Wani sabon nau'in Gram-negative, aerobic, mai haƙuri gishiri, mai aiki, mai siffar sanda, da ƙwayoyin cuta ASxL5T an keɓe shi daga tafkin takin saniya a Nottinghamshire, Ingila, kuma ya yi amfani da Campylobacter a matsayin ganima. Daga baya, an gano wasu nau'in Campylobacter da membobin dangin Enterobacteriaceae a matsayin ganima. Bayan subculture ba tare da sel mai masauki ba, an sami ci gaban aseptic rauni akan Jikowar Zuciya Agar. Mafi kyawun yanayin girma shine 37 ° C kuma pH shine 7. Ƙwararren ƙwayoyin lantarki ya bayyana wasu abubuwan da ba a saba gani ba game da samuwar ganima. Binciken phylogenetic ta amfani da jerin kwayoyin halittar 16S rRNA ya nuna cewa keɓe yana da alaƙa da memba na dangin Marine Spirulina, amma ba za a iya rarraba shi a fili azaman memba na kowane sanannen jinsi ba. Jerin-genome gabaɗaya na ASxL5T ya tabbatar da dangantaka da membobin spirochetes na ruwa. Binciken bayanai ya nuna cewa ASxL5Ts da yawa suna raba jerin kwayoyin halittar 16S rRNA tare da ƙwayoyin cuta da yawa waɗanda ba a al'ada ba daga teku, saman ƙasa da ruwan ƙasa. Muna ba da shawarar cewa nau'in ASxL5T yana wakiltar sabon nau'in a cikin sabon nau'in halitta. Muna ba da shawarar sunan Venatorbacter cucullus gen. Nuwamba, sp. A watan Nuwamba, an yi amfani da ASxL5T azaman nau'in iri.
Bakteriya masu cin zarafi ƙwayoyin cuta ne waɗanda ke nuna ikon farauta da kashe sauran ƙwayoyin cuta masu rai don samun kayan biosynthetic da kuzari. Wannan ya sha bamban da yadda ake dawo da sinadirai daga matattun kwayoyin halitta, haka nan kuma ya sha bamban da mu’amalar parasitic, inda kwayoyin cuta ke kulla alaka ta kut-da-kut da mai gidansu ba tare da kashe su ba. Kwayoyin cuta masu cin zarafi sun samo asali daban-daban na rayuwa don cin gajiyar albarkatun abinci masu yawa a cikin wuraren da aka samo su (kamar mazaunin ruwa). Ƙungiya ce daban-daban na haraji, waɗanda ke da alaƙa kawai ta keɓaɓɓen yanayin rayuwa na haifuwa1. An samo misalan ƙwayoyin cuta a cikin phyla daban-daban, ciki har da: Proteobacteria, Bacteroides, da Chlorella.3. Duk da haka, mafi yawan ƙwararrun ƙwayoyin cuta masu lalata su ne Bdellovibrio da Bdellovibrio-da-kamar kwayoyin halitta (BALOs4). Kwayoyin cuta masu saurin kamuwa da cuta sune tushen tushe na sabbin mahadi masu aiki da ilimin halitta da magungunan kashe kwayoyin cuta5.
An yi imani da ƙwayoyin cuta masu lalata suna haɓaka bambance-bambancen ƙananan ƙwayoyin cuta kuma suna da tasiri mai kyau akan lafiyar muhalli, yawan aiki da kwanciyar hankali6. Duk da wadannan halaye masu kyau, an sami 'yan bincike kan sabbin kwayoyin cuta masu farauta saboda wahalar al'adar kwayoyin cuta da kuma bukatar lura da mu'amalar tantanin halitta a hankali don fahimtar hadaddun yanayin rayuwarsu. Wannan bayanin ba shi da sauƙi a samu daga nazarin kwamfuta.
A cikin zamanin da ake ƙara yawan juriya na ƙwayoyin cuta, ana nazarin sabbin dabaru don yin niyya ga ƙwayoyin cuta, kamar yin amfani da ƙwayoyin cuta da ƙwayoyin cuta7,8. An ware kwayoyin cutar ASxL5T a cikin 2019 ta amfani da fasahar keɓewar phage daga takin saniya da aka tattara daga Cibiyar Kiwo ta Jami'ar Nottingham, Nottinghamshire. Manufar binciken shine ware kwayoyin halitta masu yuwuwar a matsayin jami'an sarrafa kwayoyin halitta. Campylobacter hyointestinalis cuta ce ta zonotic, wacce ke daɗa alaƙa da cututtukan hanjin ɗan adam10. Yana cikin ko'ina a cikin magani kuma ana amfani dashi azaman mai masaukin baki.
Kwayar cutar ASxL5T ta keɓe daga jelly na naman sa saboda an lura cewa plaques da ta yi a kan lawn na C. hyointestinalis sun yi kama da waɗanda bacteriophages ke samarwa. Wannan wani bincike ne da ba a zata ba, saboda wani ɓangare na tsarin keɓewar phage ya ƙunshi tacewa ta hanyar tacewa 0.2 µm, wanda aka ƙera don cire ƙwayoyin cuta. Binciken da ba a gani ba na kayan da aka ciro daga plaque ya nuna cewa ƙananan ƙwayoyin cuta masu lanƙwasa mai siffar gram gram ba su tara polyhydroxybutyrate (PHB). An gano al'adun aseptic mai zaman kansa daga sel ganima akan matsakaici mai ƙarfi (kamar jiko na zuciya agar (BHI) da agar jini (BA)), kuma haɓakar sa yana da rauni. Ana samun shi bayan subculture tare da ingantaccen inoculum mai nauyi. Yana girma daidai da kyau a ƙarƙashin microaerobic (7% v/v oxygen) da yanayin iskar oxygen na yanayi, amma ba a cikin yanayin anaerobic ba. Bayan sa'o'i 72, diamita na yankin ya kasance ƙanƙanta, ya kai 2 mm, kuma yana da launin fata, mai haske, zagaye, convex da sheki. Daidaitaccen gwajin kwayoyin halitta yana da cikas saboda ASxL5T ba za a iya al'ada ta dogara a cikin kafofin watsa labarai na ruwa ba, wanda ke nuna cewa yana iya dogara ga hadadden tsarin rayuwa na samuwar biofilm. Koyaya, dakatarwar farantin ya nuna cewa ASxL5T na iska ne, tabbatacce ga oxidase da catalase, kuma yana iya jure wa 5% NaCl. ASxL5T yana da juriya ga 10 μg streptomycin, amma yana kula da duk sauran maganin rigakafi da aka gwada. Kwayoyin kwayoyin cutar ASxL5T an bincika su ta TEM (Hoto 1). Lokacin girma ba tare da sel ganima akan BA, ƙwayoyin ASxL5T ƙananan Campylobacter ne, tare da matsakaicin tsayi na 1.63 μm (± 0.4), faɗin 0.37 μm (± 0.08), da tsayi guda ɗaya (har zuwa 5 μm). Tutar jima'i. Kusan 1.6% na sel suna bayyana suna da faɗin ƙasa da μm 0.2, wanda zai ba da izinin wucewa ta na'urar tacewa. An sami tsawaita tsarin da ba a saba gani ba a saman wasu sel, kama da fa'ida (Latin cucullus) (duba kibau a 1D, E, G). Wannan yana da alama yana kunshe da wuce gona da iri, wanda zai iya kasancewa saboda saurin raguwar girman ambulaf ɗin periplasmic, yayin da murfin waje ya kasance cikakke, yana nuna bayyanar "sako". Al'ada ASxL5T a cikin rashin abinci mai gina jiki (a cikin PBS) na dogon lokaci a 4 ° C ya haifar da mafi yawan (amma ba duka ba) kwayoyin da ke nuna kwayar halittar coccal (Figure 1C). Lokacin da ASxL5T yayi girma tare da Campylobacter jejuni a matsayin ganima na tsawon awanni 48, matsakaicin girman tantanin halitta yana da tsayi da kunkuntar fiye da sel waɗanda ke girma ba tare da mai watsa shiri ba (Table 1 da Figure 1E). Sabanin haka, lokacin da ASxL5T ya girma tare da E. coli a matsayin ganima na tsawon sa'o'i 48, matsakaicin girman tantanin halitta ya fi tsayi kuma ya fi girma fiye da lokacin da yake girma ba tare da ganima ba (Table 1), kuma tsawon tantanin halitta yana canzawa, yawanci yana nuna filamentous (Figure 1F). Lokacin da aka haɗa shi da Campylobacter jejuni ko E. coli a matsayin ganima na tsawon awanni 48, ƙwayoyin ASxL5T sun nuna babu flagella kwata-kwata. Tebur 1 yana taƙaita abubuwan lura na canje-canje a girman tantanin halitta bisa ga kasancewar, rashi, da nau'in ganima na ASxL5T.
Nunin TEM na ASx5LT: (A) ASx5LT yana nuna doguwar bulala; (B) baturi ASx5LT na al'ada; (C) cocci ASx5LT Kwayoyin bayan dogon shiryawa ba tare da gina jiki ba; (D) rukuni na sel ASx5LT suna nuna rashin daidaituwa (E) ƙungiyar tantanin halitta ASx5LT wanda aka haɗa tare da ganima na Campylobacter ya nuna tsayin tantanin halitta idan aka kwatanta da waɗanda ba tare da ci gaban ganima ba (D) kuma sun nuna tsarin apical; (F) Manyan Filamentous flagella, ASx5LT Kwayoyin, bayan shiryawa tare da E. coli ganima; (G) Tantanin halitta ASx5LT guda ɗaya bayan shiryawa tare da E. coli, yana nuna wani sabon tsari na sama. Bar yana wakiltar 1 μm.
Ƙayyade jerin jerin abubuwan 16S rRNA (lambar shiga MT636545.1) yana ba da damar bincike na bayanai don kafa jerin abubuwa masu kama da waɗanda ke cikin ajin Gammaproteobacteria, kuma sun fi kusa da ƙwayoyin cuta na ruwa a cikin dangin spirillum na ruwa (Hoto 2), kuma su ne membobin Thalassolituus genus genus. Mafi kusancin dangi zuwa Marine Bacillus. Jerin jinsin 16S rRNA ya bambanta a fili da ƙwayoyin cuta masu farauta na dangin Bdelvibrionaceae (Deltaproteobacteria). Kwatanta biyu na B. bacteriovorus HD100T (nau'in iri, DSM 50701) da B. bacteriovorus DM11A sun kasance 48.4% da 47.7%, kuma ga B. exovorus JSS ya kasance 46.7%. Kwayoyin ASxL5T suna da kwafi 3 na kwayar halittar 16S rRNA, biyu daga cikinsu iri ɗaya ne da juna, kuma na ukun yana da tushe 3. Wasu ɓangarorin ƙwayoyin cuta guda biyu (ASx5S da ASx5O; 16S rRNA lambobin shiga jinsin su ne MT636546.1 da MT636547.1, bi da bi) waɗanda ke da halaye iri ɗaya da yanayin halitta daga wuri ɗaya ba iri ɗaya ba ne, amma sun bambanta da ASxL5T da ƙwayoyin cuta marasa al'ada. An tattara jerin bayanan bayanai tare da sauran nau'ikan a ciki Oceanospirillaceae (Hoto na 2). An ƙaddara gabaɗayan jerin kwayoyin halitta na ASxL5T kuma an adana su a cikin bayanan NCBI, kuma lambar shiga ita ce CP046056. Halin halittar ASxL5T ya ƙunshi chromosome madauwari na 2,831,152 bp tare da rabon G + C na 56.1%. Jerin kwayoyin halitta ya ƙunshi 2653 CDS (duka), wanda 2567 an annabta don ɓoye sunadaran, wanda 1596 za a iya sanya su azaman ayyukan sakawa (60.2%). Kwayoyin halitta sun ƙunshi kwayoyin halittar RNA guda 67, gami da 9rRNAs (3 kowanne don 5S, 16S, da 23S) da 57 tRNAs. An kwatanta halayen genomic na ASxL5T tare da samuwan kwayoyin halittar nau'in nau'in dangi mafi kusa da aka gano daga jerin kwayoyin halittar 16S rRNA (Table 2). Yi amfani da ainihin amino acid (AAI) don kwatanta duk abubuwan da ke akwai na Thalassolituus zuwa ASxL5T. Mafi kusa samuwa (bai cika) jerin kwayoyin halitta wanda AAI ya ƙaddara shine Thalassolituus sp. C2-1 (ƙara NZ_VNIL01000001). An keɓe wannan nau'in daga maɓuɓɓugar ruwa mai zurfi na Mariana Trench, amma a halin yanzu babu wani bayani mai ban mamaki game da wannan nau'in don kwatantawa. Idan aka kwatanta da ASxL5T's 2.82 Mb, kwayoyin halittar kwayoyin halitta sun fi girma a 4.36 Mb. Matsakaicin girman genome na spirochetes na ruwa yana kusan 4.16 Mb (± 1.1; n = 92 cikakken binciken kwayoyin halittar da aka bincika daga https://www.ncbi.nlm.nih.gov/assembly), don haka genome na ASxL5T yana cikin layi tare da oda Idan aka kwatanta da sauran membobin, ƙananan ne. Yi amfani da GToTree 1.5.54 don samar da ƙididdiga mafi girman yiwuwar itacen phylogenetic tushen tushen kwayoyin halitta (Hoto 3A), ta amfani da jeri na amino acid masu alaƙa da haɗin kai na 172 kwafi guda ɗaya takamaiman ga Gammaproteobacteria 11,12,13,14,15,16, 17,18. Binciken ya nuna cewa yana da alaƙa da Thalassolituus, Bacterial Plane, da Bacterium na Marine. Koyaya, waɗannan bayanan sun nuna cewa ASxL5T ya bambanta da danginsa a cikin Marine Spirulina kuma ana samun bayanan jerin kwayoyin halittar sa.
Bishiyar phylogenetic ta amfani da jerin kwayoyin 16S rRNA yana nuna matsayi na ASxL5T, ASxO5, da ASxS5 (tare da guts) dangane da nau'in kwayoyin da ba a yi ba da kuma na ruwa a cikin Marine Spirulinaceae. Lambar shiga ta Genbank tana bin sunan iri a cikin baka. Yi amfani da ClustalW don daidaita jeri, kuma yi amfani da mafi girman hanyar yuwuwar da samfurin Tamura-Nei don ba da alaƙar halittu, da aiwatar da kwafi guda 1000 a cikin shirin MEGA X. Lambar da ke kan reshe tana nuna cewa ƙimar kwafin jagorar ta fi 50%. An yi amfani da Escherichia coli U/541T azaman rukuni.
(A) Bishiyar phylogenetic dangane da kwayoyin halitta, yana nuna dangantakar dake tsakanin kwayoyin cutar Spirospiraceae na ruwa ASxL5T da danginsa na kusa, E. coli U 5/41T a matsayin ƙungiya. (B) Idan aka kwatanta da T. oleivorans MIL-1T, ana hasashen rarraba nau'ikan nau'ikan nau'ikan kwayoyin halitta bisa ga rukunin orthologous (COG) na furotin ASx5LT. Hoton da ke hagu yana nuna adadin kwayoyin halitta a cikin kowane nau'in COG mai aiki a cikin kowane kwayar halitta. Hoton da ke hannun dama yana nuna adadin adadin kwayoyin halittar da ke ƙunshe a cikin kowace ƙungiyar COG mai aiki. (C) Idan aka kwatanta da T. oleiverans MIL-1T, nazarin cikakken KEGG (Kyoto Encyclopedia of Genes and Genomes) hanya madaidaiciya na ASxL5T.
Yin amfani da bayanan KEGG don bincika kwayoyin halittar da ke cikin kwayoyin halittar ASxL5T sun bayyana hanyar rayuwa ta yau da kullun na gamma Proteus aerobic. ASxL5T ya ƙunshi jimillar kwayoyin halitta 75 da aka sanya wa sunadaran motsa jiki na kwayan cuta, gami da kwayoyin halittar da ke cikin chemotaxis, taron flagella, da nau'in tsarin fimbriae na IV. A cikin rukuni na ƙarshe, 9 cikin 10 kwayoyin halitta ne ke da alhakin karkatar da motsi na wasu kwayoyin halitta. Halin halittar ASxL5T ya ƙunshi cikakken tetrahydropyrimidine biosynthetic hanya wanda ke shiga cikin martanin kariyar ga osmotic stress20, kamar yadda ake tsammani ga halophiles. Har ila yau kwayoyin halitta sun ƙunshi cikakkun hanyoyi masu yawa don masu haɗin gwiwa da bitamin, ciki har da hanyoyin haɗin riboflavin. Ko da yake alkane 1-monooxygenase (alkB2) gene yana cikin ASxL5T, hanyar amfani da hydrocarbon ba ta cika ba. A cikin jerin kwayoyin halitta na ASxL5T, homologues na kwayoyin halittar da aka gano suna da alhakin lalata hydrocarbons a cikin T. oleiverans MIL-1T21, kamar TOL_2658 (alkB) da TOL_2772 (giya dehydrogenase) a fili ba su nan. Hoto na 3B yana nuna kwatancen rarraba kwayoyin halitta a cikin nau'in COG tsakanin ASxL5T da Man Zaitun MIL-1T. Gabaɗaya, ƙaramin ƙwayar halittar ASxL5T yana ƙunshe da ƴan ƙayyadaddun kwayoyin halitta daga kowane nau'in COG idan aka kwatanta da mafi girman kwayoyin halitta. Lokacin da aka bayyana adadin kwayoyin halitta a cikin kowane nau'in aiki a matsayin kashi na kwayoyin halitta, ana lura da bambance-bambance a cikin yawan adadin kwayoyin halitta a cikin fassarar, tsarin ribosomal da nau'o'in biogenesis, da samar da makamashi da nau'o'in aikin juyawa, wanda ya zama mafi girma ASxL5T. genome Adadin an kwatanta shi da rukunin guda ɗaya da ke cikin T. oleiverans MIL-1T genome. Sabanin haka, idan aka kwatanta da kwayoyin halittar ASxL5T, T. oleivorans MIL-1T yana da kashi mafi girma na kwayoyin halitta a cikin kwafi, sake haɗawa da gyarawa, da nau'ikan rubutun. Abin sha'awa, babban bambanci a cikin abun ciki na kowane nau'in aiki na kwayoyin halittar biyu shine adadin kwayoyin halittar da ba a san su ba a cikin ASxL5T (Hoto 3B). An gudanar da nazarin haɓaka kayan aikin KEGG, inda kowane ƙirar KEGG ke wakiltar saiti na ƙayyadaddun raka'o'in ayyuka da hannu don bayani da fassarar nazarin halittu na bayanan jeri na kwayoyin halitta. Kwatancen rarraba kwayoyin halitta a cikin cikakkiyar hanyar KOG module na ASxL5T da zaitun MIL-1T ana nuna su a cikin Hoto 3C. Wannan bincike ya nuna cewa ko da yake ASxL5T yana da cikakkiyar hanyar sulfur da nitrogen metabolism, T. oleiverans MIL-1T baya. Sabanin haka, T. oleiverans MIL-1T yana da cikakkiyar hanyar cysteine ​​​​da methionine, amma bai cika ba a ASxL5T. Saboda haka, ASxL5T yana da sifa mai siffa don assimilation na sulfate (wanda aka bayyana azaman saitin kwayoyin halitta waɗanda za a iya amfani da su azaman alamomin phenotypic, kamar ƙarfin rayuwa ko cuta; https://www.genome.jp/kegg/module.html) A cikin T MIL-1T. Kwatanta abun ciki na kwayoyin halitta na ASxL5T tare da jerin kwayoyin halittar da ke ba da shawarar salon rayuwa mara kyau. Ko da yake waaL gene wanda ke ɓoye ligase da ke hade da O antigen polysaccharide zuwa ainihin yana cikin ASxL5T genome (amma yana da yawa a yawancin kwayoyin cutar Gram), tryptophan 2,3-dioxygenase (TDO) Genes na iya haɗawa da 60 amino. Yankunan acid da aka fi samu a cikin ƙwayoyin cuta masu farauta waɗanda ba su nan. Babu wasu dabi'un dabi'un dabi'un halitta a cikin kwayoyin halittar ASxL5T, gami da wadanda ke sanyawa enzymes da ke cikin biosynthesis na isoprenoid a cikin hanyar mevalonate. Yi la'akari da cewa babu wani nau'i na gntR na rubutawa a cikin rukunin mafarauta da aka bincika, amma ana iya gano nau'ikan gntR guda uku a cikin ASxL5T.
An taƙaita halayen phenotypic na ASxL5T a cikin Table 3 kuma idan aka kwatanta da halayen phenotypic na jinsin 23, 24, 25, 26, da 27 da aka ruwaito a cikin wallafe-wallafe. Warewa daga T. marinus, T. olevorans, B. sanyensis, da Oceanobacter kriegii suna aiki, masu jure gishiri, jikin oxidase-tabbataccen sanda mai siffa, amma ba su da kusan wasu halaye na phenotypic tare da ASxL5T. Matsakaicin pH na teku shine 8.1 (https://ocean.si.edu/ocean-life/invertebrates/ocean-acidification#section_77), wanda ke nunawa a cikin T. marinus, T. olevorans, B. sanyensis da O. kirigi. ASxL5T ya dace da mafi girman kewayon pH (4-9) na nau'in nau'in da ba na ruwa ba. Halayen Phenotypic na Thalassolituus sp. C2-1. Ba a sani ba. Matsakaicin zafin girma na ASxL5T gabaɗaya ya fi na nau'ikan nau'ikan ruwa (4-42 ° C), kodayake wasu amma ba duka keɓancewar T. marinus ba ne masu jure zafi. Rashin iya girma ASxL5T a cikin kafofin watsa labarai na broth ya hana ƙarin sifa na phenotypic. Yi amfani da API 20E don gwada kayan da aka goge daga farantin BA, ONPG, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, amfani da citrate, urease, tryptophan deaminase, gelatin hydrolysis Enzyme, sakamakon gwajin duka mara kyau ne, amma babu indole, acetoin da H2S. aka samar. Carbohydrates maras yisti sun haɗa da: glucose, mannose, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin da arabinose. Idan aka kwatanta da nau'ikan tunani masu alaƙa da aka buga, bayanin martabar fatty acid na salula na nau'in ASxL5T yana nunawa a cikin Table 4. Babban fatty acids na salula sune C16: 1ω6c da / ko C16: 1ω7c, C16: 0 da C18: 1ω9. Hydroxy fatty acids C12:0 3-OH da C10:0 3-OH suma sun wanzu. Matsakaicin C16:0 a cikin ASxL5T ya fi ƙimar da aka ruwaito na jinsin da ke da alaƙa. Ya bambanta, idan aka kwatanta da rahoton T. marinus IMCC1826TT, an rage rabon C18: 1ω7c da / ko C18: 1ω6c a cikin ASxL5T. oleivorans MIL-1T da O. kriegii DSM 6294T, amma ba a gano su a cikin B. sanyensis KCTC 32220T ba. Kwatanta bayanan martabar fatty acid na ASxL5T da ASxLS sun bayyana bambance-bambance masu wayo a cikin adadin fatty acid guda ɗaya tsakanin nau'ikan nau'ikan biyu, waɗanda suka yi daidai da jerin kwayoyin halittar DNA na nau'in iri ɗaya. Ba a gano barbashi na poly-3-hydroxybutyrate (PHB) ta amfani da gwajin baƙar fata na Sudan.
An yi nazarin ayyukan tsinkewar ƙwayoyin cuta na ASxL5T don tantance yawan ganima. Wannan kwayoyin cuta na iya samar da plaques akan nau'in Campylobacter, ciki har da: Campylobacter suis 11608T, Campylobacter jejuni PT14, Campylobacter jejuni 12662, Campylobacter jejuni NCTC 11168T; Escherichia coli NCTC 12667; C. helveticus NCTC 12472; C lari NCTC 11458 da C. upsaliensis NCTC 11541T. Yi amfani da al'adun da aka jera a cikin sashin tantance kewayon mai masaukin baki na hanyar don gwada ɗimbin kewayon ƙwayoyin cuta na Gram-negative da Gram-positive. Sakamakon ya nuna cewa ana iya amfani da ASxL5T a cikin Escherichia coli NCTC 86 da Citrobacter freundii NCTC 9750T. Plaques da aka kafa akan Klebsiella oxytoca 11466. Ana nuna hulɗar TEM tare da E. coli NCTC 86 a cikin Hoto 4A-D, da kuma hulɗa tare da Campylobacter jejuni PT14 da Campylobacter suis S12 an nuna su a cikin Hoto 4E-H na tsakiya. Tsarin harin yana da alama ya bambanta tsakanin nau'ikan ganima da aka gwada, tare da ɗaya ko fiye da ƙwayoyin E. coli waɗanda aka haɗe zuwa kowane tantanin halitta ASxL5T kuma an sanya su a gefe tare da tsayin tantanin halitta kafin tallatawa. Sabanin haka, ASxL5T ya bayyana yana haɗawa zuwa Campylobacter ta hanyar lamba ɗaya, yawanci yana hulɗa da koli na kwayar halitta da kuma kusa da kololuwar tantanin halitta na Campylobacter (Hoto 4H).
TEM yana nuna hulɗar tsakanin ASx5LT da ganima: (AD) da E. coli ganima; (EH) da C. jejuni ganima. (A) Tantanin halitta ASx5LT da aka haɗa da tantanin halitta E. coli (EC); (B) Filamentous ASx5LT haɗe zuwa kwayar EC guda ɗaya; (C) Tantanin halitta na filamentous ASx5LT da aka haɗa da ƙwayoyin EC da yawa; (D) Haɗe Ƙananan ƙwayoyin ASx5LT akan kwayar E. coli (EC) guda ɗaya; (E) kwayar ASx5LT guda ɗaya da aka haɗa zuwa tantanin halitta na Campylobacter jejuni (CJ); (F) ASx5LT yana kai hari ga ƙwayoyin C. hyointestinalis (CH); (G) guda biyu ASx5LT tantanin halitta sun kai hari ga cell CJ; (H) Ra'ayi na kusa da abin da aka makala ASx5LT, kusa da koli na tantanin halitta CJ (bar 0.2 μm). Bar yana wakiltar 1 μm a cikin (A-G).
Kwayoyin cuta masu cin nama sun samo asali ne don cin gajiyar albarkatu masu yawa na ganima. Babu shakka, suna yaɗuwa a wurare daban-daban. Saboda kunkuntar girman membobin jama'a, yana yiwuwa a ware kwayoyin cutar ASxL5T daga slurry ta amfani da hanyar rabuwar phage. Dangantakar kwayoyin halitta na ASxL5T ga membobin dangin oceanospirillaceae na kwayoyin cuta na ruwa abin mamaki ne, ko da yake kwayar halitta tana da jurewar gishiri kuma tana iya girma akan matsakaici mai dauke da 5% gishiri. Binciken ingancin ruwa na slurry ya nuna cewa abun ciki na sodium chloride bai wuce 0.1%. Saboda haka, laka ya yi nisa da yanayin ruwa - a yanayin ƙasa da kuma na sinadarai. Kasancewar keɓe masu alaƙa guda uku amma daban-daban daga tushe ɗaya yana ba da shaida cewa waɗannan mafarauta suna bunƙasa a cikin wannan yanayin da ba na ruwa ba. Bugu da ƙari, nazarin microbiome (fayil ɗin bayanan da ake samu daga https://www.ebi.ac.uk/ena/browser/view/PRJEB38990) ya nuna cewa jerin nau'in 16S rRNA guda ɗaya yana cikin manyan 50 mafi yawan aiki taxa (OTU). ) A cikin ƴan tazara na samfur na laka. An sami wasu ƙwayoyin cuta marasa al'ada da yawa a cikin bayanan Genbank, waɗanda ke da jerin kwayoyin halittar 16S rRNA kama da kwayoyin ASxL5T. Waɗannan jerin jeri, tare da jerin ASxL5T, ASxS5, da ASxO5, da alama suna wakiltar nau'i daban-daban waɗanda suka rabu da Thalassolituus da Oceanobacter (Hoto 2). Bakteriya iri uku (GQ921362, GQ921357 da GQ921396) an ware su daga ruwan fissure a zurfin kilomita 1.3 a ma'adanin zinare na Afirka ta Kudu a shekarar 2009, sauran biyun (DQ256320 da DQ337006) sun kasance a cikin ruwan kasa na Afirka ta Kudu. a shekara ta 2005). Jerin jinsin 16S rRNA wanda ya fi kusanci da ASxL5T wani bangare ne na jerin kwayoyin halittar 16S rRNA da aka samu daga al'adun wadatar yashi da aka samu daga rairayin bakin teku na arewacin Faransa a 2006 (lambar shiga AM29240828). Wani jerin kwayoyin halittar 16S rRNA mai alaka da shi daga kwayar cutar HQ183822.1 da ba a yi amfani da ita ba an samo shi daga tankin tattarawa da aka kwaso daga wani wurin sharar gida na kasar Sin. A bayyane yake, kwayoyin ASxL5T ba su da wakilci sosai a cikin bayanan haraji, amma waɗannan jeri daga ƙwayoyin cuta marasa al'ada suna iya wakiltar kwayoyin halitta kamar ASxL5T, waɗanda aka rarraba a duk faɗin duniya, yawanci a cikin yanayi masu kalubale. Daga dukkanin nazarin kwayoyin halittar kwayoyin halitta, dangi mafi kusa da ASxL5T shine Thalassolituus sp. C2-1, T. marinus, T. oleivorans. Kuma O. kriegii 23, 24, 25, 26, 27. Thalassolituus memba ne na marine wajibi hydrocarbon fragmentation kwayoyin (OHCB), wanda shi ne tartsatsi a cikin marine da terrestrial yanayi, kuma yawanci ya zama rinjaye bayan hydrocarbon gurbatawa events30,31. Kwayoyin cuta na ruwa ba mambobi ne na ƙungiyar OHCB ba, amma an keɓe su daga yanayin ruwa.
Bayanai na Phenotypic sun nuna cewa ASxL5T sabon nau'i ne kuma memba na jinsin da ba a san shi ba a baya a cikin dangin spirospiraceae na ruwa. A halin yanzu babu takamaiman ma'auni don rarrabuwar sabbin keɓantattun nau'ikan zuwa sabon nau'in halitta. An yi ƙoƙari don ƙayyade iyakokin jinsin duniya, alal misali, dangane da adadin kwayoyin halittar furotin mai ra'ayin mazan jiya (POCP), ana ba da shawarar cewa ƙimar yankewa ta kasance 50% daidai da nau'in tunani33. Wasu suna ba da shawarar amfani da ƙimar AAI, waɗanda ke da fa'ida akan POCP saboda ana iya samun su daga genomes34 da bai cika ba. Marubucin ya yi imanin cewa idan darajar AAI ta kasa da 74% idan aka kwatanta da nau'in nau'in nau'in nau'in samfurin, nau'in yana wakiltar wani nau'i na daban. Halin samfurin a cikin marine spirillaceae shine marine spirillum, kuma nau'in samfurin shine O. linum ATCC 11336T. Darajar AAI tsakanin ASxL5T da O. linum ATCC 11336T shine 54.34%, kuma darajar AAI tsakanin ASxL5T da T. oleivorans MIL-1T (nau'in nau'in nau'in nau'in jinsi) shine 67.61%, yana nuna cewa ASxL5T yana wakiltar sabon jinsin da ya bambanta da Thalassolituus. Yin amfani da jerin kwayoyin halittar 16S rRNA azaman ma'auni na rarrabuwa, iyakar ƙayyadaddun jinsin da aka ba da shawarar shine 94.5%35. Ana iya sanya ASxL5T a cikin jinsin Thalassolituus, yana nuna 95.03% 16S rRNA ainihi tare da T. oleivorans MIL-1T da 96.17%. marinus IMCC1826T. Duk da haka, kuma za a sanya shi a cikin nau'in Bacteroides wanda ke da 94.64% 16S rRNA asalin asalin tare da B. sanyensis NV9, yana nuna cewa yin amfani da kwayar halitta guda ɗaya kamar 16S rRNA gene zai iya haifar da rarrabuwa da aiki na sabani. Wata hanyar da aka ba da shawarar tana amfani da ANI da Genome Alignment Score (AF) don bincika tarin wuraren bayanai daga kowane nau'i da nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan da ke akwai. Marubucin ya ba da shawarar hada iyakar jinsi tare da juzu'i na ƙididdiga na ƙididdiga na musamman ga harajin da ake nazarin. Koyaya, idan babu isassun cikakkun jerin kwayoyin halitta daga keɓewar Thalassolituus, ba zai yuwu a tantance ko ASxL5T na cikin halittar Thalassolituus ta wannan hanyar ba. Saboda ƙayyadaddun samuwa na cikakkun jerin kwayoyin halitta don bincike, ya kamata a fassara duk bishiyar phylogenetic genome tare da taka tsantsan. Abu na biyu, gabaɗayan hanyoyin kwatancen kwayoyin halitta ba za su iya ƙididdige ɗimbin bambance-bambance a cikin girman kwatancen kwayoyin halitta ba. Sun auna kamanni na ainihin asalin kwafi guda ɗaya tsakanin jinsin da ke da alaƙa, amma ba su yi la'akari da adadi mai yawa na kwayoyin halitta waɗanda ba su nan a cikin ƙaramin ƙwayar halittar ASxL5T. Babu shakka, ASxL5T da ƙungiyoyin da suka haɗa da Thalassolituus, Oceanobacter, da Bacterioplanes suna da kakanni guda ɗaya, amma juyin halitta ya ɗauki wata hanya ta daban, wanda ke haifar da raguwa a cikin kwayoyin halitta, wanda zai iya zama dacewa da salon rayuwa. Wannan ya bambanta da T. oleivorans MIL-1T, wanda ya fi girma 28% kuma ya samo asali a ƙarƙashin matsi daban-daban na muhalli don amfani da hydrocarbons23,30. Ana iya kwatanta kwatance mai ban sha'awa tare da wajabta parasites na cikin salula da symbionts, irin su Rickettsia, Chlamydia, da Buchnera. Girman kwayoyin halittar su kusan 1 Mb. Ƙarfin yin amfani da ƙwayoyin ƙwayoyin ƙwayoyin ƙwayoyin ƙwayoyin cuta yana haifar da asarar kwayoyin halitta, don haka An sami gagarumin lalacewa na kwayoyin halitta. Canje-canjen juyin halitta daga kwayoyin halitta na sinadarai na ruwa zuwa salon rayuwa na yau da kullun na iya haifar da raguwa iri ɗaya a girman kwayoyin halitta. Binciken COG da KEGG yana nuna adadin kwayoyin halittar da aka yi amfani da su don takamaiman ayyuka da kuma bambance-bambancen duniya a cikin hanyoyin genomic tsakanin ASxL5T da T. oleivorans MIL-1T, waɗanda ba saboda yaɗuwar abubuwan kwayoyin halitta ta hannu ba. Bambanci a cikin rabon G + C na dukkanin kwayoyin halitta na ASxL5T shine 56.1%, kuma na T. oleivorans MIL-1T shine 46.6%, wanda kuma yana nuna cewa an ware shi.
Gwajin abun ciki na coding na kwayoyin halittar ASxL5T yana ba da fa'idodin aiki cikin halayen phenotypic. Kasancewar kwayoyin halitta nau'in IV fimbriae (Tfp) yana da ban sha'awa na musamman domin suna haɓaka motsin tantanin halitta, wanda ake kira gliding ko jijjiga, ba tare da flagella a saman ba. A cewar rahotanni, Tfp yana da wasu ayyuka, ciki har da tsinkaya, pathogenesis, samuwar biofilm, ɗaukar DNA na halitta, tarawa ta atomatik da haɓaka38. Kwayar halittar ASxL5T tana ƙunshe da kwayoyin halitta 18 da ke ɓoye diguanylate cyclase (wani enzyme wanda ke haifar da jujjuyawar 2 guanosine triphosphate zuwa guanosine 2 phosphate da cyclic diGMP) da kuma 6 genes da ke ɓoye madaidaicin diguanylate cyclase phosphate diguanylate. Halittar kwayoyin halitta don esterase (catalyzing lalacewar cyclic di-GMP zuwa guanosine monophosphate) yana da ban sha'awa saboda cycl-di-GMP shine manzo na biyu mai mahimmanci wanda ke da hannu a cikin ci gaban biofilm da rabuwa, motsi, haɗe-haɗe da ƙwayoyin cuta 39, 40 a cikin tsari. Hakanan ya kamata a lura da cewa a cikin Bdellovibrio bacteriovorus, an nuna GMP na cyclic sau biyu don sarrafa canji tsakanin rayuwa ta kyauta da salon rayuwa na yau da kullun41.
Yawancin bincike akan ƙwayoyin cuta masu farauta sun mayar da hankali kan Bdellovibrio, Bdellovibrio-kamar kwayoyin halitta, da nau'in Myxococcus. Waɗannan da sauran sanannun misalan ƙwayoyin cuta masu farauta suna haifar da rukuni daban-daban. Duk da wannan bambance-bambancen, saitin iyalai na sinadarai masu siffa waɗanda ke nuna nau'ikan nau'ikan ƙwayoyin cuta 11 da aka san su da yawa an gano su3,22. Duk da haka, kawai kwayoyin halittar O antigen ligase (waaL) ne aka gano, wanda ya zama ruwan dare a cikin kwayoyin Gram-negative. Wannan nau'i na bincike ba shi da taimako wajen zayyana ASxL5T a matsayin mafarauci, mai yiwuwa saboda yana amfani da dabarun kai hari. Samar da ƙarin nau'ikan ƙwayoyin cuta na ƙwayoyin cuta na ƙwayoyin cuta zai taimaka haɓaka mafi kyawun nazarin ƙuduri waɗanda ke la'akari da shaidar aiki da bambance-bambancen muhalli tsakanin membobin ƙungiyar. Misalan ƙwayoyin cuta masu lalata da ba a haɗa su a cikin wannan binciken sun haɗa da membobin Cupriavidus necator42 da Bradymonabacteria43, saboda kamar yadda masu bincike ke bincikar al'ummomin microbial daban-daban, an kafa ƙarin harajin cin abinci.
Mafi kyawun fasalin kwayoyin ASxL5T da hoton TEM ya kama shine na musamman da sassauƙan yanayin halittarsa, wanda zai iya haɓaka hulɗa tare da ƙwayoyin ganima. Nau'in hulɗar da aka gani ya bambanta da sauran ƙwayoyin cuta masu kama da ba a taɓa gano su ba ko ba da rahoto. An nuna tsarin zagayowar rayuwar mafarauta na ASxL5T a cikin Hoto na 5. Akwai 'yan misalai a cikin wallafe-wallafen masu irin wannan tsarin apical kamar yadda muke ba da rahoto a nan, amma waɗannan misalan sun haɗa da Terasakiispira papahanaumokuakeensis, kwayoyin spirillum na ruwa tare da kara girma na lokaci-lokaci 44, da Alphaproteobacteria, Terasakiella pus. , wanda ya kasance na asalin halittar Oceanospirillum, nunin abin da ake kira "fim ɗin polar" 45. Ana lura da siffofin Cocci sau da yawa a cikin tsofaffin al'adu, musamman ga kwayoyin cuta tare da nau'i mai lankwasa, irin su Vibrio, Campylobacter, da Helicobacter 46, 47, 48, wanda zai iya wakiltar ƙasƙanci jihar. Ana buƙatar ƙarin aiki don fayyace madaidaicin tsarin rayuwa na kwayoyin ASxL5T. Don sanin yadda yake kamawa da ganima, da kuma ko kwayar halittarsa ​​ta ƙunshi mahadi masu aiki da ilimin halitta waɗanda za a iya amfani da su don dalilai na likita ko ilimin halittu.
Bayanin Venatorbacter Gen. Nuwamba Venatorbacter (Ven.a.tor, ba'c.ter, L. ya ƙunshi venators daga L. n. venator,'mafarauta' da Gr. n. bacter,' sanda'. Venatorbacter,'a farauta Rod' Kwayoyin suna aerobic, gishiri-haƙuri, mai lankwasa Gram tabo, motsa jiki sanda da PHB ba ya tarawa Yanayin zafin jiki na 4 zuwa 42 ° C Ingrown pH na 4-9 ba sabon abu bane A cikin katantanwa na ruwa, yawancin acidic acid sune C16: 1ω6c da / ko C16: 1ω7c. C18: 1ω9 ; C12: 0 3-OH da C10: 0 3-OH ana samun su azaman hydroxy fatty Acids .
Bayanin Venatorbacter cucullus sp. Nuwamba Venatorbacter cucullus (cu'cull.us.; L. n. cucullus yana nufin adalci).
Bugu da kari, siffar siffa ta wannan nau'in ita ce lokacin girma akan BA ko BHI, sel suna da tsayin 1.63 µm da faɗin 0.37 µm. Mallakan da ke kan agar BHI ƙanana ne, sun kai 2 mm a diamita bayan sa'o'i 72. Su ne m, translucent, zagaye, convex da sheki. Membobin wannan nau'in na iya amfani da Escherichia coli da Klebsiella. Campylobacter da wasu kwayoyin cutar Gram-korau suna zama ganima.
An keɓe nau'in nau'in ASxL5T na yau da kullun daga madarar naman sa a Nottinghamshire, UK, kuma an adana shi a cikin Tarin Al'adun Nau'in Ƙasa (Birtaniya): lambar shiga NCTC 14397 da lambar shigar da Al'adun Bacterial na Netherlands (NCCB) lambar NCCB 100775. Cikakken jerin kwayoyin halitta na ASxL5T An saka shi a cikin Genbank bisa ga ƙari na CP046056.
Kwayoyin ASxL5T an ware su daga madarar naman sa ta amfani da fasahar keɓewar phage9,49. An narkar da slurry 1: 9 (w / v) a cikin SM buffer (50 mM Tris-HCl [pH 7.5], 0.1 M NaCl, 8 mM MgSO4.7H2O da 0.01% gelatin; Sigma Aldrich, Gillingham, UK), Sa'an nan incubate a 4°C na tsawon awanni 24, yana juyawa a hankali don fitar da mafarauta cikin ma'ajiya. An dakatar da dakatarwar a 3000g na minti 3. An tattara ma'auni kuma an sanya shi a cikin 13,000g a karo na biyu na minti 5. Daga nan aka wuce da ma'auni ta hanyar tacewa na 0.45 µm membrane (Minisart; Sartorius, Gottingen, Jamus) da tacewa 0.2 µm membrane (Minisart) don cire duk sauran ƙwayoyin ƙwayoyin cuta. ASxL5T na iya wuce waɗannan masu tacewa. An shirya lawn agar mai laushi na Campylobacter enterosus S12 (lambar shiga NCBI CP040464) daga wannan slurry ta amfani da daidaitattun dabaru. An rarraba slurry ɗin da aka tace akan kowane ɗayan waɗannan faranti tantanin halitta a cikin ɗigo 10 µl a cikin sau uku kuma a bar shi ya bushe. An shigar da farantin a cikin tanki na microaerophilic a 37 ° C na tsawon awanni 48 a ƙarƙashin yanayin microaerobic (5% O2, 5% H2, 10% CO2, da 80% N2). An fitar da plaque na bayyane da aka samu a cikin SM buffer kuma an canza shi zuwa sabon lawn na C. hyointestinalis S12 don kara yaduwa da kwayoyin halitta. Da zarar an ƙaddara cewa ƙwayoyin cuta sune sanadin lytic plaque kuma ba phage ba, gwada girma kwayoyin halitta ba tare da mai watsa shiri ba kuma a kara kwatanta shi. An yi al'adun aerobic a 37 ° C tare da 5% v/v defibrinated jinin doki (TCS Biosciences Lt, Buckingham, UK, kari). Bisa ga jagororin Kwamitin Ma'auni na Clinical na Ƙasa, ana amfani da hanyar rarraba diski don gwajin kamuwa da ƙwayoyin cuta. An haɓaka BHI agar a 37 ° C ta amfani da diski mai ɗauke da maganin rigakafi masu zuwa (Oxoid) don al'adun aerobic: amoxicillin da clavulanic acid 30 µg; cefotaxime 30 µg; streptomycin - 10 g; ciprofloxacin 5 µg; Ceftazidime 30 µg Nalidixic acid 30 µg; 10 µg; Azithromycin 15 g; Chloramphenicol 30 g; Cefoxitin 30 g; Tetracycline 30 g; Nitrofurantoin 300 g; Aztreonam 30 µg; ampicillin 10 g; Cefpodoxime 10 µg; Trimethoprim-Sulfamethoxazole 25 μg. An kafa haƙurin gishiri ta hanyar incubation aerobic akan faranti agar BHI a 37 ° C. An ƙara ƙarin NaCl zuwa faranti agar BHI don samar da kewayon maida hankali har zuwa 10% w/v. An ƙaddara kewayon pH ta al'adun aerobic akan faranti agar BHI a 37 ° C, inda aka daidaita kewayon pH zuwa tsakanin 4 da 9 tare da HCl bakararre ko NaOH bakararre, kuma an tabbatar da ƙimar pH da aka yi niyya kafin zuba farantin. Don nazarin fatty acid na salula, an haɓaka ASxL5T akan agar BHI na kwanaki 3 da aerobic a 37 ° C. Bisa ga MIDI (Sherlock Microbial Identification System, sigar 6.10) daidaitaccen ka'idar FERA Science Ltd, (York, UK), an fitar da fatty acids, an shirya kuma an bincika.
Don TEM, ASxL5T an horar da aerobic ta hanyar yada iri ɗaya akan BA a 37 ° C na tsawon awanni 24, sannan an girbe shi cikin 1 ml na 3% (v/v) glutaraldehyde a cikin 0.1 M cacodylate buffer a dakin da zafin jiki Gyara don awa 1, sannan centrifuge a 10,000 g na minti 3. Sa'an nan kuma a hankali sake dakatar da pellet a cikin 600 μl 0.1 M cacodylate buffer. Canja wurin kafaffen dakatarwar ASxL5T zuwa fim ɗin Formvar/carbon akan grid jan ragamar raga 200. An lalata kwayoyin cutar da 0.5% (w/v) uranyl acetate na minti 1 kuma TEM ta bincika ta amfani da TEI Tecnai G2 12 microscope na Biotwin. Kamar yadda aka ambata a sama, hada adadin ganima da mafarauta a cikin NZCYM broth (BD Difco ™, Fisher Scientific UK Ltd, Loughborough) da kuma sanyawa na tsawon sa'o'i 48 a ƙarƙashin yanayin microaerobic na Campylobacter ko Campylobacter a 37 ° C, hulɗar mafarauta da ganima. TEM kuma ta bincika. Yanayin Aerobic don Escherichia coli. Yi nazarin ganima da ƙwayoyin cuta masu kama da kansu don sanin kowane canje-canje a cikin ilimin halittar tantanin halitta saboda tsinkaya. An yi amfani da hanyar baƙar fata ta Sudan don nazarin gani na gani na tarin PHB.
Shuka al'adun ASxL5T na dare ta hanyar shafa girma akan faranti na BHI ko BA tare da swab mara kyau. Tattara ƙwayoyin ASxL5T kuma a dakatar da su a cikin MRD (CM0733, Oxoid), sa'an nan kuma sanya su a 4 ° C na kwanaki 7 don yunwar sel. Tunanin NCTC ko dakin gwaje-gwaje na al'adun ƙwayoyin cuta an shigar da su cikin broth BHI ko No. 2 na gina jiki broth (CM007, Oxoid), incubated na dare, centrifuged a 13,000g kuma restapended a MRD har OD600 ya kasance 0.4. Al'adu: Bacillus subtilis NCTC 3610T, Citrobacter freundii NCTC 9750T, Enterobacter aerogenes NCTC 10006T, Enterococcus faecalis NCTC 775T, Escherichia coli NCTC 86, Klebsiella 46octoca NCTC 10817, Listeria Special kwayoyin NCTC 4885, Bacillus macerans NCTC 6355T, Providencia stuartsii NCTC 10318, Pseudomonas fluorescens SMDL, Rhodococcus submarine hamburger NCTC 1621T, Bacteria NCTC NCTC intestinal 10861, Staphylococcus aureus NCTC 8532T, Streptococcus pneumoniae NCTC 7465T, Yersinia enterocolitica NCTC 10460. Mai watsa shiri na Campylobacter ya kasance microaerobically a kan faranti BA a 37 ° C kuma an dakatar da shi a cikin broth N. Rundunar Campylobacter da aka gwada su ne: C. coli 12667 NCTC, C. jejuni 12662, C. jejuni PT14, C. jejuni NCTC 11168T, C. helveticus NCTC 12472, C. lari NCTC 11445NC, C.11458TC. PT14, C... Tattara sel a cikin MRD, centrifuge a 13,000g kuma a sake dawowa cikin MRD har OD600 shine 0.4. Add aliquot na 0.5 ml dakatar zuwa 5 ml narke NZCYM top agar (0.6% agar) da kuma zuba shi a kan 1.2% NZCYM farantin kasa. Bayan warkewa da bushewa, an rarraba ASxL5T da aka diluted azaman ɗigon ɗigon 20 µl akan kowane katako a cikin sau uku. Yanayin zafin al'ada da yanayin sun dogara da buƙatun kwayoyin gwajin.
Yi amfani da Kit ɗin DNA na ƙwayoyin cuta na GenElute™ (Sigma Aldridge) don shirya DNA daga keɓewar ƙwayoyin cuta. An yi amfani da daidaitattun hanyoyin don haɓaka PCR na 16S rRNA gene da ƙayyadaddun tsarin samfur ta amfani da sinadarai na ƙarewa (Sabis ɗin Karatun ƙimar Yurofins, Jamus). Yi amfani da shirin BLAST-N don kwatanta waɗannan jeri-jerun tare da wasu jeri na 16S rRNA don ganowa da tattara nau'ikan da ke da alaƙa. Waɗannan an daidaita su ta amfani da ClustalW a cikin shirin MEGA X. An sake gina bishiyar phylogenetic ta amfani da MEGA X ta amfani da mafi girman hanyar da za ta iya dogara da tsarin Tamura-Nei, tare da kwafi 1000 jagora54. Yi amfani da PureLink™ Genomic DNA Kit (Fisher Scientific, Loughborough, UK) don fitar da DNA don jerin kwayoyin halitta gaba ɗaya. An ƙaddara jerin kwayoyin halitta na ASxL5T ta amfani da haɗin Illumina MiSeq, wanda ya ƙunshi 250 bp biyu-ƙarshen karantawa wanda ya ƙunshi ɗakin karatu wanda aka shirya ta amfani da kayan lakabi na Nextera da 2 zuwa 20 kb dogon karantawa daga dandalin PacBio. Genomics DNA Sequencing Research Facility a Jami'ar Sembia. An haɗa kwayar halittar ta amfani da CLC Genomics Workbench 12.0.3 (Qiagen, Aarhus, Denmark). Ana adana al'adun ASxL5T a cikin Tarin Al'adun Nau'in Ƙasa (Birtaniya) da Tarin Al'adun Bacterial na Netherlands (NCCB). Kwayoyin halittar halittu masu alaƙa da aka yi amfani da su don kwatanta su ne: Thalassolituus oleivorans MIL-1T (lambar shiga HF680312, cikakke); Bacterioplanes sanyensis KCTC 32220T (lambar hanyar samun damarY01000001, bai cika ba); Oceanobacter kriegii DSM 6294T (lambar shiga NZ_AUGV00000000, bai cika ba); Marinamonas Community DSM 5604T (ƙara ASM436330v1, bai cika ba), Oceanospirullum linum ATCC 11336T (ƙara MTSD02000001, bai cika ba) da Thalassolituus sp. C2-1 (ƙara NZ_VNIL01000001, bai cika ba). Yi amfani da JGI Genome Portal36 a https://img.jgi.doe.gov//cgi-bin/mer/main.cgi?section=ANI&page= don tantance makin daidaitawa (AF) da matsakaicin asalin nucleic acid (ANI). Biyu. An yi amfani da hanyar Rodriguez-R & Konstantinidis55 don tantance ainihin amino acid (AAI). Yi amfani da GToTree 1.5.5411,12,13,14,15,16,17,18 don samar da mafi girman yiwuwar bishiyar phylogenetic. Halin shigar da kwayoyin halitta wanda ke wakiltar nau'in kwayar halitta da ake da shi an zaɓi shi daga nau'ikan tunani da aka gano yana da alaƙa da ASxL5T daga 16S rRNA phylogeny. An ƙirƙira itacen ta amfani da kayan aikin haɗin gwiwar bishiyar rayuwa (https://itol.embl.de/). Ana aiwatar da bayanin aiki da nazarin kwayoyin halittar ASxL5T ta amfani da kayan aikin kan layi na BlastKOALA KEGG ta amfani da KEGG (Kyoto Encyclopedia of Genes and Genomes) na rarraba wadatar kayayyaki. Rarraba nau'ikan COG (ƙungiyoyin orthologus) an ƙaddara ta amfani da kayan aikin kan layi na eggNOG-mapper.
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