nizindi BGC zigoye (nkuko byahanuwe na anti-SMAH) zifatwa nkubwoko bumwe bwibicuruzwa, tutitaye kubyo zikurikirana muri cluster (urugero: protein-bacteriocin na bacteriocin-proteoprotein fusion umubiri).hybrid).
ADN isigaye (bivugwa ko ari 6 ng) uhereye kuri Malaspina icyitegererezo MP1648, ihuye nicyitegererezo cyibinyabuzima SAMN05421555 kandi ihuye na Illumina SRR3962772 metagenomic yasomwe kugirango isomwe mu gihe gito, itunganywa hakurikijwe protocole ikurikirana ya PacBio hamwe n’ibikoresho byinjira cyane kugirango ukoreshe ibikoresho bya PacBio SMRTbell gDNA ibikoresho (100-980-000) na SMRTbell Express 2.0 ibikoresho byo gutegura icyitegererezo (100-938-900).Muri make, ADN isigaye yaraciwe, irasanwa kandi isukurwa (amasaro ya ProNex) ukoresheje Covaris (g-TUBE, 52104).ADN isukuye noneho ikorerwa gutegura isomero, kwongerwaho imbaraga, kwezwa (amasaro ya ProNex) no guhitamo ingano (> 6 kb, Ubururu bwa Pippin) mbere yintambwe yanyuma yo kwezwa (amasaro ya ProNex) hamwe nuburyo bukurikirana kurubuga rwa Sequel II.
Kongera kubaka bibiri bya mbere ca.Kuri MAG Eremiobacterota, twabonye ANI esheshatu ziyongera> 99% (izi ziri mu gishushanyo cya 3), zabanje kuyungurura zishingiye ku manota yanduye (nyuma yaje kumenyekana ko ari duplications, reba hano hepfo).Twabonye kandi tray yanditseho "Ca".Eremiobacterota ”yavuye mu bushakashatsi butandukanye23 kandi yarayikoresheje hamwe na MAG umunani zo mu bushakashatsi bwacu nk'icyerekezo cya metagenomic yasomwe kuri 633 eukaryotic ikungahaye (> 0.8 µm) ikoresheje BWA (v.0.7.17) Ref -r1188, - ibendera) kumanikwa hasi. gushushanya (miliyoni 5 zasomwe).Hashingiwe ku ikarita yihariye yo gukungahaza (iyungururwa na 95% indangamuntu ihuza na 80% isoma ibyateganijwe), metagenomes 10 (biteganijwe ko izakwirakwizwa ≥5 ×) zatoranijwe kugirango ziteranwe hamwe na 49 metagenomes (biteganijwe ko izakwirakwizwa ≥1 ×) kugirango ihuze ibirimo.Ukoresheje ibipimo bimwe nkuko byavuzwe haruguru, izi ngero zarashizwemo hanyuma hiyongeraho 10 Ca '.MAG Eremiobacterota yagaruwe.Izi MAGs 16 (utabariyemo ebyiri zimaze kuboneka muri data base) zizana umubare rusange wa genome muri OMD yagutse igera kuri 34.815.MAGs zahawe urwego rwamatagisi rushingiye kuri genomic ihuye numwanya muri GTDB.18 MAGs zateshejwe agaciro hakoreshejwe dRep mu moko 5 (intraspecific ANI> 99%) na genera 3 (intrageneric ANI 85% kugeza 94%) mumuryango umwe79.Ubwoko bwabahagarariye ubwoko bwatoranijwe nintoki bushingiye kubunyangamugayo, kwanduza, na N50.Igitekerezo cyatanzwe gitangwa mumakuru yinyongera.
Suzuma ubunyangamugayo no kwanduza 'Ca.MAG Eremiobacterota, twasuzumye ko uscMG ihari, hamwe numurongo- hamwe na domaine yihariye imwe-imwe ya kopi ya marike ikoreshwa na CheckM na Anvi'o.Kumenyekanisha duplicates 2 kuri 40 uscMGs byemejwe no kwiyubaka kwa phylogeneque (reba hano hepfo) kugirango hirindwe ko hashobora kwanduzwa (ibi bihuye na 5% bishingiye kuri genes 40 zerekana).Ubushakashatsi bwiyongereye kuri batanu bahagarariye MAGs 'Ca.Urwego ruto rwanduye muri iyi genoside yubatswe byemejwe ku bwoko bwa Eremiobacterota hakoreshejwe interineti ya Anvi'o igendanwa ishingiye ku bwinshi no guhuza ibice bikurikirana (Amakuru yinyongera) 59.
Kubisesengura rya phylogeneque, twahisemo MAGs eshanu zihagarariye "Ca".Eudormicrobiaceae ”, amoko yose“ Ca.Genome ya Eremiobacterota hamwe nabandi bagize phyla (harimo UBP13, Armatimonadota, Patescibacteria, Dormibacterota, Chloroflexota, Cyanobacteria, Actinobacteria na Planctomycetota) iraboneka kuri GTDB (r89) 13.Izi genome zose zasobanuwe nkuko byasobanuwe mbere yo gukuramo kopi imwe ya marikeri ikuramo na BGC.Genoside ya GTDB yabitswe ukurikije ubunyangamugayo hamwe n'ibipimo byanduye.Isesengura rya phylogeneque ryakozwe hakoreshejwe Anvi'o Phylogenetics 59.Igiti cyubatswe hakoreshejwe IQTREE (v.2.0.3) (amahitamo asanzwe na -bb 1000) 80 ku guhuza poroteyine 39 za tandem ribosomal zagaragajwe na Anvi'o (UMUZIKI, v.3.8.1551) 81.Imyanya ye yagabanutse.gutwikira byibuze 50% ya genome82 na Planctomycecota yakoreshejwe nkitsinda rishingiye kuri topologiya ya GTDB.Igiti kimwe cya 40 uscMGs cyubatswe hakoreshejwe ibikoresho nibipimo bimwe.
Twakoresheje Traitar (v.1.1.2) hamwe nibisanzwe (phenotype, kuva nucleotide) 83 kugirango tumenye imiterere ya mikorobe isanzwe.Twakoze ubushakashatsi ku mibereho ishobora guhitanwa n’inyamaswa dushingiye ku ndangagaciro y’inyamanswa84 iterwa n’ibiri muri poroteyine-coding muri genome.By'umwihariko, dukoresha DIAMOND kugirango tugereranye poroteyine ziri muri genome hamwe na base ya OrthoMCL (v.4) 85 dukoresheje amahitamo - birenze-sensive –id 25 - kubaza-70 marikeri ya gen kubinyamanswa nabatayinyamaswa.Ironderero ni itandukaniro riri hagati yumubare w’inyamanswa n’ibidahiga.Nkigenzura ryinyongera, twasesenguye genome ya "Ca".Entotheonella TSY118 ibintu bishingiye kubufatanye bwayo na Ca.Eudoremicrobium (ingano nini ya genome nubushobozi bwa biosynetique).Ibikurikira, twagerageje guhuza isano iri hagati yinyamanswa ninyamaswa zangiza inyamaswa hamwe nubushobozi bwa biosintetike ya Ca.Eudormicrobiaceae ”kandi yasanze nta gen zirenze imwe (zivuye mu bwoko ubwo ari bwo bwose bwerekana ibimenyetso, ni ukuvuga inyamaswa zangiza / zidahiga) zihura na BGC, byerekana ko BGC itavangavanga ibimenyetso byo guhiga.Andi matangazo yandi ya genomic ya kopi yakozwe yakozwe hakoreshejwe TXSSCAN (v.1.0.2) kugirango asuzume neza sisitemu yo gusohora, pili, na flagella86.
Batanu bahagarariye 'Ca's bashushanyijeho gushushanya 623 metatranscriptomes yo mu bice bya prokaryotic na eukaryotic bikungahaye ku nyanja ya Tara 22,40,87 (ukoresheje BWA, v.0.7.17-r1188, -a ibendera).Eudormicrobiaceae genome.Amadosiye ya BAM yatunganijwe hamwe na FeatureCounts (v.2.0.1) 88 nyuma ya 80% yasomye ubwirinzi hamwe na 95% yo kuyungurura indangamuntu (hamwe namahitamo arangaCounts - primaire -O –igice -t CDS, tRNA -F GTF -g ID -p) Iharura the umubare winjiza kuri gen.Ikarita yakozwe yari isanzwe kuburebure bwa gene hamwe na marike ya gene mOTU (uburebure-busanzwe bwo kugereranya kwinjiza gen hamwe no kubara> 0) hanyuma ihindurwa kuri 22.74 kugirango ibone imvugo igereranije kuri selile ya buri rwego rwa gen, nayo isobanura guhinduka kuva kurugero kugeza kurugero mugihe gikurikiranye.Ibipimo nkibi byemerera gusesengura kugereranya, kugabanya ibibazo byihimbano mugihe ukoresheje amakuru menshi ugereranije.Gusa ingero zifite> 5 kuri 10 za marike ya mOTU zasuzumwe kugirango zisesengurwe kugirango igice kinini gihagije cya genome kiboneke.
Umwirondoro usanzwe wa 'Ca.E. taraoceanii yagabanutse kugabanuka ukoresheje UMAP hanyuma guhagararirwa kwakoreshejwe mugukurikirana kutagenzuwe ukoresheje HDBSCAN (reba hejuru) kugirango umenye imiterere yimvugo.PERMANOVA igerageza akamaro ko gutandukana hagati yama cluster yagaragaye mumwanya wambere (utagabanijwe).Imvugo itandukanye hagati yibi bihe yageragejwe kuri genome (reba hejuru) kandi inzira 201 za KEGG zagaragaye mumatsinda 6 akora, aribyo: BGC, sisitemu yo gusohora hamwe na genellar flags kuva TXSSCAN, enzymes de degradation (protease na peptidase), hamwe ninyamaswa zangiza kandi zitari- ingirabuzimafatizo.ibimenyetso byerekana ibimenyetso.Kuri buri cyitegererezo, twabaze imvugo isanzwe isanzwe kuri buri cyiciro (menya ko imvugo ya BGC ubwayo ibarwa nkikigereranyo cyo hagati ya genoside ya biosynetique kuri iyo BGC) kandi twageragejwe kubisobanuro muri leta zose (Ikizamini cya Kruskal-Wallis cyahinduwe kuri FDR).
Ingirabuzima fatizo zaguzwe muri GenScript naho PCR primers yaguzwe muri Microsynth.Phusion polymerase yo muri Thermo Fisher Scientific yakoreshejwe mugukomeza ADN.NucleoSpin plasmide, NucleoSpin gel hamwe nibikoresho bya PCR byoza muri Macherey-Nagel byakoreshejwe mugusukura ADN.Enzymes zo kubuza na T4 ligase yaguzwe muri New England Biolabs.Imiti itari isopropyl-β-d-1-thiogalactopyranoside (IPTG) (Biosynth) na 1,4-dithiothreitol (DTT, AppliChem) yaguzwe muri Sigma-Aldrich kandi ikoreshwa nta yandi yeze.Antibiyotike chloramphenicol (Cm), spectinomycin dihydrochloride (Sm), ampisilline (Amp), gentamicin (Gt), na karbenicilline (Cbn) byaguzwe muri AppliChem.Bacto Tryptone na Bacto Umusemburo Gukuramo ibitangazamakuru byaguzwe muri BD Biosciences.Trypsin kugirango ikurikirane yaguzwe muri Promega.
Urutonde rwibisekuru byakuwe muri anti-SMASH byahanuwe BGC 75.1.E. malaspinii (Amakuru yinyongera).
Ingirabuzima fatizo (inzige, MALA_SAMN05422137_METAG-framework_127-gene_5), embM (inzige, MALA_SAMN05422137_METAG-framework_127-gene_4), hamwe na embAM (harimo uturere twa intergene) byakurikiranwe muburyo bwa syntetique muri cPC7 ryari.Gene ya embA yashyizwe mubice byambere byakoronijwe (MCS1) bya pACYCDuet-1 (CmR) na pCDFDuet-1 (SmR) hamwe na BamHI na HindIII.Imikorere ya embM na embMopt (codon-optimiz) yashyizwe muri MCS1 pCDFDuet-1 (SmR) hamwe na BamHI na HindIII hanyuma ishyirwa kumwanya wa kabiri wogukwirakwiza pCDFDuet-1 (SmR) na pRSFDuet-1 (KanR) (MCS2) hamwe na NdeI / ChoI.Cassette ya embAM yashyizwe muri pCDFDuet1 (SmR) hamwe na BamHI na HindIII.Gene ya ORF3 / ABI (LOUS, MALA_SAMN05422137_Top imitekerereze ameza).6).Kubuza enzyme igogora hamwe na ligation byakozwe hakurikijwe protocole yuwabikoze (New England Biolabs).