Uhlobo olusha lwe-Gram-negative, i-aerobic, ukumelana nosawoti, okusebenzayo, okumise okwenduku, kanye namagciwane adlayo i-ASxL5T yahlukaniswa nedamu lobulongwe benkomo e-Nottinghamshire, e-England, futhi yasebenzisa i-Campylobacter njengesisulu sayo. Kamuva, ezinye izinhlobo ze-Campylobacter kanye namalungu omndeni wakwa-Enterobacteriaceae atholakala njengezisulu. Ngemuva kwe-subculture ngaphandle kwamaseli okusingatha, ukukhula okubuthakathaka kwe-aseptic kwafinyelelwa ku-Brain Heart Infusion Agar. Izimo ezikahle zokukhula ngu-37 °C kanti i-pH ingu-7. I-electron microscopy yokudlulisa yembule izici ezithile ezingavamile kakhulu ze-morphological ezihlobene nokutholakala kwenyamazane. Ukuhlaziywa kwe-Phylogenetic kusetshenziswa ukulandelana kwezakhi zofuzo ze-16S rRNA kubonise ukuthi i-isolate ihlobene nelungu lomndeni we-Marine Spirulina, kodwa ayikwazi ukufakwa ngokucacile njengelungu lanoma yiluphi uhlobo lwezinhlobo ezaziwayo. Ukulandelana kwegenome okuphelele kwe-ASxL5T kuqinisekise ubudlelwano namalungu ama-marine spirochetes. Ukusesha kusizindalwazi kwembule ukuthi ama-ASxL5T amaningana ahlanganyela ukulandelana kofuzo lwe-16S rRNA namagciwane amaningana angakhuliswanga avela olwandle, umhlaba kanye namanzi angaphansi komhlaba. Siphakamisa ukuthi uhlobo lwe-ASxL5T lumele uhlobo olusha ohlotsheni olusha. Sincoma igama elithi Venatorbacter cucullus gen. Novemba, sp. NgoNovemba, i-ASxL5T yasetshenziswa njengohlobo lohlobo.
Amabhaktheriya azidlayo amabhaktheriya abonisa ikhono lokuzingela futhi abulale amanye amagciwane aphilayo ukuze athole izinto ze-biosynthetic namandla. Lokhu kuhlukile ekubuyiseleni okuvamile kwemisoco kuma-microorganisms afile, futhi kuhlukile ekusebenzelaneni kwe-parasitic, lapho amabhaktheriya enza ubuhlobo obuseduze nomsingathi wawo ngaphandle kokuwabulala. Amagciwane ahlaselayo aguqule imijikelezo yokuphila eyahlukene ukuze asebenzise imithombo yokudla eningi ezindaweni lapho atholakala khona (njengezindawo zasolwandle). Bayiqembu elihlukahlukene ngokwe-taxonomically, elixhunywe kuphela ngomjikelezo wabo wokuphila oyingqayizivele wokuvala inzalo1. Izibonelo zamabhaktheriya azidlayo ziye zatholakala kuma-phyla amaningana ahlukene, okuhlanganisa: I-Proteobacteria, i-Bacteroides, ne-Chlorella.3. Kodwa-ke, amagciwane azidlayo afundelwe kahle kakhulu yi-Bdellovibrio kanye ne-Bdellovibrio-and-like organisms (BALOs4). Amagciwane ahlaselayo angumthombo othembisayo wezinhlanganisela ezintsha ze-biologically asebenzayo kanye nama-antibacterial agents5.
Amagciwane ahlaselayo kukholakala ukuthi athuthukisa ukuhlukahluka kwamagciwane futhi abe nomthelela omuhle empilweni ye-ecosystem, ukukhiqiza kanye nokuzinza6. Naphezu kwalezi zimfanelo ezinhle, zimbalwa izifundo mayelana namagciwane amasha adlayo ngenxa yobunzima bokukhulisa amagciwane kanye nesidingo sokubheka ngokucophelela ukusebenzisana kwamangqamuzana ukuqonda imijikelezo yawo yokuphila eyinkimbinkimbi. Lolu lwazi akulula ukuluthola ekuhlaziyeni ikhompyutha.
Esikhathini sokwanda kokumelana ne-antimicrobial, amasu amasha okukhomba izifo ezibangelwa amagciwane ayafundwa, njengokusetshenziswa kwe-bacteriophages kanye nama-predatory bacteria7,8. Amagciwane e-ASxL5T ahlukaniswa ngo-2019 kusetshenziswa ubuchwepheshe bokuhlukanisa i-phage kubulongwe bezinkomo obuqoqwe eSikhungo Sobisi sase-University of Nottingham, e-Nottinghamshire. Inhloso yophenyo wukuhlukanisa izinto eziphilayo ezinamandla njengama-ejenti okulawula ebhayoloji. I-Campylobacter hyointestinalis i-zoonotic pathogen, evame ukuhlotshaniswa nezifo zamathumbu omuntu10. Itholakala yonke indawo ku-serum futhi isetshenziswa njengomsingathi oqondiwe.
Ibhaktheriya ye-ASxL5T yahlukaniswa nejeli yenkomo ngoba kwaqashelwa ukuthi izingcezu ezakha otshanini be-C. hyointestinalis ayefana nalawo akhiqizwa ama-bacteriophages. Lokhu ukutholwa okungalindelekile, ngoba ingxenye yenqubo yokuhlukanisa i-phage ihlanganisa ukuhlunga ngesihlungi esingu-0.2 µm, esiklanyelwe ukususa amaseli ebhaktheriya. Ukuhlolwa kwe-Microscopic yezinto ezikhishwe oqwembeni kwembula ukuthi amagciwane amise okwenduku egobile igram-negative awazange aqongelele i-polyhydroxybutyrate (PHB). Isiko le-aseptic elizimele kumaseli azidlayo litholakala endaweni ecebile eqinile (njenge-brain heart infusion agar (BHI) ne-blood agar (BA)), futhi ukukhula kwayo kubuthakathaka. Itholakala ngemuva kwe-subculture nge-inoculum esindayo. Ikhula kahle ngokulinganayo ngaphansi kwe-microaerobic (7% v/v oxygen) kanye nezimo ze-oxygen yasemkhathini, kodwa hhayi endaweni ye-anaerobic. Ngemva kwamahora angu-72, ububanzi bekoloni babuncane kakhulu, bufinyelela ku-2 mm, futhi laliyi-beige, liguquguqukayo, liyindilinga, liyi-convex futhi licwebezela. Ukuhlolwa okujwayelekile kwe-biochemical kuyakhinyabezwa ngoba i-ASxL5T ayikwazi ukukhuliswa ngendlela enokwethenjelwa emithonjeni ye-liquid, okuphakamisa ukuthi ingase ithembele kumjikelezo wempilo oyinkimbinkimbi wokwakheka kwe-biofilm. Kodwa-ke, ukumiswa kwepuleti kubonise ukuthi i-ASxL5T i-aerobic, i-positive for oxidase ne-catalase, futhi ingabekezelela i-5% NaCl. I-ASxL5T ayizweli ku-10 µg streptomycin, kodwa izwela kuwo wonke amanye ama-antibiotic ahloliwe. Amaseli ebhaktheriya e-ASxL5T ahlolwe yi-TEM (Umfanekiso 1). Uma ekhuliswe ngaphandle kwamaseli ezinyamazane ku-BA, amaseli e-ASxL5T mancane i-Campylobacter, enobude obuyisilinganiso esingu-1.63 μm (± 0.4), ububanzi obungu-0.37 μm (± 0.08), kanye nesigxobo esisodwa eside (kufika ku-5 μm). I-flagella yocansi. Cishe u-1.6% wamaseli abonakala enobubanzi obungaphansi kuka-0.2 μm, okuzovumela ukudlula kudivayisi yesihlungi. Isandiso sesakhiwo esingavamile sibonwe phezulu kwamanye amaseli, afana ne-fairing (Latin cucullus) (bona imicibisholo kokuthi 1D, E, G). Lokhu kubonakala sengathi kwakhiwe ulwelwesi lwangaphandle oludlulele, okungenzeka lubangelwa ukuncipha ngokushesha kobukhulu bemvilophu ye-periplasmic, kuyilapho ulwelwesi lwangaphandle luhlala luqinile, lubonisa ukubukeka "okuxekethile". Ukuhlakulela i-ASxL5T ngokungabikho kwezakhi (ku-PBS) isikhathi eside ku-4 ° C kubangele amaseli amaningi (kodwa hhayi wonke) abonisa i-coccal morphology (Umfanekiso 1C). Lapho i-ASxL5T ikhula ne-Campylobacter jejuni njengesisulu samahora angu-48, isilinganiso sosayizi weseli sikhulu kakhulu futhi sincane kunamaseli akhule ngaphandle komsingathi (Ithebula 1 kanye noMfanekiso 1E). Ngokuphambene, lapho i-ASxL5T ikhula ne-E. coli njengesisulu samahora angu-48, isilinganiso sosayizi weseli sikhulu futhi sibanzi kunalapho sikhula ngaphandle kwenyamazane (Ithebula 1), futhi ubude beseli buyahlukahluka, ngokuvamile bubonisa i-filamentous (Figure 1F). Lapho efukanyelwe ne-Campylobacter jejuni noma i-E. coli njengesisulu samahora angu-48, amaseli e-ASxL5T awabonisanga nhlobo i-flagella. Ithebula 1 lifingqa ukubonwa koshintsho kusayizi weseli ngokusekelwe ebukhoneni, ukungabikho, kanye nohlobo lokudla lwe-ASxL5T.
Ukuboniswa kwe-TEM kwe-ASx5LT: (A) I-ASx5LT ibonisa isiswebhu eside; (B) ibhethri elijwayelekile le-ASx5LT; (C) amaseli e-cocci ASx5LT ngemva kokufukamela isikhathi eside ngaphandle kwezakhi; (D) iqembu lamaseli e-ASx5LT abonisa ukungavamile (E) Iqembu leseli le-ASx5LT elifakwe inyamazane ye-Campylobacter libonise ubude beseli okwandayo uma kuqhathaniswa nalawo angenawo ukukhula kwezilwane (D) aphinde abonise isakhiwo se-apical; (F) I-flagella enkulu ye-Filamentous, amaseli e-ASx5LT, ngemva kokufukamela nge-E. coli inyamazane; (G) Iseli elilodwa le-ASx5LT ngemva kokufukamela nge-E. coli, ebonisa isakhiwo esingaphezulu esingajwayelekile. Ibha imele u-1 μm.
Ukunquma ukulandelana kofuzo lwe-16S rRNA (inombolo yokufinyelela ethi MT636545.1) kuvumela ukusesha kwemininingwane ukuthi kutholwe ukulandelana okufana nalokhu okusesigabeni se-Gammaproteobacteria, futhi kuseduze kakhulu namagciwane asolwandle emndenini we-marine spirillum (Umfanekiso 2), futhi angamalungu ohlobo lwe-Thalassolituus Isihlobo esiseduze ne-Marine Bacillus. Ukulandelana kofuzo lwe-16S rRNA kuhluke ngokusobala kubhaktheriya ehlaselayo yomndeni we-Bdelvibrionaceae (Deltaproteobacteria). Ukuqhathaniswa kokubili kwe-B. bacteriovorus HD100T (uhlobo lwe-strain, i-DSM 50701) kanye ne-B. bacteriovorus DM11A bekungu-48.4% no-47.7%, futhi ku-B. exovorus JSS kube ngu-46.7%. Amabhaktheriya e-ASxL5T anamakhophi angu-3 ofuzo lwe-16S rRNA, amabili awo afana namanye, kanti eyesithathu izisekelo ezi-3 ngokuhlukana. Amanye ama-bacterial isolate amabili abulalayo (ASx5S ne-ASx5O; 16S rRNA izinombolo zofuzo MT636546.1 kanye MT636547.1, ngokulandelana) ezinezici ezifanayo ze-morphological kanye ne-phenotypic ezivela endaweni efanayo azifani, kodwa zihlukile ku-ASxL5T kanye ne-uncultured Bact. ukulandelana kwedathabhesi kuhlanganiswe ndawonye nezinye izinhlobo I-Oceanospirillaceae (Umfanekiso 2). Lonke ukulandelana kofuzo lwe-ASxL5T kunqunyiwe futhi kwalondolozwa kusizindalwazi se-NCBI, futhi inombolo yokungena ithi CP046056. I-genome ye-ASxL5T iqukethe i-chromosome eyindilinga engu-2,831,152 bp enesilinganiso se-G + C esingu-56.1%. Ukulandelana kofuzo kuqukethe ama-CDS angu-2653 (inani), angu-2567 okubikezelwa ukuthi azohlanganisa amaprotheni, angu-1596 anganikezwa njengemisebenzi yokubeka (60.2%). I-genome iqukethe izakhi zofuzo ze-RNA-coding ezingama-67, okuhlanganisa ama-rRNA angu-9 (3 ngalinye ku-5S, 16S, kanye no-23S) kanye nama-tRNA angu-57. Izici ze-genomic ze-ASxL5T ziqhathaniswe nama-genomes atholakalayo ezinhlobo zohlobo oluseduze oluhlonzwe kusukela kuchungechunge lofuzo lwe-16S rRNA (Ithebula 2). Sebenzisa i-amino acid identity (AAI) ukuze uqhathanise wonke ama-genome e-Thalassolituus atholakalayo ne-ASxL5T. Ukulandelana kofuzo okuseduze okutholakalayo (okungaphelele) okunqunywe yi-AAI yi-Thalassolituus sp. I-C2-1 (engeza i-NZ_VNIL01000001). Lolu hlobo lwahlukaniswa kusukela ekujuleni kolwandle lwe-Mariana Trench, kodwa okwamanje alukho ulwazi lwe-phenotypic mayelana nalolu hlobo ukuze luqhathaniswe. Uma kuqhathaniswa ne-ASxL5T's 2.82 Mb, i-genome yento ephilayo inkulu ngo-4.36 Mb. Usayizi ojwayelekile wofuzo wama-spirochetes olwandle cishe u-4.16 Mb (± 1.1; n = 92 ama-genome aphelele ayireferensi aphenywa kusukela ku-https://www.ncbi.nlm.nih.gov/assembly), ukuze i-genome ye-ASxL5T ihambisane ukuhleleka Uma kuqhathaniswa namanye amalungu, kuncane kakhulu. Sebenzisa i-GToTree 1.5.54 ukuze ukhiqize isihlahla se-phylogenetic esilinganiselwe esisekelwe ku-genome esiphezulu (Umfanekiso 3A), usebenzisa ukulandelana kwe-amino acid eqondaniswe futhi exhunyiwe yezakhi zofuzo zekhophi eyodwa eziyi-172 eziqondene ne-Gammaproteobacteria 11,12,13,14,15,16, 17 ,18. Ukuhlaziywa kubonise ukuthi ihlobene eduze ne-Thalassolituus, i-Bacterial Plane, ne-Marine Bacterium. Kodwa-ke, le datha ikhombisa ukuthi i-ASxL5T ihlukile ezihlotsheni zayo e-Marine Spirulina futhi idatha yayo yokulandelana kwe-genome iyatholakala.
Isihlahla se-phylogenetic esisebenzisa ukulandelana kofuzo lwe-16S rRNA sigqamisa isikhundla sezinhlobo ze-ASxL5T, ASxO5, kanye ne-ASxS5 (enezibilini) ngokuhlobene nezinhlobo zamabhaktheriya angalinywanga nasolwandle e-Marine Spirulinaceae. Inombolo yokungena ye-Genbank ilandela igama le-strain kubakaki. Sebenzisa i-ClustalW ukuze uqondanise ukulandelana, futhi usebenzise indlela enkulu yokungenzeka kanye nemodeli ye-Tamura-Nei ukuze uqonde ubudlelwano be-phylogenetic, futhi wenze iziphindaphindo eziqondisiwe eziyi-1000 kuhlelo lwe-MEGA X. Inombolo esegatsheni ikhombisa ukuthi inani lekhophi eqondisiwe likhulu kuno-50%. I-Escherichia coli U/541T isetshenziswe njengeqembu elingaphandle.
(A) Isihlahla se-phylogenetic esisekelwe ku-genome, esibonisa ubudlelwano phakathi kwebhaktheriya yasolwandle i-Spirospiraceae ASxL5T kanye nezihlobo zayo eziseduze, i-E. coli U 5/41T njengeqembu elingaphandle. (B) Uma kuqhathaniswa ne-T. oleivorans MIL-1T, ukusabalalisa kwesigaba sokusebenza kofuzo kubikezelwa ngokusekelwe kuqoqo le-orthologous group (COG) le-ASx5LT protein. Isibalo esingakwesokunxele sibonisa inani lezakhi zofuzo esigabeni ngasinye esisebenzayo se-COG ku-genome ngayinye. Igrafu engakwesokudla ibonisa amaphesenti ama-genome aqukethwe eqenjini ngalinye elisebenzayo le-COG. (C) Uma kuqhathaniswa ne-T. oleiverans MIL-1T, ukuhlaziywa kwe-KEGG ephelele (i-Kyoto Encyclopedia of Genes and Genomes) indlela eyimodulayo ye-ASxL5T.
Ukusebenzisa imininingo egciniwe ye-KEGG ukuhlola isakhi sofuzo esikhona kugenome ye-ASxL5T kwembule indlela ye-metabolic ye-aerobic gamma Proteus. I-ASxL5T iqukethe isamba sezakhi zofuzo ezingama-75 ezinikezwe amaprotheni enjini yebhaktheriya, okuhlanganisa nofuzo oluhilelekile ku-chemotaxis, ukuhlanganisa i-flagella, kanye nohlelo lwe-IV fimbriae. Esigabeni sokugcina, izakhi zofuzo eziyi-9 kweziyi-10 zinesibopho sokunyakazisa uhla lwezinye izinto eziphilayo. I-genome ye-ASxL5T iqukethe indlela ephelele ye-tetrahydropyrimidine biosynthetic ebamba iqhaza ekuphenduleni okuvikelekile ku-osmotic stress20, njengoba kulindelekile kuma-halophile. I-genome futhi iqukethe izindlela eziningi eziphelele zama-cofactors namavithamini, kufaka phakathi izindlela ze-riboflavin synthesis. Nakuba ufuzo lwe-alkane 1-monooxygenase (alkB2) lukhona ku-ASxL5T, indlela yokusebenzisa i-hydrocarbon ayiphelele. Ngokulandelana kofuzo kwe-ASxL5T, ama-homologue ezakhi zofuzo ezikhonjwe ngokuyinhloko abangela ukonakala kwama-hydrocarbon ku-T. oleiverans MIL-1T21, njenge-TOL_2658 (alkB) ne-TOL_2772 (alcohol dehydrogenase) ngokusobala awekho. Umfanekiso 3B ubonisa ukuqhathaniswa kokusatshalaliswa kofuzo esigabeni se-COG phakathi kwe-ASxL5T namafutha e-Olive MIL-1T. Sekukonke, igenome encane ye-ASxL5T iqukethe izakhi zofuzo ezimbalwa ngokulinganayo ezivela esigabeni ngasinye se-COG uma kuqhathaniswa negenome enkulu ehlobene. Lapho inani lezakhi zofuzo esigabeni ngasinye sokusebenza livezwa njengephesenti le-genome, umehluko uyaphawulwa kumaphesenti ezakhi zofuzo ezigabeni zokuhumusha, ukwakheka kwe-ribosomal kanye nezigaba ze-biogenesis, kanye nezigaba zomsebenzi wokukhiqiza nokuguqulwa kwamandla, akha i-ASxL5T enkulu. igenome Iphesenti iqhathaniswa neqembu elifanayo elikhona kugenome ye-T. oleiverans MIL-1T. Ngokuphambene, uma kuqhathaniswa ne-ASxL5T genome, i-T. oleivorans MIL-1T inamaphesenti aphezulu ofuzo ezigabeni zokuphindaphinda, ukuhlanganisa nokulungisa, kanye nezigaba zokuloba. Kuyathakazelisa ukuthi umehluko omkhulu kokuqukethwe kwesigaba ngasinye sokusebenza kwama-genomes amabili yinombolo yezakhi zofuzo ezingaziwa ezikhona ku-ASxL5T (Umfanekiso 3B). Ukuhlaziywa kokunothisa kwamamojula e-KEGG kwenziwa, lapho imojula ngayinye ye-KEGG imelela isethi yamayunithi asebenzayo achazwe ngesandla esichasiselweni kanye nokuchazwa kwebhayoloji yedatha yokulandelana kofuzo. Ukuqhathaniswa kokusatshalaliswa kofuzo endleleni yemojuli ye-KOG ephelele ye-ASxL5T kanye ne-Olive MIL-1T kuboniswa kuMfanekiso 3C. Lokhu kuhlaziya kubonisa ukuthi nakuba i-ASxL5T inomzila ophelele wesulfure ne-nitrogen, i-T. oleiverans MIL-1T ayinawo. Ngokuphambene, i-T. oleiverans MIL-1T inomzila ophelele we-cysteine ​​​​ne-methionine, kodwa ayiphelele ku-ASxL5T. Ngakho-ke, i-ASxL5T inemojula yesici yokwenziwa kwe-sulfate (echazwa njengesethi yezakhi zofuzo ezingasetshenziswa njengezimaka ze-phenotypic, ezifana nomthamo we-metabolic noma i-pathogenicity; https://www.genome.jp/kegg/module.html) Ku-T . oleiverans MIL-1T. Ukuqhathanisa izakhi zofuzo ze-ASxL5T nohlu lwezakhi zofuzo eziphakamisa indlela yokuphila edlayo akuhlangani. Nakuba isakhi sofuzo se-waaL esifaka i-ligase ehlotshaniswa ne-O antigen polysaccharide kuya kumongo sikhona ku-ASxL5T genome (kodwa sivamile kuma-bacterium amaningi e-Gram-negative), i-tryptophan 2,3-dioxygenase (TDO ) Ufuzo lungase luhlanganise i-amino engama-60. izifunda ezine-asidi ezivame ukutholakala kumabhaktheriya ahlaselayo angekho. Azikho ezinye izinhlobo zofuzo eziyisici ku-ASxL5T genome, okuhlanganisa nalawo ma-encoding enzymes abandakanyeka ku-isoprenoid biosynthesis kumzila we-mevalonate. Qaphela ukuthi alukho uhlobo lofuzo olulawula ukulotshwa kwe-gntR eqenjini lezilwane ezizingelayo elihloliwe, kodwa izakhi zofuzo ezintathu ezifana ne-gntR zingabonakala ku-ASxL5T.
Izici ze-phenotypic ze-ASxL5T zifinyezwa kuThebula 3 futhi ziqhathaniswa nezici ze-phenotypic zegenera elihlobene elingu-23, 24, 25, 26, ne-27 elibikwe ezincwadini. I-Isolates kusukela ku-T. marinus, T. olevorans, B. sanyensis, ne-Oceanobacter kriegii ayasebenza, abekezelela usawoti, imizimba emise okwenduku e-oxidase-positive, kodwa cishe ayinazo ezinye izici ze-phenotypic nge-ASxL5T. I-pH evamile yolwandle ingu-8.1 (https://ocean.si.edu/ocean-life/invertebrates/ocean-acidification#section_77), eboniswa ku-T. marinus, T. olevorans, B. sanyensis no-O. ikriegi. I-ASxL5T ifanele i-pH enkulu (4-9) ejwayelekile yezinhlobo ezingezona ezasolwandle. Izici ze-Phenotypic ze-Thalassolituus sp. C2-1. Akwaziwa. Izinga lokushisa elikhulayo le-ASxL5T livamise ukuba banzi kunalelo lezinhlobo zasolwandle (4–42 °C), nakuba ezinye kodwa kungezona zonke izingxenye ze-T. marinus ezikwazi ukumelana nokushisa. Ukungakwazi ukukhulisa i-ASxL5T kumidiya yomhluzi kuvimbele ukucaciswa okwengeziwe kwe-phenotypic. Sebenzisa i-API 20E ukuhlola izinto ezikhishwe epuletini le-BA, i-ONPG, i-arginine dihydrolase, i-lysine decarboxylase, i-ornithine decarboxylase, ukusetshenziswa kwe-citrate, urease, i-tryptophan deaminase, i-gelatin hydrolysis Enzyme, imiphumela yokuhlolwa yonke ibingemihle, kodwa ayikho indole, i-acetoin ne-H2. zakhiqizwa. Ama-carbohydrate angenamvubelo afaka: i-glucose, i-mannose, inositol, i-sorbitol, i-rhamnose, i-sucrose, i-melibiose, i-amygdalin ne-arabinose. Uma kuqhathaniswa nezinhlobo zereferensi ezihambisanayo ezishicilelwe, iphrofayili ye-fatty acid yeselula yohlobo lwe-ASxL5T iboniswa kuThebula 4. Ama-fatty acids amaselula ayinhloko yi-C16:1ω6c kanye/noma i-C16:1ω7c, i-C16:0 ne-C18:1ω9. Ama-Hydroxy fatty acids C12:0 3-OH kanye ne-C10:0 3-OH nawo akhona. Isilinganiso se-C16:0 ku-ASxL5T siphezulu kunevelu elibikiwe lesizukulwane esihlobene. Ngokuphambene, uma kuqhathaniswa ne-T. marinus IMCC1826TT ebikiwe, isilinganiso se-C18:1ω7c kanye/noma i-C18:1ω6c ku-ASxL5T sinciphile. i-oleivorans MIL-1T ne-O. kriegii DSM 6294T, kodwa ayitholwa ku-B. sanyensis KCTC 32220T. Ukuqhathanisa amaphrofayili e-fatty acid ye-ASxL5T ne-ASxLS yembula umehluko ocashile enanini lamafutha e-acids ngamanye phakathi kwezinhlobo ezimbili, ezihambisana nokulandelana kwe-genomic DNA yohlobo olufanayo. Azikho izinhlayiya ze-poly-3-hydroxybutyrate (PHB) ezitholwe kusetshenziswa ukuhlolwa okumnyama kwaseSudan.
Umsebenzi we-predation we-ASxL5T amagciwane wacwaningwa ukuze kutholakale uhla lwezilwane ezizingelayo. Leli gciwane lingakha ama-plaque ezinhlotsheni zeCampylobacter, okuhlanganisa: Campylobacter suis 11608T, Campylobacter jejuni PT14, Campylobacter jejuni 12662, Campylobacter jejuni NCTC 11168T; I-Escherichia coli NCTC 12667; C. helveticus NCTC 12472; I-C lari NCTC 11458 kanye ne-C. upsaliensis NCTC 11541T. Sebenzisa amasiko asohlwini lwesigaba sokunquma ibanga lomsingathi wendlela ukuze uhlole uhla olubanzi lwebhaktheriya ye-Gram-negative kanye ne-Gram-positive. Imiphumela ibonisa ukuthi i-ASxL5T ingasetshenziswa naku-Escherichia coli NCTC 86 kanye ne-Citrobacter freundii NCTC 9750T. Ama-plaque akhiwe ku-Klebsiella oxytoca 11466. Ukusebenzisana kwe-TEM ne-E. coli NCTC 86 kuboniswa kuMfanekiso 4A-D, futhi ukusebenzelana ne-Campylobacter jejuni PT14 kanye ne-Campylobacter suis S12 kuboniswa kuMfanekiso 4E-H phakathi nendawo. Indlela yokuhlasela ibonakala yehlukile phakathi kwezinhlobo zezilwane ezizingelayo ezihloliwe, ngeseli ye-E. coli eyodwa noma amaningi anamathiselwe kuseli ngalinye le-ASxL5T futhi abekwe eceleni eduze kweseli enwetshiwe ngaphambi kokukhangiswa. Ngokuphambene, i-ASxL5T ibonakala inamathisela ku-Campylobacter ngephuzu elilodwa lokuxhumana, ngokuvamile ngokuthintana ne-apex yeseli yezilwane ezizingelayo futhi eduze nendawo ephezulu ye-Campylobacter cell (Umfanekiso 4H).
I-TEM ebonisa ukusebenzisana phakathi kwe-ASx5LT nenyamazane: (AD) kanye ne-E. coli inyamazane; (EH) kanye noC. jejuni inyamazane. (A) Iseli elijwayelekile le-ASx5LT elixhunywe kuseli eyodwa ye-E. coli (EC); (B) I-ASx5LT ene-filamentous enamathiselwe kuseli elilodwa le-EC; (C) Iseli ye-ASx5LT ene-filamentous exhunywe kumaseli amaningi e-EC; (D) Okunamathiselwe kumaseli Amancane we-ASx5LT kuseli eyodwa ye-E. coli (EC); (E) iseli elilodwa le-ASx5LT elixhunywe kuseli ye-Campylobacter jejuni (CJ); (F) I-ASx5LT ihlasela amaseli e-C. hyointestinalis (CH); (G) amaseli amabili e-One ASx5LT ahlasele iseli le-CJ; (H) Ukubuka okuseduze kwephoyinti lokunamathiselwe le-ASx5LT, eduze nendawo ephezulu yeseli ye-CJ (ibha 0.2 μm). Ibha imele u-1 μm ku-(A–G).
Amabhaktheriya abulalayo aguquke ukuze asebenzise imithombo eminingi yezilwane ezizingelayo. Ngokusobala, zikhona kabanzi ezindaweni eziningi ezahlukene. Ngenxa yobukhulu obuncane bamalungu abantu, kungenzeka ukuhlukanisa amabhaktheriya e-ASxL5T ku-slurry usebenzisa indlela yokuhlukanisa i-phage. Ukuhlobana kwe-genomic kwe-ASxL5T kumalungu omndeni we-oceanospirillaceae yamagciwane olwandle kuyamangaza, nakuba isilwane sibekezelela usawoti futhi singakhula endaweni equkethe usawoti ongu-5%. Ukuhlaziywa kwezinga lamanzi le-slurry kubonise ukuthi okuqukethwe kwe-sodium chloride kwakungaphansi kuka-0.1%. Ngakho-ke, udaka luqhelelene nemvelo yasolwandle—ngokwendawo nangamakhemikhali. Ukuba khona kweziqhingi ezintathu ezihlobene kodwa ezihlukene ezivela emthonjeni ofanayo kunikeza ubufakazi bokuthi lezi zilwane ezidla ezinye ziyachuma kule ndawo engeyona eyasolwandle. Ngaphezu kwalokho, ukuhlaziywa kwe-microbiome (amafayela edatha atholakala ku-https://www.ebi.ac.uk/ena/browser/view/PRJEB38990) kubonise ukuthi ukulandelana kwezakhi zofuzo ze-16S rRNA kutholakala ku-50 ephezulu yentela yokusebenza (OTU) ) Ezinkathini ezimbalwa zesampula zodaka. Kutholwe amagciwane amaningana angakhuliswanga kusizindalwazi se-Genbank, anama-gene alandelanayo angu-16S rRNA afana namagciwane e-ASxL5T. Lokhu kulandelana, kanye nokulandelana kwe-ASxL5T, ASxS5, kanye ne-ASxO5, kubonakala sengathi kumelela izigaba ezihlukene ezihlukaniswe ku-Thalassolituus ne-Oceanobacter (Umfanekiso 2). Izinhlobo ezintathu zamabhaktheriya angakhuliswanga (GQ921362, GQ921357 kanye ne-GQ921396) ahlukaniswa emanzini aqhekekile ekujuleni okungamakhilomitha angu-1.3 emayini yegolide yaseNingizimu Afrika ngo-2009, kanti amanye amabili (DQ256320 kanye ne-DQ337006) ayesuka emanzini angaphansi komhlaba aseNingizimu Afrika (also). ngo-2005). Ukulandelana kofuzo lwe-16S rRNA okuhlobene kakhulu ne-ASxL5T kuyingxenye yokulandelana kofuzo lwe-16S rRNA etholwe kusiko lokunothisa lezinsalela zesihlabathi ezitholwe emabhishi asenyakatho yeFrance ngo-2006 (inombolo yokufinyelela AM29240828). Okunye ukulandelana kofuzo okuhlobene eduze kwe-16S rRNA okuvela kubhaktheriya engakhuliswanga i-HQ183822.1 kutholwe ethangini eliqoqwe elikhishwe endaweni yokulahla imfucumfucu kamasipala e-China. Ngokusobala, amabhaktheriya e-ASxL5T awameleli kakhulu kudathabhethi ye-taxonomic, kodwa lokhu kulandelana okuvela kubhaktheriya angafundiwe kungenzeka kumelele izinto eziphilayo ezifana ne-ASxL5T, ezisatshalaliswa emhlabeni wonke, ngokuvamile ezindaweni eziyinselele. Kusuka kukho konke ukuhlaziywa kwe-phylogenetic yofuzo, isihlobo esiseduze kakhulu ne-ASxL5T i-Thalassolituus sp. C2-1, T. marinus, T. oleivorans. Futhi u-O. kriegii 23, 24, 25, 26, 27. I-Thalassolituus iyilungu le-marine obligate hydrocarbon fragmentation bacteria (OHCB), elisabalele ezindaweni zasolwandle nasemhlabeni, futhi ngokuvamile liba elibusayo ngemva kwezigameko zokungcoliswa kwe-hydrocarbon30,31. Amagciwane olwandle awawona amalungu eqembu le-OHCB, kodwa ahlukanisiwe nemvelo yasolwandle.
Idatha ye-Phenotypic ibonisa ukuthi i-ASxL5T iwuhlobo olusha futhi iyilungu lohlobo olwalungaziwa ngaphambili emndenini we-marine spirospiraceae. Okwamanje alikho izinga elicacile lokuhlukanisa izinhlobo ezisanda kuhlukaniswa zibe uhlobo olusha. Imizamo yenziwe yokunquma imingcele yegenera yomhlaba wonke, isibonelo, ngokusekelwe kumaphesenti e-genome ye-conservative protein (POCP), kunconywa ukuthi inani lokunqanyulwa lifane no-50% wenkomba33. Abanye baphakamisa ukusebenzisa amanani e-AAI, anezinzuzo kune-POCP ngoba angatholakala kuma-genomes34 angaphelele. Umbhali ukholelwa ukuthi uma inani le-AAI lingaphansi kuka-74% uma liqhathaniswa nohlobo lwemodeli yohlobo lwemodeli, uhlobo lumelela uhlobo oluhlukile. Imodeli yohlobo lwe-marine spirillaceae i-marine spirillum, futhi uhlobo lwemodeli yi-O. linum ATCC 11336T. Inani le-AAI eliphakathi kwe-ASxL5T ne-O. linum ATCC 11336T lingu-54.34%, futhi inani le-AAI phakathi kwe-ASxL5T ne-T. oleivorans MIL-1T (uhlobo lohlobo lwezinhlobo) lingu-67.61%, okubonisa ukuthi i-ASxL5T imele uhlobo olusha oluhlukile kune-Thalassolituus. Kusetshenziswa ukulandelana kofuzo lwe-16S rRNA njengezinga lokuhlukanisa, umngcele ophakanyisiwe wokunqanyulwa kohlobo ngu-94.5%35. I-ASxL5T ingase ibekwe kuhlobo lwe-Thalassolituus, ebonisa ubunikazi bokulandelana obungu-95.03% 16S rRNA nge-T. oleivorans MIL-1T kanye no-96.17%. I-marinus IMCC1826T. Kodwa-ke, izophinde ibekwe ohlotsheni lwe-Bacteroides olune-94.64% 16S rRNA identity yofuzo ene-B. sanyensis NV9, okubonisa ukuthi ukusetshenziswa kofuzo olulodwa olufana nofuzo lwe-16S rRNA kungaholela ekuhlukaniseni ngokungafanele kanye nomsebenzi. Enye indlela ephakanyisiwe isebenzisa i-ANI kanye ne-Genome Alignment Score (AF) ukuze ihlole ukuqoqwa kwamaphoyinti edatha kuzo zonke izinhlobo nezinhlobo ezingezona zohlobo lwegenera ekhona. Umbhali uncoma ukuthi kuhlanganiswe umngcele wohlobo nephuzu lokuguquguquka lohlobo olulinganiselwe oluqondene nentela ehlaziywayo. Kodwa-ke, uma kungekho ukulandelana kwegenome okuphelele okwanele okuvela ku-Thalassolituus isolate, akunakwenzeka ukucacisa ukuthi i-ASxL5T ingeyohlobo lwe-Thalassolituus ngale ndlela. Ngenxa yokutholakala okulinganiselwe kokulandelana okuphelele kwe-genome ukuze kuhlaziywe, sonke isihlahla se-genome phylogenetic kufanele sihunyushwe ngokuqapha. Okwesibili, izindlela zokuqhathanisa zofuzo eziphelele azikwazi ukulandisa ngomehluko omkhulu kusayizi wama-genome aqhathaniswayo. Balinganise ukufana kwezakhi zofuzo eziwumgogodla wekhophi eyodwa phakathi kofuzo oluhlobene, kodwa abazange bacabangele inani elikhulu lezakhi zofuzo ezingekho ku-genome encane kakhulu ye-ASxL5T. Ngokusobala, i-ASxL5T namaqembu ahlanganisa i-Thalassolituus, i-Oceanobacter, ne-Bacterioplanes inokhokho oyedwa, kodwa ukuziphendukela kwemvelo kuthathe indlela ehlukile, okuholela ekunciphiseni kwe-genome, okungase kube ukuzivumelanisa nendlela yokuphila edlayo. Lokhu kuphambene ne-T. oleivorans MIL-1T, enkulu ngo-28% futhi ethuthuke ngaphansi kwezingcindezi ezehlukene zemvelo ukuze kusetshenziswe ama-hydrocarbons23,30. Ukuqhathanisa okuthakazelisayo kungenziwa nama-parasite e-intracellular kanye nama-symbionts, njenge-Rickettsia, i-Chlamydia, ne-Buchnera. Usayizi wabo we-genome cishe u-1 Mb. Ikhono lokusebenzisa i-metabolite yeseli elibambayo liholela ekulahlekelweni kwezakhi zofuzo, ngakho-ke Kwenziwa ukuwohloka kwe-genomic okuphawulekayo kokuziphendukela kwemvelo. Izinguquko zokuziphendukela kwemvelo zisuka ezintweni eziphila olwandle ezinomsoco ziye ezindleleni ezidla izilwane zingase zibangele ukuncipha okufanayo kosayizi wofuzo. Ukuhlaziywa kwe-COG ne-KEGG kugqamisa inani lezakhi zofuzo ezisetshenziselwa imisebenzi ethile kanye nomehluko womhlaba wonke kumzila we-genomic phakathi kwe-ASxL5T ne-T. oleivorans MIL-1T, okungabangelwa ukutholakala okubanzi kwezinto zofuzo zamaselula. Umehluko esilinganisweni se-G + C salo lonke i-genome ye-ASxL5T ngu-56.1%, kanti leyo ye-T. oleivorans MIL-1T ingu-46.6%, okuphinde kubonise ukuthi ihlukanisiwe.
Ukuhlolwa kokuqukethwe kwekhodi ye-ASxL5T genome kunikeza imininingwane esebenzayo ezicini ze-phenotypic. Ukuba khona kohlobo lombhalo wekhodi we-IV fimbriae (Tfp) kuthakazelisa ngokukhethekile ngoba akhuthaza ukunyakaza kwamaseli, okubizwa ngokuthi ukugxuma komphakathi noma ukudlikizeka, ngaphandle kwe-flagella ngaphezulu. Ngokwemibiko, i-Tfp ineminye imisebenzi, okuhlanganisa i-predation, i-pathogenesis, ukwakheka kwe-biofilm, ukuthatha i-DNA yemvelo, ukuhlanganisa amaseli okuzenzakalelayo kanye nokuthuthukiswa38. Igenome ye-ASxL5T iqukethe izakhi zofuzo ezingu-18 ezifaka ikhodi ye-diguanylate cyclase (i-enzyme eyenza ukuguqulwa kwe-2 guanosine triphosphate ibe i-guanosine 2 phosphate kanye ne-cyclic diGMP) kanye nofuzo olu-6 olufaka ikhodi ehambisanayo ye-diguanylate cyclase phosphate diguanylate. Isakhi sofuzo se-esterase (okudala ukuwohloka kwe-cyclic di-GMP kuya ku-guanosine monophosphate) siyathakazelisa ngoba i-cycl-di-GMP iyisithunywa sesibili esibalulekile esibandakanyeka ekuthuthukisweni nasekuhlukaniseni i-biofilm, ukunyakaza, ukunamathiselwa kweseli kanye ne-virulence 39, 40 kule nqubo. Kufanele futhi kuqashelwe ukuthi ku-Bdellovibrio bacteriovorus, i-cyclic double GMP ikhonjiswe ukulawula uguquko phakathi kwempilo yamahhala nendlela yokuphila edla ezinye41.
Ucwaningo oluningi ngamabhaktheriya adlayo lugxile ku-Bdellovibrio, i-Bdellovibrio-like organisms, kanye nezinhlobo ze-Myxococcus. Lezi nezinye izibonelo ezaziwayo zamabhaktheriya azidlayo zakha iqembu elihlukahlukene. Naphezu kwalokhu kuhlukahluka, iqoqo lemindeni ye-protein yesici ekhombisa ama-phenotypes we-11 eyaziwa ngamabhaktheriya adlayo ahlonziwe3,22. Kodwa-ke, kuphela izakhi zofuzo ezifaka ikhodi ye-O antigen ligase (waaL) ezihlonziwe, ezivame kakhulu kubhaktheriya we-Gram-negative. Lolu hlobo lokuhlaziya alulona usizo ekukhetheni i-ASxL5T njengesilwane esizingelayo, mhlawumbe ngoba sisebenzisa isu lokuhlasela elinoveli. Ukutholakala kwama-genome amagciwane ahlukahlukene ahlukahlukene kuzosiza ukuthuthukisa ukuhlaziya kokuxazulula okucolisekileyo okucabangela ubufakazi bomehluko wokusebenza nokwemvelo phakathi kwamalungu eqembu. Izibonelo zamagciwane adla ezinye ezingafakiwe kulokhu kuhlaziya zifaka amalungu e-Cupriavidus necator42 kanye ne-Bradymonabacteria43, ngoba njengoba abacwaningi baphenya imiphakathi ehlukene yamagciwane, kumiswa intela eningi edlayo.
Isici esiphawuleka kakhulu samabhaktheriya e-ASxL5T athwetshulwe isithombe se-TEM i-morphology yayo eyingqayizivele futhi eguquguqukayo, engakhuthaza ukusebenzisana namabhaktheriya adla inyama. Uhlobo lokusebenzelana olubonwayo luhlukile kwamanye amagciwane adla ezinye futhi alukatholwa noma alubikwa. Umjikelezo wokuphila ohlongozwayo we-ASxL5T uboniswa kuMfanekiso 5. Kunezibonelo ezimbalwa ezincwadini ezinezakhiwo ezifanayo ze-apical njengoba sibika lapha, kodwa lezi zibonelo zifaka i-Terasakiispira papahanaumokuakeensis, i-marine spirillum bacterium ene-apex enlargement 44, kanye ne-Alphaproteobacteria, i-Teterasaki. , ngaphambili engowohlobo lwe-Oceanospirillum, ukubonisa Okubizwa ngokuthi "ifilimu ye-polar" 45. Amafomu e-Cocci avame ukubonwa emasikweni amadala, ikakhulukazi kumagciwane anamafomu agobile, njenge-Vibrio, i-Campylobacter, ne-Helicobacter 46, 47, 48, okungenzeka imele isimo esibi. Kudingeka omunye umsebenzi ukuze kucaciswe umjikelezo wokuphila onembile wamabhaktheriya e-ASxL5T. Ukunquma ukuthi ithwebula futhi idla kanjani, kanye nokuthi i-genome yayo ifaka ikhodi yini inhlanganisela yezinto eziphilayo ezingasetshenziselwa izinjongo zezokwelapha noma ze-biotechnological.
Incazelo ye-Venatorbacter gen. November Venatorbacter (Ven.a.tor, ba'c.ter, L. yakhiwe ama-venator avela ku-L. n. venator,'hunter' and Gr. n. bacter,'a rod'. Venatorbacter,'a hunting Rod' . Amaseli ane-aerobic, ayakwazi ukumelana nosawoti, amabala asontekile e-Gram, umsebenzi we-Catalase kanye ne-oxidase awukwenzi buthelela emazingeni okushisa angama-4 kuya ku-42 °C Ububanzi be-pH obungu-4-9 buyinto engavamile Eminenke yasolwandle, iningi alikwazi ukubekezelela i-asidi ene-asidi eyinhloko yi-C16:1ω6c kanye/noma i-C16:1ω7c. I-C16:0 kanye ne-C18:1ω9 ; I-3-OH itholakala njenge-hydroxy fatty acids Ayikhuli emithonjeni yomhluzi Okuqukethwe kwe-DNA G + C yi-56.1 mol%. isikhundla salolu hlobo sisemndenini.
Incazelo ye-Venatorbacter cuculus sp. November Venatorbacter cucullus (cu'cull.us.; L. n. cucullus kusho fairing).
Ukwengeza, isici esichazayo salolu hlobo ukuthi uma ekhuliswe ku-BA noma e-BHI, amaseli angu-1.63 µm ubude no-0.37 µm ububanzi. Amakoloni ku-BHI agar mancane kakhulu, afinyelela ku-2 mm ububanzi ngemva kwamahora angu-72. Ziyi-beige, ziguquguqukayo, ziyindilinga, zi-convex futhi ziyacwebezela. Amalungu alolu hlobo angasebenzisa i-Escherichia coli ne-Klebsiella. I-Campylobacter namanye amagciwane amaningana angenayo i-Gram-negative asebenza njengezisulu.
Uhlobo olujwayelekile lwe-ASxL5T lwahlukaniswa nobisi lwenyama yenkomo e-Nottinghamshire, e-UK, futhi lwafakwa ku-National Type Culture Collection (UK): inombolo yokungena i-NCTC 14397 kanye ne-Netherlands Bacterial Culture Collection (NCCB) inombolo yokungena i-NCCB 100775. Ukulandelana okuphelele kwegenome kwe-ASxL5T ifakwe e-Genbank ngokusho kwe-CP046056.
Amagciwane e-ASxL5T ahlukaniswa nobisi lwenkomo esebenzisa ubuchwepheshe bokuhlukanisa i-phage9,49. I-slurry ihlanjululwe ngo-1:9 (w/v) ku-SM buffer (50 mM Tris-HCl [pH 7.5], 0.1 M NaCl, 8 mM MgSO4.7H2O kanye no-0.01% gelatin; Sigma Aldrich, Gillingham, UK), Bese ifukamela ku-4°C amahora angu-24, izungeza kancane ukuze ikhiphe izilwane ezizingelayo ku-buffer. I-suspension yayiyi-centrifuged ku-3000g imizuzu engu-3. I-supernatant yaqoqwa futhi yafakwa phakathi ku-13,000g okwesibili imizuzu emi-5. I-supernatant ibe isidluliswa ngesihlungi se-membrane esingu-0.45 µm (Minisart; Sartorius, Gottingen, Germany) kanye nesihlungi se-membrane esingu-0.2 µm (Minisart) ukuze kukhishwe noma imaphi amaseli ebhaktheriya asele. I-ASxL5T ingadlula lezi zihlungi. I-agar lawn ethambile ye-Campylobacter enterosus S12 (inombolo yokungena ye-NCBI CP040464) evela ku-slurry efanayo yalungiswa kusetshenziswa amasu ajwayelekile. I-slurry ehlungiwe yasatshalaliswa ku-host cell plate ngayinye ngamaconsi angu-10 µl ngokuphindwe kathathu futhi avunyelwe ukuba ome. Ipuleti yafakwa ku-microaerophilic tank ku-37 ° C amahora angu-48 ngaphansi kwezimo ze-microaerobic (5% O2, 5% H2, 10% CO2, kanye ne-80% N2). Uqwembe olubonakalayo olutholiwe lwakhishelwa ku-SM buffer futhi lwadluliselwa otshanini obusha be-C. hyointestinalis S12 ukuze kuqhutshekwe kusabalaliswe izinto eziphila emanzini. Uma sekunqunywa ukuthi amabhaktheriya ayimbangela ye-lytic plaque hhayi i-phage, zama ukukhulisa i-organism ngokuzimela ngaphandle komninikhaya futhi uqhubeke uyichaza. Isiko le-aerobic lenziwe ku-37 °C ngegazi lehhashi elingenalutho elingu-5% v/v (TCS Biosciences Lt, Buckingham, UK, supplement). Ngokweziqondiso ze-National Clinical Standards Committee, indlela yokusabalalisa i-disc isetshenziselwa ukuhlola ukutholakala kwe-antibacterial susceptibility. I-BHI agar yakhuliswa ku-37 °C kusetshenziswa idiski equkethe ama-antibiotic alandelayo (i-Oxoid) yesiko le-aerobic: i-amoxicillin ne-clavulanic acid 30 µg; cefotaxime 30 µg; streptomycin 10 µg; i-ciprofloxacin 5 g; Ceftazidime 30 µg Nalidixic acid 30 µg; Imipenem 10 µg; I-Azithromycin 15 μg; I-Chloramphenicol 30 μg; Cefoxitin 30 µg; Tetracycline 30 µg; I-Nitrofurantoin 300 µg; I-Aztreonam 30 µg; I-Ampicillin 10 µg; I-Cefpodoxime 10 μg; I-Trimethoprim-Sulfamethoxazole 25 µg. Ukubekezelela usawoti kwasungulwa yi-aerobic incubation kumapuleti e-agar e-BHI ku-37 °C. I-NaCl eyengeziwe yengezwe kumapuleti e-agar e-BHI ukuze kuhlinzekwe uhla lokuhlushwa olungafika ku-10% w/v. Ibanga le-pH linqunywa isiko le-aerobic kumapuleti e-agar e-BHI angu-37°C, lapho ububanzi be-pH bushintshwe baba phakathi kuka-4 no-9 nge-HCl eyinyumba noma i-NaOH eyinyumba, futhi inani le-pH eliqondiwe liyaqinisekiswa ngaphambi kokuthelwa kwepuleti . Ukuze kuhlaziywe i-cell fatty acid, i-ASxL5T yenziwe nge-BHI agar izinsuku ezi-3 kanye ne-aerobic ku-37 °C. Ngokwe-MIDI (Sherlock Microbial Identification System, version 6.10) standard protocol ye-FERA Science Ltd, (York, UK), ama-cell fatty acids akhishwa, alungiswa futhi ahlaziywa.
Ku-TEM, i-ASxL5T yenziwe i-aerobics ethuthukisiwe ngokusabalalisa ngokufanayo ku-BA ku-37°C amahora angu-24, bese ivunwa ibe ngu-1 ml we-3% (v/v) i-glutaraldehyde ku-0.1 M cacodylate buffer ekamelweni lokushisa Lungisa ihora elingu-1, bese i-centrifuge. ku-10,000 g imizuzu emi-3. Bese umisa kabusha i-pellet ngobumnene ku-600 μl 0.1 M ye-cacodylate buffer. Dlulisela ukumiswa kwe-ASxL5T okungaguquki kufilimu ye-Formvar/carbon kugridi yethusi enemeshi engu-200. Amagciwane ayegcotshwe nge-0.5% (w/v) i-uranyl acetate iminithi elingu-1 futhi ahlolwa yi-TEM kusetshenziswa isibonakhulu se-TEI Tecnai G2 12 Biotwin. Njengoba kushiwo ngenhla, hlanganisa inani elifanayo lesilwane esizingelayo kanye nesilwane esizingelayo ku-NZCYM broth (BD Difco™, Fisher Scientific UK Ltd, Loughborough) futhi ufukamele amahora angu-48 ngaphansi kwezimo ezincane ze-Campylobacter noma i-Campylobacter ku-37°C, Ukusebenzisana kwesilwane esizingelayo kanye nenyamazane. yabuye yahlolwa yi-TEM. Izimo ze-Aerobic ze-Escherichia coli. Hlola ngokuzimela inyamazane kanye namagciwane adlayo ukuze unqume noma yiziphi izinguquko ku-cell morphology ngenxa yokudliwa. Indlela emnyama yaseSudan yasetshenziselwa i-optical microscopy yokunqwabelana kwe-PHB.
Khulisa amasiko e-ASxL5T ngobusuku obubodwa ngokugcoba ukukhula kumapuleti e-BHI noma e-BA ngeswabhu eyinyumba. Qoqa amaseli e-ASxL5T futhi uwamise ku-MRD (CM0733, Oxoid), bese uwabeka ku-4°C izinsuku ezingu-7 ukuze ulambe amaseli. Ireferensi ye-NCTC noma isiko sebhaktheriya sesitoko selabhorethri yafakwa ku-BHI umhluzi noma umhluzi we-nutrient No. Amasiko: Bacillus subtilis NCTC 3610T, Citrobacter freundii NCTC 9750T, Enterobacter aerogenes NCTC 10006T, Enterococcus faecalis NCTC 775T, Escherichia coli NCTC 86, Klebsiella 1uTC1co4 oxysto6 10817, Listeria Amagciwane akhethekile NCTC 4885, Bacillus macerans NCTC 6355T, Providencia stuartsii NCTC 10318, Pseudomonas fluorescens SMDL, Rhodococcus submarine hamburger NCTC 1621T, TC7 Salmonellagecilateville TC7 Salmonellagecilateville I-NCTC 10861, Staphylococcus aureus NCTC 8532T, Streptococcus pneumoniae NCTC 7465T, Yersinia enterocolitica NCTC 10460. Umsingathi weCampylobacter wayefakwe microaerobically kumapuleti e-BA ku-37°C futhi amiswe i-brownNZ. Abasingathi be-Campylobacter abahloliwe yilena: C. coli 12667 NCTC, C. jejuni 12662, C. jejuni PT14, C. jejuni NCTC 11168T, C. helveticus NCTC 12472, C. lari NCTC 114558, C. PT14, C... Qoqa amaseli ku-MRD, i-centrifuge ku-13,000g bese umisa futhi ku-MRD kuze kube i-OD600 ingu-0.4. Engeza i-aliquot yokumiswa okungu-0.5 ml ku-5 ml encibilikisiwe i-NZCYM top agar (0.6% agar) bese uwuthela epuletini elingaphansi le-NZCYM elingu-1.2%. Ngemva kokwelapha nokomisa, i-ASxL5T ehlanjululwe ngokulandelanisa yasatshalaliswa njengamaconsi angu-20 µl ebhodini lotshani ngalinye ngokuphindwe kathathu. Izinga lokushisa kwesiko kanye nomkhathi kuncike ezidingweni zamabhaktheriya okuhlola.
Sebenzisa i-GenElute™ Bacterial Genomic DNA Kit (Sigma Aldridge) ukuze ulungiselele i-DNA ekuhlukaniseni amagciwane. Kusetshenziswe izindlela ezijwayelekile zokukhulisa i-PCR yofuzo lwe-16S rRNA kanye nokunqunywa kokulandelana komkhiqizo kusetshenziswa ikhemistri yokuqeda udayi (Isevisi Yokufunda I-Eurofins Value, Germany). Sebenzisa uhlelo lwe-BLAST-N ukuze uqhathanise lokhu kulandelana nokunye ukulandelana kofuzo kwe-16S rRNA ukuze uhlonze futhi uqoqe izinhlobo zezinhlobo ezihlobene eduze. Lezi ziqondaniswe kusetshenziswa i-ClustalW kuhlelo lwe-MEGA X. Isihlahla se-phylogenetic sakhiwa kabusha kusetshenziswa i-MEGA X kusetshenziswa indlela enkulu yokuba nokwenzeka ngokusekelwe kumodeli ye-Tamura-Nei, enamakhophi aqondisiwe angu-100054. Sebenzisa i-PureLink™ Genomic DNA Kit (Fisher Scientific, Loughborough, UK) ukuze ukhiphe i-DNA yokulandelana kwegenome lonke. Ukulandelana kofuzo lwe-ASxL5T kwanqunywa kusetshenziswa inhlanganisela ye-Illumina MiSeq, equkethe ukufundwa okuphindwe kabili okungu-250 bp okwakhiwe umtapo wolwazi olungiselelwe kusetshenziswa ikhithi yokulebula ye-Nexdera kanye no-2 kuya ku-20 kb ofundwe isikhathi eside kusukela kuplathifomu ye-PacBio. I-Genomics DNA Sequencing Research Facility eSembia University. I-genome yahlanganiswa kusetshenziswa i-CLC Genomics Workbench 12.0.3 (Qiagen, Aarhus, Denmark). Amasiko e-ASxL5T afakwe Kuqoqo Likazwelonke Lohlobo LweSiko (UK) kanye ne-Netherlands Bacterial Culture Collection (NCCB). Ama-genome ezinto eziphilayo ezihlobene asetshenziselwa ukuqhathanisa yilezi: I-Thalassolituus oleivorans MIL-1T (inombolo yokufinyelela i-HF680312, iphelele); Ama-bacterioplanes sanyensis KCTC 32220T (inombolo yokufinyelela BMYY01000001, ayiphelele); I-Oceanobacter kriegii DSM 6294T (inombolo yokufinyelela NZ_AUGV00000000, ayiphelele); Umphakathi waseMarinamonas DSM 5604T (ungezwe i-ASM436330v1, engaphelele), i-Oceanospirullum linum ATCC 11336T (ingezwe i-MTSD02000001, engaphelele) kanye ne-Thalassolituus sp. I-C2-1 (engeza i-NZ_VNIL01000001, ayiphelele). Sebenzisa i-JGI Genome Portal36 kokuthi https://img.jgi.doe.gov//cgi-bin/mer/main.cgi?section=ANI&page= ukuze unqume amaphuzu wokuqondanisa (AF) kanye ne-nucleic acid identity (ANI). Ngababili. Indlela kaRodriguez-R & Konstantinidis55 yasetshenziselwa ukunquma ubunikazi be-amino acid (AAI). Sebenzisa i-GToTree 1.5.5411,12,13,14,15,16,17,18 ukuze ukhiqize isilinganiso esiphezulu sesihlahla se-phylogenetic esingenzeka. I-genome yokufaka emelela i-genome eyireferensi etholakalayo ikhethwa esizukulwaneni sereferensi esihlonzwe njengesihlobene ne-ASxL5T kusukela ku-16S rRNA phylogeny. Chaza isihlahla kusetshenziswa ithuluzi le-inthanethi lesihlahla sokuphila (https://itol.embl.de/). Isichasiselo esisebenzayo kanye nokuhlaziywa kofuzo lwe-ASxL5T lwenziwa kusetshenziswa ithuluzi le-inthanethi le-BlastKOALA KEGG kusetshenziswa i-KEGG (Kyoto Encyclopedia of Genes and Genomes) ukusatshalaliswa okunothisayo kwemojuli. Ukusatshalaliswa kwezigaba ze-COG (amaqembu e-orthologous) kunqunywa kusetshenziswa ithuluzi le-inthanethi le-eggNOG-mapper.
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