304 ʻO ke kila kila wiliwili ʻia i ka paipu wili ʻia / tubing zhemical zomponent, Biosynthetic Potential o ka Global Marine Microbiome

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Hōʻike kikoʻī huahana

304 ʻO ke kila kila wiliwili ʻia i ka paipu / paipu
1. Hōʻike: ʻO ke kila kila wili paipu / tubing
2. ʻAno: welded a seamless
3. Kūlana: ASTM A269, ASTM A249
4. Kuhiliʻole kila coil paipu OD: 6mm i 25.4MM
5. Ka lōʻihi: 600-3500MM aiʻole e like me ka makemake o ka mea kūʻai.
6. Pā mānoanoa: 0.2mm i 2.0mm.

7. Hoʻomanawanui: OD: +/-0.01mm;Mānoanoa: +/-0.01%.

8. Coil i loko o ka lua nui: 500MM-1500MM (hiki ke hoʻololi e like me nā koi o ka mea kūʻai aku)

9. Coil kiʻekiʻe: 200MM-400MM (hiki ke hoʻololi e like me nā koi o ka mea kūʻai aku)

10. Ili: Bright a annealed
11. Mea: 304, 304L, 316L, 321, 301, 201, 202, 409, 430, 410, huila 625, 825, 2205, 2507, etc.
12. Hoʻopili: ʻeke i ulana ʻia i ka pahu lāʻau, pallet lāʻau, lāʻau lāʻau, a i ʻole e like me ka makemake o ka mea kūʻai aku.
13. Ho'āʻo: ʻāpana kemika, ikaika hua, ikaika tensile, ana paʻakikī
14. Hōʻoiaʻiʻo: ʻO ke kolu o ka ʻaoʻao (no ka laʻana: SGS TV ) nānā, etc.
15. Hoʻohana: Mea hoʻonani, mea lako, lawe aila, mea hoʻololi wela, hana railing, hana pepa, kaʻa, kaʻai ʻana, lāʻau lapaʻau, etc.

He ʻano phylogenetically a me ka metabolically nā kaiāulu microbial kūlohelohe.Ma waho aʻe o nā pūʻulu o nā organisms1 i hoʻonaʻauao ʻia, paʻa kēia ʻokoʻa i kahi waiwai nui no ka ʻike ʻana i nā enzymes koʻikoʻi a me ka biotechnologically nui a me nā pūhui biochemical2,3.Eia nō naʻe, ʻo ke aʻo ʻana i kēia ʻokoʻa e hoʻoholo ai i nā ala genomic e synthesize i ia mau pūhui a hoʻopaʻa iā lākou i kā lākou mau pūʻali paʻa.ʻAʻole ʻike nui ʻia ka hiki biosynthetic o nā microorganism ma ke kai ākea ma muli o nā palena o ka nānā ʻana i ka ʻikepili hoʻonā genome holoʻokoʻa ma ka pae honua.Ma ʻaneʻi, ʻimi mākou i ka ʻokoʻa a me ka ʻokoʻa o nā pūʻulu gene biosynthetic i loko o ke kai ma o ka hoʻohui ʻana ma kahi o 10,000 microbial genomes mai nā cell moʻomeheu a me nā cell hoʻokahi me ka ʻoi aku ma mua o 25,000 hou i kūkulu hou ʻia nā genomes mai ka 1,000 mau ʻano wai kai.Ua ʻike ʻia kēia mau hoʻoikaika ʻana ma kahi o 40,000 putative ka hapanui o nā pūʻulu gene biosynthetic hou, ʻo kekahi o ia mau mea i loaʻa i nā hui phylogenetic i manaʻo ʻole ʻia.I loko o kēia mau heluna kanaka, ua ʻike mākou i kahi moʻopuna i hoʻonui ʻia i loko o nā pūʻulu gene biosynthetic (“Candidatus Eudormicrobiaceae”) i pili i kahi phylum bacteria i kanu ʻole ʻia a ua komo kekahi o nā microorganisms biosynthetically like ʻole i kēia kaiapuni.ʻO kēia mau mea, ua hōʻike mākou i nā ala phosphatase-peptide a me pytonamide, e ʻike ana i nā ʻano o ka hoʻohuihui bioactive ʻokoʻa a me ka enzymology, kēlā me kēia.I ka hopena, hōʻike kēia haʻawina pehea e hiki ai i nā hoʻolālā microbiome-based ke hiki i ka ʻimi ʻana i nā enzyme i wehewehe ʻole ʻia ma mua a me nā meaʻai kūlohelohe i loko o kahi microbiota a me ka honua i maopopo ʻole.
Hoʻokele nā ​​microbes i nā pōʻai biogeochemical honua, mālama i nā pūnaewele meaʻai, a mālama pono i nā mea kanu a me nā holoholona5.ʻO kā lākou nui phylogenetic, metabolic and functional diversity e hōʻike ana i ka waiwai waiwai no ka loaʻa ʻana o nā taxa1 hou, nā enzymes a me nā pūhui biochemical, me nā huahana kūlohelohe6.I loko o nā kaiaulu kaiaola, hāʻawi kēia mau molekala i nā microorganisms me nā ʻano hana physiological a me ka ékological, mai ke kamaʻilio ʻana a hiki i ka hoʻokūkū 2, 7.Ma waho aʻe o kā lākou mau hana kumu, hāʻawi kēia mau huahana kūlohelohe a me kā lākou genetically coded production pathways i nā hiʻohiʻona no nā noi biotechnological a therapeutic2,3.Ua maʻalahi ka ʻike ʻana o ia mau ala a me nā pilina ma ke aʻo ʻana i nā microbes moʻomeheu.Eia naʻe, ua hōʻike ʻia nā haʻawina taxonomic o nā kaiapuni kūlohelohe ʻaʻole i mahi ʻia ka hapa nui o nā microorganisms8.Hoʻopili kēia ʻano moʻomeheu i ko mākou hiki ke hoʻohana i ka ʻokoʻa hana i hoʻopaʻa ʻia e nā microbes4,9.
No ka lanakila ʻana i kēia mau palena, ua ʻae ka holomua ʻenehana i nā makahiki he ʻumi i hala aku nei i ka poʻe noiʻi e hoʻonohonoho pololei (ʻo ia hoʻi, me ka ʻole o ka moʻomeheu mua) i nā ʻāpana DNA microbial mai nā kaiāulu holoʻokoʻa (metagenomics) a i ʻole nā ​​​​pūnaewele hoʻokahi.ʻO ka hiki ke hōʻuluʻulu i kēia mau ʻāpana i loko o nā ʻāpana genome nui a kūkulu hou i nā genomes metagenomically assembled genomes (MAGs) a i ʻole single amplified genomes (SAGs), i kēlā me kēia, e wehe i kahi manawa nui no nā haʻawina taxocentric o ka microbiome (ʻo ia hoʻi, nā kaiāulu microbial a me ka microbiome).e hoala i na ala hou.pono'ī i loko o kahi kaiapuni i hāʻawi ʻia) 10,11,12.ʻOiaʻiʻo, ua hoʻonui nui nā haʻawina hou i ka hōʻike phylogenetic o ka ʻokoʻa microbial ma Earth1, 13 a ua hōʻike i ka nui o ka ʻokoʻa hana i loko o nā kaiāulu microbial pākahi i uhi ʻole ʻia e nā moʻomeheu microorganism reference genome sequences (REFs)14.He mea koʻikoʻi ka hiki ke kau i ka ʻokoʻa hana i ʻike ʻole ʻia i loko o ka pōʻaiapili o ka genome host (ʻo ia hoʻi, genome resolution) he mea koʻikoʻi no ka wānana ʻana i nā laina microbial i ʻike ʻole ʻia e hoʻopili paha i nā huahana kūlohelohe hou15,16 a i ʻole no ka huli ʻana i ia mau pūhui i kā lākou mea hana mua17.No ka laʻana, ua alakaʻi ʻia kahi ʻano loiloi metagenomic a me hoʻokahi-cell genomic i ka ʻike ʻia ʻana o Candidatus Entotheonella, he pūʻulu o nā maʻi bacteria e pili ana i ka huaʻai metabolically waiwai, ma ke ʻano he mea hana i nā ʻano lāʻau like ʻole18.Eia nō naʻe, ʻoiai nā hoʻāʻo hou ʻana i ka ʻimi genomic o nā kaiāulu microbial like ʻole, 16,19 ʻoi aku ma mua o ʻelua hapakolu o ka ʻikepili metagenomic honua no ka moana nui loa o ka honua kaiaola16,20 e nalowale ana.No laila, ma ka laulā, ʻike nui ʻia ka hiki ke biosynthetic o ka microbiome moana a me kona hiki ke hoʻopaʻa ʻia o ka enzymatic novel a me nā huahana kūlohelohe.
No ka ʻimi ʻana i ka hiki ke biosynthetic o nā microbiomes moana ma ka pae honua, ua hoʻohui mua mākou i nā genomes microbial moana i loaʻa me ka hoʻohana ʻana i nā ʻano moʻomeheu a me nā ʻano moʻomeheu ʻole e hana i kahi waihona nui o nā phylogenetics a me ka hana gene.Ua hōʻike ʻia i ka nānā ʻana i kēia ʻikepili i nā ʻano hui like ʻole biosynthetic gene clusters (BGC), ʻo ka hapa nui o ia mau mea no nā ʻohana ʻohana uncharacterized gene cluster (GCF).Eia hou, ua ʻike mākou i kahi ʻohana bacteria i ʻike ʻole ʻia e hōʻike ana i ka ʻokoʻa kiʻekiʻe loa o nā BGC i ka moana ākea a hiki i kēia lā.Ua koho mākou i ʻelua ala ribosomal synthesis a me post-translationally modified peptide (RiPP) ala no ka hōʻoia hoʻokolohua ma muli o kā lākou ʻokoʻa genetic mai nā ala i ʻike ʻia i kēia manawa.Ua hōʻike ʻia ke ʻano hana o kēia mau ala i nā hiʻohiʻona i manaʻo ʻole ʻia o ka enzymology a me nā pūhui maʻamau ʻole me ka hana pale protease.
I ka wā mua, ua manaʻo mākou e hana i kahi waiwai ʻikepili honua no ka nānā ʻana i ka genome, e kālele ana i kāna mau mea bacteria a archaeal.No kēia hopena, ua hoʻohui mākou i ka ʻikepili metagenomic a me 1038 mau laʻana wai kai mai 215 mau wahi laʻana o ka honua (latitude range = 141.6°) a me kekahi mau papa hohonu (mai ka 1 a 5600 m ka hohonu, e uhi ana i nā ʻāpana pelagic, mesopelagic a me abyssal).Background21,22,23 (Fig. 1a, kaʻikepili hoʻonui, Fig. 1a a me ka Papa Hoʻohui 1).Ma waho aʻe o ka hāʻawi ʻana i kahi uhi ākea ākea, ua ʻae kēia mau mea kānana i koho ʻia e hoʻohālikelike i nā ʻāpana like ʻole o ka microbiome moana, e komo pū ana me ka virus-rich (<0.2 µm), prokaryotic-rich (0.2-3 µm), particle-rich (0.8 µm). ).–20 µm) a me nā kolo i hoʻopau ʻia (>0.2 µm).
a, He 1038 mau genomes i loaʻa i ka lehulehu (metagenomics) o nā kaiaulu microbial moana i hōʻiliʻili ʻia mai 215 mau wahi i hoʻolaha ʻia ma ka honua (62°S a 79°N a me 179°W a 179°E.).Palapala palapala © Esri.Nā Puna: GEBCO, NOAA, CHS, OSU, UNH, CSUMB, National Geographic, DeLorme, NAVTEQ, a me Esri.b, ua hoʻohana ʻia kēia mau metagenomes no ke kūkulu hou ʻana i nā MAGs (nā ʻano a me nā ʻike hou aʻe), i ʻokoʻa i ka nui a me ka maikaʻi (nā ʻano) i nā datasets (hōʻailona ʻia i ke kala).Hoʻohui ʻia nā MAG i kūkulu hou ʻia me nā genomes i loaʻa i ka lehulehu (waho), me ka MAG26, SAG27 a me REF.27 Houluulu OMD.c, ke hoʻohālikelike ʻia me nā hōʻike mua e pili ana i ka SAG (GORG)20 a i ʻole MAG (GEM)16, hoʻomaikaʻi ʻo OMD i ke ʻano genomic o nā kaiāulu microbial moana (metagenomic read mapping rate; method) e ʻelua a ʻekolu mau manawa me ka hōʻike kūlike i ka hohonu a latitu..<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, ʻO ka hui ʻana o OMD i loko o nā pae puʻupuʻu ʻano (95% mean nucleotide identity) e hōʻike ana i ka huina o ma kahi o 8300 mau ʻano, ʻoi aku ma mua o ka hapalua o ia mau mea ʻaʻole i hōʻike mua ʻia e like me nā annotations taxonomic me ka hoʻohana ʻana i ka GTDB (version 89) e, ka hoʻokaʻawale ʻana o nā ʻano ma ke ʻano genome i hōʻike ʻia ua hoʻokō maikaʻi nā MAG, SAG a me REF i kekahi i kekahi i ka noʻonoʻo ʻana i ka ʻokoʻa phylogenetic o ka microbiome moana.ʻO ka mea kūikawā, 55%, 26% a me 11% o nā ʻano mea kikoʻī no MAG, SAG a me REF, kēlā me kēia.BATS, Bermuda Atlantic Time Series;GEM, nā genomes o ka microbiome o ka Honua;ʻO GORG, ka genome kuhikuhi moana honua;HOT, Hawaiian Ocean time series.
Me ka hoʻohana ʻana i kēia ʻikepili, ua kūkulu hou mākou i ka huina o 26,293 MAGs, ka hapa nui o ka bacteria a me ka archaeal (Fig. 1b a me ka ʻikepili hoʻonui, Fig. 1b).Ua hana mākou i kēia mau MAG mai nā hui ʻokoʻa ma mua o ka hui ʻana i nā laʻana metagenomic i mea e pale ai i ka hāʻule ʻana o ka hoʻololi ʻana o ke kaʻina kūlohelohe ma waena o nā laʻana mai nā wahi like ʻole a i ʻole nā ​​manawa manawa (nā ala).Eia hou, ua hui pū mākou i nā ʻāpana genomic e pili ana i kā lākou prevalence correlations ma waena o ka nui o nā laʻana (mai 58 a 610 mau laʻana, ma muli o ka noiʻi; ʻano hana).Ua ʻike mākou he step24 hoʻopau manawa akā koʻikoʻi kēia i hoʻokuʻu ʻia i kekahi mau hana hana hou MAG16, 19, 25 nui a hoʻomaikaʻi nui i ka nui (2.7-paʻi ma ka awelika) a me ka maikaʻi (+ 20% ma ka awelika) o ka genome.hana hou ʻia mai ka metagenome moana i aʻo ʻia ma ʻaneʻi (nā ʻikepili hoʻonui, Fig. 2a a me nā ʻike hou aʻe).ʻO ka holoʻokoʻa, ua hopena kēia mau hana i ka piʻi ʻana o 4.5-fold i nā MAGs microbial moana (6-fold inā ʻike ʻia nā MAG kiʻekiʻe wale nō) i hoʻohālikelike ʻia i ka punawai MAG piha loa i loaʻa i kēia lā16 (Methods).Ua hui pū ʻia kēia set MAG i hana hou ʻia me 830 mau MAG26 i koho lima, 5969 SAG27 a me 1707 REF.He iwakāluakūmāhiku mau ʻano o ka bacteria moana a me archaea i hui pū ʻia he 34,799 genomes (Fig. 1b).
A laila, loiloi mākou i ka waiwai hou i hana ʻia e hoʻomaikaʻi i kona hiki ke hōʻike i nā kaiāulu microbial moana a nānā i ka hopena o ka hoʻohui ʻana i nā ʻano genome like ʻole.Ma ka awelika, ua ʻike mākou ua uhi ʻia ma kahi o 40-60% o ka ʻikepili metagenomic moana (Figure 1c), ʻelua a ʻekolu mau manawa o ka uhi ʻana o nā hōʻike MAG-wale nō ma mua i ka hohonu a me ka latitude More serial 16 a i ʻole SAG20.Eia kekahi, no ke ana ʻana i ka ʻokoʻa ʻauhau ma nā hōʻiliʻili i hoʻokumu ʻia, ua hōʻike mākou i nā genome āpau me ka hoʻohana ʻana i ka pahu hana Genome Taxonomy Database (GTDB) a hoʻohana i ka ʻike nucleotide genome-ākea o 95%.28 e ʻike i 8,304 mau pūʻulu ʻano (nā ʻano).ʻAʻole i ʻike mua ʻia ʻelua hapakolu o kēia mau ʻano (me nā clades hou) i ka GTDB, kahi i ʻike ʻia ai ka 2790 me ka hoʻohana ʻana i ka MAG i kūkulu hou ʻia i kēia haʻawina (Fig. 1d).Eia kekahi, ua ʻike mākou he hoʻohui nui nā ʻano genomes: 55%, 26%, a me 11% o nā ʻano i haku ʻia me MAG, SAG, a me REF, kēlā me kēia (Fig. 1e).Eia kekahi, ua uhi ʻo MAG i nā ʻano 49 a pau i loaʻa i ka kolamu wai, ʻoiai ʻo SAG a me REF wale nō ka 18 a me 11 o lākou.Eia naʻe, ʻoi aku ka maikaʻi o ka SAG i ka ʻokoʻa o nā clades maʻamau (ka ʻikepili i hoʻonui ʻia, Fig. 3a), e like me Pelagic Bacteriales (SAR11), me ka SAG e uhi ana ma kahi o 1300 mau ʻano a me MAG wale nō 390 mau ʻano.ʻO ka mea nui, ʻaʻole i hoʻopili ʻia nā REF me MAG a i ʻole SAG ma ka pae ʻano a ua hōʻike ʻia ma kahi o 95% o ka 1000 genomes ʻaʻole i loaʻa i loko o nā pūʻulu metagenomic moana ākea i aʻo ʻia ma ʻaneʻi, ma muli o ka launa pū ʻana me nā ʻano mea like ʻole o ka moana i hoʻokaʻawale ʻia (e laʻa. .aiʻole hoa hoʻokipa).No ka hoʻolako ākea ʻana i ke kaiāulu ʻepekema, hiki ke hoʻohālikelike ʻia kēia punawai genome moana, kahi i hoʻopaʻa ʻia i nā ʻāpana unclassified (e laʻa, mai nā phages wānana, nā mokupuni genomic, a me nā ʻāpana genome i lawa ʻole ai ka ʻikepili no ka hana hou ʻana o MAG), hiki ke hoʻohālikelike ʻia me ka ʻikepili taxonomic. .E kiʻi i nā hōʻike me ka hana gene a me nā ʻāpana ʻokoʻa ma ka Ocean Microbiology Database (OMD; https://microbiomics.io/ocean/).
A laila hoʻomaka mākou e ʻimi i ka waiwai a me ka mea hou o ka hiki ke biosynthetic i nā microbiome moana ākea.No kēia hopena, ua hoʻohana mua mākou i ka antiSMASH no nā MAG, SAG, a me REF i loaʻa ma 1038 marine metagenomes (nā ʻano) e wānana i ka huina o 39,055 BGC.A laila hoʻohui mākou i kēia mau mea i loko o 6907 GCFs non-redundant a me 151 gene cluster populations (GCCs; Supplementary Table 2 a me nā ʻano) no ka helu ʻana i ka redundancy inherent (ʻo ia hoʻi, hiki ke hoʻopili ʻia ka BGC i nā genome he nui) a me ka metagenomic data Fragmentation of concentrated BGCs.ʻAʻole i hoʻonui nui ʻia nā BGC i hoʻopiha ʻole ʻia, inā loaʻa kekahi (Supplementary Information), ka helu o GCFs a me GCCs, i kēlā me kēia, aia ma ka liʻiliʻi hoʻokahi lālā BGC paʻa i 44% a me 86% o nā hihia.
Ma ka pae GCC, ua ʻike mākou i nā ʻano like ʻole o nā RiPP i wānana ʻia a me nā huahana kūlohelohe ʻē aʻe (Fig. 2a).I waena o lākou, no ka laʻana, nā arylpolenes, carotenoids, ectoines, a me siderophores no nā GCC me kahi pālahalaha phylogenetic ākea a me ka nui o nā metagenomes moana, e hōʻike ana i kahi hoʻololi ākea o nā microorganisms i ke kaiapuni moana, me ke kūʻē ʻana i nā ʻano oxygen reactive. oxidative a me ka osmotic stress..aiʻole ka hao hao (ʻike hou aku).Hoʻohālikelike kēia ʻokoʻa hana me kahi loiloi hou o kahi 1.2 miliona BGC ma waena o 190,000 genomes i mālama ʻia i ka waihona NCBI RefSeq (BiG-FAM/RefSeq, i kapa ʻia ma hope nei ʻo RefSeq)29, ka mea i hōʻike i ka nonribosomal Synthetase peptides (NRPS) a me ka polyketide synthase. (PKS) BGCs (ʻIke Hoʻohui).Loaʻa iā mākou he 44 (29%) GCC pili wale i kekahi RefSeq BGC (\(\bar{d}\)RefSeq> 0.4; Fig. 2a a me nā ala) a me 53 (35%) GCC wale nō ma MAG, e hōʻike ana i ka hiki. e ʻike i nā kemika i wehewehe ʻole ʻia ma OMD.Ma muli o kēlā me kēia o kēia mau GCC e hōʻike ana i nā hana biosynthetic ʻokoʻa loa, ua kālailai hou mākou i ka ʻikepili ma ka pae GCF i ka hoʻāʻo ʻana e hāʻawi i kahi hui kikoʻī o nā BGC i wānana ʻia e code no nā huahana kūlohelohe like29.ʻO ka huina o 3861 (56%) i ʻike ʻia ʻaʻole i uhi ʻia nā GCF me RefSeq, a ʻoi aku ka 97% o nā GCF ʻaʻole i loaʻa i MIBiG, kekahi o nā waihona nui loa o nā BGC i hoʻopaʻa ʻia i ka hoʻokolohua (Figure 2b).ʻOiai ʻaʻole ia he mea kupanaha ka ʻike ʻana i nā ala hou e hiki mai ana i nā hoʻonohonoho ʻaʻole i hōʻike maikaʻi ʻia e ka genome reference, ʻo kā mākou ala no ka hoʻopau ʻana i nā BGC i GCF ma mua o ka hoʻohālikelike ʻana he ʻokoʻa ia mai nā hōʻike mua 16 a hiki iā mākou ke hāʻawi i kahi loiloi kūpono ʻole o ka mea hou.ʻO ka hapa nui o nā ʻokoʻa hou (3012 GCF a i ʻole 78%) pili i nā terpenes i wānana ʻia, RiPP a i ʻole nā ​​​​huahana kūlohelohe ʻē aʻe, a ʻo ka hapa nui (1815 GCF a i ʻole 47%) ua hoʻopili ʻia i nā ʻano ʻike ʻole ma muli o ko lākou hiki ke biosynthetic.ʻAʻole like me nā pūʻulu PKS a me NRPS, ʻoi aku ka liʻiliʻi o kēia mau BGC paʻa i ka wā o ka hui metagenomic 31 a hāʻawi i ka manawa a me ka waiwai-koʻikoʻi o kā lākou huahana.
Ua hui pū ʻia he 39,055 BGC i 6,907 GCF a me 151 GCC.a, hōʻike ʻikepili (waho i loko).ʻO ka hui ʻana o ka hierarchical o nā mamao BGC e pili ana i ka GCC, 53 o ia mau mea i hoʻopaʻa ʻia e MAG wale nō.Aia i loko o ka GCC nā BGC mai nā ʻauhau like ʻole (ln-transformed gate frequency) a me nā papa BGC like ʻole (kūlike ka nui o ka pōʻai i kona alapine).No kēlā me kēia GCC, hōʻike ka papa waho i ka helu o nā BGC, ka prevalence (pakeneka o nā laʻana), a me ka mamao (ka palena liʻiliʻi BGC cosine (min(dMIBiG))) mai BiG-FAM a i BGC.Hōʻike ʻia nā GCC me nā BGC e pili kokoke ana i nā BGC i hōʻoia ʻia i ka hoʻokolohua (MIBiG) me nā pua.b Ka hoʻohālikelike ʻana i ka GCF me nā BGC i wānana ʻia (BiG-FAM) a me nā BGC i hōʻoia ʻia i ka hoʻokolohua, 3861 mau GCF hou (d–>0.2) i loaʻa.ʻO ka hapa nui (78%) o kēia code no RiPP, terpenes, a me nā huahana kūlohelohe ʻē aʻe.c, ua waiho 'ia nā genomes a pau i ka OMD i loa'a ma 1038 marine metagenomes i loko o ka kumu kumu kumu GTDB e hō'ike i ka uhi phylogenetic o ka OMD.Hōʻike ʻia nā clades me ka ʻole o nā genome ma ka OMD i ka hina.Pili ka helu o nā BGC i ka helu nui loa o nā BGC i wānana ʻia no kēlā me kēia genome ma kahi clade i hāʻawi ʻia.No ka akaka, ua hāʻule ka 15% hope loa o nā nodes.Hōʻike nā pua i nā ʻāpana waiwai i BGC (>15 BGC), koe wale ʻo Mycobacterium, Gordonia (ʻelua wale nō iā Rhodococcus), a me Crocosphaera (ʻelua wale nō iā Synechococcus).d, ʻAʻole ʻike ʻia c.Ua hōʻike ʻo Eremiobacterota i ke ʻano biosynthetic kiʻekiʻe loa (Shannon index e pili ana i ke ʻano huahana kūlohelohe).Hōʻike kēlā me kēia hui i ka genome me ka nui o nā BGC i ka ʻano.T1PKS, PKS ʻano I, T2/3PKS, PKS ʻano II a me ke ʻano III.
Ma waho aʻe o ka waiwai a me ka mea hou, ʻimi mākou i ke ʻano biogeographic o ka hiki biosynthetic o ka microbiome moana.ʻO ka hui pū ʻana o nā laʻana ma ka awelika metagenomic GCF kope helu puʻupuʻu (Methods) hōʻike i ka haʻahaʻa-latitude, surface, prokaryotic-rich and virus- poor community, ʻo ka hapa nui mai ka ʻili a i ʻole ka wai hohonu o ka lā, ua waiwai i nā terpenes RiPP a me BGC.ʻO ka hoʻokaʻawale ʻana, ua pili nā polar, hohonu-kai, virus-a me nā kaiāulu waiwai me ka nui o ka NRPS a me PKS BGC (nā ʻikepili i hoʻonui ʻia, Fig. 4 a me nā ʻike hou aʻe).ʻO ka mea hope loa, ua ʻike mākou ʻo nā kaiāulu tropical a me pelagic i aʻo maikaʻi ʻia nā kumu hoʻohiki ʻoi loa o nā terpenes hou (Augmented Data Figure).Loaʻa kiʻekiʻe loa no PKS, RiPP a me nā huahana kūlohelohe ʻē aʻe (Figure 5a me ka ʻikepili hoʻonui).
No ka hoʻokō ʻana i kā mākou noiʻi ʻana i ka hiki ke biosynthetic o nā microbiomes moana, manaʻo mākou e palapala i kā lākou puʻupuʻu phylogenetic a ʻike i nā clades hou i hoʻonui ʻia BGC.No kēia hopena, ua kau mākou i nā genomes o nā microbes moana i loko o kahi kumulāʻau bacterial a me archaeal phylogenetic GTDB13 maʻamau a uhi i nā ala biosynthetic putative a lākou e hoʻopili ai (Fig. 2c).Ua ʻike maʻalahi mākou i kekahi mau clades i hoʻonui ʻia i ka BGC (i hōʻike ʻia e nā 15 BGCs) i nā laʻana wai kai (nā ʻano) i ʻike ʻia no ko lākou hiki ke biosynthetic, e like me cyanobacteria (Synechococcus) a me Proteus bacteria, e like me Tistrella32,33, a i ʻole i huki ʻia i ka nānā no kā lākou. huahana kūlohelohe .e like me Myxococcota (Sandaracinaceae), Rhodococcus a me Planctomycetota34,35,36.ʻO ka mea mahalo, ua ʻike mākou i kekahi mau laina i ʻike ʻole ʻia ma kēia mau clades.No ka laʻana, ʻo kēlā mau ʻano i loaʻa ka waiwai biosynthetic i loko o ka phyla Planctomycetota a me Myxococcota no nā kauoha moho a me nā genera i hoʻokaʻawale ʻole ʻia.Hoʻohui pū ʻia, hōʻike kēia e hāʻawi ka OMD i ka ʻike i ka ʻike phylogenetic i ʻike mua ʻole ʻia, me nā microorganisms, e hōʻike ana i nā pahuhopu hou no ka enzyme a me ka ʻike ʻana i nā huahana kūlohelohe.
Ma hope aʻe, ua hōʻike mākou i ka clade i hoʻonui ʻia i ka BGC ma ka helu ʻana i ka helu kiʻekiʻe o nā BGC i hoʻopaʻa ʻia e kona mau lālā, akā me ka loiloi ʻana i ka ʻokoʻa o kēia mau BGC, e wehewehe ana i ka pinepine o nā ʻano huahana moho kūlohelohe (Fig. 2c a me nā ʻano hana. )..Ua ʻike mākou ua hōʻike ʻia ka nui o nā ʻano like ʻole biosynthetically e nā MAG bacteria i hana ʻia ma kēia noiʻi.Aia kēia mau bacteria i ka phylum i kanu ʻole ʻia ʻo Candidatus Eremiobacterota, kahi i ʻike ʻole ʻia ma waho o kekahi mau haʻawina genomic37,38.He mea mahalo ia "ca.Ua kālailai ʻia ka genus Eremiobacterota ma kahi kaiapuni honua39 a ʻaʻole ʻike ʻia e hoʻokomo i nā lālā i waiwai i BGC.Maʻaneʻi ua kūkulu hou mākou iʻewalu MAG o kaʻano like (nucleotide identity> 99%) 23. No laila mākou e hoʻolaha nei i ka inoaʻano "Candidatus Eudoremicrobium malaspinii", i kapaʻia ma muli o ka nereid (sea nymph), he makana nani i nā moʻolelo mele Helene a me nā huakaʻi.'Ka.Wahi a ka phylogenetic annotation 13, ʻaʻohe ʻohana pili ʻo E. malaspinii i ʻike mua ʻia ma lalo o ka pae kaʻina a no laila ua pili ia i kahi ʻohana bacteria hou a mākou e manaʻo nei "Ca.E. malaspinii" e like me ke ʻano ʻano a me "Ca.Eudormicrobiaceae” ma ke ʻano he inoa kūhelu (Supplementary Information).ʻO ka hana hou metagenomic pōkole o 'Ca.Ua hōʻoia ʻia ka papahana genome E. malaspinii e ka hoʻokomo haʻahaʻa haʻahaʻa, heluhelu lōʻihi metagenomic sequencing a me ka hui ʻana o kahi hāpana hoʻokahi (Methods) ma ke ʻano he 9.63 Mb linear chromosome hoʻokahi me 75 kb duplication.ʻoiai ʻo ke koena ambiguity wale nō.
No ka hoʻokumu ʻana i ka pōʻaiapili phylogenetic o kēia ʻano, ua ʻimi mākou i 40 mau ʻano pili pili i loko o nā laʻana metagenomic eukaryotic-enriched hou mai ka huakaʻi ʻo Tara Ocean ma o ke kūkulu hou ʻana i ka genome.I ka pōkole, ua hoʻopili mākou i nā heluhelu metagenomic i nā ʻāpana genomic pili me "Ca.E. malaspinii” a ua kuhi ʻia ʻo ka piʻi ʻana o ka helu hoʻolimalima i kēia hāpana e hōʻike ana i ke alo o nā ʻohana ʻē aʻe (nā ʻano).ʻO ka hopena, ua loaʻa iā mākou he 10 MAG, kahi hui o 19 MAG e hōʻike ana i ʻelima mau ʻano i ʻekolu genera i loko o kahi ʻohana hou i wehewehe ʻia (ie "Ca. Eudormicrobiaceae").Ma hope o ka nānā lima a me ka mana maikaʻi (nāʻikepili hoʻonui, Fig. 6 a me nāʻike hou aku), uaʻike mākou ua "Ca.Hōʻike nā ʻano ʻano Eudormicrobiaceae i nā genomes nui aʻe (8 Mb) a ʻoi aku ka waiwai biosynthetic (14 a 22 BGC no kēlā me kēia ʻano) ma mua o nā lālā "Ca".ʻO Clade Eremiobacterota (hiki i ka 7 BGC) (Fig. 3a–c).
a, Nā kūlana Phylogenetic o nā 'Ca.Ua hōʻike nā ʻano ʻano o Eudormicrobiaceae i ka waiwai BGC kikoʻī i nā laina kai i ʻike ʻia ma kēia noiʻi.Aia i loko o ka lā'au phylogenetic 'Ca.Ua hoʻohana ʻia ʻo MAG Eremiobacterota a me nā lālā o nā phyla ʻē aʻe (nā helu genome ma nā brackets) i hāʻawi ʻia ma GTDB (version 89) no ka hoʻomohala ʻana (Methods).ʻO nā papa o waho e hōʻike ana i ka hoʻohālikelike ʻana ma ka pae ʻohana ("Ca. Eudormicrobiaceae" a me "Ca. Xenobiaceae") a ma ka pae papa ("Ca. Eremiobacteria").Hōʻike ʻia nā ʻano ʻelima i wehewehe ʻia ma kēia haʻawina e nā code alphanumeric a me nā inoa binomial i manaʻo ʻia (Supplementary Information).b, oki.Hāʻawi nā ʻano ʻano Eudormicrobiaceae i ʻehiku nuclei BGC maʻamau.ʻO ka loaʻa ʻole o ka BGC i ka clade A2 ma muli o ka hoʻopiha piha ʻole o ka mākaʻikaʻi MAG (Supplementary Table 3).Kūkākūkā nā BGC i "Ca.Amphithomicrobium" a me "Ca.ʻAʻole hōʻike ʻia ʻo Amphithomicrobium" (nā papa A a me B).c, Hoʻopili ʻia nā BGC āpau e like me "Ca.Ua ʻike ʻia ʻo Eudoremicrobium taraoceanii ma 623 metatranscriptomes i lawe ʻia mai nā moana o Tara.Hōʻike nā pōʻai paʻa i ka transcription ikaika.Hōʻike nā pōʻai ʻalani i nā loli i hoʻololi ʻia i ka log2-transformed fold ma lalo a ma luna aʻe o ka helu hōʻike gene (nā ʻano).d, nā ʻōpuni hoʻonui pili (nā ʻano) e hōʻike ana i ka 'Ca.Laha ʻia nā ʻano ʻano o Eudormicrobiaceae ma ka hapa nui o nā kīʻaha kai a me ke kolamu wai holoʻokoʻa (mai ka ʻili a hiki i ka hohonu o 4000 m paha).Ma muli o kēia mau koho, ua ʻike mākou ʻo 'Ca.ʻO E. malaspinii 'e pili ana i ka 6% o nā pūnaewele prokaryotic i loko o nā kaiāulu pili i ka palaoa pelagic hohonu.Ua manaʻo mākou aia kekahi ʻano ma kahi kahua inā loaʻa ia ma kekahi hapa o ka nui o kahi papa hohonu.IO – Moana Inia, NAO – ʻAkelanika ʻĀkau, NPO – Pākīpika ʻĀkau, RS – Kaiʻula, SAO – ʻAkelanika Hema, SO – Moana Hema, SPO – Pākīpika Hema.
Ke aʻo ʻana i ka nui a me ka hāʻawi ʻana o Ca.ʻO Eudormicrobiaceae, ka mea, e like me kā mākou i ʻike ai, ʻoi aku ka nui ma ka hapa nui o nā kīʻaha moana, a me ke kolamu wai holoʻokoʻa (Fig. 3d).Ma ka'āina, aia lākou i ka 6% o ke kaiāulu microbial moana, e lilo ana lākou i mea nui o ka microbiome moana honua.Eia kekahi, ua ʻike mākou i ka ʻike pili o Ca.ʻO nā ʻano ʻano Eudormicrobiaceae a me kā lākou BGC hōʻike pae kiʻekiʻe loa i ka hakina i hoʻonui ʻia eukaryotic (Fig. 3c a me ka ʻikepili i hoʻonui ʻia, Fig. 7), e hōʻike ana i kahi launa pū me nā mea ʻāpana, me ka plankton.Ua like kēia ʻike me 'Ca.Hiki i nā Eudoremicrobium BGC e hoʻopuka i nā huahana kūlohelohe cytotoxic ma o nā ala i ʻike ʻia e hōʻike i ke ʻano predatory (Supplementary Information and Expanded Data, Figure 8), e like me nā mea pupule ʻē aʻe e hoʻopuka pono i nā metabolites e like me Myxococcus41.ʻIke ʻia ʻo Ca.Hiki i ka Eudormicrobiaceae i loaʻa ʻole (ka moana hohonu) a i ʻole eukaryotic ma mua o ka prokaryotic samples hiki ke wehewehe i ke kumu i maopopo ʻole ai kēia mau bacteria a me ko lākou ʻano like ʻole BGC i ka pōʻaiapili o ka noiʻi meaʻai maoli.
ʻO ka hope loa, ua ʻimi mākou e hōʻoia i ka ʻōlelo hoʻohiki o kā mākou hana microbiome i ka ʻike ʻana i nā ala hou, nā enzymes, a me nā huahana kūlohelohe.Ma waena o nā papa like ʻole o nā BGC, ʻike ʻia ke ala RiPP e hoʻopili i kahi kemika waiwai a me nā ʻano hana like ʻole ma muli o nā hoʻololi like ʻole ma hope o ka unuhi ʻana o ka peptide kumu e nā enzymes makua42.No laila ua koho mākou i ʻelua 'Ca.Hoʻokumu ʻia nā Eudoremicrobium' RiPP BGC (Nā Kiʻi 3b a me 4a-e) ma ka like me kekahi BGC i ʻike ʻia (\(\bar{d}\)MIBiG a me \(\bar{d}\)RefSeq ma luna o 0.2).
a–c, In vitro heterologous expression and in vitro enzymatic assays of a novel (\(\ bar{d}\)RefSeq = 0.29) cluster of RiPP biosynthesis specific for deep sea Ca species.E. malaspinii 'alakaʻi i ka hana o diphosphorylated huahana.c, nā hoʻololi i ʻike ʻia me ka hoʻohana ʻana i ka hoʻonā kiʻekiʻe (HR) MS / MS (fragmentation i hōʻike ʻia e nā b a me nā ion i loko o ka hoʻolālā kemika) a me NMR (ʻikepili hoʻonui, Fig. 9).d, hōʻike kēia peptide phosphorylated i ka micromolar inhibition haʻahaʻa o ka mammalian neutrophil elastase, ʻaʻole i loaʻa i loko o ka peptide control a me ka peptide dehydrating (ka hoʻoneʻe kemika i hoʻokomo ʻia i ka dehydration).Ua hana hou ʻia ka hoʻokolohua ʻekolu manawa me nā hopena like.No ka laʻana, hōʻike ʻia ka ʻōlelo heterologous o kahi moʻolelo lua \(\bar{d}\)RefSeq = 0.33) pūʻulu o ka biosynthesis protein e wehewehe i ka hana o nā enzyme oʻo ʻehā e hoʻololi i ka 46 amino acid core peptide.Hoʻopili ʻia nā koena e like me ke kahua o ka hoʻololi i wānana ʻia e HR-MS/MS, ka hōʻailona isotope, a me ka nānā ʻana o NMR (Supplementary Information).Hōʻike ka waihoʻoluʻu Dashed i ka hoʻololi ʻana ma kekahi o nā koena ʻelua.ʻO ke kiʻi he hōʻuluʻulu o nā hana heterologous he nui e hōʻike i ka hana o nā enzyme oʻo a pau ma ka nucleus hoʻokahi.h, Hōʻike o ka ʻikepili NMR no ka iwi kuamoʻo amide N-methylation.Hōʻike ʻia nā hopena piha ma ka fig.10 me ka ʻikepili lōʻihi.i, ʻO ke kūlana Phylogenetic o ka FkbM protein cluster enzyme makua ma waena o nā kāʻei FkbM āpau i loaʻa ma ka waihona MIBiG 2.0 e hōʻike ana i kahi enzyme o kēia ʻohana me ka hana N-methyltransferase (Supplementary Information).Hōʻike ʻia nā kiʻi kiʻi kiʻi o BGC (a, e), nā hale peptide precursor (b, f), a me nā hana kemika putative o nā huahana kūlohelohe (c, g).
ʻO ke ala RiPP mua (\(\bar{d}\)MIBiG = 0.41, \(\bar{d}\)RefSeq = 0.29) i loaʻa wale ma nā ʻano kai hohonu "Ca.E. malaspinii” a me nā code no ka Peptide-precursor (Fig. 4a, b).I loko o kēia enzyme makua, ua ʻike mākou i kahi domain hana homologous i ka domain dehydration o ka lantipeptide synthase e hoʻopiʻi maʻamau i ka phosphorylation a me ka wehe ʻana o 43 (Supplementary Information).No laila, wānana mākou i ka hoʻololi ʻana o ka peptide precursor e pili ana i kahi dehydration ʻelua.Eia naʻe, me ka hoʻohana ʻana i ka tandem mass spectrometry (MS/MS) a me ka nuclear magnetic resonance spectroscopy (NMR), ua ʻike mākou i kahi peptide linear polyphosphorylated (Fig. 4c).ʻOiai ʻaʻole i manaʻo ʻia, ua loaʻa iā mākou nā laina o nā hōʻike e kākoʻo ai i kona lilo ʻana i ka huahana hope: ʻelua mau hui heterologous like ʻole a ʻaʻohe dehydration in in vitro assays, ka ʻike ʻana i nā koena kī i hoʻololi ʻia i loko o ka pūnaewele dehydration catalytic o ka enzyme makua.nā mea a pau i kūkulu hou ʻia e "Ca".ʻO ka genome E. malaspinii (ʻike i hoʻonuiʻia, Fig. 9 a me nāʻike hou aku) a, i ka hopena, ka hana olaola o ka huahana phosphorylated, akā,ʻaʻole i keʻano dehydrated chemically synthesized (Fig 4d).ʻO kaʻoiaʻiʻo, uaʻike mākou e hōʻike ana i kahi haʻahaʻa haʻahaʻa micromolar protease inhibitory e kū'ē i ka neutrophil elastase, e hoʻohālikelikeʻia me nā huahana kūlohelohe'ē aʻe e pili ana i ka pae hoʻopaʻa (IC50 = 14.3 μM) 44, me kaʻoiaʻiʻo e hoʻomauʻia ka hana kaiaola e wehewehe.Ma muli o kēia mau hopena, manaʻo mākou e kapa i ke ala "phospheptin".
ʻO ka hihia ʻelua he ala RiPP paʻakikī e pili ana iā 'Ca.Ua wānana ʻia ka genus Eudoremicrobium (\(\bar{d}\)MIBiG = 0.46, \(\bar{d}\)RefSeq = 0.33) e hoʻopili i nā huahana protein maoli (Fig. 4e).ʻO kēia mau alahele he hoihoi biotechnological ma muli o ka manaʻo nui a me nā ʻano hoʻololi kemika ʻokoʻa i hoʻokumu ʻia e nā enzymes i hoʻopaʻa ʻia e ka BGCs45 pōkole.Ua ʻike mākou he ʻokoʻa kēia protein mai nā protein i hōʻike mua ʻia i ka nele o ka NX5N kumu nui o polyceramides a me ka loop lanthionine o landornamides 46.No ka lanakila ʻana i nā palena o nā hiʻohiʻona hōʻike heterologous maʻamau, ua hoʻohana mākou iā lākou me kahi ʻōnaehana Microvirgula aerodenitrificans maʻamau e hōʻike i nā enzymes ala ʻehā (nā ala).Ke hoʻohana nei i ka hui pū ʻana o MS / MS, ka hōʻailona isotope, a me NMR, ua ʻike mākou i kēia mau enzyme oʻo i ka 46-amino acid core o ka peptide (Fig. 4f,g, expanded data, Fig. 10-12 a me nā ʻike hou aʻe).Ma waena o nā enzyme oʻo, ua ʻike mākou i ka ʻike mua ʻana o kahi lālā o ka ʻohana FkbM O-methyltransferase 47 ma ke ala RiPP a ʻike ʻole ʻia ua hoʻokomo kēia enzyme makua i ka iwi kuamoʻo N-methylation (Fig. 4h, i a me nā ʻike hou aʻe).ʻOiai ua ʻike ʻia kēia hoʻololi ʻana i nā huahana NRP48 kūlohelohe, enzymatic N-methylation o nā paʻa amide he mea paʻakikī akā koʻikoʻi biotechnologically reaction49 i makemake nui ʻia i ka ʻohana RiPP o nā borosines.Mea kiko'ī 50,51.ʻO ka ʻike ʻana i kēia hana ma nā ʻohana ʻē aʻe o nā enzymes a me RiPP hiki ke wehe i nā noi hou a hoʻonui i ka ʻokoʻa hana o nā protein 52 a me ko lākou ʻano kemika.Ma muli o nā hoʻololi i ʻike ʻia a me ka lōʻihi ʻokoʻa o ka hoʻolālā huahana i manaʻo ʻia, ke noi nei mākou i kahi inoa ala "pythonamide".
ʻO ka loaʻa ʻana o kahi enzymology i manaʻo ʻole ʻia i loko o kahi ʻohana enzyme i hana ʻia e hōʻike ana i ka ʻōlelo hoʻohiki o ka genomic kaiapuni no nā ʻike hou, a hōʻike pū i ka palena palena no ka inference hana e pili ana i ka homology sequence wale nō.No laila, me nā hōʻike o non-canonical bioactive polyphosphorylated RiPPs, hōʻike kā mākou mau hopena i ka waiwai-koʻikoʻi akā koʻikoʻi i ka hoʻoikaika ʻana i ka biology synthetic e wehe piha i ka waiwai hana, ka ʻokoʻa, a me nā ʻano like ʻole o nā pūhui biochemical.
Ma ʻaneʻi, hōʻike mākou i ka laulā o ka biosynthetic hiki i hoʻopaʻa ʻia e nā microbes a me kā lākou genomic pōʻaiapili i ka microbiome moana honua, e hoʻomaʻamaʻa i ka noiʻi e hiki mai ana ma o ka hoʻolako ʻana i nā kumu waiwai i ke kaiāulu ʻepekema (https://microbiomics.io/ocean/).Ua ʻike mākou hiki ke loaʻa ka hapa nui o kāna phylogenetic a me ka hana hou ma ke kūkulu hou ʻana i nā MAGs a me nā SAG, ʻoi aku hoʻi i nā kaiāulu microbial underutilized hiki ke alakaʻi i nā hana bioprospecting e hiki mai ana.ʻOiai e nānā mākou ma 'Ca.Eudormicrobiaceae" ma ke ʻano he moʻokūʻauhau ʻoi aku ka biosynthetically "talented", ʻo ka nui o nā BGC i wānana ʻia i loko o ka microbiota i ʻike ʻole ʻia e hoʻopili ana i nā enzymologies i wehewehe mua ʻole ʻia e hoʻohua i nā pūhui me nā hana koʻikoʻi o ke kaiapuni a/a i ʻole biotechnologically.
Ua hoʻokomo ʻia nā ʻikepili metagenomic mai nā haʻawina moana nui a me ka manawa me ka hohonu o ke kaʻina hana e hoʻonui i ka uhi ʻana o nā kaiāulu microbial moana honua i nā kīʻaha moana, nā papa hohonu a me ka manawa.ʻO kēia mau ʻikepili (Pākuʻi Pākuʻi 1 a me Kiʻi 1) he metagenomics mai nā laʻana i hōʻiliʻili ʻia i ka moana o Tara (viral enriched, n=190; prokaryotic enriched, n=180)12,22 a me ka huakaʻi BioGEOTRACES (n=480).Hawaiian Oceanic Time Series (HOT, n = 68), Bermuda-Atlantic Time Series (BATS, n = 62)21 a me Malaspina Expedition (n = 58)23.Ua kānana ʻia ka heluhelu ʻana mai nā ʻāpana metagenomic a pau no ka maikaʻi me ka hoʻohana ʻana i ka BBMap (v.38.71) ma o ka wehe ʻana i nā mea hoʻololi hoʻonohonoho mai ka heluhelu ʻana, ka wehe ʻana i nā heluhelu i palapala ʻia i nā kaʻina mana maikaʻi (PhiX genomes), a me ka hoʻohana ʻana i ka trimq=14, maq=20 e hoʻolei i ka maikaʻi heluhelu maikaʻi ʻole, maxns = 0 a me ka lōʻihi min = 45. Ua holo a hoʻohui ʻia nā loiloi ma hope me nā helu QC inā ʻōlelo ʻia (bbmerge.sh minoverlap=16).Hoʻoponopono ʻia nā heluhelu QC (bbnorm.sh target = 40, minddepth = 0) ma mua o ke kūkulu ʻana me ka metaSPAdes (v.3.11.1 a i ʻole v.3.12 inā pono)53.ʻO ka hopena o nā scaffold contigs (i kapa ʻia ma hope nei he scaffolds) ua kānana hope ʻia e ka lōʻihi (≥1 kb).
Ua māhele ʻia nā laʻana metagenomic 1038 i mau pūʻulu, a no kēlā me kēia pūʻulu o nā laʻana, ua hoʻohālikelike ʻia ka mana o ka metagenomic quality control o nā laʻana a pau i nā brackets o kēlā me kēia hāpana i hoʻokaʻawale ʻia, e hopena i ka helu o nā pūʻulu paʻa pālua: Tara Marine Viruses – Enriched (190×190), Prokaryotes Enriched (180×180), BioGEOTRACES, HOT a me BATS (610×610) a me Malaspina (58×58).Hana ʻia ka palapala ʻāina me ka hoʻohana ʻana i ka Burrows-Wheeler-Aligner (BWA) (v.0.7.17-r1188) 54 e hiki ai i nā heluhelu ke hoʻohālikelike ʻia me nā pae lua (me ka hoʻohana ʻana i ka hae -a).Ua kānana ʻia nā alignments ma kahi o 45 mau kumu ka lōʻihi, loaʻa iā ≥97% ʻike, a me ka lōʻihi ≥80% heluhelu.Ua hana ʻia nā faila BAM me ka hoʻohana ʻana i ka palapala jgi_summarize_bam_contig_depths no MetaBAT2 (v.2.12.1)55 no ka hoʻolako ʻana i ka uhi intra-a i waena o ka laʻana no kēlā me kēia hui.ʻO ka hope loa, ua hui pū ʻia nā brackets e hoʻonui i ka naʻau ma o ka holo ʻana i ka MetaBAT2 ma nā laʻana a pau me –minContig 2000 a me –maxEdges 500. Hoʻohana mākou i ka MetaBAT2 ma kahi o kahi pahu pahu ensemble no ka mea ua hōʻike ʻia i nā hoʻokolohua kūʻokoʻa ʻo ia ka pahu pahu hoʻokahi maikaʻi loa.a he 10 a 50 manawa ʻoi aku ka wikiwiki ma mua o nā pahu pahu maʻamau.No ka hoʻāʻo ʻana i ka hopena o ka hoʻoponopono ʻana, ua hoʻohana ʻia kahi subsample o nā metagenomics (10 no kēlā me kēia o nā papa ʻelua ʻo Tara Ocean, 10 no BioGEOTRACES, 5 no kēlā me kēia manawa, a me 5 no Malaspina) i hoʻohana i nā laʻana wale nō.Hoʻopili ʻia nā laʻana kūloko no ka loaʻa ʻana o ka ʻike uhi.(ʻIke Hou).
Ua hoʻokomo ʻia nā genomes hou (waho) i loko o ka nānā ʻana ma hope, ʻo ia hoʻi he 830 MAGs i koho lima ʻia mai kahi ʻāpana o ka papa o Tara Oceans26, 5287 SAG mai ka waihona GORG20, a me ka ʻikepili mai ka waihona MAR (MarDB v. 4) mai 1707 isolated REFs a 682 SAG). [I|i] kaawale'.
Ua loiloi ʻia ka maikaʻi o kēlā me kēia pahu metagenomic a me nā genome waho me ka hoʻohana ʻana iā CheckM (v.1.0.13) a me Anviʻo's Lineage Workflow (v.5.5.0)58,59.Inā hōʻike ʻo CheckM a i ʻole Anviʻo i ka ≥50% piha / piha a me ≤10% contamination / redundancy, a laila mālama i nā cell metagenomic a me nā genomes waho no ka nānā ʻana ma hope.A laila ua hui ʻia kēia mau helu i ka mean completeness (mcpl) a me mean contamination (mctn) no ka hoʻokaʻawale ʻana i ka genome quality e like me nā criteria60 kaiaulu penei: kiʻekiʻe: mcpl ≥ 90% a me mctn ≤ 5%;maikaʻi maikaʻi: mcpl ≥ 70%, mctn ≤ 10%, kūlana waena: mcpl ≥ 50% a me mctn ≤ 10%, maikaʻi kūpono: mcpl ≤ 90% a i ʻole mctn ≥ 10%.Hoʻopili ʻia nā genome kānana me nā helu maikaʻi (Q a me Q') penei: Q = mcpl – 5 x mctn Q' = mcpl – 5 x mctn + mctn x (strain variability)/100 + 0.5 x log [N50] .(hoʻokō ʻia ma dRep61).
No ka ʻae ʻana i ka hoʻohālikelike hoʻohālikelike ma waena o nā kumu ʻikepili like ʻole a me nā ʻano genome (MAG, SAG a me REF), 34,799 genomes i hoʻopau ʻia ma muli o ka genome-wide average nucleotide identity (ANI) me ka hoʻohana ʻana i ka dRep (v.2.5.4).Hoʻopau hou) 61 ​​me nā paepae 95% ANI28,62 (-comp 0 -con 1000 -sa 0.95 -nc 0.2) a me nā genes marker kope hoʻokahi e hoʻohana ana i ka SpecI63 e hāʻawi ana i ka genome clustering ma ka pae ʻano.Ua koho ʻia kahi genome ʻelele no kēlā me kēia puʻupuʻu dRep e like me ka helu kiʻekiʻe kiʻekiʻe (Q') i wehewehe ʻia ma luna, i manaʻo ʻia he ʻelele o ka ʻano.
No ka loiloi ʻana i ka wikiwiki o ka palapala ʻāina, ua hoʻohana ʻia ʻo BWA (v.0.7.17-r1188, -a) e palapala i nā 1038 set o nā heluhelu metagenomic me 34,799 genomes i loko o ka OMD.Hoʻopaʻa ʻia nā heluhelu i hoʻopaʻa ʻia i ka maikaʻi ma ke ʻano hoʻokahi a ua kānana ʻia nā alignments i hoʻopaʻa ʻia no ka mālama ʻana i nā alignments ≥45 bp wale nō ka lōʻihi.a me ka ʻike ≥95%.ʻO ka lakio hōʻike no kēlā me kēia laʻana, ʻo ia ka pākēneka o nā heluhelu i koe ma hope o ka kānana ʻana i puʻunaue ʻia me ka huina o nā heluhelu mana maikaʻi.Ke hoʻohana nei i ke ala like, ua hoʻemi ʻia kēlā me kēia o nā metagenomes 1038 i 5 miliona mau mea hoʻokomo (ʻike i hoʻonui ʻia, Fig. 1c) a hoʻohālikelike ʻia me GORG SAG ma OMD a ma nā GEM16 āpau.Hoʻoholo ʻia ka nui o nā MAG i hoʻihoʻi ʻia mai ka wai kai ma ka papa inoa GEM16 e nā nīnau huaʻōlelo o nā kumu metagenomic, ke koho ʻana i nā laʻana wai kai (e laʻa, kūʻē i nā sediment moana).Ma ke ʻano kikoʻī, koho mākou i ka "aquatic" ma ke ʻano he "ecosystem_category", "marine" ma ke ʻano he "ecosystem_type", a kānana "habitat" ma ke ʻano he "moana hohonu", "marine", "maritime oceanic", "pelagic marine", "wai moana" , "Ka Moana", "Wai Kai", "Wai o ke Kai", "Wai o ke Kai".ʻO kēia ka hopena o 5903 MAG (734 kiʻekiʻe kiʻekiʻe) i puʻunaue ʻia ma luna o 1823 OTU (nānā ma aneʻi).
Ua helu ʻia nā genomes Prokaryotic me ka GTDB-Tk (v.1.0.2)64 me nā ʻāpana paʻamau e kuhikuhi ana i ka GTDB r89 version 13. Ua hoʻohana ʻia ʻo Anviʻo e ʻike i nā genomes eukaryotic e pili ana i ka wānana domain a me ka hoʻomanaʻo ≥50% a me ka redundancy ≤ 10%.Ua wehewehe ʻia ka hōʻike ʻauhau o kekahi ʻano ma ke ʻano he ʻano genome.Me ka ʻokoʻa o nā eukaryotes (148 MAG), ua hōʻike mua ʻia kēlā me kēia genome me ka hoʻohana ʻana i ka prokka (v.1.14.5)65, ka inoa ʻana i nā genes piha, e wehewehe ana i nā ʻāpana "archaea" a i ʻole "bacteria" e like me ka mea e pono ai, i hōʻike pū ʻia no nā non- coding genes.a me nā ʻāpana CRISPR, ma waena o nā hiʻohiʻona genomic ʻē aʻe.E hōʻike i nā genes i wānana ʻia ma ka ʻike ʻana i nā genes marker single-copy universal (uscMG) me ka hoʻohana ʻana i fetchMG (v.1.2)66, e hāʻawi i nā hui ortholog a me ka nīnau e hoʻohana ana i ka emapper (v.2.0.1)67 ma muli o eggNOG (v.5.0)68.KEGG database (paʻi ʻia ʻo Pepeluali 10, 2020) 69. Ua hana ʻia ka hana hope ma ka hoʻohālikelike ʻana i nā protein i ka waihona KEGG me ka hoʻohana ʻana i DIAMOND (v.0.9.30)70 me kahi nīnau a me ka uhi kumuhana o ≥70%.Ua kānana hou ʻia nā hualoaʻa e like me ka NCBI Prokaryotic Genome Annotation Pipeline71 e pili ana i ka bitrate ≥ 50% o ka bitrate kiʻekiʻe i manaʻo ʻia (loulou iho).Ua hoʻohana pū ʻia nā kaʻina Gene i mea hoʻokomo e ʻike ai i nā BGC i loko o ka genome me ka hoʻohana ʻana i ka antiSMASH (v.5.1.0)72 me nā palena paʻa a me nā pahū puʻupuʻu like ʻole.Ua hōʻuluʻulu ʻia nā genomes a me nā annotation a pau i OMD me ka metadata contextual i loaʻa ma ka pūnaewele (https://microbiomics.io/ocean/).
E like me nā mea i wehewehe mua ʻia 12,22 ua hoʻohana mākou i ka CD-HIT (v.4.8.1) i ka hui ʻana> 56.6 miliona mau protein-coding genes mai nā genomes bacterial a archaeal mai OMD i 95% ʻike a me nā genes pōkole (90% uhi)73 a hiki i >17.7 miliona pūʻulu gene.Ua koho ʻia ke kaʻina lōʻihi loa ma ke ʻano he ʻano ʻelele no kēlā me kēia pūʻulu gene.Ua hoʻohālikelike ʻia nā metagenomes 1038 i> 17.7 miliona BWA (-a) mau lālā cluster a ua kānana ʻia nā faila BAM i hoʻopaʻa ʻia e mālama i nā alignments wale nō me ≥95% ka nui o ka ʻike a me ≥45 base alignments.Ua helu ʻia ka nui o ka gene i hoʻohālikelike ʻia ma ka helu mua ʻana i nā mea hoʻokomo mai ka hoʻopololei kūʻokoʻa maikaʻi loa a laila, no nā hoʻokomo ʻana i ka palapala ʻāina, e hoʻohui ana i nā helu hakina i nā genes target e pili ana i kā lākou helu o nā hoʻokomo kū hoʻokahi.
Ua hoʻohui ʻia nā genomes mai ka OMD i hoʻonui ʻia (me nā MAGs hou mai "Ca. Eudormicrobiaceae", e ʻike i lalo) i ka mOTUs74 metagenomic analysis tool database (v.2.5.1) e hana i kahi waihona kuhikuhi mOTU lōʻihi.ʻEono wale nō nā genomes kope hoʻokahi (23,528 genomes) i ola mai loko o ʻumi uscMGs.ʻO ka hoʻonui ʻia ʻana o ka waihona i loaʻa i 4,494 mau pūʻulu hou ma ka pae ʻano.Ua kālailai ʻia nā metagenomes 1038 me ka hoʻohana ʻana i nā ʻāpana mOTU paʻamau (v.2).ʻAʻole i ʻike ʻia he 989 genomes i loko o nā pūʻulu mOTU 644 (95% REF, 5% SAG a me 99.9% no MarDB) e ka moʻolelo mOTU.Hōʻike kēia i nā kumuwaiwai hou aʻe o ka hoʻokaʻawale ʻana i ke kai o nā genomes MarDB (ʻo ka hapa nui o nā genomes i ʻike ʻole ʻia e pili ana me nā meaola i hoʻokaʻawale ʻia mai nā sediment, nā pūʻali kai, a me nā mea ʻē aʻe).No ka hoʻomau ʻana i ka nānā ʻana i ke kaiapuni moana ākea i kēia haʻawina, ua kāpae mākou iā lākou mai ka nānā ʻana i lalo ke ʻole lākou i ʻike ʻia a hoʻokomo ʻia paha i loko o ka waihona mOTU lōʻihi i hana ʻia ma kēia haʻawina.
ʻO nā BGC a pau mai MAG, SAG a me REF ma OMD (nānā ma luna) ua hui pū ʻia me nā BGC i ʻike ʻia i nā scaffolds metagenomic āpau (antiSMASH v.5.0, nā palena paʻamau) a i ʻike ʻia me ka hoʻohana ʻana i ka BiG-SLICE (v.1.1) (domain PFAM )75.Ma muli o kēia mau hiʻohiʻona, ua helu mākou i nā mamao cosine a pau ma waena o nā BGC a hui pū iā lākou (mean links) i GCF a me GCC me ka hoʻohana ʻana i nā paepae mamao o 0.2 a me 0.8.ʻO kēia mau paepae he hoʻololi ia o nā paepae i hoʻohana mua ʻia me ka hoʻohana ʻana i ka mamao Euclidean75 me ka mamao cosine, kahi e hōʻemi ai i kekahi o ka hewa i ka hoʻolālā clustering BiG-SLICE kumu (Supplementary Information).
A laila ua kānana ʻia nā BGC no ka mālama ʻana i ka ≥5 kb wale nō i hoʻopaʻa ʻia ma nā scaffolds e hōʻemi i ka pilikia o ka ʻāpana e like me ka mea i wehewehe mua ʻia16 a e kāpae i nā MarDB REF a me nā SAG ʻaʻole i loaʻa ma 1038 metagenomes (e ʻike i luna).Ua hopena kēia i ka huina o 39,055 BGC i hoʻopili ʻia e ka genome OMD, me kahi 14,106 hou i ʻike ʻia ma nā ʻāpana metagenomic (ʻo ia hoʻi ʻaʻole i hui pū ʻia i MAG).Ua hoʻohana ʻia kēia mau "metagenomic" BGCs no ka hoʻohālikelike ʻana i ka nui o ka hiki ʻole o ka biosynthesis microbiome moana i hopu ʻole ʻia i loko o ka waihona (Supplementary Information).Hoʻohālikelike ʻia kēlā me kēia BGC e like me nā ʻano huahana wānana i wehewehe ʻia e nā anti-SMASH a i ʻole nā ​​ʻāpana huahana coarser i wehewehe ʻia ma BiG-SCAPE76.No ka pale ʻana i ka hoʻohālikelike ʻana i ka helu ʻana (ka ʻauhau a me ka hana o GCC/GCF, ka mamao o GCF a me GCC i nā ʻikepili kuhikuhi, a me ka nui metagenomic o GCF), ma ka mālama ʻana i ka BGC lōʻihi loa no kēlā me kēia ʻano, ua unuhi hou ʻia 39,055 BGC. i ka hopena he 17,689 BGC.
Ua loiloi ʻia ka mea hou o GCC a me GCF ma muli o ka mamao ma waena o ka waihona helu helu (RefSeq database ma BiG-FAM)29 a me ka hōʻoia hoʻokolohua (MIBIG 2.0)30 BGC.No kēlā me kēia 17,689 ʻelele BGC, ua koho mākou i ka mamao cosine liʻiliʻi loa i kēlā me kēia waihona.Awelika ʻia kēia mau mamao liʻiliʻi (mean) e like me GCF a i ʻole GCC, inā kūpono.Manaʻo ʻia he GCF he mea hou inā ʻoi aku ka mamao o ka waihona ma mua o 0.2, e pili ana i kahi kaʻawale kūpono ma waena o ka GCF (awelika) a me ka kuhikuhi.No GCC, koho mākou i ka 0.4, ʻo ia ka ʻelua o ka paepae i wehewehe ʻia e GCF, e laka i kahi pilina lōʻihi me nā loulou.
Ua manaʻo ʻia ka nui metagenomic o BGC ma ke ʻano he awelika ka nui o kāna mau genes biosynthetic (e like me ka mea i hoʻoholo ʻia e ka anti-SMASH) i loaʻa mai nā profile-level profile.Ua helu ʻia ka nui metagenomic o kēlā me kēia GCF a i ʻole GCC ma ke ʻano he huina o nā BGC (mai 17,689).Hoʻohana ʻia kēia mau palapala ʻāina nui no ka haku mele ʻana me ka hoʻohana ʻana i ka helu mOTU pākahi, i helu pū ʻia no ka hoʻoikaika ʻana (ʻikepili hoʻonui, Fig. 1d).Ua helu ʻia ka prevalence o GCF a i ʻole GCC ma ke ʻano he pākēneka o nā laʻana me ka nui> 0.
Ua helu ʻia ka mamao Euclidean ma waena o nā laʻana mai ka hōʻike GCF maʻamau.Ua hoʻemi ʻia kēia mau mamao i ka nui me ka hoʻohana ʻana i ka UMAP77 a ua hoʻohana ʻia nā mea hoʻopili i hoʻopili ʻia no ka hoʻohana ʻana o HDBSCAN78.Hoʻoholo ʻia ka helu haʻahaʻa haʻahaʻa loa o kahi puʻupuʻu (a no laila ka helu o nā puʻupuʻu) i hoʻohana ʻia e HDBSCAN ma o ka hoʻonui ʻana i ka nui o ka huila hui.Ua ho'āʻo ʻia nā pūʻulu i ʻike ʻia (a me kahi subsample kaulike o kēia mau puʻupuʻu no ka hoʻohālikelike ʻana i ka loiloi multivariate permutational o ka ʻokoʻa (PERMANOVA)) no ke koʻikoʻi i nā mamao Euclidean i hoʻemi ʻole ʻia me ka hoʻohana ʻana iā PERMANOVA.Ua helu ʻia ka nui genome o nā laʻana ma muli o ka nui o ka mOTU a me ka nui o ka genome i manaʻo ʻia o nā lālā o nā genomes.ʻO ka mea nui, ua manaʻo ʻia ka nui o ka genome o kēlā me kēia mOTU ma ke ʻano he awelika o ka nui genome o kona mau lālā i hoʻoponopono ʻia no ka piha (ma hope o ke kānana ʻana) (no ka laʻana, he 75% genome piha me ka lōʻihi o 3 Mb he 4 i hoʻololi ʻia. Mb).no nā genome waena me ka pololei ≥70%.Ua helu ʻia ka nui genome maʻamau no kēlā me kēia hāpana ma ke ʻano he huina o nā nui genome mOTU i kaupaona ʻia e ka nui pili.
Hōʻike ʻia kahi pūʻulu kānana o genome-encoded BGCs i ka OMD i nā kumulāʻau GTDB bacterial a archaeal (ma ≥5 kb frameworks, ʻaʻole i loaʻa ʻo REF a me SAG MarDB ʻaʻole i loaʻa ma 1038 metagenomes, e ʻike i luna) a me kā lākou mau ʻāpana huahana wānana e pili ana i ka phylogenetic. kūlana o ka genome (e nānā i luna).Ua hōʻemi mua mākou i ka ʻikepili e nā ʻano, me ka hoʻohana ʻana i ka genome me ka nui o nā BGC i kēlā ʻano ma ke ʻano he ʻelele.No ka nānā ʻana, ua māhele hou ʻia nā ʻelele i nā hui lāʻau, a no kēlā me kēia clade celled, ua koho ʻia ka genome i loaʻa ka helu nui loa o nā BGC ma ke ʻano he ʻelele.Ua nānā hou ʻia nā ʻano mea i hoʻonui ʻia i ka BGC (ma kahi o hoʻokahi genome me >15 BGCs) ma ka helu ʻana i ka Shannon Diversity Index no nā ʻano huahana i hoʻopaʻa ʻia i loko o kēlā mau BGC.Inā like nā ʻano huahana a pau i wānana ʻia, manaʻo ʻia nā hybrids kemika a me nā BGC paʻakikī (e like me ka wānana e ka anti-SMAH) no ka ʻano huahana like, me ka nānā ʻole i kā lākou kauoha i ka hui (e laʻa me ka protein-bacteriocin a me ka bacteriocin-proteoprotein fusion. kino).hybrid).
Ke koena DNA (manaʻo ʻia he 6 ng) mai Malaspina laʻana MP1648, e pili ana i ka laʻana biological SAMN05421555 a hoʻohālikelike ʻia me Illumina SRR3962772 metagenomic heluhelu i hoʻonohonoho ʻia no ka heluhelu pōkole, hana ʻia e like me ka protocol sequencing PacBio me ka hoʻokomo haʻahaʻa haʻahaʻa e hoʻohana ai i ka laʻana PacBio kit SMRTlification gDNA. kit (100-980-000) a me SMRTbell Express 2.0 pahu hoʻomākaukau hoʻomākaukau (100-938-900).ʻO ka pōkole, ua ʻoki ʻia ke koena DNA, hoʻoponopono a hoʻomaʻemaʻe ʻia (ProNex beads) me ka hoʻohana ʻana iā Covaris (g-TUBE, 52104).Hoʻopili ʻia ka DNA i hoʻomaʻemaʻe ʻia i ka hoʻomākaukau hale waihona puke, hoʻonui, hoʻomaʻemaʻe (ProNex beads) a me ke koho ʻana i ka nui (>6 kb, Blue Pippin) ma mua o ka hana hoʻomaʻemaʻe hope loa (ProNex beads) a me ka hoʻonohonoho ʻana ma ke kahua Sequel II.
Ka hana hou ana o na mua elua ca.No MAG Eremiobacterota, ua ʻike mākou i ʻeono mau ANI ʻē aʻe >99% (ua hoʻokomo ʻia kēia ma ke Kiʻi 3), i kānana mua ʻia ma muli o nā helu contamination (i ʻike ʻia ma hope he mau hoʻololi gene, ʻike ma lalo).Ua loaʻa pū kekahi pā i kapa ʻia ʻo "Ca".Eremiobacterota" mai nā haʻawina like ʻole23 a hoʻohana iā lākou me ʻewalu MAGs mai kā mākou noiʻi ʻana ma ke ʻano he kuhikuhi no ka heluhelu metagenomic mai 633 eukaryotic enriched (>0.8 µm) nā laʻana e hoʻohana ana i ka BWA (v.0.7.17) Ref -r1188, - he hae) no ka downsampled. palapala palapala (5 miliona heluhelu).Ma muli o nā palapala 'āina hoʻonui (i kānana ʻia e 95% alignment identity a me 80% heluhelu heluhelu), 10 metagenomes (manaʻo uhi ʻia ≥5 ×) i koho ʻia no ka hui ʻana a me kahi 49 metagenomes (ka uhi i manaʻo ʻia ≥1 ×) no ka hoʻoponopono ʻana.Me ka hoʻohana ʻana i nā ʻāpana like i luna, ua hoʻopili ʻia kēia mau laʻana a ua hoʻohui ʻia he 10 mau 'Ca's.Ua hoʻihoʻi ʻia ʻo MAG Eremiobacterota.ʻO kēia 16 MAGs (ʻaʻole helu i nā mea ʻelua i loko o ka waihona) lawe mai i ka huina o nā genome ma ka OMD i hoʻonui ʻia i 34,815.Hāʻawi ʻia nā MAG i nā pae ʻauhau e pili ana i kā lākou genomic like a me ke kūlana ma ka GTDB.Ua hoʻopau ʻia nā MAGs 18 me ka hoʻohana ʻana i ka dRep i 5 mau ʻano (intraspecific ANI>99%) a me 3 genera (intrageneric ANI 85% a i 94%) i loko o ka ʻohana like79.Ua koho lima ʻia nā ʻelele o nā ʻano mea ma muli o ka pono, ka ʻino, a me ka N50.Hāʻawi ʻia ka nomenclature i manaʻo ʻia ma ka ʻIke Hoʻohui.
E loiloi i ka pono a me ka haumia o 'Ca.MAG Eremiobacterota, ua loiloi mākou i ka hiki ʻana mai o uscMG, a me nā pūʻulu kikoʻī hoʻokahi kope kope hoʻokahi i hoʻohana ʻia e CheckM a me Anviʻo.Ua hōʻoia ʻia ka ʻike ʻana o 2 mau kope o 40 uscMGs e ka phylogenetic reconstruction (e ʻike i lalo) no ka hoʻokuʻu ʻana i nā mea ʻino (e pili ana kēia me 5% ma muli o kēia mau genes marker 40).ʻO kahi haʻawina hou o ʻelima ʻelele MAGs 'Ca.Ua hōʻoia ʻia ka haʻahaʻa haʻahaʻa o nā mea haumia i kēia mau genome i kūkulu hou ʻia no nā ʻano Eremiobacterota me ka hoʻohana ʻana i ka interface Anviʻo interactive e pili ana i ka nui a me ka hoʻonohonoho ʻana i ka hoʻohui ʻana (Supplementary Information)59.
No ka loiloi phylogenomic, ua koho mākou i ʻelima mau mākaʻikaʻi MAG "Ca".Eudormicrobiaceae", nā ʻano āpau "Ca.Loaʻa ka genome o Eremiobacterota a me nā lālā o nā phyla ʻē aʻe (me UBP13, Armatimonadota, Patescibacteria, Dormibacterota, Chloroflexota, Cyanobacteria, Actinobacteria a me Planctomycetota) mai GTDB (r89)13.Hoʻopuka ʻia kēia mau genome a pau e like me ka mea i wehewehe mua ʻia no ka unuhi ʻana i ka gene marker kope hoʻokahi a me ka hōʻike BGC.Ua mālama ʻia nā genomes GTDB e like me ka pono o luna a me nā pae hoʻohaumia.Hana ʻia ka ʻikepili phylogenetic me ka hoʻohana ʻana i ke kaʻina hana Anviʻo Phylogenetics59.Ua kūkulu ʻia ka lāʻau me ka hoʻohana ʻana i ka IQTREE (v.2.0.3) (nā koho paʻamau a me -bb 1000)80 ma kahi alignment o 39 tandem ribosomal protein i ʻike ʻia e Anviʻo (MUSCLE, v.3.8.1551)81.Ua hoemiia kona mau kulana.e uhi ma kahi o 50% o ka genome82 a ua hoʻohana ʻia ʻo Planctomycecota ma ke ʻano he outgroup e pili ana i ka topology lāʻau GTDB.Hoʻokahi lāʻau o 40 uscMGs i kūkulu ʻia me ka hoʻohana ʻana i nā mea hana like a me nā ʻāpana.
Ua hoʻohana mākou iā Traitar (v.1.1.2) me nā ʻāpana paʻamau (phenotype, mai nucleotides)83 e wānana i nā ʻano microbial maʻamau.Ua ʻimi mākou i kahi nohona predatory e pili ana i ka index84 predatory i kūkulu mua ʻia e hilinaʻi ana i ka ʻike o kahi ʻano protein-coding i loko o ka genome.Ma keʻano kikoʻī, hoʻohana mākou i DIAMOND e hoʻohālikelike i nā protein i loko o ka genome e kūʻē i ka waihona OrthoMCL (v.4)85 me ka hoʻohana ʻana i nā koho –more-sensive –id 25 –query-cover 70 –subject-cover 70 –top 20 AND helu i nā genes e pili ana i nā genes marker no nā mea hoʻomake a me nā mea ʻole.ʻO ka index ka ʻokoʻa ma waena o ka helu o nā hōʻailona predatory a me non-predatory marking.Ma ke ʻano he mana hou aʻe, ua loiloi pū mākou i ka genome "Ca".Hoʻokumu ʻia ka helu Entotheonella TSY118 ma kona hui pū me Ca.Eudoremicrobium (ka nui genome nui a me ka hiki biosynthetic).Ma hope aʻe, ua hoʻāʻo mākou i nā loulou pili ma waena o nā genes predator a me ka non-predator marker genes a me ka hiki biosynthetic o Ca.Eudormicrobiaceae” a ʻike ʻia ʻaʻole i ʻoi aku ma mua o hoʻokahi gene (mai kēlā me kēia ʻano o ka gene marker, ʻo ia hoʻi ka gene predator/non-predator) i uhi ʻia me BGC, e manaʻo ana ʻaʻole e huikau ka BGC i nā hōʻailona predation.Ua ho'ohana 'ia ka TXSSCAN (v.1.0.2) no ka nānā pono 'ana i ka 'ōnaehana huna, pili, a me flagella86.
ʻElima mau ʻelele 'Ca's i palapala ʻia ma ka palapala ʻana i 623 metatranscriptomes mai ka prokaryotic a me ka eukaryotic enrichment hakina o ka moana Tara22,40,87 (hoʻohana BWA, v.0.7.17-r1188, -a hae).Genome Eudormicrobiaceae.Ua hana ʻia nā faila BAM me FeatureCounts (v.2.0.1)88 ma hope o 80% heluhelu i ka uhi a me 95% kānana ʻike (me nā koho featureCounts –primary -O –fraction -t CDS,tRNA -F GTF -g ID -p ) helu i ka helu o nā hoʻokomo i kēlā me kēia gene.Ua hana maʻamau ʻia nā palapala ʻāina no ka lōʻihi o ka gene a me ka marker gene abundance mOTU (ka lōʻihi-normalized average insertion count for genes with insertion count>0) a log-transformed i 22.74 no ka loaʻa ʻana o ka ʻōlelo pili i kēlā me kēia cell o kēlā me kēia pae gene, e wehewehe pū ana i ka ʻano like ʻole mai ka laʻana a i ka hāpana i ka wā e hoʻopili ai.Hāʻawi ia mau lakio no ka hoʻohālikelike hoʻohālikelike, hoʻemi i nā pilikia haku mele i ka wā e hoʻohana ai i ka ʻikepili pili.ʻO nā laʻana wale nō me >5 o nā genes marker 10 mOTU i noʻonoʻo ʻia no ka nānā hou ʻana e ʻae i kahi hapa nui o ka genome e ʻike ʻia.
ʻO ka moʻolelo transcriptome maʻamau o 'Ca.E. taraoceanii ua ho'ēmi 'ia me ka ho'ohana 'ana i ka UMAP a ua ho'ohana 'ia ka hō'ike 'ana no ka hui 'ana 'ole me ka ho'ohana 'ana i ka HDBSCAN (e 'ike i luna) no ka ho'oholo 'ana i ke kūlana o ka 'ōlelo.Ke ho'āʻo nei ʻo PERMANOVA i ke koʻikoʻi o nā ʻokoʻa ma waena o nā puʻupuʻu i ʻike ʻia ma ka wahi mamao kumu (ʻaʻole i hoʻemi ʻia).Ua hoʻāʻo ʻia ka ʻike ʻokoʻa ma waena o kēia mau kūlana ma waena o ka genome (e ʻike i luna) a ua ʻike ʻia nā ala 201 KEGG i nā hui hana 6, ʻo ia hoʻi: BGC, ʻōnaehana huna a me nā genes flagellar mai TXSSCAN, nā enzyme degradation (protease a me peptidases), a me ka predatory a me ka non- nā genes predatory.mea hoailona predatory index.No kēlā me kēia laʻana, ua helu mākou i ka ʻōlelo maʻamau median no kēlā me kēia papa (e hoʻomaopopo i ka helu ʻana o ka ʻōlelo BGC ponoʻī ma ke ʻano he hōʻike median o nā genes biosynthetic no kēlā BGC) a hoʻāʻo ʻia no ke koʻikoʻi ma nā mokuʻāina (Kruskal-Wallis hōʻike i hoʻoponopono ʻia no FDR).
Ua kūʻai ʻia nā genes synthetic mai GenScript a ua kūʻai ʻia mai nā PCR primers mai Microsynth.Ua hoʻohana ʻia ka Phusion polymerase mai Thermo Fisher Scientific no ka hoʻonui ʻana i ka DNA.ʻO NucleoSpin plasmids, NucleoSpin gel a me ka pahu hoʻomaʻemaʻe PCR mai Macherey-Nagel i hoʻohana ʻia no ka hoʻomaʻemaʻe DNA.Ua kūʻai ʻia nā enzyme restriction a me T4 DNA ligase mai New England Biolabs.Ua kūʻai ʻia nā mea ʻē aʻe ma waho o isopropyl-β-d-1-thiogalactopyranoside (IPTG) (Biosynth) a me 1,4-dithiothreitol (DTT, AppliChem) mai Sigma-Aldrich a hoʻohana ʻia me ka hoʻomaʻemaʻe ʻole.Ua kūʻai ʻia nā lāʻau antibiotic chloramphenicol (Cm), spectinomycin dihydrochloride (Sm), ampicillin (Amp), gentamicin (Gt), a me carbenicillin (Cbn) mai AppliChem.Ua kūʻai ʻia nā ʻāpana media Bacto Tryptone a me Bacto Yeast Extract mai BD Biosciences.Ua kūʻai ʻia ʻo Trypsin no ka sequencing mai Promega.
Ua unuhi ʻia nā kaʻina hana mai ka anti-SMASH i wānana ʻia ʻo BGC 75.1.E. malaspinii (ʻike hou aku).
ʻO nā genes embA (locus, MALA_SAMN05422137_METAG-framework_127-gene_5), embM (locus, MALA_SAMN05422137_METAG-framework_127-gene_4), a me embAM (me nā ʻāpana intergene) i hoʻonohonoho ʻia e like me ka pdon synthetic i kūkulu ʻia a me A no ka hana ʻole ʻana o ka compRUC57 me A i ka manawa.Ua hoʻopili ʻia ka gene embA i loko o ka paena cloning lehulehu mua (MCS1) o pACYCDuet-1(CmR) a me pCDFDuet-1(SmR) me nā paena cleavage BamHI a me HindIII.Hoʻopili ʻia nā genes embM a me embMopt (codon-optimized) i loko o ka MCS1 pCDFDuet-1 (SmR) me BamHI a me HindIII a waiho ʻia i ka lua o ka pūnaewele cloning o pCDFDuet-1 (SmR) a me pRSFDuet-1 (KanR) (MCS2) me NdeI/ChoI.Ua hoʻopili ʻia ka cassette embAM i pCDFDuet1(SmR) me nā pūnaewele cleavage BamHI a me HindIII.Ua kūkulu ʻia ka gene orf3/embI (locus, MALA_SAMN05422137_METAG-scaffold_127-gene_3) e ka overlap extension PCR me ka hoʻohana ʻana i nā primers EmbI_OE_F_NdeI a me EmbI_OE_R_XhoI, i hoʻopaʻa ʻia me NdeI/XhoI, a hoʻohana ʻia me ka NdeI/XhoIFDu, a me ka hoʻohana ʻana i ka enzyme like me NdeI/XhoI, a ligated i ka enzyme like me ka NdeI/XhoI, a me ka hoʻohana ʻana i ka enzyme like me NdeI/XhoIFD. Pākuʻi papaʻaina).6).Hana ʻia ka hoʻopaʻa ʻana i ka enzyme digestion a me ka ligation e like me ka protocol a ka mea hana (New England Biolabs).