Uhlobo olutsha lwe-Gram-negative, i-aerobic, i-salt-tolerant, esebenzayo, i-rod-shaped, kunye neebhaktheriya ezihlaselayo i-ASxL5T yahlukaniswa kwidama lobulongwe benkomo e-Nottinghamshire, eNgilani, kwaye yasebenzisa i-Campylobacter njengexhoba layo. Emva koko, ezinye iintlobo zeCampylobacter kunye namalungu osapho lwe-Enterobacteriaceae zafunyanwa njengexhoba. Emva kwe-subculture ngaphandle kweeseli ze-host, ukukhula okubuthathaka kwe-aseptic kwafezekiswa kwi-Brain Heart Infusion Agar. Ezona meko zokukhula zingcono ngama-37 °C kunye ne-pH sisi-7. I-electron microscopy yothumelo iveze iimpawu ezingaqhelekanga kakhulu zemorphological ezinxulumene nokufumaneka kwexhoba. Uhlalutyo lwe-Phylogenetic usebenzisa i-16S rRNA i-gene sequence ibonise ukuba i-isolate inxulumene nelungu lentsapho yaseMarine Spirulina, kodwa ayikwazi ukuchazwa ngokucacileyo njengelungu lalo naluphi na uhlobo oluziwayo. Ukulandelelana kwe-genome yonke ye-ASxL5T iqinisekisile ubudlelwane namalungu e-spirochetes yolwandle. Ukhangelo lwesiseko sedatha luveze ukuba ii-ASxL5T ezininzi zabelana nge-16S rRNA ulandelelwano lwemfuza kunye neebhaktheriya ezininzi ezingakhuliswanga ezisuka elwandle, umhlaba kunye namanzi aphantsi komhlaba. Sicebisa ukuba uhlobo lwe-ASxL5T lumele uhlobo olutsha kuhlobo olutsha. Sincoma igama elithi Venatorbacter cucullus gen. NgoNovemba, sp. NgoNovemba, i-ASxL5T isetyenziswe njengohlobo loxinzelelo.
Iibhaktheriya ezihlaselayo ziibhaktheriya ezibonisa amandla okuzingela kunye nokubulala ezinye iibhaktheriya eziphilayo ukuze ufumane izinto ze-biosynthetic kunye namandla. Oku kwahluke ekubuyiselweni ngokubanzi kwezondlo ezivela kwi-microorganisms ezifileyo, kwaye ihluke kwintsebenziswano ye-parasitic, apho iibhaktheriya zenza ubudlelwane obusondeleyo kunye nomkhosi wazo ngaphandle kokubabulala. Iibhaktiriya ezihlaselayo ziye zavela kwimijikelo yobomi eyahlukeneyo ukuze zithathe ithuba lokutya okuninzi kwiindawo ezifumaneka kuzo (ezinje ngeendawo zokuhlala zaselwandle). Baliqela elahlukileyo ngokwerhafu, elidityaniswe kuphela ngumjikelo wabo wobomi obukhethekileyo bokuvala inzala1. Imizekelo yeebhaktheriya ezihlaselayo ifunyenwe kwiiphyla ezininzi ezahlukeneyo, eziquka: Proteobacteria, Bacteroides, kunye neChlorella.3. Nangona kunjalo, ezona bhaktiriya ezizingelayo ezifundwe kakuhle yiBdellovibrio kunye neBdellovibrio-and-like organisms (BALOs4). Iibhaktheriya ezihlaselayo zingumthombo othembisayo weekhompawundi ezintsha zebhayoloji ezisebenzayo kunye nee-antibacterial agents5.
Iibhaktheriya ezihlaselayo kukholelwa ukuba zongeza iyantlukwano yeentsholongwane kwaye zinempembelelo entle kwimpilo ye-ikhosistim, imveliso kunye nozinzo6. Ngaphandle kwezi mpawu zintle, kukho izifundo ezimbalwa kwiibhaktheriya ezintsha ezihlaselayo ngenxa yobunzima bokukhulisa iibhaktheriya kunye nesidingo sokujonga ngononophelo ukusebenzisana kweeseli ukuqonda imijikelo yabo yobomi entsonkothileyo. Olu lwazi alufumaneki lula kuhlalutyo lwekhompyuter.
Ngexesha lokunyuka kwe-antimicrobial resistance, izicwangciso ezintsha zokujolisa kwiintsholongwane zebhaktheriya zifundwa, ezifana nokusetyenziswa kwe-bacteriophages kunye ne-predatory bacteria7,8. Iibhaktheriya ze-ASxL5T zabekwa zodwa ngo-2019 zisebenzisa itekhnoloji yokwahlula i-phage kubulongwe benkomo obuqokelelwe kwiZiko loDairy leYunivesithi yaseNottingham, eNottinghamshire. Injongo yophando kukwahlula izinto eziphilayo ezinokubakho njengee-arhente zolawulo lwebhayoloji. I-Campylobacter hyointestinal yi-pathogen ye-zoonotic, eyandayo ehambelana nezifo zamathumbu omntu10. Ifumaneka kuyo yonke indawo kwiserum kwaye isetyenziswa njengomamkeli ekujoliswe kuwo.
Ibhaktheriya ye-ASxL5T yayibekwe yodwa kwijeli yenyama yenkomo kuba yabonwa ukuba iipleyiti ezenzileyo kwingca ye-C. hyointestinalis zazifana nezo ziveliswa yi-bacteriophages. Oku kukufunyaniswa okungalindelekanga, kuba inxalenye yenkqubo yokwahlulwa kwephage ibandakanya ukuhluzwa ngesihluzo se-0.2 µm, esiyilelwe ukususa iiseli zebhaktiriya. Ukuhlolwa kweMicroscopic yezinto ezikhutshwe kwi-plaque kubonise ukuba ibhaktheriya encinci ye-gram-negative enentonga egobile ayizange iqokelele i-polyhydroxybutyrate (PHB). Inkcubeko ye-aseptic ezimeleyo kwiiseli zexhoba ibonwa kwindawo etyebileyo eqinileyo (efana ne-brain heart infusion agar (BHI) kunye ne-agar yegazi (BA)), kwaye ukukhula kwayo kubuthathaka. Ifunyenwe emva kwe-subculture kunye nokuphucula i-inoculum enzima. Ikhula kakuhle ngokulinganayo phantsi kwe-microaerobic (7% v/v oxygen) kunye neemeko ze-oxygen emoyeni, kodwa hayi kwi-anaerobic atmosphere. Emva kweeyure ezingama-72, ububanzi bekholoni babuncinci kakhulu, bufikelela kwi-2 mm, kwaye yayiyi-beige, i-translucent, ijikelezileyo, i-convex kunye ne-shiny. Uvavanyo oluqhelekileyo lwe-biochemical luyaphazamiseka kuba i-ASxL5T ayinakukhuliswa ngokuthembekileyo kumajelo eendaba, nto leyo ecebisa ukuba inokuxhomekeka kumjikelo wobomi ontsonkothileyo wokwenziwa kwe-biofilm. Nangona kunjalo, ukumiswa kweplate kubonise ukuba i-ASxL5T i-aerobic, i-positive for oxidase kunye ne-catalase, kwaye inokunyamezela i-5% ye-NaCl. I-ASxL5T iyaxhathisa kwi-10 µg streptomycin, kodwa inovakalelo kuzo zonke ezinye izibulala-ntsholongwane ezivavanyiweyo. Iiseli zebhaktheriya ze-ASxL5T zihlolwe yi-TEM (Umfanekiso 1). Xa ikhulile ngaphandle kweeseli zexhoba kwi-BA, iiseli ze-ASxL5T zincinci zeCampylobacter, ezinobude obuyi-1.63 μm (± 0.4), ububanzi be-0.37 μm (± 0.08), kunye nobude obunye (ukuya kwi-5 μm). I-flagella yesondo. Ngokumalunga ne-1.6% yeeseli zibonakala zinobubanzi obungaphantsi kwe-0.2 μm, eziza kuvumela ukudlula kwisixhobo sokucoca. Ulwandiso lwesakhiwo esingaqhelekanga lwabonwa phezulu kwezinye iiseli, ezifana ne-fairing (Latin cucullus) (jonga iintolo ku-1D, E, G). Oku kubonakala ngathi kufakwe kwi-membrane yangaphandle engaphezulu, enokuthi ibe ngenxa yokunciphisa ngokukhawuleza ubungakanani bemvulophu ye-periplasmic, ngelixa i-membrane yangaphandle ihlala injalo, ibonisa ukubonakala "okukhululekile". Ukuhlakulela i-ASxL5T ngokungabikho kwezondlo (kwi-PBS) ixesha elide kwi-4 ° C kubangele ezininzi (kodwa kungekhona zonke) iiseli ezibonisa i-coccal morphology (Umfanekiso 1C). Xa i-ASxL5T ikhula kunye neCampylobacter jejuni njengexhoba leeyure ze-48, ubungakanani beseli obuqhelekileyo bude kakhulu kwaye buncinci kuneeseli ezikhulile ngaphandle komninimzi (Itheyibhile 1 kunye ne-Figure 1E). Ngokwahlukileyo, xa i-ASxL5T ikhula kunye ne-E. coli njengexhoba leeyure ze-48, ubukhulu beseli obuqhelekileyo bude kwaye bubanzi kunokuba bukhula ngaphandle kwexhoba (Itheyibhile 1), kunye nobude beseli buguquguqukayo, ngokuqhelekileyo bubonisa i-filamentous (Figure 1F). Xa ifakwe kwi-Campylobacter jejuni okanye i-E. coli njengexhoba leeyure ze-48, iiseli ze-ASxL5T zabonisa ukuba akukho flagella kwaphela. Itheyibhile 1 ishwankathela ukuqwalaselwa kweenguqu kwisayizi yeseli ngokusekelwe kubukho, ukungabikho, kunye nohlobo lwexhoba le-ASxL5T.
Umboniso we-TEM we-ASx5LT: (A) I-ASx5LT ibonisa i-whip ende; (B) ibhetri ye-ASx5LT eqhelekileyo; (C) iiseli ze-cocci ASx5LT emva kokufukanyelwa ixesha elide ngaphandle kwezondlo; (D) iqela leeseli ze-ASx5LT zibonisa ukungaqhelekanga (E) Iqela le-ASx5LT leseli elifakwe kwi-Campylobacter ixhoba libonise ubude beeseli ezongeziweyo xa kuthelekiswa nalabo abangenalo ukukhula kwexhoba (D) nalo libonise isakhiwo se-apical; (F) I-flagella enkulu ye-Filamentous, iiseli ze-ASx5LT, emva kokufakwa kunye ne-E. coli ixhoba; (G) Iseli enye ye-ASx5LT emva kokufakwa kunye ne-E. coli, ebonisa isakhiwo esiphezulu esingaqhelekanga. Ibha imele i-1 μm.
Ukumisela ulandelelwano lwemfuza ye-16S rRNA (inombolo yofikelelo MT636545.1) yenza ukuba uphendlo lwesiseko sedatha ukuseka ulandelelwano olufana nelo lweklasi yeGammaproteobacteria, kwaye ikufutshane kwiibhaktheriya zaselwandle kusapho lwe-marine spirillum (Umfanekiso 2), kwaye ngamalungu eThalassolituus genus Esona sihlobo sisondeleyo kuMarine Bacillus. Ulandelelwano lwemfuza ye-16S rRNA yahluke ngokucacileyo kwiibhaktheriya ezihlaselayo zosapho lwe-Bdelvibrionaceae (Deltaproteobacteria). Uthelekiso lwesibini lwe-B. bacteriovorus HD100T (uhlobo lwe-strain, i-DSM 50701) kunye ne-B. bacteriovorus DM11A yayingama-48.4% kunye nama-47.7%, kwaye kwi-B. exovorus JSS yayiyi-46.7%. Iibhaktheriya ze-ASxL5T zineekopi ezi-3 ze-16S rRNA gene, ezimbini zazo zifana enye kwenye, kwaye okwesithathu ziziseko ezi-3 ngaphandle. Ezinye izinto ezimbini ezisecaleni kwebhaktiriya (i-ASx5S kunye ne-ASx5O; i-16S rRNA amanani okufikelela kwimfuza yi-MT636546.1 kunye ne-MT636547.1, ngokulandelanayo) kunye neempawu ezifanayo ze-morphological kunye ne-phenotypic ezivela kwindawo enye azifani, kodwa zahlukile kwi-ASxL5T kunye ne-Bacterial uncultured ulandelelwano lwesiseko sedatha zidityanisiwe kunye nezinye iigenera I-Oceanospirillaceae (Umfanekiso 2). Ukulandelelana kwe-genome yonke ye-ASxL5T imiselwe kwaye igcinwe kwi-database ye-NCBI, kunye nenombolo yokungena yi-CP046056. I-genome ye-ASxL5T iqulethe i-chromosome ejikelezayo ye-2,831,152 bp kunye nomlinganiselo we-G + C we-56.1%. Ulandelelwano lwe-genome luqulethe i-2653 CDS (itotali), apho i-2567 iqikelelwa ukuba ifake iiprotheni, apho i-1596 inokwabelwa njengemisebenzi yokubeka (60.2%). I-genome iqulethe i-67 ye-RNA-coding genes, kuquka i-9 rRNAs (i-3 nganye ye-5S, i-16S, kunye ne-23S) kunye ne-57 tRNAs. Iimpawu ze-genomic ze-ASxL5T zifaniswa ne-genomes ekhoyo yeentlobo zohlobo olusondeleyo oluchongiweyo ukusuka kwi-16S rRNA ukulandelelana kofuzo (Itheyibhile 2). Sebenzisa isazisi se-amino acid (AAI) ukuthelekisa zonke iigenome zeThalassolituus ezikhoyo kwi-ASxL5T. Ulandelelwano lwegenome olusondeleyo olukhoyo (olungaphelelanga) olugqitywe yi-AAI yiThalassolituus sp. C2-1 (yongeza NZ_VNIL01000001). Olu xinzelelo lwahlukaniswa kwintlenga yolwandle enzulu yeMariana Trench, kodwa okwangoku akukho lwazi lwephenotypic malunga nolu hlobo lokuthelekisa. Xa kuthelekiswa ne-ASxL5T's 2.82 Mb, i-genome ye-organism inkulu kwi-4.36 Mb. Umyinge wesayizi ye-genome ye-spirochetes yaselwandle imalunga ne-4.16 Mb (± 1.1; n = 92 i-genome yereferensi epheleleyo ephandwe ku-https://www.ncbi.nlm.nih.gov/assembly), ngoko ke i-genome ye-ASxL5T ihambelana umyalelo xa kuthelekiswa namanye amalungu, incinci kakhulu. Sebenzisa i-GToTree 1.5.54 ukuvelisa umthi we-phylogenetic oqikelelweyo osekelwe kwi-genome (Figure 3A), usebenzisa ulandelelwano lwe-amino acid edibeneyo kunye ne-172 yekopi enye ye-Gammaproteobacteria 11,12,13,14,15,16, 17 ,18. Uhlalutyo lubonise ukuba luhambelana ngokusondeleyo ne-Thalassolituus, i-Bacterial Plane, kunye ne-Marine Bacterium. Nangona kunjalo, ezi datha zibonisa ukuba i-ASxL5T ihluke kwizihlobo zayo kwi-Marine Spirulina kunye nedatha yokulandelelana kwe-genome iyafumaneka.
Umthi we-phylogenetic usebenzisa i-16S rRNA i-gene sequence igxininisa indawo ye-ASxL5T, ASxO5, kunye ne-ASxS5 strains (kunye ne-guts) ngokumalunga neentlobo zebhaktheriya ezingalinywanga kunye nolwandle kwi-Marine Spirulinaceae. Inombolo yokungena yeGenbank ilandela igama loxinzelelo kwizibiyeli. Sebenzisa i-ClustalW ukulungelelanisa ulandelelwano, kwaye usebenzise indlela yokunokwenzeka ephezulu kunye nemodeli ye-Tamura-Nei ukuthobela ubudlelwane be-phylogenetic, kwaye wenze ii-replications ezikhokelwayo ze-1000 kwinkqubo ye-MEGA X. Inani elikwisebe libonisa ukuba ixabiso lekopi ekhokelwayo likhulu kuno-50%. I-Escherichia coli U/541T yasetyenziswa njengeqela elingaphandle.
(A) Umthi wephylogenetic osekwe kwigenome, ebonisa ubudlelwane phakathi kwebhaktiriya yaselwandle iSpirospiraceae ASxL5T kunye nezihlobo zayo ezisondeleyo, i-E. coli U 5/41T njengeqela elingaphandle. (B) Xa kuthelekiswa ne-T. oleivorans MIL-1T, ukuhanjiswa kodidi olusebenzayo lweejeni kuqikelelwa ngokusekelwe kwiqela le-orthologous (COG) ye-ASx5LT protein. Umzobo ongasekhohlo ubonisa inani leejini kwicandelo ngalinye elisebenzayo le-COG kwi-genome nganye. Igrafu esekunene ibonisa ipesenti yeegenomes eziqulethwe kwiqela ngalinye elisebenzayo leCOG. (C) Xa kuthelekiswa ne-T. oleiverans MIL-1T, uhlalutyo lwe-KEGG epheleleyo (i-Kyoto Encyclopedia of Genes kunye neGenomes) indlela yemodyuli ye-ASxL5T.
Ukusebenzisa i-database ye-KEGG ukuphonononga izakhi zofuzo ezikhoyo kwi-ASxL5T genome zibonise indlela ye-metabolic ye-aerobic gamma Proteus. I-ASxL5T iqulethe isimbuku se-75 yofuzo eyabelwe iiproteni zeemoto zebhaktiriya, kubandakanywa ufuzo olubandakanyekayo kwi-chemotaxis, i-flagella assembly, kunye nohlobo lwe-IV fimbriae system. Kudidi lokugqibela, i-9 kwi-10 yemfuza inoxanduva lokushukuma kwentshukumo yoluhlu lwezinye izinto eziphilayo. I-genome ye-ASxL5T iqulethe indlela epheleleyo ye-tetrahydropyrimidine biosynthetic ethatha inxaxheba kwimpendulo yokukhusela kwi-osmotic stress20, njengoko kulindelwe kwi-halophiles. I-genome iqulethe iindlela ezininzi ezipheleleyo ze-cofactors kunye neevithamini, kuquka i-riboflavin synthesis pathways. Nangona i-alkane 1-monooxygenase (alkB2) gene ikhona kwi-ASxL5T, indlela yokusetyenziswa kwe-hydrocarbon ayiphelelanga. Kulandelelwano lwe-genome ye-ASxL5T, ii-homologues zofuzo ezichongiweyo njengeyona nto inoxanduva lokuthotywa kwee-hydrocarbons kwi-T. oleiverans MIL-1T21, njenge-TOL_2658 (alkB) kunye ne-TOL_2772 (i-alcohol dehydrogenase) azikho ngokucacileyo. Umzobo we-3B ubonisa ukuthelekiswa kokusasazwa kofuzo kwicandelo le-COG phakathi kwe-ASxL5T kunye ne-Olive oil MIL-1T. Ngokubanzi, igenome encinci ye-ASxL5T iqulathe ngokwemilinganiselo yemfuza embalwa kudidi ngalunye lweCOG xa kuthelekiswa negenome enkulu enxulumeneyo. Xa inani leemfuza kwicandelo ngalinye elisebenzayo lichazwa njengepesenti ye-genome, ukungafani kuphawulwe kwipesenti yezakhi zofuzo kuguqulelo, isakhiwo se-ribosomal kunye neentlobo ze-biogenesis, kunye nemveliso yamandla kunye neendidi zomsebenzi wokuguqulwa, ezenza i-ASxL5T enkulu. genome Ipesenti ithelekiswa neqela elifanayo elikhoyo kwi-T. oleiverans MIL-1T genome. Ngokwahlukileyo, xa kuthelekiswa ne-ASxL5T genome, i-T. oleivorans i-MIL-1T inepesenti ephezulu yemfuza kwi-replication, ukudibanisa kunye nokulungiswa, kunye neendidi zokukhutshelwa. Kuyathakazelisa ukuba umahluko omkhulu kumxholo wecandelo ngalinye elisebenzayo lee-genomes ezimbini linani leejeni ezingaziwayo ezikhoyo kwi-ASxL5T (Umfanekiso 3B). Uhlalutyo lokutyebisa kwiimodyuli ze-KEGG lwenziwa, apho imodyuli nganye ye-KEGG imele iseti yeeyunithi ezichazwe ngesandla ezisebenzayo zochazo kunye nokutolika kwebhayoloji yedatha yokulandelelana kwegenome. Ukuthelekiswa kokusasazwa kofuzo kwindlela epheleleyo yemodyuli ye-KOG ye-ASxL5T kunye nomnquma i-MIL-1T iboniswe kuMfanekiso 3C. Olu hlalutyo lubonisa ukuba nangona i-ASxL5T ine-sulfure epheleleyo kunye ne-nitrogen indlela ye-metabolic, i-T. oleiverans i-MIL-1T ayinayo. Ngokwahlukileyo, i-T. oleiverans MIL-1T ine-cysteine ​​​​kunye ne-methionine indlela ye-metabolic, kodwa ayiphelelanga kwi-ASxL5T. Ke ngoko, i-ASxL5T inemodyuli yeempawu ze-sulfate assimilation (echazwa njengeseti yeejini ezinokuthi zisetyenziswe njengeziphawuli ze-phenotypic, ezifana nomthamo we-metabolic okanye i-pathogenicity; https://www.genome.jp/kegg/module.html) Kwi-T . oleiverans MIL-1T. Ukuthelekisa umxholo wemfuza we-ASxL5T kunye noluhlu lwezakhi zofuzo ezibonisa indlela yokuphila ekhohlakeleyo ayigqibeki. Nangona i-waaL gene encoding i-ligase ehambelana ne-O antigen polysaccharide kwi-core ikhona kwi-ASxL5T genome (kodwa iqhelekile kwiibhaktheriya ezininzi ze-Gram-negative), i-tryptophan 2,3-dioxygenase (TDO ) I-Gene ingabandakanya i-60 amino. imimandla yeasidi edla ngokufumaneka kwiibhaktheriya ezihlaselayo ezingekhoyo. Azikho ezinye iijini zeempawu ezihlaselayo kwigenome ye-ASxL5T, kubandakanywa nezo enzymes ze-encoding ezibandakanyekayo kwi-isoprenoid biosynthesis kwindlela ye-mevalonate. Qaphela ukuba akukho geni yolawulo yokubhala i-gntR kwiqela lezilwanyana ezivavanyayo, kodwa iijini ezintathu ezifana ne-gntR zinokuchongwa kwi-ASxL5T.
Iimpawu ze-phenotypic ze-ASxL5T zishwankathelwa kwiThebhile 3 kwaye zifaniswa neempawu ze-phenotypic zegenera ezinxulumene ne-23, i-24, i-25, i-26 kunye ne-27 echazwe kwiincwadi. I-Isolates ukusuka ku-T. marinus, T. olevorans, B. sanyensis, kunye ne-Oceanobacter kriegii ziyasebenza, zinyamezela ityuwa, imizimba enentonga ene-oxidase-positive, kodwa ayinazo ezinye iimpawu ze-phenotypic kunye ne-ASxL5T. Umyinge we-pH yolwandle yi-8.1 (https://ocean.si.edu/ocean-life/invertebrates/ocean-acidification#section_77), ebonakala kwi-T. marinus, T. olevorans, B. sanyensis kunye ne-O. ikriegii. I-ASxL5T ilungele uluhlu lwe-pH olukhulu (4-9) oluqhelekileyo lweentlobo ezingezizo zaselwandle. Iimpawu zePhenotypic zeThalassolituus sp. C2-1. Akwaziwa. Uluhlu lweqondo lokushisa lokukhula lwe-ASxL5T lubanzi ngokubanzi kunelo lweentlobo zaselwandle (4-42 ° C), nangona ezinye kodwa kungekhona zonke i-T. marinus i-isolate ziyakwazi ukunyamezela ubushushu. Ukungakwazi ukukhula kwe-ASxL5T kwimidiya yomhluzi kuthintele ukubonakaliswa okungaphezulu kwe-phenotypic. Sebenzisa i-API 20E ukuvavanya izinto ezikhutshiweyo kwi-BA plate, i-ONPG, i-arginine dihydrolase, i-lysine decarboxylase, i-ornithine decarboxylase, ukusetyenziswa kwe-citrate, urease, i-tryptophan deaminase, i-gelatin hydrolysis Enzyme, iziphumo zovavanyo zonke bezingalunganga, kodwa akukho indole, i-acetoin kunye ne-H2S. zaveliswa. Iicarbohydrates ezingenagwele ziquka: i-glucose, i-mannose, inositol, i-sorbitol, i-rhamnose, i-sucrose, i-melibiose, i-amygdalin kunye ne-arabinose. Xa kuthelekiswa neentlobo zereferensi ezipapashiweyo, iprofayili ye-fatty acid ye-ASxL5T iboniswe kwiThebhile 4. Ii-acids eziphambili ze-cellular fatty zi-C16: 1ω6c kunye / okanye i-C16: 1ω7c, i-C16: 0 kunye ne-C18: 1ω9. I-Hydroxy fatty acids C12: 0 3-OH kunye ne-C10: 0 3-OH nazo zikhona. Umlinganiselo we-C16: 0 kwi-ASxL5T uphezulu kunexabiso elixeliweyo lejenari ehambelanayo. Ngokwahlukileyo, xa kuthelekiswa nengxelo ye-T. marinus IMCC1826TT, umlinganiselo we-C18: 1ω7c kunye / okanye i-C18: 1ω6c kwi-ASxL5T iyancitshiswa. oleivorans MIL-1T kunye O. kriegii DSM 6294T, kodwa ayibhaqwa B. sanyensis KCTC 32220T. Ukuthelekisa i-fatty acid profiles ye-ASxL5T kunye ne-ASxLS ibonise ukungafani okufihlakeleyo kwinani le-fatty acids nganye phakathi kweentlobo ezimbini, ezihambelana nokulandelelana kwe-DNA ye-genomic yohlobo olufanayo. Akukho masuntswana e-poly-3-hydroxybutyrate (PHB) afunyenwe kusetyenziswa uvavanyo olumnyama lwaseSudan.
Umsebenzi we-predation we-ASxL5T ibhaktiriya waphononongwa ukumisela uluhlu lwexhoba. Le bhaktheriya inokwenza amacwecwe kwiintlobo zeCampylobacter, ezibandakanya: 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 kunye C. upsaliensis NCTC 11541T. Sebenzisa iinkcubeko ezidweliswe kwicandelo lokumisela uluhlu lomkhosi wendlela yokuvavanya uluhlu olubanzi lwe-Gram-negative kunye ne-Gram-positive bacteria. Iziphumo zibonisa ukuba i-ASxL5T inokusetyenziswa kwakhona kwi-Escherichia coli NCTC 86 kunye neCitrobacter freundii NCTC 9750T. Iiplagi ezenziwe kwi-Klebsiella oxytoca 11466. Ukusebenzisana kwe-TEM kunye ne-E. coli NCTC 86 kuboniswe kwi-Figure 4A-D, kunye nokusebenzisana neCampylobacter jejuni PT14 kunye neCampylobacter suis S12 iboniswe kwi-Figure 4E-H phakathi. Indlela yokuhlasela ibonakala yahlukile phakathi kweentlobo zexhoba elivavanyiweyo, kunye neseli enye okanye ngaphezulu ye-E. coli edityaniswe kwiseli nganye ye-ASxL5T kwaye ibekwe ecaleni ecaleni kweseli eyandisiweyo phambi kwe-adsorption. Ngokwahlukileyo, i-ASxL5T ibonakala inamathele kwiCampylobacter ngendawo enye yoqhagamshelwano, ngokuqhelekileyo idibanisa ne-apex ye-predator cell kunye kufuphi ne-apex ye-Campylobacter cell (Figure 4H).
I-TEM ebonisa intsebenziswano phakathi kwe-ASx5LT kunye nexhoba: (AD) kunye ne-E. coli ixhoba; (EH) kunye neC. jejuni ixhoba. (A) Iseli ye-ASx5LT eqhelekileyo exhunywe kwi-cell E. coli (EC) enye; (B) I-ASx5LT ene-filamentous edityaniswe kwiseli enye ye-EC; (C) Iseli ye-ASx5LT ye-filamentous edibeneyo kwiiseli ezininzi ze-EC; (D) I-Attachment Smaller ASx5LT iiseli kwi-cell E. coli (EC) enye; (E) iseli enye ye-ASx5LT exhunywe kwi-Campylobacter jejuni (CJ) iseli; (F) I-ASx5LT ihlasela iiseli ze-C. hyointestinalis (CH); (G) ezimbini Iseli enye ye-ASx5LT yahlasela iseli ye-CJ; (H) Umbono osondeleyo we-ASx5LT yokunamathisela indawo, kufuphi ne-apex yeseli ye-CJ (bar 0.2 μm). Ibha imele i-1 μm ku (A–G).
Iibhaktheriya ezihlaselayo ziye zavela ukuze zisebenzise imithombo eninzi yamaxhoba. Ngokucacileyo, zikho ngokubanzi kwiindawo ezininzi ezahlukeneyo. Ngenxa yobukhulu obuncinci bamalungu abantu, kunokwenzeka ukuba uhlukanise iibhaktheriya ze-ASxL5T kwi-slurry usebenzisa indlela yokwahlula i-phage. Ukufaneleka kwe-genomic ye-ASxL5T kumalungu osapho lwe-oceanospirillaceae yebhaktiriya yaselwandle iyamangalisa, nangona i-organism ikwazi ukunyamezela ityuwa kwaye ingakhula kwindawo equkethe i-5% yetyuwa. Uhlalutyo lomgangatho wamanzi we-slurry lubonise ukuba umxholo we-sodium chloride wawungaphantsi kwe-0.1%. Ngoko ke, udaka lukude nendawo engqongileyo yaselwandle-ngokwejografi nangemichiza. Ubukho beendawo ezikwanti ezintathu ezinxulumeneyo kodwa ezahlukeneyo eziphuma kumthombo omnye zibonelela ngobungqina bokuba ezi ziqwenga zichumile kule ndawo ingekhoyo elwandle. Ukongeza, uhlalutyo lwe-microbiome (iifayile zedatha zifumaneka kwi-https://www.ebi.ac.uk/ena/browser/view/PRJEB38990) ibonise ukuba ulandelelwano olufanayo lwe-16S rRNA gene lubekwe kwi-50 ephezulu kakhulu yetaxi yokusebenza (OTU) ) Kwizithuba ezimbalwa zeesampuli zodaka. Iibhaktheriya ezininzi ezingaxutywanga zifunyenwe kwi-database ye-Genbank, ene-16S rRNA i-gene sequences efana ne-ASxL5T ibhaktheriya. Olu luhlu, kunye nokulandelelana kwe-ASxL5T, i-ASxS5, kunye ne-ASxO5, kubonakala ngathi imele i-clades eyahlukileyo eyahlulwe kwi-Thalassolituus kunye ne-Oceanobacter (Umfanekiso 2). Iintlobo ezintathu zebhaktiriya ezingakhuliswanga (GQ921362, GQ921357 kunye ne-GQ921396) zabekwa zodwa kumanzi aqhekeka kubunzulu be-1.3 yeekhilomitha kumgodi wegolide waseMzantsi Afrika ngo-2009, kwaye ezinye ezimbini (DQ256320 kunye ne-DQ337006) zazisuka eMzantsi Afrika emanzini aphantsi komhlaba (also). ngo-2005). Ulandelelwano lwemfuza ye-16S rRNA esondele kakhulu kwi-ASxL5T yinxalenye yolandelelwano lwemfuza ye-16S rRNA efunyenwe kwinkcubeko yokutyebisa yentlenga yesanti efunyenwe kumanxweme asemantla eFransi ngo-2006 (inombolo yofikelelo AM29240828). Olunye ulandelelwano lofuzo olusondeleyo lwe-16S rRNA oluvela kwibhaktiriya engakhuliswanga i-HQ183822.1 yafunyanwa kwitanki yengqokelela evuthiswe kwindawo yokulahla inkunkuma kamasipala e-China. Ngokucacileyo, iibhaktheriya ze-ASxL5T azimelanga kakhulu kuluhlu lwedatha ye-taxonomic, kodwa olu landelelwano lusuka kwiibhaktheriya ezingakhuliswanga kusenokwenzeka ukuba zimele izinto eziphilayo ezifana ne-ASxL5T, ezisasazwa kwihlabathi liphela, ngokwesiqhelo kwiindawo ezinomngeni. Ukusuka kulo lonke uhlalutyo lwe-phylogenetic ye-genome, isalamane esisondeleyo kwi-ASxL5T yi-Thalassolituus sp. C2-1, T. marinus, T. oleivorans. Kwaye O. kriegii 23, 24, 25, 26, 27. I-Thalassolituus lilungu le-marine obligate hydrocarbon fragmentation bacteria (OHCB), exhaphake kakhulu kwiindawo zaselwandle kunye nomhlaba, kwaye ngokuqhelekileyo iba yinto ephambili emva kweziganeko zongcoliseko lwe-hydrocarbon30,31. Iibhaktheriya zaselwandle azikho amalungu eqela le-OHCB, kodwa zihlukanisiwe kwindawo yolwandle.
Idatha ye-Phenotypic ibonisa ukuba i-ASxL5T luhlobo olutsha kunye nelungu le-genus eyayingaziwa ngaphambili kwi-marine spirospiraceae family. Okwangoku akukho mgangatho ucacileyo wokuhlela iintlobo ezisanda kukwahlulwa zibe yi-genus entsha. Iinzame zenziwe ukumisela imida yegenera yehlabathi jikelele, umzekelo, ngokusekelwe kwipesenti ye-genome yeprotheni yolondolozo (i-POCP), kucetyiswa ukuba ixabiso lokunqunyulwa liyi-50% lifana ne-reference strain33. Abanye bacebisa ukuba kusetyenziswe amaxabiso e-AAI, anenzuzo ngaphezulu kwe-POCP kuba inokufumaneka kwiigenomes34 ezingaphelelanga. Umbhali ukholelwa ukuba ukuba ixabiso le-AAI lingaphantsi kwe-74% xa kuthelekiswa noxinzelelo lwemodeli yeentlobo zemodeli, i-strain imele i-genera eyahlukileyo. Imodeli yohlobo lwe-marine spirillaceae yi-marine spirillum, kunye nohlobo lomzekelo yi-O. linum ATCC 11336T. Ixabiso le-AAI phakathi kwe-ASxL5T kunye ne-O. linum ATCC 11336T yi-54.34%, kunye nexabiso le-AAI phakathi kwe-ASxL5T kunye ne-T. oleivorans MIL-1T (i-genus type strains) yi-67.61%, ebonisa ukuba i-ASxL5T imele i-genus entsha eyahlukileyo kwi-Thalassolituus. Ukusebenzisa i-16S rRNA ulandelelwano lwemfuza njengomgangatho wokuhlelwa, umda wokusikelwa kwe-genus ophakanyisiweyo yi-94.5%35. I-ASxL5T inokubekwa kwi-Thalassolituus genus, ebonisa i-95.03% ye-16S rRNA yolandelelwano yesazisi kunye ne-T. oleivorans MIL-1T kunye ne-96.17%. marinus IMCC1826T. Nangona kunjalo, kuya kufakwa kwakhona kwi-genus ye-Bacteroides ene-94.64% ye-16S ye-rRNA i-gene identity kunye ne-B. sanyensis NV9, ebonisa ukuba ukusetyenziswa kwe-gene eyodwa efana ne-16S rRNA gene kunokukhokelela ekuhleleni ngokungaqhelekanga kunye nesabelo. Enye indlela ephakanyisiweyo isebenzisa i-ANI kunye neGenome Alignment Score (AF) ukuhlola ukuhlanganiswa kwamanqaku edatha kuzo zonke iintlobo kunye nohlobo olungelulo uhlobo lwe-genera ekhoyo. Umbhali uncoma ukudibanisa umda we-genus kunye ne-inflection point ye-genus eqikelelweyo ngqo kwi-taxa ehlalutywayo. Nangona kunjalo, ukuba akukho ulandelelwano olupheleleyo lwe-genome oluvela kwi-Thalassolituus isolates, akunakwenzeka ukumisela ukuba i-ASxL5T yeye-Thalassolituus genus ngale ndlela. Ngenxa yokufumaneka okulinganiselweyo kolandelelwano olupheleleyo lwe-genome ukuhlalutya, yonke i-genome phylogenetic tree kufuneka itolikwe ngononophelo. Okwesibini, iindlela zokuthelekisa i-genome epheleleyo ayinakuphendula umahluko omkhulu kubungakanani beegenome ezithelekisayo. Balinganise ukufana kwe-core conserved core-copy genes phakathi kwegenera ehambelanayo, kodwa abazange bathathele ingqalelo inani elikhulu leemfuza ezingekho kwi-genome encinci ye-ASxL5T. Ngokucacileyo, i-ASxL5T kunye namaqela aquka i-Thalassolituus, i-Oceanobacter, kunye ne-Bacterioplanes inokhokho oqhelekileyo, kodwa ukuziphendukela kwemvelo kuye kwathatha indlela eyahlukileyo, ekhokelela ekunciphiseni i-genome, enokuthi ilungelelanise nendlela yokuphila ekhohlakeleyo. Oku kuchasene ne-T. oleivorans MIL-1T, eyi-28% enkulu kwaye iye yavela phantsi koxinzelelo oluhlukeneyo lokusingqongileyo ukusebenzisa i-hydrocarbons23,30. Uthelekiso olunomdla lunokwenziwa kunye ne-obligate intracellular parasites kunye ne-symbionts, njenge-Rickettsia, i-Chlamydia, kunye ne-Buchnera. Ubungakanani begenome babo bumalunga ne-1 Mb. Ukukwazi ukusebenzisa i-metabolites yeeseli ezibambayo kukhokelela ekulahlekeni kwemfuza, ngenxa yoko Kwenziwa ukuthotywa okubalulekileyo kwe-genomic. Utshintsho lwendalo olusuka kwizinto eziphila elwandle lwezondlo zekhemikhali ukuya kwindlela yokuphila eyingozi lunokubangela ukuncitshiswa okufanayo kobukhulu begenome. Uhlalutyo lwe-COG kunye ne-KEGG lubonisa inani leemfuza ezisetyenziselwa imisebenzi ethile kunye nokungafani kwehlabathi kwiindlela ze-genomic phakathi kwe-ASxL5T kunye ne-T. oleivorans MIL-1T, engekho ngenxa yokufumaneka ngokubanzi kwezinto ze-mobile genetic elements. Umahluko kwi-G + C ye-genome yonke ye-ASxL5T yi-56.1%, kunye ne-T. oleivorans MIL-1T yi-46.6%, ebonisa kwakhona ukuba iyahlula.
Ukuvavanywa komxholo wekhowudi ye-ASxL5T genome inikezela ngengqiqo esebenzayo kwiimpawu ze-phenotypic. Ubukho bohlobo lwe-encoding type IV fimbriae (Tfp) bunomdla kakhulu kuba bakhuthaza ukuhamba kweeseli, okubizwa ngokuba yi-social gliding okanye ukuxhuzula, ngaphandle kwe-flagella phezu komhlaba. Ngokweengxelo, i-Tfp ineminye imisebenzi, kubandakanywa i-predation, i-pathogenesis, ukubunjwa kwe-biofilm, ukuthathwa kwe-DNA yendalo, ukuhlanganiswa kweeseli ngokuzenzekelayo kunye nophuhliso38. I-ASxL5T genome iqulethe ii-genes ze-18 ze-encoding diguanylate cyclase (i-enzyme eyenza ukuguqulwa kwe-2 guanosine triphosphate ibe yi-guanosine 2 phosphate kunye ne-cyclic diGMP) kunye nee-genes ezi-6 ezidibanisa i-diguanylate cyclase phosphate diguanylate. I-gene ye-esterase (i-catalyzing degradation ye-cyclic di-GMP kwi-guanosine monophosphate) inomdla kuba i-cycl-di-GMP sisithunywa sesibini esibalulekileyo esibandakanyekayo ekuphuhlisweni kwe-biofilm kunye nokwahlula, ukunyakaza, ukudibanisa iseli kunye ne-virulence 39, 40 kwinkqubo. Kufuneka kwakhona kuqatshelwe ukuba kwi-Bdellovibrio bacteriovorus, i-GMP ye-cyclic ephindwe kabini ibonakaliswe ukulawula utshintsho phakathi kobomi obukhululekile kunye nendlela yokuphila ekhohlakeleyo41.
Uninzi lophando kwiibhaktheriya ezihlaselayo lujolise kwi-Bdellovibrio, i-Bdellovibrio-like organisms, kunye neentlobo ze-Myxococcus. Le kunye neminye imizekelo eyaziwayo yeebhaktheriya ezihlaselayo zenza iqela elahlukileyo. Nangona le ntlukwano, isethi yeentsapho zeprotheyini ezibonisa iimpawu ze-phenotypes ze-11 iibhaktheriya ezizingelayo ezaziwayo ziye zachongwa3,22. Nangona kunjalo, ii-genes kuphela ezifakela i-O antigen ligase (waaL) zichongiwe, ezixhaphake kakhulu kwiibhaktheriya ze-Gram-negative. Olu hlobo lohlalutyo aluloncedo ekukhetheni i-ASxL5T njengexhoba, mhlawumbi ngenxa yokuba isebenzisa isicwangciso sohlaselo lwenoveli. Ukufumaneka kwee-genomes zebhaktiriya ezahlukahlukeneyo kuya kunceda ukuphuhlisa uhlalutyo lwesisombululo esithathela ingqalelo ubungqina bokungafani okusebenzayo kunye nokusingqongileyo phakathi kwamalungu eqela. Imizekelo yeebhaktheriya ezihlaselayo ezingabandakanyiyo kolu hlalutyo ziquka amalungu e-Cupriavidus necator42 kunye ne-Bradymonabacteria43, kuba njengoko abaphandi baphanda uluntu oluhlukeneyo lwe-microbial, i-taxa engaphezulu ye-predatory isungulwe.
Eyona nto iphawulekayo yebhaktheriya ye-ASxL5T ebanjwe ngumfanekiso we-TEM yi-morphology yayo ekhethekileyo kunye neguquguqukayo, enokukhuthaza ukusebenzisana kunye neebhaktheriya zexhoba. Uhlobo lwentsebenziswano olubonwayo lwahlukile kwezinye iibhaktheriya ezihlaselayo kwaye aluzange lufunyanwe ngaphambili okanye luxelwe. Umjikelo ocetywayo wobomi bobutshaba be-ASxL5T uboniswe kuMfanekiso 5. Kukho imizekelo embalwa kuncwadi olunezakhiwo ezifanayo ze-apical njengoko sichaza apha, kodwa le mizekelo ibandakanya iTerasakiispira papahanaumokuakeensis, ibhaktiriya ye-marine spirillum ene-apex enlargement 44, kunye ne-Alphaproteobacteria, i-Terasaki. , owayekade engowohlobo lwe-Oceanospirillum, ukubonisa Okubizwa ngokuba yi "polar film" 45. Iifom zeCocci zivame ukubonwa kwiinkcubeko ezindala, ngakumbi kwiibhaktheriya ezineefom ezigobileyo, ezifana neVibrio, iCampylobacter, kunye neHelicobacter 46, 47, 48, enokuthi imele imeko ethotyiweyo. Umsebenzi owongezelelweyo uyafuneka ukucacisa umjikelo wobomi obuchanekileyo bebhaktiriya ye-ASxL5T. Ukumisela ukuba ibamba kwaye ibambe njani, kwaye ingaba i-genome yayo ifaka iikhompawundi zebhayoloji ezisebenzayo ezinokuthi zisetyenziselwe iinjongo zonyango okanye ze-biotechnological.
Inkcazelo yeVenatorbacter gen. NgoNovemba iVenatorbacter (iVen.a.tor, ba'c.ter, L. yenziwe ngabavelisi abavela kwi-L. n. venator,'hunter' kunye neGr. n. bacter,'a rod'. Venatorbacter,'a hunting Rod' Iiseli zi-aerobic, ziyakwazi ukunyamezela ityuwa, i-Gram stain negative, umsebenzi we-Catalase kunye ne-oxidase ayenzi ziqokelele Kuluhlu lweqondo lokushisa lwe-4 ukuya kwi-42 °C Uluhlu lwe-pH lwe-4-9 aluqhelekanga kwi-snails yaselwandle, ininzi i-acidic acidic acids yi-C16: 1ω6c. C16: 0 kunye neC18: 1ω9 ; I-3-OH ifunyenwe njenge-hydroxy fatty acids Abakhuli kwimidiya yomhluzi I-DNA G + C i-56.1 mol%. indawo yolu didi kusapho.
Inkcazelo yeVenatorbacter cuculus sp. NgoNovemba iVenatorbacter cucullus (cu'cull.us.; L. n. cucullus ithetha ukwenza okulungileyo).
Ukongeza, into echazayo yolu genus kukuba xa ikhuliswe kwi-BA okanye kwi-BHI, iiseli ziyi-1.63 µm ubude kunye ne-0.37 µm ububanzi. Iikoloni kwi-BHI agar zincinci kakhulu, zifikelela kwi-2 mm ububanzi emva kweeyure ezingama-72. Ziyi-beige, ziguquguqukayo, zingqukuva, ziconvex kwaye ziyabengezela. Amalungu alolu hlobo angasebenzisa i-Escherichia coli kunye ne-Klebsiella. I-Campylobacter kunye nezinye iibhaktheriya ezininzi ze-Gram-negative zisebenza njengexhoba.
Uxinzelelo oluqhelekileyo lwe-ASxL5T lwahlukaniswa nobisi lwenkomo e-Nottinghamshire, e-UK, kwaye lufakwe kwiNgqokelela yeNkcubeko yeSizwe (i-UK): inombolo yokufikelela kwi-NCTC 14397 kunye ne-Netherlands Bacterial Culture Collection (NCCB) inombolo yokufikelela kwi-NCCB 100775. Ukulandelelana kwe-genome epheleleyo ye-ASxL5T ifakwe kwi-Genbank ngokongezwa kwe-CP046056.
Iibhaktheriya ze-ASxL5T zahlukaniswa kubisi lwenkomo zisebenzisa i-phage isolation technology9,49. I-slurry yaxutywa nge-1: 9 (w/v) kwi-SM buffer (50 mM Tris-HCl [pH 7.5], 0.1 M NaCl, 8 mM MgSO4.7H2O kunye ne-0.01% gelatin; Sigma Aldrich, Gillingham, UK), Emva koko ifukamele ku-4°C kangangeeyure ezingama-24, ijikeleza kancinane ukukhupha amarhamncwa kwisithinteli. Ukunqunyanyiswa kwe-centrifuged kwi-3000g imizuzu emi-3. I-supernatant yaqokelelwa kwaye i-centrifuged kwi-13,000g okwesibini imizuzu emi-5. I-supernatant yagqithiswa nge-0.45 µm inwebu yokucoca (Minisart; Sartorius, Gottingen, Germany) kunye ne-0.2 µm inwebu yesihluzo (i-Minisart) ukususa naziphi iiseli zebhaktiriya eziseleyo. I-ASxL5T inokudlula ezi zihluzo. I-agar lawn ethambileyo yeCampylobacter enterosus S12 (inombolo yokufikelela kwi-NCBI CP040464) ukusuka kwi-slurry efanayo yalungiswa ngokusebenzisa iindlela eziqhelekileyo. I-slurry ehluziweyo yasasazwa kwipleyiti nganye ye-host cell ngo-10 µl amathontsi aphindwe kathathu kwaye avunyelwe ukuba ome. I-plate yafakwa kwi-tank ye-microaerophilic kwi-37 ° C kwiiyure ze-48 phantsi kweemeko ze-microaerobic (5% O2, 5% H2, 10% CO2, kunye ne-80% N2). Icwecwe elibonakalayo elifunyenweyo lakhutshelwa kwi-SM buffer laza latshintshelwa kwingca entsha ye-C. hyointestinalis S12 ukusasaza ngakumbi izinto eziphila kwi-lysed. Emva kokuba kunqunywe ukuba ibhaktheriya yimbangela ye-lytic plaque kwaye kungekhona i-phage, zama ukukhulisa i-organism ngokuzimeleyo kwinginginya kwaye uyibonakalise ngakumbi. Inkcubeko ye-aerobic yenziwa kwi-37 ° C kunye ne-5% ye-v / v i-defibrinated yegazi yehashe (TCS Biosciences Lt, Buckingham, UK, supplement). Ngokwezikhokelo zeKomiti yeSizwe yeMigangatho yeKlinikhi, indlela yokusabalalisa i-disc isetyenziselwa uvavanyo lwe-antibacterial susceptibility test. I-BHI agar yakhuliswa kwi-37 °C isebenzisa i-disc equkethe i-antibiotics elandelayo (i-Oxoid) yenkcubeko ye-aerobic: i-amoxicillin kunye ne-clavulanic acid 30 µg; cefotaxime 30 µg; streptomycin 10 µg; ciprofloxacin 5 µg; Ceftazidime 30 µg Nalidixic acid 30 µg; Imipenem 10 µg; Azithromycin 15 µg; I-Chloramphenicol 30 µg; Cefoxitin 30 µg; Tetracycline 30 µg; Nitrofurantoin 300 µg; Aztreonam 30 µg; Ampicillin 10 µg; I-Cefpodoxime 10 µg; Trimethoprim-Sulfamethoxazole 25 µg. Ukunyamezela ityuwa kwasekwa ngokufukanyelwa kwe-aerobic kwi-BHI agar plates kuma-37 °C. I-NaCl eyongezelelweyo yongezwa kwiipleyiti ze-agar ze-BHI ukubonelela ngoluhlu lokugxininiswa ukuya kwi-10% w / v. Uluhlu lwe-pH luchongwa yinkcubeko ye-aerobic kwiipleyiti ze-agar ze-BHI kwi-37 ° C, apho uluhlu lwe-pH luye lwahlengahlengiswa phakathi kwe-4 kunye ne-9 kunye ne-HCl eyinyumba okanye i-NaOH eyinyumba, kwaye ixabiso le-pH elijoliswe kuyo liqinisekisiwe ngaphambi kokugalela ipleyiti. Uhlalutyo lwe-asidi enamafutha eselula, i-ASxL5T yakhuliswa kwi-BHI agar iintsuku ezi-3 kunye ne-aerobic kwi-37 °C. Ngokutsho kwe-MIDI (i-Sherlock Microbial Identification System, i-version 6.10) ye-protocol esemgangathweni ye-FERA Science Ltd, (eYork, e-UK), i-cell fatty acids yakhutshwa, yalungiswa kwaye yahlalutywa.
Kwi-TEM, i-ASxL5T yayikhuliswe i-aerobic ngokusasazwa ngokufanayo kwi-BA kwi-37 ° C kwiiyure ze-24, kwaye emva koko ivunwe kwi-1 ml ye-3% (v / v) i-glutaraldehyde kwi-0.1 M cacodylate buffer kwiqondo lobushushu begumbi Lungisa iyure eyi-1, emva koko i-centrifuge kwi-10,000 g yemizuzu emi-3. Emva koko phinda umise ngobunono ipellet kwi-600 μl 0.1 M cacodylate buffer. Dlulisa ukumiswa kwe-ASxL5T esisigxina kwi-Formvar/carbon film kwigridi yobhedu enemesh engama-200. Iibhaktheriya zihlanjululwe nge-0.5% (w / v) i-uranyl acetate ye-1 iminithi kwaye ihlolwe yi-TEM isebenzisa i-TEI Tecnai G2 12 Biotwin microscope. Njengoko kukhankanyiwe ngasentla, hlanganisa inani elifanayo lexhoba kunye ne-predator kwi-NZCYM broth (BD Difco™, Fisher Scientific UK Ltd, Loughborough) kwaye ufukamele iiyure ezingama-48 phantsi kweemeko ze-microaerobic zeCampylobacter okanye iCampylobacter kwi-37 ° C , Ukusebenzisana kwe-predator kunye nexhoba. yavavanywa kwakhona yi-TEM. Iimeko zeAerobic ze-Escherichia coli. Ngokuzimeleyo vavanya ixhoba kunye neebhaktheriya ezihlaselayo ukufumanisa naluphi na utshintsho kwi-cell morphology ngenxa yokuhlaselwa. Indlela emnyama yaseSudan yayisetyenziselwa i-optical microscopy yokuqokelela kwe-PHB.
Khulisa iinkcubeko ze-ASxL5T ngobusuku ngokuthambisa ukukhula kwiipleyiti ze-BHI okanye ze-BA ngeswabhu enyumba. Ukuqokelela iiseli ze-ASxL5T kwaye uzimise kwi-MRD (CM0733, Oxoid), uze uzibeke kwi-4 ° C iintsuku ze-7 ukuze zilambe iiseli. Ireferensi ye-NCTC okanye i-laboratory stock bacterial culture inoculated kwi-BHI broth okanye No. 2 umhluzi wezondlo (CM007, Oxoid), ifakwe ubusuku bonke, i-centrifuged kwi-13,000g kwaye iphinde imiswe kwi-MRD de kube yi-OD600 ibe yi-0.4. Inkcubeko: I-Bacillus subtilis NCTC 3610T, Citrobacter freundii NCTC 9750T, Enterobacter aerogenes NCTC 10006T, Enterococcus faecalis NCTC 775T, Escherichia coli NCTC 86, Klebsiella 1uTCtoca6 Oxysto6 10817, Listeria Iibhaktheriya ezikhethekileyo NCTC 4885, Bacillus macerans NCTC 6355T, Providencia stuartsii NCTC 10318, Pseudomonas fluorescens SMDL, Rhodococcus submarine hamburger NCTC 1621T, Salmonella NC7 Salmonellagecilateville TC Salmonellateville 5 NCTC 10861, Staphylococcus aureus NCTC 8532T, Streptococcus pneumoniae NCTC 7465T, Yersinia enterocolitica NCTC 10460. Umkhosi weCampylobacter wawufakwe kwi-microaerobically incubated kwi-BA plates kwi-37 ° C kunye ne-brownCYM ye-suspended. Imikhosi evavanyiweyo yeCampylobacter yile: C. coli 12667 NCTC, C. jejuni 12662, C. jejuni PT14, C. jejuni NCTC 11168T, C. helveticus NCTC 12472, C. lari NCTC 114548, C. PT14, C... Qokelela iiseli kwi-MRD, i-centrifuge kwi-13,000g kwaye uphinde umise kwi-MRD kude kube yi-OD600 yi-0.4. Yongeza i-aliquot ye-0.5 ml yokumiswa kwi-5 ml enyibilike i-NZCYM ephezulu ye-agar (i-0.6% ye-agar) kwaye uyithululele kwi-1.2% ye-NZCYM yeplate ephantsi. Emva kokunyanga nokomisa, i-ASxL5T ehlanjululweyo ngokulandelelana yasasazwa njengamathontsi angama-20 µl kwibhodi yengca nganye ngokuphindwe kathathu. Ubushushu benkcubeko kunye nomoya uxhomekeke kwiimfuno zebhaktheriya yovavanyo.
Sebenzisa i-GenElute ™ i-Bacterial Genomic DNA Kit (i-Sigma Aldridge) ukulungisa i-DNA kwiindawo ezizimeleyo zebhaktiriya. Iindlela ezisemgangathweni zisetyenziselwe ukukhulisa i-PCR ye-16S rRNA gene kunye nokuzimisela kokulandelelana kwemveliso usebenzisa i-chemistry yokuphelisa idayi (i-Eurofins Value Read Service, eJamani). Sebenzisa inkqubo ye-BLAST-N ukuthelekisa olu landelelwano kunye nezinye i-16S rRNA ulandelelwano lwemfuza ukuchonga nokuqokelela iintlobo ezinxulumene ngokusondeleyo. Ezi zilungelelaniswa kusetyenziswa i-ClustalW kwinkqubo ye-MEGA X. Umthi we-phylogenetic waphinda wakhiwa kusetyenziswa i-MEGA X usebenzisa eyona ndlela iphezulu yokunokwenzeka ngokusekelwe kumzekelo weTamura-Nei, kunye neekopi ezikhokelwayo ze-100054. Sebenzisa iPureLink™ Genomic DNA Kit (Fisher Scientific, Loughborough, UK) ukukhupha iDNA yolandelelwano lwegenome epheleleyo. Ukulandelelana kwe-genome ye-ASxL5T kunqunywe ngokusebenzisa i-Illumina MiSeq indibaniselwano, equkethe i-250 bp ephindwe kabini efundwayo eyenziwe yilayibrari elungiselelwe usebenzisa i-kit ye-labeling ye-Nexedera kunye ne-2 ukuya kwi-20 kb ende efundwayo kwi-platform ye-PacBio. IGenomics DNA Sequencing Research Facility kwiYunivesithi yaseSembia. I-genome yahlanganiswa kusetyenziswa i-CLC Genomics Workbench 12.0.3 (Qiagen, Aarhus, Denmark). Iinkcubeko ze-ASxL5T zifakwe kwiNgqokelela yeNkcubeko yeSizwe (e-UK) kunye neNgqokelela yeNkcubeko yeBhaktheriya yaseNetherlands (NCCB). Iigenome zezinto ezinxulumeneyo ezisetyenziselwa ukuthelekisa zezi: Thalassolituus oleivorans MIL-1T (inombolo yofikelelo HF680312, epheleleyo); Ii-Bacterioplanes sanyensis KCTC 32220T (inombolo yofikelelo BMYY01000001, ayiphelelanga); I-Oceanobacter kriegii DSM 6294T (inombolo yofikelelo NZ_AUGV00000000, engaphelelanga); Uluntu lwaseMarinamonas i-DSM 5604T (eyongeziweyo i-ASM436330v1, engagqibekanga), i-Oceanospirullum linum ATCC 11336T (eyongeziweyo MTSD02000001, engaphelelanga) kunye neThalassolituus sp. I-C2-1 (yongeza i-NZ_VNIL01000001, ayigqitywanga). Sebenzisa i-JGI Genome Portal36 apha https://img.jgi.doe.gov//cgi-bin/mer/main.cgi?section=ANI&page= ukumisela amanqaku olungelelwaniso (AF) kunye ne-avareji ye-nucleic acid identity (ANI). Ngokwepere. Indlela kaRodriguez-R & Konstantinidis55 yayisetyenziselwa ukumisela i-amino acid identity (AAI). Sebenzisa iGToTree 1.5.5411,12,13,14,15,16,17,18 ukuvelisa umthi oqikelelwayo wokuba nokwenzeka kwephylogenetic. I-genome yegalelo emele i-reference genome ekhoyo ikhethiwe kwi-reference genera echongiweyo njengenxulumene ne-ASxL5T ukusuka kwi-16S rRNA phylogeny. Uchaze umthi usebenzisa umthi wobomi osebenzisanayo kwi-intanethi (https://itol.embl.de/). Inkcazo esebenzayo kunye nohlalutyo lwe-ASxL5T genome luqhutywa kusetyenziswa isixhobo se-intanethi ye-BlastKOALA KEGG kusetyenziswa i-KEGG (i-Kyoto Encyclopedia of Genes and Genomes) yokusabalalisa imodyuli yokutyebisa. Ukuhanjiswa kweendidi ze-COG (amaqela e-orthologous) kunqunywe ngokusebenzisa i-eggNOG-mapper isixhobo se-intanethi.
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