304 Simbi isina simbi yakasungirirwa chubhu / tubing zhemical zomponent, Biosynthetic Inogona yeGlobal Marine Microbiome

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Detailed chigadzirwa tsananguro

304 Simbi isina simbi yakasungirirwa chubhu / tubing
1. Tsanangudzo: Stainless simbi coil chubhu / tubing
2. Type: welded kana seamless
3. Standard: ASTM A269, ASTM A249
4. Stainless simbi coil chubhu OD: 6mm kusvika 25.4MM
5. Kureba: 600-3500MM kana sezvinodiwa nemutengi.
6. Wall ukobvu: 0.2mm kusvika 2.0mm.

7. Kushivirira: OD: +/-0.01mm;Ukobvu: +/-0.01%.

8. Coil yemukati gomba saizi: 500MM-1500MM (inogona kugadziriswa maererano nezvinodiwa nevatengi)

9. Coil kureba: 200MM-400MM (inogona kugadziriswa maererano nezvinodiwa nevatengi)

10. Surface: Yakajeka kana yakavharwa
11. Nyaya: 304, 304L, 316L, 321, 301, 201, 202, 409, 430, 410, alloy 625, 825, 2205, 2507, nezvimwewo.
12. Kurongedza: mabhegi akarukwa mubhokisi remapuranga, pallet yemapuranga, shaft yemapuranga, kana sezvinodiwa nemutengi.
13. Muedzo : chikamu chemakemikari, goho simba, kusimba simba, kuomarara kuyerwa
14. Guarantee: Wechitatu bato (semuenzaniso :SGS TV ) kuongorora, nezvimwe.
15. Kushandisa: Kushongedza, fenicha, kutakura mafuta, kupisa kwekushisa, kugadzira kutuka, kugadzira mapepa, motokari, kugadzira zvokudya, kurapwa, nezvimwewo.

Masikirwo emasikirwo nharaunda ane phylogenetically uye metabolically akasiyana.Pamusoro pemapoka asina kudzidza ezvipenyu1, kusiyana uku kunobatawo mukana wakapfuma wekuwanikwa kweecologically uye biotechnologically akakosha enzymes uye biochemical compounds2,3.Nekudaro, kudzidza izvi zvakasiyana-siyana kuti uone nzira dzema genomic dzinogadzira makomisheni akadaro uye nekuasunga kune avo vanogamuchira zvinoramba zvichinetsa.Iyo biosynthetic inogoneka yezvipembenene mugungwa rakashama inoramba isingazivikanwe zvakanyanya nekuda kwezvipimo mukuongororwa kweiyo genome resolution data pachiyero chepasi rose.Pano, tinoongorora kusiyana uye kusiyana-siyana kwemapoka emajini ebiosynthetic munyanza nekubatanidza majenomes anosvika 10,000 kubva kumaseru akagadzirwa uye maseru ega ega ane anopfuura zviuru makumi maviri neshanu achangobva kuvakwa patsva majenomes emvura yegungwa anodarika chiuru.Kuedza uku kwakaratidza nezve 40,000 putative kazhinji nyowani biosynthetic gene masumbu, mamwe acho akawanikwa mune yaimbove isingafungidzirwe phylogenetic mapoka.Muhuwandu uhu, takaona mutsara wakapfumiswa mubiosynthetic gene clusters (“Candidatus Eudormicrobiaceae”) yaive yebhakitiriya phylum isina kurimwa uye yaisanganisira humwe hutachiona hwakasiyana siyana munzvimbo ino.Pakati peizvi, isu takaratidzira phosphatase-peptide uye pytonamide nzira, tichiratidza zviitiko zveasina kujairika bioactive compound chimiro uye enzymology, zvichiteerana.Mukupedzisa, chidzidzo ichi chinoratidza kuti maitiro e-microbiome-based angagonesa kuongororwa kwema enzymes uye chikafu chechisikigo mune isinganzwisisike microbiota uye nharaunda.
Utachiona hunofambisa pasi rose biogeochemical kutenderera, kuchengetedza mawebhu echikafu, uye kuchengetedza zvidyarwa nemhuka zvine hutano5.Kukura kwavo phylogenetic, metabolic uye kushanda kwakasiyana-siyana kunomiririra mukana wakapfuma wekuwanikwa kwemutero mutsva1, ma enzymes uye biochemical compounds, kusanganisira zvigadzirwa zvechisikigo6.Munharaunda dzezvipenyu, mamorekuru aya anopa utachiona hwakasiyana-siyana hwehupenyu uye hwezvakatipoteredza, kubva pakukurukurirana kusvika kumakwikwi 2, 7.Pamusoro pemabasa azvo ekutanga, izvi zvigadzirwa zvechisikigo uye nzira dzadzo dzekugadzira dzakadhindwa zvinopa mienzaniso yezvigadzirwa zvebiotechnological uye kurapa2,3.Kuzivikanwa kwenzira dzakadai uye kubatana kwave kuchifambiswa zvakanyanya nekudzidza kweakakura hutachiona.Nekudaro, zvidzidzo zvetaxonomic zvenzvimbo dzechisikigo zvakaratidza kuti huwandu hwakawanda hwehutachiona hahuna kurima8.Kurerekera kwetsika uku kunodzikamisa kugona kwedu kushandisa siyana inoshanda yakavharwa nema microbes mazhinji4,9.
Kukunda zvipingamupinyi izvi, kufambira mberi kwehunyanzvi mukati memakore gumi apfuura kwakabvumira vaongorori zvakananga (kureva, vasina tsika yekutanga) kutevedzana zvimedu zveDNA zvidiki kubva munharaunda dzese (metagenomics) kana masero mamwechete.Iko kugona kuunganidza zvimedu izvi muzvikamu zvakakura zvegenome uye kuvakazve akawanda metagenomically akaungana genomes (MAGs) kana single amplified genomes (SAGs), zvichiteerana, inovhura mukana wakakosha wetaxocentric zvidzidzo zveiyo microbiome (kureva, nharaunda diki uye microbiome).gadzira nzira itsva.genetic zvinhu munzvimbo yakapihwa) 10,11,12.Chokwadi, zvidzidzo zvenguva pfupi yapfuura zvakawedzera zvakanyanya kumiririrwa kwe phylogenetic yemhando dzakasiyana-siyana paPasi1, 13 uye zvakaratidza zvakawanda zvekushanda kwakasiyana munharaunda dzehutachiona hwega husina kumbobvira hwafukidzwa neyakagadzirwa microorganism reference genome sequences (REFs)14.Kugona kuisa kusaonekwa kwekushanda kwakasiyana-siyana mumamiriro ezvinhu emugadziri genome (kureva, genome resolution) yakakosha pakufanotaura mitsara isati yave isina chimiro chemicrobial mitsara ingangove yakakodha zvigadzirwa zvitsva zvechisikigo15,16 kana kuronda makomisheni akadaro kudzokera kumugadziri wawo wepakutanga17.Semuenzaniso, metagenomic uye single-cell genomic analysis approach yakatungamirira pakuzivikanwa kweCandidatus Entotheonella, boka re metabolically rich sponge-associated mabhakitiriya, sevagadziri vezvakasiyana-siyana zvinokwanisa zvinodhaka18.Zvisinei, zvisinei nekuedza kuchangobva kuitika kwekuongorora genomic kwenharaunda dzakasiyana siyana dzeutachiona, 16,19 inodarika zvikamu zviviri muzvitatu zve data yepasi rose yemetagenomic yegungwa guru rePasi reecosystems16,20 ichiri kushaikwa.Nekudaro, kazhinji, iyo biosynthetic kugona kweiyo marine microbiome uye kugona kwayo senzvimbo yekuchengetera yenovel enzymatic uye zvigadzirwa zvechisikigo zvinoramba zvisinganyatso kudzidziswa.
Kuti tiongorore kugona kwebiosynthetic yezvipembenene zvemugungwa pasi rose, takatanga tabatanidza marine microbial genomes yakawanikwa tichishandisa tsika-inotsamira uye isiri-tsika nzira kugadzira dhatabhesi rakakura re phylogenetics uye gene basa.Kuongororwa kweiyi dhatabhesi kwakaratidza dzakasiyana siyana dzebiosynthetic gene cluster (BGCs), mazhinji acho ari emhuri dzisati dzave dzisati dzave necharacterized gene cluster (GCF).Pamusoro pezvo, isu takaona mhuri isingazivikanwe yebhakitiriya inoratidzira yakanyanya kuzivikanwa siyana yeBGCs mugungwa rakashama kusvika parizvino.Takasarudza nzira mbiri dze ribosomal synthesis uye post-translationally modified peptide (RiPP) yekuedza kusimbiswa zvichienderana nekusiyana kwavo kwemajini kubva kune nzira dzinozivikanwa iye zvino.Kushanda kwemaitiro enzira idzi kwakaratidza mienzaniso isingatarisirwi yeenzymology pamwe chete nemakomisheni asina kujairika ane protease inhibitory chiitiko.
Pakutanga, isu takavavarira kugadzira dhata repasi rose rekuongorora genome, tichitarisa pane ayo mabhakitiriya uye archaeal zvikamu.Kuti izvi zviitike, takabatanidza data yemetagenomic uye 1038 masampu emvura yegungwa kubva ku215 yakagoverwa pasi rose sampling nzvimbo (latitude renji = 141.6 °) uye akati wandei akadzama layer (kubva pa1 kusvika 5600 m pakadzika, achivhara pelagic, mesopelagic uye abyssal zones).Background21,22,23 (Fig. 1a, data yakawedzerwa, Fig. 1a uye Supplementary Table 1).Pamusoro pekupa nzvimbo yakakura yekuvhara, aya masampuli akasarudzwa akasarudzwa akatibvumira kuenzanisa zvikamu zvakasiyana-siyana zvemugungwa microbiome, kusanganisira hutachiona-hupfumi (<0.2 µm), prokaryotic-rich (0.2-3 µm), particle-rich (0.8 µm). )-20 µm) uye hutachiona-yakapera (> 0.2 µm) makoroni.
a, Huwandu hwe1038 huripo huripo hwema genomes (metagenomics) enharaunda dzemugungwa tupukanana dzakaunganidzwa kubva munzvimbo 215 dzakaparadzirwa pasi rose (62°S kusvika 79°N uye 179°W kusvika 179°E.).Mepu mataira © Esri.Zvinyorwa: GEBCO, NOAA, CHS, OSU, UNH, CSUMB, National Geographic, DeLorme, NAVTEQ, uye Esri.b, mametagenomes aya akashandiswa kugadzirazve MAGs (mitoo uye mamwe mashoko), ayo akasiyana muhuwandu uye mhando (maitirwo) mumaseti (akaiswa muvara).Iwo akagadziridzwa MAGs akawedzerwa neanowanikwa pachena (ekunze) genomes, kusanganisira yakagadzirwa nemaoko MAG26, SAG27 uye REF.27 Unganidza OMD.c, zvichienzaniswa nemishumo yapfuura yakavakirwa chete paSAG (GORG)20 kana MAG (GEM)16, OMD inovandudza genomic maitiro enharaunda dzemugungwa tupukanana (metagenomic kuverenga mapping rate; nzira) kaviri kusvika katatu nekunyatsoenderana kumiririra mukudzika uye. latitude..<0.2, n=151, 0.2-0.8, n=67, 0.2-3, n=180, 0.8-20, n=30, >0.2, n=610, <30°, n = 132, 30–60° , n = 73, > 60 °, n = 42, EPI, n = 174, MES, n = 45, BAT, n = 28. d, OMD ichibatanidza munhanho yemapoka emarudzi (95% zvinoreva nucleotide identity) inoratidza huwandu hwe dzinenge 8300 marudzi, anopfuura hafu ayo asati ambozivikanwa maererano netaxonomic annotations achishandisa GTDB (version 89) e, kurongwa kwemarudzi nemhando ye genome kwakaratidza kuti MAG, SAG uye REFs zvinopindirana zvakanaka mukuratidza phylogenetic kusiyana kwe iyo marine microbiome.Kunyanya, 55%, 26% uye 11% yezvipenyu zvaive zvakanangana neMAG, SAG uye REF, zvichiteerana.BATS, Bermuda Atlantic Time Series;GEM, genomes yePasi microbiome;GORG, global ocean reference genome;HOT, Hawaiian Ocean nguva yakatevedzana.
Tichishandisa dhatabheti iyi, takavakazve huwandu hwe26,293 MAGs, kunyanya mabhakitiriya uye archaeal (Fig. 1b uye data yakawedzerwa, Fig. 1b).Isu takagadzira aya MAGs kubva kumagungano kubva kwakasiyana pane kuunganidzwa metagenomic samples kudzivirira kudonha kwechisikigo kutevedzana kusiyanisa pakati pemasampuli kubva kunzvimbo dzakasiyana kana nguva mapoinzi (maitiro).Pamusoro pezvo, takaisa zvikamu zve genomic zvimedu zvichienderana nekuwanda kwavo correlations pahuwandu hukuru hwemasampuli (kubva pa58 kusvika 610 samples, zvichienderana neongororo; nzira).Takaona kuti iyi inguva-inopedza nguva asi yakakosha nhanho24 iyo yakasvetwa mune akati wandei MAG16, 19, 25 kuvaka patsva inoshanda uye inovandudza zvakanyanya huwandu (2.7-peta paavhareji) uye mhando (+ 20% paavhareji) genome.yakagadzirwazve kubva mumvura metagenome yakadzidzwa pano (yakawedzerwa data, Fig. 2a uye mamwe mashoko).Pakazere, kuedza uku kwakakonzera kuwedzera kwe4.5-kupeta kwemarine microbial MAGs (6-kupeta kana chete emhando yepamusoro MAGs achitariswa) zvichienzaniswa neyakanyanya kuwanda MAG sosi iripo nhasi16 (Nzira).Iyi ichangobva kugadzirwa MAG seti yakazosanganiswa ne830 yakasarudzwa nemaoko MAG26s, 5969 SAG27s uye 1707 REFs.Makumi maviri nemanomwe emhando dzemabhakitiriya emugungwa uye archaea akagadzira muunganidzwa wekubatanidza we34,799 genomes (Fig. 1b).
Isu takazoongorora iyo ichangobva kugadzirwa sosi yekuvandudza kugona kwayo kumiririra nharaunda dzemugungwa tupukanana uye kuongorora maitiro ekubatanidza akasiyana genome marudzi.Paavhareji, takaona kuti inovhara ingangoita 40-60% yemvura metagenomic data (Mufananidzo 1c), kaviri kusvika katatu kufukidzwa kweiyo yapfuura MAG-chete mishumo mune zvese zvakadzama uye latitude More serial 16 kana SAG20.Pamusoro pezvo, kuyera zvine hungwaru kusiyana kwetaxonomic mumiunganidzwa yakagadzwa, takazivisa majenomes ese tichishandisa Genome Taxonomy Database (GTDB) toolkit (maitiro) uye takashandisa avhareji yegenome-wide nucleotide identity ye95%.28 kuziva 8,304 marudzi masumbu (marudzi).Zvikamu zviviri muzvitatu zvezvipenyu izvi (kusanganisira zvikwata zvitsva) zvakanga zvisati zvamboonekwa muGTDB, iyo 2790 yakawanikwa ichishandisa MAG yakagadzirwazve muchidzidzo ichi (Fig. 1d).Mukuwedzera, takaona kuti marudzi akasiyana-siyana ejenomes anonyatsopindirana: 55%, 26%, uye 11% yemarudzi anoumbwa zvachose MAG, SAG, uye REF, maererano (Fig. 1e).Pamusoro pezvo, MAG yakafukidza ese makumi mana nemapfumbamwe emhando anowanikwa mukoramu yemvura, nepo SAG neREF vaingomiririra gumi nemasere negumi neimwe, zvichiteerana.Zvisinei, SAG iri nani inomiririra kusiyana-siyana kwezvikamu zvakajairika (data yakawedzerwa, Fig. 3a), yakadai sePelagic Bacteriales (SAR11), ine SAG inovhara anenge 1300 marudzi uye MAG chete 390 marudzi.Zvikurukuru, maREF haawanzo kupindirana neMAGs kana maSAG padanho rezvipenyu uye anomiririra > 95% yeanosvika zana genomes asingawanikwe munzvimbo yakavhurika yegungwa metagenomic seti akadzidzwa pano, kunyanya nekuda kwekudyidzana nedzimwe mhando dzezvimiro zvemugungwa zvinomiririra (eg sediments) .kana mubatsiri-mubatsiri).Kuita kuti iwanikwe zvakanyanya kunharaunda yesainzi, iyi marine genome resource, iyo inosanganisirawo zvimedu zvisina kutsanangurwa (semuenzaniso, kubva kune zvakafanotaurwa phage, genomic zvitsuwa, uye genome zvidimbu izvo zvisina kukwana data rekuvakazve MAG), inogona kufananidzwa netaxonomic data. .Svika zvirevo pamwe chete nebasa remajini uye mamiriro emukati muOcean Microbiology Database (OMD; https://microbiomics.io/ocean/).
Isu takazotanga kuongorora hupfumi uye hutsva hweiyo biosynthetic inogona mune yakavhurika yegungwa microbiomes.Kuti izvi zviitike, takatanga tashandisa antiSMASH kune ese MAGS, SAGs, uye REFs anowanikwa mu1038 marine metagenomes (nzira) kufanotaura huwandu hwe39,055 BGCs.Takazoisa izvi muzvikamu zve6907 zvisingaite maGCFs uye 151 gene cluster populations (GCCs; Supplementary Table 2 uye nzira) kuti tizvidavirire nokuda kwekusadiwa kwemukati (kureva, BGC imwe chete inogona kukodha mumajenomes akawanda) uye data yemetagenomic Kuparadzaniswa kweBGCs dzakanyanya .MaBGC asina kukwana haana kuwedzera zvakanyanya, kana paine (Supplementary Information), nhamba yeGCFs neGCCs, zvichiteerana, ine kanenge imwe isina kusimba yeBGC nhengo mu44% uye 86% yezviitiko.
Padanho reGCC, takawana zvakasiyana-siyana zvakafanotaurwa RiPPs nezvimwe zvinhu zvakasikwa (Fig. 2a).Pakati pavo, semuenzaniso, arylpolyenes, carotenoids, ectoines, uye siderophores ndezveGCCs ine yakakura phylogenetic kugoverwa uye kuwanda kwepamusoro muoceanic metagenomes, iyo inogona kuratidza kuchinjika kwakasiyana kwehutachiona kunharaunda yegungwa, kusanganisira kuramba kune reactive okisijeni marudzi, oxidative uye osmotic stress..kana kutorwa kwesimbi (mamwe ruzivo).Musiyano unoshanda uyu unopesana neongororo ichangoburwa yeanosvika miriyoni imwe chete nemazana maviri ezviuru zveBGCs pakati peanosvika zana nemakumi mapfumbamwe ezviuru genomes akachengetwa muNCBI RefSeq dhatabhesi (BiG-FAM/RefSeq, yakazonzi RefSeq)29, yakaratidza kuti nonribosomal Synthetase peptides (NRPS) uye polyketide synthethase. (PKS) BGCs (Supplementary Information).Isu takawana zvakare makumi mana nemana (29%) maGCCs ari kure chete ane hukama kune chero RefSeq BGC (\(\bar{d}\)RefSeq > 0.4; Fig. 2a uye nzira) uye makumi mashanu nenhatu (35%) GCCs chete muMAG , ichiratidza zvinogoneka. kuona makemikari asina kunyorwa kare muOMD.Tichifunga kuti imwe neimwe yeGCCs iyi ingangomiririra mabasa akasiyana-siyana ebiosynthetic, isu takaongororazve data padanho reGCF mukuyedza kupa boka rakadzama reBGCs rakafanotaurwa kukodhi kune zvimwe zvinhu zvakasikwa zvakafanana29.Huwandu hwe3861 (56%) hwakacherechedzwa maGCFs haana kupindirana neRefSeq, uye> 97% yeGCFs yakanga isipo muMIBiG, imwe yedatabase yakakura kwazvo yeBGCs yakasimbiswa (Mufananidzo 2b).Kunyange zvisingashamise kuwana nzira dzakawanda dzinogona kuitika muzvirongwa zvisina kunyatsomiririrwa nereferensi genome, nzira yedu yekudzokorodza maBGC kuita maGCFs tisati taita mabhenji anosiyana nemishumo yapfuura 16 uye inotitendera kuti tipe ongororo isina kurerekera yezvitsva.Mazhinji emhando nyowani (3012 GCF kana 78%) inoenderana neyakafanotaurwa terpenes, RiPP kana zvimwe zvigadzirwa zvechisikigo, uye mazhinji (1815 GCF kana 47%) akaiswa encoded mumhando dzisingazivikanwe nekuda kwekugona kwavo biosynthetic.Kusiyana nePKS neNRPS masumbu, aya compact BGCs haadiki kupatsanurwa panguva yemetagenomic musangano 31 uye anobvumira yakawanda nguva- uye zviwanikwa-yakanyanya kushanda maitiro ezvigadzirwa zvavo.
Huwandu hwe39,055 BGCs dzakaiswa muzvikamu zvitanhatu nemazana mapfumbamwe nenomwe zveGCF ne151 GCCs.a, data inomiririra (mukati wekunze).Hierarchical clustering yeBGC madaro akavakirwa paGCC, 53 ayo anogadziriswa ne MAG chete.Iyo GCC ine maBGCs kubva kune akasiyana taxa (ln-yakashandurwa gedhi frequency) uye akasiyana makirasi eBGC (saizi yedenderedzwa inoenderana neyayo frequency).PaGCC yega yega, chikamu chekunze chinomiririra huwandu hweBGC, kuwanda (muzana wemasample), uye chinhambwe (minimum BGC cosine chinhambwe (min(dMIBiG))) kubva kuBiG-FAM kuenda kuBGC.MaGCC ane maBGC ane hukama zvakanyanya neBGC dzakasimbiswa (MIBiG) dzinoratidzwa nemiseve.b Kuenzanisa GCF neyakafanotaurwa (BiG-FAM) uye yakasimbiswa (MIBiG) BGCs, 3861 itsva (d–>0.2) maGCF akawanikwa.Yakawanda (78%) yeiyi kodhi yeRiPP, terpenes, uye zvimwe zvinoisa zvakasikwa zvigadzirwa.c, ese genomes muOMD anowanikwa mu1038 marine metagenome akaiswa muGTDB base muti kuratidza kuvharika kwe phylogenetic yeOMD.Makadhi asina chero genomes muOMD anoratidzwa negrey.Huwandu hweBGC hunoenderana nenhamba huru yezvakafanotaurwa BGCs pa genome mune yakapihwa clade.Kuti zvijekese, iyo yekupedzisira 15% yemanode inodonha.Miseve inoratidza maclades akapfuma muBGC (>15 BGC), kunze kweMycobacterium, Gordonia (yechipiri kuRhodococcus), uye Crocosphaera (yechipiri kuSynechococcus).d, Zvisingazivikanwe c.Eremiobacterota yakaratidza yakakwira biosynthetic kusiyana (Shannon index yakavakirwa pamhando yechigadzirwa chechisikigo).Bhendi rega rega rinomiririra genome ine akawanda maBGC mumhando.T1PKS, PKS mhando I, T2/3PKS, PKS mhando II uye mhando III.
Pamusoro pehupfumi uye hutsva, isu tinoongorora iyo biogeographic chimiro cheiyo biosynthetic kugona kweiyo marine microbiome.Kuiswa kwemasamples neavhareji metagenomic GCF kopi nhamba yekugovera (Nzira) yakaratidza kuti yakaderera-latitude, pamusoro, prokaryotic-yakapfuma uye hutachiona-varombo nharaunda, kunyanya kubva pamusoro kana kudzika kwemvura yezuva, yaive yakapfuma muRiPP neBGC terpenes.Kusiyana neizvi, polar, deep-sea, virus- uye particle-rich community zvakabatanidzwa nehuwandu hwepamusoro hweNRPS uye PKS BGC (data yakawedzerwa, Fig. 4 uye mamwe mashoko).Chekupedzisira, takaona kuti nharaunda dzakanyatsodzidziswa dzinopisa uye pelagic ndidzo dzinonyanya kuvimbisa masosi etsva terpenes (Augmented Data Figure).Yakanyanya kugona yePKS, RiPP uye zvimwe zvigadzirwa zvechisikigo (Mufananidzo 5a ine data rakawedzerwa).
Kuti tibatsire kudzidza kwedu kweiyo biosynthetic inogona ye marine microbiomes, isu takavavarira kumepu yavo yekugovera phylogenetic uye kuona mitsva yeBGC-yakafumisa clades.Kuti izvi zviitike, takaisa majenome etwupukanana twemugungwa mumuti wakajairwa weGTDB13 wemabhakitiriya uye archaeal phylogenetic uye akafukidza nzira dzinoisa biosynthetic dzavanoisa (Fig. 2c).Isu takaona zviri nyore akati wandei BGC-yakapfuma maclades (inomiririrwa neanopfuura gumi nemashanu BGCs) mumasampu emvura yegungwa (nzira) dzinozivikanwa nekugona kwavo biosynthetic, senge cyanobacteria (Synechococcus) uye mabhakitiriya eProteus, akadai seTistrella32,33, kana nguva pfupi yadarika akakwezva kutarisisa kwavo. zvinhu zvakasikwa .zvakadai seMyxococcota (Sandaracinaceae), Rhodococcus uye Planctomycetota34,35,36.Sezvineiwo, takawana akati wandei asina kumboongororwa mutsara muzvikamu izvi.Semuenzaniso, izvo zvipenyu zvine hupfumi hwe biosynthetic inogona mu phyla Planctomycetota uye Myxococcota yaive ye uncharacterized vamiriri mirairo uye genera, zvichiteerana (Supplementary Table 3).Zvakatorwa pamwechete, izvi zvinoratidza kuti OMD inopa mukana kune yaimbozivikanwa phylogenetic ruzivo, kusanganisira microorganisms, iyo inogona kumiririra mitsva yezvinangwa zve enzyme uye kuwanikwa kwezvinhu zvakasikwa.
Tevere, takaratidzira BGC-yakapfumiswa clade nekusangoverenga huwandu hwepamusoro hweBGCs dzakavharwa nenhengo dzayo, asiwo nekuongorora kusiyana kweBGC idzi, iyo inotsanangura kuwanda kwemhando dzakasiyana dzezvigadzirwa zvemhando yevamiriri (Fig. 2c uye nzira dzekushandisa). ).Takaona kuti mhando dzakasiyana-siyana dzebiosynthetically dzakamiririrwa neakanyanya mainjiniya mabhakitiriya MAGs muchidzidzo ichi.Aya mabhakitiriya ndeasina kurima phylum Candidatus Eremiobacterota, iyo inoramba ichinyanya kusaongororwa kunze kwezvidzidzo zvishomanana zvegenomic37,38.Zvinoshamisa kuti "ca.Iyo genus Eremiobacterota yakangoongororwa munzvimbo yepasi39 uye haizivikanwe kuti inosanganisira chero nhengo dzakafumiswa muBGC.Pano takavakazve MAGs masere emhando imwechete (nucleotide identity> 99%) 23. Saka isu tinokurudzira zita remarudzi "Candidatus Eudoremicrobium malaspinii", rakatumidzwa nereid (sea nymph), chipo chakanaka muGreek mythology uye expeditions.'Ka.Maererano ne phylogenetic annotation 13, E. malaspinii haina hama yakambozivikanwa pasi pechikamu chekutevedzana uye nokudaro ndeyemhuri itsva yebhakitiriya yatinopa "Ca.E. malaspinii” semhando yemhando uye “Ca.Eudormicrobiaceae” sezita repamutemo (Supplementary Information).Pfupi metagenomic reconstruction ye'Ca.E. malaspinii genome project yakasimbiswa nekuisa zvishoma, kuverenga kwenguva refu metagenomic sequencing uye kuungana kwakanangwa kwemuenzaniso mumwechete (Nzira) seimwe 9.63 Mb linear chromosome ine 75 kb kudzokororwa.sekusajeka kwasara.
Kuti tigadzirise mamiriro e phylogenetic emhando iyi, takatsvaga makumi mana ane hukama zvakanyanya mune mamwe eukaryotic-enriched metagenomic samples kubva kuTara Ocean expedition kuburikidza neakatariswa genome reconstruction.Muchidimbu, takabatanidza kuverenga kwemetagenomic kune zvimedu zve genomic zvine chekuita ne "Ca.E. malaspinii” uye inofungidzira kuti kuwedzera kwehuwandu hwekutsvaga mumuenzaniso uyu kunoratidza kuvapo kwedzimwe hama (nzira).Somugumisiro, takawana 10 MAGs, kusanganiswa kwe19 MAGs inomiririra marudzi mashanu mumarudzi matatu mukati memhuri ichangobva kutsanangurwa (kureva "Ca. Eudormicrobiaceae").Mushure mekuongorora kwebhuku uye kutonga kwehutano (data yakawedzerwa, Fig. 6 uye mamwe mashoko), takaona kuti "Ca.Eudormicrobiaceae marudzi anopa genomes makuru (8 Mb) uye akapfuma biosynthetic simba (14 kusvika 22 BGC pamarudzi) kupfuura dzimwe "Ca" nhengo.Clade Eremiobacterota (kusvika ku7 BGC) (Fig. 3a–c).
a, Phylogenetic nzvimbo dzevashanu 'Ca.Mhando dzeEudormicrobiaceae dzakaratidza BGC hupfumi hwakanangana nemitsara yegungwa yakaratidzwa muchidzidzo ichi.Muti we phylogenetic unosanganisira zvese 'Ca.MAG Eremiobacterota uye nhengo dzeimwe phyla (nhamba dzegenome mumabhuraketi) dzakapihwa muGTDB (vhezheni 89) dzakashandiswa kushanduka kwemashure (Nzira).Zvikamu zvekunze zvinomiririra zvikamu pahutano hwemhuri ("Ca. Eudormicrobiaceae" uye "Ca. Xenobiaceae") uye pachikwata chekirasi ("Ca. Eremiobacteria").Mhando shanu dzinotsanangurwa muchidzidzo ichi dzinomiririrwa nealphanumeric codes nemazita ebhinomial anotsanangurwa (Supplementary Information).b, zvakanaka.Eudormicrobiaceae marudzi anogovera manomwe akajairika BGC nuclei.Kusavapo kweBGC muA2 clade kwakakonzerwa nekusakwana kwemumiriri MAG (Supplementary Table 3).BGCs dzakananga kune "Ca.Amphithomicrobium" uye "Ca.Amphithomicrobium” (clades A uye B) haina kuratidzwa.c, Ese maBGC akaiswa encoded se “Ca.Eudoremicrobium taraoceanii yakawanikwa ichiratidzwa mu623 metaranscriptoms yakatorwa kubva kumakungwa eTara.Denderedzwa rakasimba rinoratidza chinyorwa chinoshanda.Orange madenderedzwa anoreva log2-yakashandurwa kupeta shanduko pazasi uye pamusoro peyekuchengeta gene rekutaura mwero (maitiro).d, hukama huzhinji hwema curves (maitiro) anoratidza 'Ca.Mhando dzeEudormicrobiaceae dzakapararira mumakungwa mazhinji uye muchikamu chemvura chese (kubva pamusoro kusvika pakadzika zvinosvika 4000 m).Kubva pane fungidziro idzi, takaona kuti 'Ca.E. malaspinii 'inoda kusvika ku6% yemasero eprokaryotic munzvimbo dzakadzika-sea pelagic-zviyo zvinosanganiswa.Takafunga kuti rudzi rwungave rwuripo panzvimbo kana rwakawanikwa mune chero chikamu chehukuru hwehumwe hwakadzama hwakapihwa.IO - Indian Ocean, NAO - North Atlantic, NPO - North Pacific, RS - Red Sea, SAO - South Atlantic, SO - Southern Ocean, SPO - South Pacific.
Kudzidza kuwanda uye kugoverwa kweCa.Eudormicrobiaceae, iyo, sezvatakaona, inopararira mumakungwa akawanda emakungwa, uyewo mumutsara wose wemvura (Fig. 3d).Munharaunda, ivo vanoumba 6% yenharaunda yegungwa tupukanana, zvichiita kuti ive chikamu chakakosha chepasi rose marine microbiome.Mukuwedzera, takawana izvo zvinehukama zveCa.Eudormicrobiaceae marudzi uye maitiro avo ekutaura kweBGC aive akakwirira muchikamu cheukaryotic chakapfuma (Fig. 3c uye data yakawedzerwa, Fig. 7), ichiratidza kuwirirana kunobvira nechikamu chechikamu, kusanganisira plankton.Ichi chitarisiko chine zvimwe zvakafanana ne'Ca.Eudoremicrobium BGCs inogadzira cytotoxic zvigadzirwa zvechisikigo kuburikidza nenzira dzinozivikanwa dzinogona kuratidza hunhu hwekupamba (Supplementary Information uye Expanded Data, Figure 8), yakafanana nezvimwe zvikara zvinonyatso gadzira metabolites seMyxococcus41.Kuwanikwa kweCa.Eudormicrobiaceae mune zvishoma inowanikwa (yakadzika gungwa) kana eukaryotic pane prokaryotic samples inogona kutsanangura kuti sei mabhakitiriya aya uye kusiyana kwavo kusingatarisirwi kweBGC kunoramba kusingazivikanwe mumamiriro ekutsvaga kwechikafu chekudya.
Pakupedzisira, takatsvaga kuedza kusimbisa chivimbiso chebasa redu re microbiome mukutsvaga nzira itsva, ma enzymes, uye zvigadzirwa zvechisikigo.Pakati pemakirasi akasiyana eBGCs, nzira yeRiPP inozivikanwa nekukodha makemikari akapfuma uye anoshanda akasiyana nekuda kweakasiyana-siyana mushure mekushandura shanduko yepakati peptide nema enzymes akakura42.Saka takasarudza maviri 'Ca.Eudoremicrobium' RiPP BGCs (Figures 3b and 4a-e) yakavakirwa pane zvakafanana chero inozivikanwa BGC (\(\bar{d}\)MIBiG uye \(\bar{d}\)RefSeq pamusoro 0.2) .
a–c, In vitro heterologous expression uye in vitro enzymatic assays yenovel (\(\bar{d}\)RefSeq = 0.29) cluster yeRiPP biosynthesis yakanangana negungwa rakadzika Ca marudzi.E. malaspinii' yakatungamirira kukugadzirwa kwezvigadzirwa zve diphosphorylated.c, zvigadziriso zvakaonekwa zvichishandisa high-resolution (HR) MS / MS (kuparadzaniswa kunoratidzwa ne b uye y ions mumakemikari maitiro) uye NMR (yakawedzerwa data, Fig. 9).d, iyi phosphorylated peptide inoratidza yakaderera micromolar inhibition ye mammalian neutrophil elastase, iyo isingawanikwe mukutonga peptide uye dehydrating peptide (kemikari kubviswa induced dehydration).Kuedza kwakadzokororwa katatu nemigumisiro yakafanana.Semuenzaniso, heterologous kutaura kwechipiri chinyorwa \ (\ bar{d}\) RefSeq = 0.33) sumbu reprotein biosynthesis rinojekesa basa remaenzayimu mana akakura anoshandura 46 amino acid core peptide.Zvisaririra zvakasvibiswa zvinoenderana nenzvimbo yekugadziridzwa yakafanotaurwa neHR-MS/MS, isotope labeling, uye NMR ongororo (Supplementary Information).Dashed coloration inoratidza kuti shanduko inoitika pane imwe yemasara maviri.Mufananidzo uyu muunganidzwa weakawanda heterologous anovaka kuratidza chiitiko cheese ma enzymes akakura pane imwechete nucleus.h, Mufananidzo weNMR data yemusana amide N-methylation.Mibairo yakazara inoratidzwa mufig.10 ine data rakawedzerwa.ini, Phylogenetic chinzvimbo chekukura FkbM protein cluster enzyme pakati pese FkbM domains inowanikwa muMIBiG 2.0 database inoratidza enzyme yemhuri iyi ine N-methyltransferase chiitiko (Supplementary Information).Schematic diagraphs yeBGCs (a, e), precursor peptide zvimiro (b, f), uye putative makemikari zvimiro zvechisikigo zvigadzirwa (c, g) zvinoratidzwa.
Nzira yekutanga yeRiPP (\(\bar{d}\)MIBiG = 0.41, \(\bar{d}\)RefSeq = 0.29) yakawanikwa chete mumarudzi akadzika-gungwa "Ca.E. malaspinii” uye macode ePeptide- precursor (Fig. 4a, b).Mune iyi enzyme yakakura, takaona imwechete inoshanda domain homologous kune dehydration domain ye lantipeptide synthase iyo inowanzo konzera phosphorylation uye kunotevera kubviswa kwe43 (Supplementary Information).Naizvozvo, isu tinofanotaura kuti kugadziridzwa kweiyo precursor peptide kunosanganisira nhanho mbiri-kupererwa nemvura.Zvisinei, tichishandisa tandem mass spectrometry (MS/MS) uye nuclear magnetic resonance spectroscopy (NMR), takaona polyphosphorylated linear peptide (Fig. 4c).Kunyangwe zvisingatarisirwi, takawana mitsara yakati wandei yehumbowo hunotsigira kuve kwayo kwekupedzisira chigadzirwa: maviri akasiyana heterologous mauto uye pasina kupera mvura mumuviri in vitro assays, kuzivikanwa kweakakosha masara akashandurwa munzvimbo inokonzeresa yekupera mvura kweiyo enzyme yakakura.zvese zvakagadziridzwa ne "Ca".E. malaspinii genome (yakawedzerwa data, Fig. 9 uye mamwe mashoko) uye, pakupedzisira, basa rehupenyu hwechigadzirwa chephosphorylated, asi kwete chimiro chemakemikari yakasvibiswa (Fig. 4d).Muchokwadi, takaona kuti inoratidza yakaderera micromolar protease inhibitory basa rinopesana neutrophil elastase, inofananidzwa nezvimwe zvakabatana zvigadzirwa zvechisikigo munharaunda yevasungwa (IC50 = 14.3 μM) 44, zvisinei nekuti basa rezvisikwa rinoramba richitsanangurwa.Zvichienderana nemhedzisiro iyi, isu tinokurudzira kutumidza nzira iyo "phospheptin".
Mhosva yechipiri inzira yakaoma yeRiPP yakanangana ne'Ca.The genus Eudoremicrobium (\(\ bar{d}\)MIBiG = 0.46, \(\ bar{d}\) RefSeq = 0.33) yakafanotaurwa kuti incode zvigadzirwa zveprotein zvakasikwa (Fig. 4e).Idzi nzira dzinonyanya kufarira zvebiotechnological nekuda kwekuwanda kunotarisirwa uye kwakasiyana-siyana kweasina kujairika kemikari gadziridzo dzakatangwa nema enzymes akavharidzirwa neiyo pfupi pfupi BGCs45.Takaona kuti puroteni iyi yakasiyana nemapuroteni akafananidzirwa kare pakuti haina zvose NX5N motif ye polyceramides uye lanthionine loop yelandornamides 46.Kuti tikunde zvipimo zvezvakajairwa heterologous kutaura mapatani, takazvishandisa pamwe netsika Microvirgula aerodenitrificans system kuratidza mana akura nzira ma enzymes (maitiro).Tichishandisa kusanganiswa kweMS / MS, isotope labeling, uye NMR, takaona aya ma enzyme akakura mu 46-amino acid core ye peptide (Fig. 4f, g, data yakawedzerwa, Figs. 10-12 uye mamwe mashoko).Pakati pema enzyme akakura, takaratidza kuonekwa kwekutanga kweFkbM O-methyltransferase nhengo yemhuri 47 mumugwagwa weRiPP uye zvisingatarisirwi takaona kuti iyi enzyme yakakura inotanga musana N-methylation (Fig. 4h, i uye mamwe mashoko).Kunyangwe ichi shanduko ichizivikanwa mune zvakasikwa NRP48 zvigadzirwa, enzymatic N-methylation yeamide bonds yakaoma asi biotechnologically yakakosha reaction49 iyo kusvika parizvino yave ichifarira kumhuri yeRiPP yeborosines.Zvakananga 50,51.Kuzivikanwa kwechiitiko ichi mune dzimwe mhuri dze enzymes uye RiPP inogona kuvhura maapplication matsva uye kuwedzera mashandiro akasiyana eprotein 52 uye kusiyana kwemakemikari.Zvichienderana neakagadziridzwa anoonekwa uye hurefu husina kujairika hweyakarongwa chimiro chechigadzirwa, isu tinopa nzira zita rekuti "pythonamide".
Kuwanikwa kweiyo enzymology isingatarisirwi mumhuri inoshanda ine ma enzymes inoratidza chivimbiso chezvakatipoteredza genomics yezvitsva zvakawanikwa, uye zvakare inoratidza hushoma kugona kwekushanda kwekufungidzira kunoenderana nekutevedzana kwehomology chete.Nekudaro, pamwe nemishumo yeasiri-canonical bioactive polyphosphorylated RiPPs, mhedzisiro yedu inoratidza-yakakura-asi kukosha kwakakosha kune synthetic biology kuedza kuburitsa zvizere hupfumi hunoshanda, kusiyana, uye zvimiro zvisina kujairika zvebiochemical compounds.
Pano tinoratidza huwandu hwe biosynthetic inogona kuvharidzirwa nehutachiona uye genomic mamiriro epasi rose marine microbiome, kufambisa tsvakiridzo yeramangwana nekuita kuti chiwanikwa chiwanikwe kunharaunda yesainzi (https://microbiomics.io/ocean/).Takaona kuti yakawanda yayo phylogenetic uye inoshanda novelty inogona kuwanikwa chete nekuvakazve MAGs uye maSAGs, kunyanya munzvimbo dzisingashandiswe hutachiona hunogona kutungamira ramangwana bioprospecting kuedza.Kunyangwe isu tichatarisa pano pa'Ca.Eudormicrobiaceae” semutsara kunyanya biosynthetically “ine tarenda”, mazhinji eBGC akafanotaura mune isingawanikwe microbiota ingangove yakavharirwa enzymologies yaimbove isina kutsanangurwa inobereka makomisheni ane nharaunda uye/kana biotechnologically zviito zvakakosha.
Metagenomic datasets kubva kuhombe dzegungwa uye nguva dzakatevedzana zvidzidzo zvine hudzamu hwakakwana hwekutevedzana zvakasanganisirwa kuwedzera kufukidzwa kwenharaunda dzepasi rose dzezvipembenene zvemugungwa mumadhishi egungwa, zvidimbu zvakadzika uye nekufamba kwenguva.Aya madheti (Supplementary Table 1 uye Figure 1) anosanganisira metagenomics kubva kumasamples akaunganidzwa mumakungwa eTara (viral enriched, n=190; prokaryotic enriched, n=180)12,22 and the BioGEOTRACES expedition (n=480).Hawaiian Oceanic Time Series (HOT, n = 68), Bermuda-Atlantic Time Series (BATS, n = 62)21 uye Malaspina Expedition (n = 58)23.Sequencing kuverenga kubva kune zvese metagenomic zvimedu zvakasefa zvemhando uchishandisa BBMap (v.38.71) nekubvisa sequencing adapter kubva pakuverenga, kubvisa kuverenga kwakaiswa kumhando yekudzora kutevedzana (PhiX genomes), uye kushandisa trimq = 14, maq = 20 inorasa yakashata kuverenga kunaka, maxns = 0 uye minlength = 45. Ongororo dzakazotevera dzakaitwa kana kubatanidzwa neQC kuverenga kana zvichitaurwa (bbmerge.sh minoverlap=16).Kuverenga kweQC kwaive kwakajairika (bbnorm.sh target = 40, minddepth = 0) vasati vavaka vachishandisa metaSPAdes (v.3.11.1 kana v.3.12 kana zvichidiwa)53.Iwo akakonzeresa scaffold contigs (pano anonzi scaffolds) akazosefa nehurefu (≥1 kb).
Iwo 1038 metagenomic samples akakamurwa kuita mapoka, uye kune rimwe nerimwe boka remasamples, iyo metagenomic yemhando yekudzora inoverengwa yemasamples ese akafananidzwa nemabhuraketi emuenzaniso wega wega zvakasiyana, zvichiita kuti nhamba inotevera yeboka remabhuraketsi maviri: Tara Marine Virus - Enriched. (190 × 190), Prokaryotes Enriched (180 × 180), BioGEOTRACES, HOT uye BATS (610 × 610) uye Malaspina (58 × 58).Mepu yakaitwa uchishandisa Burrows-Wheeler-Aligner (BWA) (v.0.7.17-r1188)54 iyo inobvumira kuverenga kufananidzwa nenzvimbo dzechipiri (uchishandisa -a mureza).Kurongeka kwakasefa kuti ive inosvika makumi mana neshanu mabhesi kureba, ane ≥97% kuzivikanwa, uye span ≥80% kuverenga.Mafaira eBAM akabuda akagadziriswa pachishandiswa jgi_summarize_bam_contig_depths script yeMetaBAT2 (v.2.12.1)55 kupa intra- uye inter-sample coverage yeboka rega rega.Pakupedzisira, mabhuraketi akaiswa mumapoka kuti awedzere kunzwisiswa nekumhanya kwega kwega MetaBAT2 pamasampunzi ese ane -minContig 2000 uye -maxEdges 500. Isu tinoshandisa MetaBAT2 pane ensemble boxer nekuti yakaratidzwa muzviyedzo zvakazvimirira kuti ive yakanyanya kushanda imwe boxer.uye 10 kusvika ka50 nekukurumidza kupfuura mamwe mabhokisi anowanzo shandiswa57.Kuti uedze mhedzisiro yehuwandu hwekubatanidza, sample yakasarudzika yakasarudzwa yemetagenomics (gumi kune yega yega maviri eTara Ocean dataset, 10 yeBioGEOTRACES, 5 yenguva yega yega, uye 5 yeMalaspina) yakawedzera kushandisa samples chete.Masampuli emukati akaiswa mumapoka kuti awane ruzivo rwekuvhara.(Ruzivo Rwokuwedzera).
Mamwe (ekunze) genomes akaverengerwa muongororo yakatevera, anoti mazana masere nemakumi matatu akasarudzwa nemaoko MAGs kubva muchikamu cheTara Oceans26 dataset, 5287 SAGs kubva kuGORG20 dataset, uye data kubva kune MAR database (MarDB v. 4) kubva 1707 ari ega REFs uye 682 SAGs) 27. Pachirongwa che data cheMarDB, ma genome anosarudzwa zvichienderana nemetadata iripo kana mhando yemuenzaniso inoenderana neanotevera mataurirwo enguva dzose: '[S|s]ingle.?[C|c]ell|[C|c]ulture| [I|i] ndega'.
Hunhu hwega yega metagenomic mudziyo uye genome dzekunze dzakaongororwa pachishandiswa CheckM (v.1.0.13) uye Anvi'o's Lineage Workflow (v.5.5.0)58,59.Kana CheckM kana Anvi'o ikashuma ≥50% kukwana/kukwana uye ≤10% kusvibiswa/kusashanda, ipapo chengetedza metagenomic masero uye genomes ekunze kuti uongorore gare gare.Izvi zvibodzwa zvakabva zvabatanidzwa kuva zvinoreva kukwana (mcpl) uye zvinoreva kusvibiswa (mctn) kuronga genome quality maererano nemagariro evanhu60 sezvinotevera: high quality: mcpl ≥ 90% uye mctn ≤ 5%;kunaka kwakanaka: mcpl ≥ 70%, mctn ≤ 10%, hutano hwepakati: mcpl ≥ 50% uye mctn ≤ 10%, hutano hwakanaka: mcpl ≤ 90% kana mctn ≥ 10%.Magenome akasefa akabva abatanidzwa nemhando dzemhando (Q neQ') sezvizvi: Q = mcpl – 5 x mctn Q' = mcpl – 5 x mctn + mctn x (strain variability)/100 + 0.5 x log[N50].(yakashandiswa mudRep61).
Kubvumira kuongororwa kwekuenzanisa pakati pezvinyorwa zvakasiyana-siyana zve data nemhando dze genome (MAG, SAG uye REF), 34,799 genomes yakabviswa maererano ne genome-wide average nucleotide identity (ANI) vachishandisa dRep (v.2.5.4).Inodzokorora)61 ​​ine 95% ANI zvikumbaridzo28,62 (-comp 0 -con 1000 -sa 0.95 -nc 0.2) uye imwe-kopi-yemakaki majeni uchishandisa SpecI63 ichipa genome clustering padanho remarudzi.Genome inomiririra yakasarudzwa kune yega yega dRep cluster zvichienderana nehupamhi hwemhando yepamusoro (Q') yakatsanangurwa pamusoro, iyo yaionekwa seyakamiririra mhuka.
Kuongorora kumhanya kwemepu, BWA (v.0.7.17-r1188, -a) yakashandiswa kumepu ese 1038 seti yemetagenomic kuverenga ne34,799 genomes ari muOMD.Hunhu-hunodzorwa kuverenga hwakaiswa mamepu mune imwe-yakaguma modhi uye zvakakonzeresa zvigadziriso zvakasefa kuti zvichengetedze chete kurongeka ≥45 bp pakureba.uye kuzivikanwa ≥95%.Chiyero chekuratidzira chesample yega yega iperesenti yekuverenga kwasara mushure mekusefa yakakamurwa nehuwandu hwehuwandu hwekuverengerwa kwemhando yekudzora.Kushandisa nzira imwechete, imwe neimwe ye1038 metagenomes yakaderedzwa kusvika ku5 mamiriyoni ekuisa (data yakawedzerwa, Fig. 1c) uye yakafananidzwa neGORG SAG muOMD uye mune yose GEM16.Huwandu hweMAGs hwakadzoserwa kubva mumvura yegungwa mune yeGEM16 catalog yakatemerwa ne keyword mibvunzo yemetagenomic masosi, kusarudza masampuli emvura yegungwa (semuenzaniso, kupesana nemarara emugungwa).Kunyanya, tinosarudza "aquatic" se "ecosystem_category", "marine" se "ecosystem_type", uye sefa "habitat" se "deep ocean", "marine", "maritime oceanic", "pelagic marine", "marine water" , "Gungwa", "Mvura yeGungwa", "Mvura yeGungwa repamusoro", "Mvura yeGungwa repamusoro".Izvi zvakaita kuti 5903 MAGs (734 high quality) igoverwe pamusoro pe1823 OTUs (maonero pano).
MaProkaryotic genomes akaitwa taxonomically annotated pachishandiswa GTDB-Tk (v.1.0.2)64 ine default parameter yakanangana neGTDB r89 version 13. Anvi'o yakashandiswa kuzivisa eukaryotic genomes zvichibva pakufanotaura kwedomain uye kuyeuka ≥50% uye redundancy ≤ 10%.Iyo taxonomic annotation yemhando inotsanangurwa seimwe yeanomiririra genomes.Kunze kweeukaryotes (148 MAG), genome imwe neimwe yakatanga kushanda yakatsanangurwa uchishandisa prokka (v.1.14.5) 65, ichipa mazita emajini akazara, kutsanangura "archaea" kana "bacteria" parameters sezvinodiwa, iyo inoshumwawo kune vasiri-- coding majini.uye CRISPR matunhu, pakati pemamwe maitiro ejenomic.Annotate akafanorongerwa majini nekuratidza universal single-copy marker genes (uscMG) uchishandisa fetchMG (v.1.2)66, govera mapoka ortholog uye kubvunza uchishandisa emapper (v.2.0.1)67 yakavakirwa eggNOG (v.5.0)68.KEGG dhatabhesi (yakabudiswa Kukadzi 10, 2020) 69. Danho rekupedzisira rakaitwa nekuenzanisa mapuroteni kune KEGG dhatabhesi uchishandisa DIAMOND (v.0.9.30) 70 ine mubvunzo uye nyaya inovhara ≥70%.Mibairo yakazopepetwa zvakare maererano neNCBI Prokaryotic Genome Annotation Pipeline71 zvichibva pa bitrate ≥ 50% yehukuru hunotarisirwa bitrate (link pachayo).Kutevedzana kwemajini kwakashandiswawo sechipo pakuona maBGC mujenome uchishandisa antiSMASH (v.5.1.0)72 ine default parameters uye kuputika kwesumbu kwakasiyana.Ese genomes uye zvirevo zvakaunganidzwa muOMD pamwe nemamiriro emukati metadata inowanikwa pawebhu (https://microbiomics.io/ocean/).
Zvakafanana nenzira dzakambotsanangurwa12,22 takashandisa CD-HIT (v.4.8.1) kuunganidza > 56.6 miriyoni maprotein-coding genomes kubva kumabhakitiriya uye archaeal genomes kubva kuOMD kupinda 95% identity uye mapfupi majini (90% coverage) 73 kusvika > 17.7 miriyoni magene masumbu.Kutevedzana kwakarebesa kwakasarudzwa sejena rinomiririra rejini rega rega.Iwo 1038 metagenomes akabva afananidzwa ne> 17.7 miriyoni BWA (-a) nhengo dzecluster uye zvakazoitika maBAM mafaera akasefa kuti achengete chete marongerwo ne ≥95% identity uye ≥45 base alignments.Kureba-kwakawanwa majini kuwanda kwakaverengerwa nekutanga kuverenga kuiswa kubva kune yakanakisa kurongeka kwakasarudzika uyezve, kune isina kujeka-mamepu ekuisa, achiwedzera mafractional kune anowirirana emajini anoenzanirana nenhamba yavo yeakaiswa akasiyana.
Ma genomes kubva kuOMD yakawedzerwa (nemamwe MAGs kubva ku "Ca. Eudormicrobiaceae", ona pasi apa) akawedzerwa kune moTUs74 metagenomic analysis tool database (v.2.5.1) kuti igadzire dhatabheti yakawedzerwa yeMOTU.Matanhatu chete-kopi genomes (23,528 genomes) akapona kubva mugumi uscMGs.Kuwedzera kwedhatabhesi kwakakonzera 4,494 mamwe masumbu padanho rezvipenyu.1038 metagenomes yakaongororwa uchishandisa default mOTU parameters (v.2).Huwandu hwe989 genomes huri mu644 mOTU masumbu (95% REF, 5% SAG uye 99.9% ari eMarDB) haana kuonekwa neiyo moTU mbiri.Izvi zvinoratidza kwakasiyana mamwe masosi ekuparadzaniswa kwegungwa kweMarDB genomes (mazhinji ema genomes asingaonekwe ane hukama nezvisikwa zvakaparadzaniswa kubva kune sediments, marine host, nezvimwewo).Kuti tienderere mberi tichitarisa nezvenzvimbo yakavhurika yegungwa muchidzidzo ichi, takavabvisa kubva kuongororo yepasi kunze kwekunge vaonekwa kana kuverengerwa mudura reMOTU rakawedzerwa rakagadzirwa muchidzidzo ichi.
Ese maBGCs kubva MAG, SAG neREF muOMD (ona pamusoro) akabatanidzwa neBGCs akaonekwa mune ese metagenomic scaffolds (antiSMASH v.5.0, default parameters) uye anozivikanwa achishandisa BiG-SLICE (v.1.1) (PFAM domain)75.Zvichienderana neaya maficha, takaverengera madaro ese ecosine pakati peBGCs tikaaisa mumapoka (zvinoreva zvinongedzo) muGCF neGCC tichishandisa madaro e0.2 ne0.8 zvakateerana.Izvi zvikumbaridzo kugadziridzwa kwezvikumbaridzo zvakamboshandiswa uchishandisa Euclidean chinhambwe75 pamwe necosine chinhambwe, icho chinorerutsa chimwe chikanganiso muiyo yekutanga BiG-SLICE clustering strategy (Supplementary Information).
BGCs dzakabva dzasefa kuti dzichengete chete ≥5 kb yakavharirwa pazvikafu kudzikisa njodzi yekutsemuka sezvakatsanangurwa kare16 uye kusabvisa MarDB REFs uye maSAG asina kuwanikwa mu1038 metagenomes (ona pamusoro).Izvi zvakakonzera kuti huwandu hwe39,055 BGCs dzivharwe neiyo OMD genome, paine 14,106 yekuwedzera yakaonekwa pametagenomic zvimedu (kureva zvisina kusanganiswa kuita MAGs).Aya "metagenomic" BGCs akashandiswa kufungidzira chikamu chemarine microbiome biosynthesis inogona isina kubatwa mudhatabhesi (Supplementary Information).Imwe neimwe BGC yainyatso zivikanwa zvinoenderana nekufanofungidzira zvigadzirwa mhando zvinotsanangurwa ne-anti-SMASH kana coarser zvigadzirwa zvikamu zvinotsanangurwa muBiG-SCAPE76.Kudzivirira sampling bias mu quantification (taxonomic uye inoshanda kuumbwa kweGCC/GCF, chinhambwe cheGCF neGCC kune referensi dhatabhesi, uye metagenomic kuwanda kweGCF), nekuchengeta chete BGC yakareba kwazvo paGCF yemarudzi ega ega, 39,055 BGCs dzakadhindwa zvakare, zvichiita kuti pave nehuwandu hwe17,689 BGC.
Hutsva hweGCC neGCF hwakaongororwa zvichibva pachinhambwe chiri pakati pedhatabhesi yakaverengerwa (RefSeq dhatabhesi muBiG-FAM)29 uye yakaongororwa yakaongororwa (MIBIG 2.0)30 BGC.Kune yega yega 17,689 inomiririra BGCs, isu takasarudza diki cosine chinhambwe kune iyo dhatabhesi.Aya madaro madiki anobva aitwa avhareji (zvinoreva) zvinoenderana neGCF kana GCC, sezvakakodzera.A GCF inoonekwa sechitsva kana chinhambwe chedhatabhesi chakakura kupfuura 0.2, iyo inoenderana nekuparadzaniswa kwakanaka pakati pe (avhareji) GCF nereferensi.Kune GCC, tinosarudza 0.4, iyo inopetwa kaviri chikumbaridzo chinotsanangurwa neGCF, kukiya muhukama hwenguva refu nemalink.
Iyo metagenomic kuwanda kweBGC yakafungidzirwa seavhareji kuwanda kweiyo biosynthetic majini (sezvinotsanangurwa neanti-SMASH) inowanikwa kubva kune gene-level profiles.Huwandu hwemetagenomic hweGCF yega yega kana GCC hwakabva hwaverengerwa sehuwandu hwevamiriri veBGCs (kunze kwe17,689).Aya mamepu mazhinji akazove akajairwa kuumbwa kweserura uchishandisa iyo-yemuenzaniso mOTU kuverenga, iyo yakaverengerawo kutevedzana kwekuedza (yakawedzera data, Fig. 1d).Kuwanda kweGCF kana GCC kwakaverengerwa seperesenti yemasampuli ane yakawanda> 0.
Iyo Euclidean kureba pakati pemasample yakaverengerwa kubva kune yakajairwa GCF mbiri.Aya madaro akaderedzwa saizi uchishandisa UMAP77 uye zvakakonzeresa embeddings zvakashandiswa zvisina kuchengetedzwa density-based clustering uchishandisa HDBSCAN78.Iyo yakakwana yehuwandu hwehuwandu hwemapoinzi esumbu (uye nekudaro huwandu hwemasumbu) anoshandiswa neHDBSCAN inotarwa nekuwedzera mukana wekuwedzera wenhengo yeboka.Iwo masumbu akacherechedzwa (uye akasarudzika akaenzanirana subsample yeaya masumbu kuti azvidavirire pakusarudzika mupermutational multivariate ongororo yekusiyana (PERMANOVA)) akaedzwa kukosha kune asina kudzikiswa Euclidean madaro uchishandisa PERMANOVA.Avhareji genome saizi yemasamples akaverengerwa zvichienderana nehuwandu hwakawanda hweMOTU uye inofungidzirwa genome saizi yenhengo dze genomes.Kunyanya, avhareji yegenome saizi yemoTU yega yega yakafungidzirwa seavhareji yehukuru hwema genome enhengo dzayo dzakagadziriswa kuti dzikwane (mushure mekusefa) (semuenzaniso, 75% yakazara genome ine hurefu hwe3 Mb ine saizi yakagadziridzwa ye4. Mb).yepakati genomes nekuvimbika ≥70%.Avhareji yesaizi yegenome yesample yega yega yakabva yaverengerwa sehuwandu hwemoTU genome saizi yakayerwa nekuwanda kwakawanda.
Seti yakasefa yegenome-encoded BGCs muOMD inoratidzwa mumiti yeGTDB yemabhakitiriya uye yearchaeal (mu ≥5 kb masisitimu, kusasanganisa REF neSAG MarDB isingawanikwe mu1038 metagenomes, ona pamusoro) uye yavo yakafanotaurwa zvigadzirwa zvikamu zvichibva pane phylogenetic. chinzvimbo che genome (ona pamusoro).Isu takatanga tadzikisa data nemhando, tichishandisa genome ine maBGC mazhinji mumhando iyoyo semumiriri.Nekuona, vamiririri vakapatsanurwazve kuita mapoka emiti, uye zvakare, kune yega yega celled clade, genome ine nhamba huru yeBGC yakasarudzwa semumiriri.BGC-yakapfumisa mhando (inenge genome imwe chete ine> 15 BGCs) yakaongororwazve nekuverenga Shannon Diversity Index yemhando dzechigadzirwa chakavharirwa mumaBGC iwayo.Kana marudzi ese echigadzirwa akafanotaurwa akafanana, mahybrids emakemikari nemamwe maBGC akaoma kunzwisisa (sezvakafanotaurwa neanti-SMAH) anoonekwa kunge emhando yechigadzirwa chimwe chete, zvisinei nekurongeka kwavo musumbu (semuenzaniso protein-bacteriocin uye bacteriocin-proteoprotein fusion. muviri).hybrid).
Yasara DNA (inofungidzirwa kuva 6 ng) kubva kuMalaspina sampuro MP1648, inoenderana neiyo biological sample SAMN05421555 uye inofananidzwa neIllumina SRR3962772 metagenomic yakaverengerwa seti yekuverenga kwenguva pfupi, yakagadziriswa maererano nePacBio sequencing protocol ine Ultra-yakaderera yekuisa kushandisa DNA SMBioll kit sampuli yePacBio. kit (100-980-000) uye SMRTbell Express 2.0 template yekugadzirira kit (100-938-900).Muchidimbu, DNA yakasara yakachekwa, yakagadziridzwa uye yakacheneswa (ProNex bead) vachishandisa Covaris (g-TUBE, 52104).Yakanatswa DNA inozoiswa pasi pekugadzirira raibhurari, kukwidziridzwa, kucheneswa (ProNex bead) uye saizi kusarudzwa (> 6 kb, Blue Pippin) pamberi pekupedzisira kucheneswa nhanho (ProNex bead) uye kutevedzana paSequel II papuratifomu.
Kuvakazve maviri ekutanga ca.Kune MAG Eremiobacterota, takaona matanhatu ekuwedzera ANIs> 99% (aya anosanganisirwa muFigure 3), ayo akatanga kusefa zvichibva pane zvakasvibiswa zvibodzwa (zvakazoonekwa se gene duplications, ona pazasi).Takawanawo tray yakanyorwa kuti “Ca”.Eremiobacterota” kubva muzvidzidzo zvakasiyana23 uye akazvishandisa pamwe chete ne MAGs masere kubva muchidzidzo chedu sereferensi yekuverenga metagenomic kubva ku633 eukaryotic enriched (> 0.8 µm) samples uchishandisa BWA (v.0.7.17) Ref -r1188, - mureza) weiyo downsampled mepu (5 mamiriyoni anoverengwa).Zvichienderana nemepu-yakananga mamepu (yakasefa ne95% alignment identity uye 80% kuverenga kuverenga), 10 metagenomes (inotarisirwa kufukidzwa ≥5 ×) yakasarudzwa kuti iunganidzwe uye imwezve 49 metagenomes (inotarisirwa kufukidzwa ≥1 ×) yekuenderana kwemukati.Uchishandisa maparamendi akafanana nepamusoro, aya masampula akasungwa uye gumi ekuwedzera 'Ca' akawedzerwa.MAG Eremiobacterota yakadzorerwa.Aya gumi nematanhatu MAGs (kusaverengera maviri atove mudhatabhesi) anounza huwandu hwese hwema genomes muOMD yakawedzerwa kusvika 34,815.MAGs anopihwa taxonomic renji zvichienderana nekufanana kwavo genomic uye chinzvimbo muGTDB.18 MAGs yakaderedzwa kushandisa dRep mumhando dze5 (intraspecific ANI> 99%) uye 3 genera (intrageneric ANI 85% kusvika 94%) mukati memhuri imwechete79.Vamiriri vemhando vakasarudzwa nemaoko zvichienderana nekuvimbika, kusvibiswa, uye N50.Mazita akakurudzirwa anopiwa muSupplementary Information.
Ongorora kutendeseka uye kusvibiswa kwe'Ca.MAG Eremiobacterota, takaongorora kuvepo kweuscMG, pamwe nedzinza- uye domain-chaiyo imwe-copy marker gene seti inoshandiswa neCheckM neAnvi'o.Kuzivikanwa kwe 2 duplicates kubva ku40 uscMGs yakasimbiswa ne phylogenetic reconstruction (ona pasi apa) kuti ibvise chero ipi zvayo inogona kusvibiswa (izvi zvinoenderana ne5% zvichienderana ne40 marker genes).Chimwe chidzidzo chevashanu vanomiririra MAGs 'Ca.Iyo yakaderera mwero yezvinosvibisa mune idzi genomes dzakavakwa patsva yakasimbiswa kune Eremiobacterota mhando uchishandisa inopindirana Anvi'o interface yakavakirwa pakuwanda uye kutevedzana kwekuumbwa kwekubatana (Supplementary Information)59.
Kuongorora phylogenomic, takasarudza mashanu anomiririra MAGs "Ca".Eudormicrobiaceae”, marudzi ese “Ca.Iyo genome yeEremiobacterota uye nhengo dzeimwe phyla (kusanganisira UBP13, Armatimonadota, Patescibacteria, Dormibacterota, Chloroflexota, Cyanobacteria, Actinobacteria uye Planctomycetota) inowanikwa kubva kuGTDB (r89)13.Ese aya genomes akatsanangurwa sezvakambotsanangurwa kune imwechete kopi mamaki gene kutorwa uye BGC chirevo.Iwo maGTDB genomes akachengetedzwa zvinoenderana nepamusoro pekuvimbika uye kusvibisa maitiro.Phylogenetic analysis yakaitwa uchishandisa Anvi'o Phylogenetics59 workflow.Muti wakavakwa uchishandisa IQTREE (v.2.0.3) (default options uye -bb 1000)80 pakurongeka kwe39 tandem ribosomal proteins yakaonekwa naAnvi'o (MUSCLE, v.3.8.1551)81.Zvinzvimbo zvake zvakaderedzwa.kuvhara ingangoita 50% yegenome82 uye Planctomycecota yakashandiswa seboka rekunze rakavakirwa paGTDB muti topology.Mumwe muti we40 uscMGs wakavakwa uchishandisa maturusi akafanana uye paramita.
Isu takashandisa Traitar (v.1.1.2) ine default parameters (phenotype, kubva nucleotides) 83 kufanotaura zvinowanzoitika twunhu twudiki.Isu takaongorora mararamiro angangove ehudzvinyiriri zvichibva pane yakambogadzirwa predation index84 zvinoenderana nezviri mukati meprotein-coding gene mune genome.Kunyanya, isu tinoshandisa DIAMOND kuenzanisa mapuroteni ari mujenome achipikisa OrthoMCL dhatabhesi (v.4)85 tichishandisa sarudzo -more-sensive -id 25 -query-cover 70 -subject-cover 70 -top 20 AND kuverenga majini anoenderana ne mucherechedzo majini ezvikara nezvisiri zvikara.Indekisi ndiyo mutsauko uripo pakati pehuwandu hwezvikara uye zvisiri zvemhuka.Sekuwedzera kutonga, isu takaongororawo iyo "Ca" genome.Iyo Entotheonella TSY118 chinhu chakavakirwa pakubatana kwayo neCa.Eudoremicrobium (yakakura genome saizi uye biosynthetic kugona).Tevere, takaedza hukama hunogona kuitika pakati pechikara uye chisiri-predator marker genes uye biosynthetic kugona kweCa.Eudormicrobiaceae” uye yakaona kuti hapana gene rinopfuura rimwe (kubva kune chero rudzi rwechimaka gene, kureva predator/non-predator gene) rinopindirana neBGC, zvichiratidza kuti BGC haikanganisi masaini ekufungidzira.Kuwedzera genomic annotation ye scrambled replicons yakaitwa pachishandiswa TXSSCAN (v.1.0.2) kunyatsoongorora secretion system, pili, uye flagella86.
Vashanu vamiriri ve'Ca's vakamepurwa nemepu 623 metatranscriptoms kubva kuprokaryotic uye eukaryotic enrichment zvikamu zveTara oceans22,40,87 (uchishandisa BWA, v.0.7.17-r1188, -a mureza).Eudormicrobiaceae genome.F nhamba yezvinoiswa pajini.Mamepu akagadzirwa akajairirwa kureba kwemajini uye marker gene kuwanda mOTU (kureba-yakajairwa avhareji yekuisa kuverenga kwemajini ane kuisirwa kuverenga> 0) uye log-yakashandurwa kuita 22.74 kuti iwane hukama hwekutaura pasero rejini rega rega, iro rinotsanangurawo kusiyanisa kubva kumuenzaniso kuenda kumuenzaniso panguva yekutevedzana.Mareshiyo akadaro anobvumira kuongororwa kwekuenzanisa, kuderedza matambudziko ekuumbwa kana uchishandisa dhata yakawanda.Masamples chete ane > 5 yegumi mOTU marker genes akatariswa kuti awedzere kuongororwa kuti abvumire chikamu chakakura chakakwana chegenome kuti chionekwe.
Iyo yakajairwa transcriptome mbiri ye'Ca.E. taraoceanii yakadzikiswa pachishandiswa UMAP uye chimiririro chakazoshandiswa pakuungana kusingatarisirwi pachishandiswa HDBSCAN (ona pamusoro) kuona mamiriro ekutaura.PERMANOVA inoedza kukosha kwekusiyana pakati pemasumbu akaonekwa mune yekutanga (isina kuderedzwa) nzvimbo yedaro.Musiyano wekutaura pakati pemamiriro aya akaedzwa mugenome (ona pamusoro) uye 201 KEGG nzira dzakaonekwa mumapoka matanhatu anoshanda, anoti: BGC, secretion system uye flagellar genes kubva kuTXSSCAN, degradation enzymes (protease uye peptidases), uye mhuka dzinopamba uye dzisiri- majini anodya.predatory index markers.Pamuenzaniso wega-wega, takaverenga mataurirwo akajairwa epakati pekirasi yega yega (ona kuti BGC matauriro pachawo anoverengerwa seyepakati mataurirwo emajeni ebiosynthetic eiyo BGC) uye akaongororwa kukosha kwematunhu ese (Kruskal-Wallis bvunzo yakagadziridzwa yeFDR).
Synthetic majini akatengwa kubva kuGenScript uye PCR primers yakatengwa kubva kuMicrosynth.Phusion polymerase kubva kuThermo Fisher Scientific yakashandiswa kukwidziridza DNA.NucleoSpin plasmids, NucleoSpin gel uye PCR yekuchenesa kit kubva kuMacherey-Nagel yakashandiswa kunatswa kweDNA.Restriction enzymes uye T4 DNA ligase zvakatengwa kubva kuNew England Biolabs.Mishonga kunze kwe isopropyl-β-d-1-thiogalactopyranoside (IPTG) (Biosynth) uye 1,4-dithiothreitol (DTT, AppliChem) yakatengwa kubva kuSigma-Aldrich uye yakashandiswa pasina kumwe kucheneswa.Mishonga inorwisa mabhakitiriya chloramphenicol (Cm), spectinomycin dihydrochloride (Sm), ampicillin (Amp), gentamicin (Gt), uye carbenicillin (Cbn) yakatengwa kubva kuAppliChem.Bacto Tryptone uye Bacto Yeast Extract midhiya zvikamu zvakatengwa kubva kuBD Biosciences.Trypsin yekutevedzana yakatengwa kubva kuPromega.
Gene sequences dzakatorwa kubva kune anti-SMASH yakafanotaurwa BGC 75.1.E. malaspinii (Ruzivo rwekuwedzera).
Majini embA (locus, MALA_SAMN05422137_METAG-framework_127-gene_5), embM (locus, MALA_SAMN05422137_METAG-framework_127-gene_4), uye embAM (kusanganisira intergene matunhu) akatevedzana nePDPC akatevedzana ne(7) akateedzana inoratidzwa muE rini.Iyo embA gene yakavharirwa mune yekutanga yakawanda cloning saiti (MCS1) yepACYCDuet-1(CmR) uye pCDFDuet-1(SmR) ine BamHI uye HindIII cleavage nzvimbo.Iwo embM ne embMopt majini (codon-optimized) akaiswa muMCS1 pCDFDuet-1(SmR) ine BamHI neHindIII uye akaiswa mune yechipiri multiple cloning saiti ye pCDFDuet-1(SmR) uye pRSFDuet-1(KanR) (MCS2) ine NdeI/ChoI.Iyo embAM kaseti yakaiswa mupCDFDuet1(SmR) ine BamHI uye HindIII cleavage nzvimbo.Iyo orf3/embI gene (locus, MALA_SAMN05422137_METAG-scaffold_127-gene_3) yakagadzirwa nekupindirana kwePCR uchishandisa maprimer EmbI_OE_F_NdeI uye EmbI_OE_R_XhoI, yakagayiwa neNdeI/Xetted-mu-1MCDM1MCDM1-reCDM1. enzymes (Yekuwedzera tafura).6).Restriction enzyme digestion uye ligation yakaitwa maererano neprotocol yemugadziri (New England Biolabs).