Mofuta o mocha oa libaktheria tsa Gram-negative, aerobic, tse mamellang letsoai, tse mafolofolo, tse bōpehileng joaloka molamu, le tse jang libaktheria ASxL5T e ile ea aroloa letamong la bolokoe ba khomo Nottinghamshire, Engelane, 'me ea sebelisa Campylobacter e le phofu ea eona. Ka mor'a moo, mefuta e meng ea Campylobacter le litho tsa lelapa la Enterobacteriaceae li ile tsa fumanoa e le phofu. Kamora "subculture" ntle le lisele tse amohelang batho, kholo e fokolang ea aseptic e fihletsoe ho Brain Heart Infusion Agar. Maemo a nepahetseng a ho hola ke 37 °C le pH ke 7. Maekrosekopu ea elektrone ea phetiso e senotse likarolo tse ling tse sa tloaelehang tsa mofokoloho tse amanang le boteng ba phofu. Tlhahlobo ea Phylogenetic e sebelisang tatellano ea liphatsa tsa lefutso tsa 16S rRNA e bontšitse hore ho itšehla thajana ho amana le setho sa lelapa la Marine Spirulina, empa ho ke ke ha hlalosoa ka ho hlaka e le setho sa mofuta ofe kapa ofe o tsejoang. Tatelano ea genome eohle ea ASxL5T e netefalitse kamano le litho tsa li-spirochete tsa metsing. Patlisiso ea database e senotse hore li-ASxL5T tse 'maloa li arolelana tatellano ea liphatsa tsa lefutso tsa 16S rRNA le libaktheria tse' maloa tse sa hlaoleheng tse tsoang leoatleng, holim'a lefatše le metsing a ka tlas'a lefatše. Re fana ka maikutlo a hore mofuta oa ASxL5T o emetse mofuta o mocha oa mofuta o mocha. Re khothaletsa lebitso Venatorbacter cucullus gen. November, sp. Ka Pulungoana, ASxL5T e sebelisitsoe joalo ka mofuta oa mofuta.
Libaktheria tse hlaselang ke libaktheria tse bonts'ang bokhoni ba ho tsoma le ho bolaea libaktheria tse ling tse phelang ho fumana lisebelisoa le matla a biosynthetic. Sena se fapane le ho hlaphoheloa ka kakaretso ha limatlafatsi ho likokoana-hloko tse shoeleng, hape li fapane le ho sebelisana le likokoana-hloko, moo libaktheria li etsang kamano e haufi le moamoheli oa tsona ntle le ho li bolaea. Likokoana-hloko tse hlaselang li fetohile mekhoa e fapaneng ea bophelo ho nka monyetla ka mehloli e mengata ea lijo libakeng tseo li fumanehang ho tsona (joalo ka libaka tsa leoatle). Ke sehlopha se fapaneng sa taxonomically, se hokahaneng feela ke bophelo ba bona bo ikhethang ba ho thibela likokoana-hloko1. Mehlala ea libaktheria tse jang liphoofolo e 'nile ea fumanoa li-phyla tse ngata tse fapaneng, ho akarelletsa: Proteobacteria, Bacteroides, le Chlorella.3. Leha ho le joalo, likokoana-hloko tse ithutoang hantle ka ho fetisisa ke Bdellovibrio le Bdellovibrio-and-like organisms (BALOs4). Likokoana-hloko tse hlaselang ke mohloli o tšepisang oa metsoako e mecha ea likokoana-hloko le li-antibacterial agents5.
Ho lumeloa hore libaktheria tse jang liphoofolo li matlafatsa mefuta-futa ea likokoana-hloko 'me li na le phello e ntle ho bophelo bo botle ba tikoloho, tlhahiso le botsitso6. Leha ho na le litšoaneleho tsena tse ntle, ho na le lithuto tse 'maloa mabapi le libaktheria tse ncha tse jang liphoofolo ka lebaka la bothata ba ho hlaolela libaktheria le tlhokeho ea ho shebella ka hloko litšebelisano tsa lisele ho utloisisa potoloho ea bona e rarahaneng ea bophelo. Litaba tsena ha li bonolo ho li fumana ho tsoa tlhahlobong ea komporo.
Nakong ea ho eketseha ha li-antimicrobial resistance, ho ntse ho ithutoa mekhoa e mecha ea ho lebisa tlhokomelo ho likokoana-hloko tsa likokoana-hloko, tse kang tšebeliso ea bacteriophages le likokoana-hloko tse hlaselang7,8. Libaktheria tsa ASxL5T li ile tsa aroloa ka selemo sa 2019 ho sebelisoa theknoloji ea ho itšehla thajana ho tloha bolokoeng ba khomo bo bokeletsoeng Setsing sa Dairy sa Univesithi ea Nottingham, Nottinghamshire. Sepheo sa lipatlisiso ke ho arola lintho tse phelang tse nang le bokhoni ba ho laola likokoana-hloko. Campylobacter hyointestinal ke kokoana-hloko ea zoonotic, e ntseng e tsoela pele ho amahanngoa le mafu a mala a motho10. E fumaneha hohle ka har'a serum mme e sebelisoa joalo ka moamoheli.
Baktheria ea ASxL5T e ne e arohane le jelly ea nama ea khomo hobane ho ile ha hlokomeloa hore li-plaque tseo li li entseng mohloeng oa C. hyointestinal li ne li tšoana le tse hlahisoang ke bacteriophages. Ena ke phumano e sa lebelloang, hobane karolo ea ts'ebetso ea ho itšehla thajana e kenyelletsa ho sefa ka filthara ea 0.2 µm, e etselitsoeng ho tlosa lisele tsa baktheria. Tlhahlobo ea microscopic ea thepa e ntšitsoeng letlapeng e ile ea senola hore libaktheria tse nyenyane tsa gram-negative tse nang le molamu o kobehileng ha lia ka tsa bokella polyhydroxybutyrate (PHB). Tloaelo ea aseptic e ikemetseng ho lisele tsa liphofu e bonoa ka mokhoa o tiileng oa "brain heart infusion agar" (BHI) le blood agar (BA)), mme kholo ea eona e fokola. E fumanoa ka mor'a subculture ka ho ntlafatsa inoculum e boima. E hola ka ho lekana ka tlasa microaerobic (7% v/v oxygen) le maemo a atmosfere ya oksijene, empa eseng sebakeng sa anaerobic. Ka mor'a lihora tse 72, bophara ba kolone bo ne bo le nyane haholo, bo fihla ho 2 mm, 'me e ne e le beige, e feto-fetoha, e pota-potile, e khanyang ebile e benya. Teko e tloaelehileng ea lik'hemik'hale ea lintho tse phelang e ea sitisoa hobane ASxL5T e ke ke ea hlahisoa ka mokhoa o ts'epahalang mecheng ea litaba ea metsi, e fanang ka maikutlo a hore e ka itšetleha ka potoloho e rarahaneng ea bophelo ea sebopeho sa biofilm. Leha ho le joalo, ho emisoa ha poleiti ho bontšitse hore ASxL5T ke aerobic, e ntle bakeng sa oxidase le catalase, 'me e ka mamella 5% NaCl. ASxL5T e hanana le 10 µg streptomycin, empa e utloa lithibela-mafu tse ling kaofela tse lekiloeng. Lisele tsa baktheria tsa ASxL5T li ile tsa hlahlojoa ke TEM (Setšoantšo sa 1). Ha e hōlile ntle le lisele tsa liphofu ho BA, lisele tsa ASxL5T li nyenyane Campylobacter, ka bolelele ba bolelele ba 1.63 μm (± 0.4), bophara ba 0.37 μm (± 0.08), le palo e le 'ngoe e telele (ho fihlela ho 5 μm). Flagella ea thobalano. Hoo e ka bang 1.6% ea lisele li bonahala li e-na le bophara bo ka tlaase ho 0.2 μm, e leng se tla lumella ho feta ka sesebelisoa sa sefe. Katoloso e sa tloaelehang ea sebopeho e ile ea bonoa kaholimo ho lisele tse ling, tse tšoanang le "Cucullus" ea Selatine (sheba metsu ho 1D, E, G). Sena se bonahala se entsoe ka lera le ka ntle le feteletseng, le ka 'nang la bakoa ke ho fokotseha ka potlako ha boholo ba enfelopo ea periplasmic, ha lesela le ka ntle le ntse le le teng, le bontšang "ponahalo e hlephileng". Ho hlaolela ASxL5T ha ho se na limatlafatsi (ho PBS) ka nako e telele ho 4 ° C ho ile ha fella ka lisele tse ngata (empa eseng kaofela) tse bontšang coccal morphology (Setšoantšo sa 1C). Ha ASxL5T e ntse e hola ka Campylobacter jejuni e le phofu ea lihora tse 48, boholo ba sele bo bolelele haholo ebile bo patisane ho feta lisele tse hōlileng ntle le moeti (Letlapa la 1 le Setšoantšo sa 1E). Ka lehlakoreng le leng, ha ASxL5T e ntse e hōla le E. coli e le phofu ea lihora tsa 48, karolelano ea boholo ba sele e telele le e pharaletseng ho feta ha e ntse e hōla ntle le phofu (Letlapa la 1), 'me bolelele ba sele bo fetoha, hangata bo bontša filamentous (Figure 1F). Ha e kenngoa ka Campylobacter jejuni kapa E. coli e le phofu ea lihora tse 48, lisele tsa ASxL5T li ne li sa bontše flagella ho hang. Letlapa la 1 le akaretsa litebello tsa liphetoho tsa boholo ba sele ho latela boteng, ho ba sieo, le mofuta oa phofu oa ASxL5T.
TEM pontšo ea ASx5LT: (A) ASx5LT e bontša sephali se selelele; (B) betri e tloaelehileng ea ASx5LT; (C) lisele tsa cocci ASx5LT ka mor'a ho kenngoa ha nako e telele ntle le limatlafatsi; (D) sehlopha sa lisele tsa ASx5LT se bonts'a ho se tloaelehe (E) ASx5LT sehlopha sa lisele se kenngoeng ka phofu ea Campylobacter se bontšitse ho eketseha ha selelele ha se bapisoa le ba se nang liphofu (D) le tsona li bontšitse sebopeho sa apical; (F) Lisele tse kholo tsa Filamentous flagella, ASx5LT lisele, ka mor'a ho kenngoa le phofu ea E. coli; (G) Sele e le 'ngoe ea ASx5LT ka mor'a ho kenngoa le E. coli, e bontšang mohaho o sa tloaelehang oa holimo. Thepa e emela 1 μm.
Ho fumana tatellano ea liphatsa tsa lefutso tsa 16S rRNA (nomoro ea tumello MT636545.1) e nolofalletsa lipatlisiso tsa database ho theha tatellano e ts'oanang le ea sehlopha sa Gammaproteobacteria, 'me e haufi haholo le libaktheria tsa leoatleng tsa lelapa la marine spirillum (Setšoantšo sa 2), hape ke litho tsa mofuta oa Thalassolituus. Leloko le haufi le Marine Bacillus. Tatelano ea liphatsa tsa lefutso tsa 16S rRNA e fapane ka ho hlaka le libaktheria tse jang liphoofolo tsa lelapa la Bdelvibrionaceae (Deltaproteobacteria). Lipapiso tse peli tsa B. bacteriovorus HD100T (mofuta oa mofuta, DSM 50701) le B. bacteriovorus DM11A e ne e le 48.4% le 47.7%, 'me bakeng sa B. exovorus JSS e ne e le 46.7%. Libaktheria tsa ASxL5T li na le likopi tse 3 tsa mofuta oa 16S rRNA, tse peli tsa tsona li tšoana, 'me ea boraro ke metheo e 3 e arohaneng. Likarolo tse ling tse peli tsa libaktheria tse hlaselang (ASx5S le ASx5O; 16S rRNA linomoro tsa ho kena liphatseng tsa lefutso ke MT636546.1 le MT636547.1, ka ho latellana) tse nang le litšobotsi tse tšoanang tsa morphological le phenotypic tse tsoang sebakeng se le seng ha li tšoane, empa li fapane le ASxL5T le Bacterial uncultured tatellano ea database e kopantsoe hammoho le mefuta e meng ho Oceanospirillaceae (Setšoantšo sa 2). Tatelano eohle ea genome ea ASxL5T e khethiloe ebile e bolokiloe polokelong ea NCBI, 'me nomoro ea ho kena ke CP046056. Genome ea ASxL5T e na le chromosome e chitja ea 2,831,152 bp e nang le karolelano ea G + C ea 56.1%. Tatelano ea liphatsa tsa lefutso e na le li-CDS tse 2653 (kakaretso), tseo 2567 ho tsona ho boleloang esale pele hore li tla kenyelletsa liprotheine, tseo 1596 ea tsona e ka abeloang e le mesebetsi ea ho beha (60.2%). Genome e na le liphatsa tsa lefutso tse 67 tsa RNA, ho kenyeletsoa li-rRNA tse 9 (3 e 'ngoe le e 'ngoe bakeng sa 5S, 16S, le 23S) le 57 tRNAs. Litšobotsi tsa genomic tsa ASxL5T li ne li bapisoa le liphatsa tsa lefutso tse fumanehang tsa mefuta e haufi ea mofuta o haufi o khethiloeng ho tsoa ho 16S rRNA gene sequence (Letlapa la 2). Sebelisa boitsebiso ba amino acid (AAI) ho bapisa liphatsa tsa lefutso tsa Thalassolituus le ASxL5T. Tatelano e haufi ka ho fetisisa e fumanehang (e sa phethahalang) ea genome e khethiloeng ke AAI ke Thalassolituus sp. C2-1 (eketsa NZ_VNIL01000001). Mofuta ona o ne o qheletsoe ka thoko ho sediments tse tebileng tsa leoatle tsa Mariana Trench, empa hajoale ha ho na tlhahisoleseling ea phenotypic mabapi le mofuta ona oa papiso. Ha ho bapisoa le ASxL5T's 2.82 Mb, genome ea ntho e phelang e kholoanyane ho 4.36 Mb. Karolelano ea boholo ba genome ea marine spirochetes e ka ba 4.16 Mb (± 1.1; n = 92 liphatsa tsa lefutso tse felletseng tse batlisisitsoeng ho tsoa ho https://www.ncbi.nlm.nih.gov/assembly), kahoo genome ea ASxL5T e tsamaisana le taelo Ha e bapisoa le litho tse ling, e nyane haholo. Sebelisa GToTree 1.5.54 ho hlahisa sefate sa phylogenetic se hakantsoeng ka genome (setšoantšo sa 3A), u sebelisa tatellano ea amino acid ea 172 ea kopi e le 'ngoe e khethehileng ho Gammaproteobacteria 11,12,13,14,15,16, 17 ,18. Tlhahlobo e bontšitse hore e amana haufi-ufi le Thalassolituus, Bacterial Plane, le Marine Bacterium. Leha ho le joalo, lintlha tsena li bontša hore ASxL5T e fapane le beng ka eona ba Marine Spirulina mme data ea eona ea tatellano ea genome e fumaneha.
Sefate sa phylogenetic se sebelisang tatellano ea liphatsa tsa lefutso tsa 16S rRNA se totobatsa boemo ba mefuta ea ASxL5T, ASxO5, le ASxS5 (ka li-guts) tse amanang le likokoana-hloko tse sa lengoang le tsa marine ho Marine Spirulinaceae. Nomoro ea ho kena ea Genbank e latela lebitso la khatello ka masakaneng. Sebelisa ClustalW ho hokahanya tatellano, 'me u sebelise mokhoa o phahameng oa monyetla le mohlala oa Tamura-Nei ho fokotsa likamano tsa phylogenetic, le ho etsa likatiso tse tataisoang tse 1000 lenaneong la MEGA X. Nomoro e lekaleng e bontša hore boleng ba kopi e tataisoang bo feta 50%. Escherichia coli U/541T e ne e sebelisoa e le sehlopha se ka ntle.
(A) Sefate sa phylogenetic se thehiloeng ho genome, se bontšang kamano pakeng tsa baktheria ea marine Spirospiraceae ASxL5T le beng ka eona ba haufi, E. coli U 5/41T e le sehlopha se ka thōko. (B) Ha ho bapisoa le T. oleivorans MIL-1T, karolo e sebetsang ea kabo ea liphatsa tsa lefutso e boleloa esale pele ho latela sehlopha sa orthologous (COG) sa protheine ea ASx5LT. Setšoantšo se ka letsohong le letšehali se bontša palo ea liphatsa tsa lefutso sehlopheng ka seng se sebetsang sa COG ho genome ka 'ngoe. Kerafo e ka ho le letona e bontša peresente ea liphatsa tsa lefutso tse teng sehlopheng ka seng se sebetsang sa COG. (C) Ha ho bapisoa le T. oleiverans MIL-1T, tlhahlobo ea KEGG e feletseng (Kyoto Encyclopedia of Genes and Genomes) tsela ea modular ea ASxL5T.
Ho sebelisa database ea KEGG ho hlahloba likarolo tsa liphatsa tsa lefutso tse teng ho ASxL5T genome ho senola tsela e tloaelehileng ea metabolic ea aerobic gamma Proteus. ASxL5T e na le kakaretso ea liphatsa tsa lefutso tse 75 tse abetsoeng liprotheine tsa makoloi a baktheria, ho kenyelletsa le liphatsa tsa lefutso tse amehang ho chemotaxis, kopano ea flagella, le mofuta oa IV fimbriae system. Mokhahlelong oa ho qetela, liphatsa tsa lefutso tse 9 ho tse 10 li ikarabella bakeng sa ho sisinyeha ha mefuta e meng ea lintho tse phelang. Genome ea ASxL5T e na le tsela e feletseng ea tetrahydropyrimidine biosynthetic e kenyang letsoho karabong ea tšireletso ho osmotic stress20, joalokaha ho lebeletsoe bakeng sa li-halophile. Genome e boetse e na le litsela tse ngata tse felletseng tsa li-cofactors le livithamini, ho kenyeletsoa litsela tsa motsoako oa riboflavin. Leha mofuta oa alkane 1-monooxygenase (alkB2) o le teng ho ASxL5T, tsela ea tšebeliso ea hydrocarbon ha e ea phethahala. Ka tatelano ea genome ea ASxL5T, li-homologue tsa liphatsa tsa lefutso tse khetholloang e le tsona tse ikarabellang haholo bakeng sa ho senyeha ha li-hydrocarbon ho T. oleiverans MIL-1T21, tse kang TOL_2658 (alkB) le TOL_2772 (alcohol dehydrogenase) ho hlakile hore ha li eo. Setšoantšo sa 3B se bonts'a papiso ea kabo ea liphatsa tsa lefutso sehlopheng sa COG lipakeng tsa ASxL5T le oli ea Olive MIL-1T. Ka kakaretso, genome e nyane ea ASxL5T e na le liphatsa tsa lefutso tse fokolang ho tsoa sehlopheng ka seng sa COG ha ho bapisoa le genome e kholo e amanang. Ha palo ea liphatsa tsa lefutso karolong e 'ngoe le e' ngoe ea ts'ebetso e hlahisoa e le peresente ea genome, phapang e hlokomeloa peresente ea liphatsa tsa lefutso phetolelong, sebopeho sa ribosomal le likarolo tsa biogenesis, le likarolo tsa ts'ebetso ea tlhahiso ea matla le phetoho, e leng karolo e kholoanyane ea ASxL5T. genome Peresente e bapisoa le sehlopha se tšoanang se teng ho genome ea T. oleiverans MIL-1T. Ka lehlakoreng le leng, ha ho bapisoa le genome ea ASxL5T, T. oleivorans MIL-1T e na le liphesente tse phahameng tsa liphatsa tsa lefutso likarolong tsa ho pheta-pheta, ho kopanya le ho lokisa, le ho ngola. Hoa thahasellisa hore phapang e kholo ka ho fetisisa litabeng tsa sehlopha ka seng se sebetsang sa liphatsa tsa lefutso tse peli ke palo ea liphatsa tsa lefutso tse sa tsejoeng tse teng ho ASxL5T (Setšoantšo sa 3B). Ho ile ha etsoa tlhahlobo ea ntlafatso ea li-module tsa KEGG, moo mojule ka mong oa KEGG o emelang sehlopha sa likarolo tse sebetsang tse hlalositsoeng ka letsoho bakeng sa tlhaloso le tlhaloso ea baeloji ea data ea tatellano ea genome. Papiso ea kabo ea liphatsa tsa lefutso tseleng e felletseng ea module ea KOG ea ASxL5T le mohloaare MIL-1T e bonts'oa ho Setšoantšo sa 3C. Tlhahlobo ena e bontša hore le hoja ASxL5T e na le tsela e feletseng ea sulfure le nitrogen metabolic, T. oleiverans MIL-1T ha e na eona. Ka lehlakoreng le leng, T. oleiverans MIL-1T e na le tsela e felletseng ea cysteine ​​​​le methionine metabolic, empa ha e ea fella ho ASxL5T. Ka hona, ASxL5T e na le mojule oa sebopeho sa sulfate assimilation (e hlalosoang e le sehlopha sa liphatsa tsa lefutso tse ka sebelisoang e le matšoao a phenotypic, joalo ka matla a metabolic kapa pathogenicity; https://www.genome.jp/kegg/module.html) Ho T . oleiverans MIL-1T. Ho bapisa liphatsa tsa lefutso tsa ASxL5T le lenane la liphatsa tsa lefutso tse fanang ka maikutlo a hore bophelo ba bosholu ha bo tsejoe. Le hoja liphatsa tsa lefutso tsa waaL tse kenyelletsang ligase tse amanang le O antigen polysaccharide ho ea mantlha li teng ho genome ea ASxL5T (empa e tloaelehile ho libaktheria tse ngata tsa Gram-negative), tryptophan 2,3-dioxygenase (TDO ) Liphatsa tsa lefutso li ka kenyelletsa 60 amino. libaka tse nang le acid hangata li fumanoa ho libaktheria tse jang tse seng teng. Ha ho na liphatsa tse ling tsa liphatsa tsa lefutso ho genome ea ASxL5T, ho kenyeletsoa le li-enzyme tse kenyang letsoho ho isoprenoid biosynthesis tseleng ea mevalonate. Hlokomela hore ha ho na mofuta oa taolo oa transcriptal gntR sehlopheng sa liphoofolo tse jang liphoofolo tse hlahlobiloeng, empa mefuta e meraro ea mofuta oa gntR e ka tsejoa ho ASxL5T.
Litšobotsi tsa phenotypic tsa ASxL5T li akaretsoa ho Lethathamo la 3 mme li bapisoa le litšobotsi tsa phenotypic tsa genera tse amanang le 23, 24, 25, 26, le 27 tse tlalehiloeng libukeng. Li-Isolates tse tsoang ho T. marinus, T. olevorans, B. sanyensis, le Oceanobacter kriegii li sebetsa, li mamella letsoai, li na le 'mele o nang le molamu o nang le oxidase, empa ha li na hoo e ka bang ha li na litšobotsi tse ling tsa phenotypic tse nang le ASxL5T. Karolelano ea pH ea leoatle ke 8.1 (https://ocean.si.edu/ocean-life/invertebrates/ocean-acidification#section_77), e hlahang ho T. marinus, T. olevorans, B. sanyensis le O. kriegii. ASxL5T e loketse mofuta o moholo oa pH (4-9) o tloaelehileng oa mefuta e seng ea metsing. Phenotypic litšobotsi tsa Thalassolituus sp. C2-1. Tse sa tsejoeng. Mocheso oa mocheso oa ho hōla oa ASxL5T ka kakaretso o pharaletse ho feta oa mefuta ea marine (4-42 ° C), le hoja tse ling empa e se li-isolates tsa T. marinus kaofela li mamella mocheso. Ho se khone ho holisa ASxL5T mecheng ea litaba tsa moro ho ile ha thibela sebopeho se eketsehileng sa phenotypic. Sebelisa API 20E ho leka lisebelisoa tse hlakotsoeng ho tloha poleiting ea BA, ONPG, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, ts'ebeliso ea citrate, urease, tryptophan deaminase, gelatin hydrolysis Enzyme, liphetho tsa liteko kaofela li ne li le mpe, empa ha ho indole, acetoin le H2. li ile tsa hlahisoa. Li-carbohydrate tse sa lomosoang li kenyelletsa: tsoekere, mannose, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin le arabinose. Ha ho bapisoa le mefuta e amanang le litšupiso tse hatisitsoeng, profil ea cellular fatty acid ea mofuta oa ASxL5T e bontšoa ho Lethathamo la 4. Mafura a ka sehloohong a mafura a cellular ke C16: 1ω6c le / kapa C16: 1ω7c, C16: 0 le C18: 1ω9. Hydroxy fatty acids C12:0 3-OH le C10:0 3-OH le tsona li teng. Karo-karolelano ea C16:0 ho ASxL5T e phahame ho feta boleng bo tlalehiloeng ba moloko o amanang. Ka lehlakoreng le leng, ha ho bapisoa le tlaleho ea T. marinus IMCC1826TT, karolelano ea C18: 1ω7c le / kapa C18: 1ω6c ho ASxL5T e fokotsehile. oleivorans MIL-1T le O. kriegii DSM 6294T, empa ha e fumanwe ho B. sanyensis KCTC 32220T. Ho bapisa li-fatty acid profiles tsa ASxL5T le ASxLS li senotse phapang e poteletseng ea palo ea mafura a mafura ka bomong pakeng tsa mefuta e 'meli, e lumellanang le tatellano ea genomic DNA ea mefuta e tšoanang. Ha ho na likaroloana tsa poly-3-hydroxybutyrate (PHB) tse fumanoeng ho sebelisoa tlhahlobo e ntšo ea Sudan.
Ts'ebetso ea predation ea libaktheria tsa ASxL5T e ile ea ithutoa ho fumana hore na liphofu li na le mefuta e mengata hakae. Baktheria ena e ka etsa li-plaque mefuteng ea Campylobacter, ho akarelletsa: 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 le C. upsaliensis NCTC 11541T. Sebelisa litloaelo tse thathamisitsoeng karolong ea sepheo sa moamoheli ea mokhoa oa ho lekola mefuta e mengata ea libaktheria tsa Gram-negative le Gram-positive. Liphetho li bontša hore ASxL5T e ka boela ea sebelisoa ho Escherichia coli NCTC 86 le Citrobacter freundii NCTC 9750T. Li-plaque tse entsoeng ka Klebsiella oxytoca 11466. Tšebelisano ea TEM le E. coli NCTC 86 e bontšoa ho Setšoantšo sa 4A-D, 'me ho sebelisana le Campylobacter jejuni PT14 le Campylobacter suis S12 ho bontšoa ho Setšoantšo sa 4E-H bohareng. Mokhoa oa ho hlasela o bonahala o fapane pakeng tsa mefuta ea liphofu tse lekiloeng, e nang le lisele tse le 'ngoe kapa tse ngata tsa E. coli tse khomaretsoeng sele e 'ngoe le e' ngoe ea ASxL5T 'me e behiloe ka thōko ho sele e atolositsoeng pele ho adsorption. Ka lehlakoreng le leng, ASxL5T e bonahala e khomarela Campylobacter ka ntlha e le 'ngoe ea ho kopana, hangata e kopana le tlhōrō ea sele ea liphoofolo tse jang liphoofolo le haufi le tlhōrō ea sele ea Campylobacter (Setšoantšo sa 4H).
TEM e bontšang tšebelisano pakeng tsa ASx5LT le phofu: (AD) le phofu ea E. coli; (EH) le C. jejuni phofu. (A) Sele e tloaelehileng ea ASx5LT e amanang le sele e le 'ngoe ea E. coli (EC); (B) ASx5LT e nang le filamentous e khomaretsoeng sele e le 'ngoe ea EC; (C) A filamentous ASx5LT sele e kopantsoeng le lisele tse ngata tsa EC; (D) Ho khomarela Lisele tse nyenyane tsa ASx5LT ka sele e le 'ngoe ea E. coli (EC); (E) sele e le 'ngoe ea ASx5LT e amanang le sele ea Campylobacter jejuni (CJ); (F) ASx5LT e hlasela lisele tsa C. hyointestinalis (CH); (G) lisele tse peli tsa One ASx5LT li ile tsa hlasela sele ea CJ; (H) Pono e haufi-ufi ea ntlha ea ho khomarela ea ASx5LT, haufi le tlhōrō ea sele ea CJ (bar 0.2 μm). Theko e emetse 1 μm ho (A–G).
Likokoana-hloko tse hlaselang li fetohile ho nka monyetla ka mehloli e mengata ea liphofu. Ho hlakile hore li teng ka bongata libakeng tse ngata tse fapaneng. Ka lebaka la boholo bo moqotetsane ba litho tsa baahi, hoa khoneha ho arola libaktheria tsa ASxL5T ho tloha slurry ho sebelisa mokhoa oa ho arohana ha phage. Tšebelisano ea genomic ea ASxL5T ho litho tsa lelapa la oceanospirillaceae ea libaktheria tsa leoatleng ea makatsa, le hoja sebōpuoa se mamella letsoai 'me se ka hōla ka mokhoa o nang le letsoai la 5%. Tlhahlobo ea boleng ba metsi ea slurry e bontšitse hore sodium chloride e ka tlaase ho 0.1%. Ka hona, seretse se hole le tikoloho ea maoatle—ka libaka le ka lik’hemik’hale. Ho ba teng ha libaka tse ka thōko tse tharo tse amanang empa tse fapaneng tse tsoang mohloling o le mong ho fana ka bopaki ba hore libatana tsena li atleha tikolohong ena eo e seng ea leoatle. Ho feta moo, tlhahlobo ea microbiome (lifaele tsa data tse fumanehang ho https://www.ebi.ac.uk/ena/browser/view/PRJEB38990) li bontšitse hore tatellano e tšoanang ea liphatsa tsa lefutso tsa 16S rRNA e fumaneha ka holimo ho 50 e ngata ka ho fetisisa ea ts'ebetso ea lekhetho (OTU). ) Likarolong tse 'maloa tsa mehlala ea seretse. Ho ile ha fumanoa likokoana-hloko tse 'maloa tse sa etsoang sebakeng sa polokelo ea litaba tsa Genbank, tse nang le liphatsa tsa lefutso tsa 16S rRNA tse tšoanang le libaktheria tsa ASxL5T. Tatelano ena, hammoho le tatelano ea ASxL5T, ASxS5, le ASxO5, e bonahala e emela lihlopha tse fapaneng tse arohaneng le Thalassolituus le Oceanobacter (Setšoantšo sa 2). Mefuta e meraro ea libaktheria tse sa lengoang (GQ921362, GQ921357 le GQ921396) li ne li qheletsoe ka thoko ho metsi a phatlohileng botebong ba lik'hilomithara tse 1.3 morafong oa khauta oa Afrika Boroa ka 2009, 'me tse ling tse peli (DQ256320 le DQ337006) li ne li tsoa Afrika Boroa ka 2005). Tatelano ea liphatsa tsa lefutso ea 16S rRNA e amanang haholo le ASxL5T ke karolo ea tatellano ea liphatsa tsa lefutso tsa 16S rRNA tse fumanoeng ho tsoa moetlong oa ho nonofatsa oa lehlabathe le fumanoeng mabopong a lebopo la Fora ka 2006 (nomoro ea tumello AM29240828). Tatelano e 'ngoe ea liphatsa tsa lefutso tsa 16S rRNA e amanang haufi-ufi ho tsoa ho baktheria e sa lengoang HQ183822.1 e fumanoe tanka ea pokello e nkiloeng thotoeng ea 'masepala ea Chaena. Ho hlakile hore libaktheria tsa ASxL5T ha li emele haholo ho li-database tsa taxonomic, empa tatelano ena e tsoang ho libaktheria tse sa rutehang e kanna ea emela lintho tse phelang tse tšoanang le ASxL5T, tse ajoang lefatšeng ka bophara, hangata libakeng tse thata. Ho tsoa tlhahlobong eohle ea genome phylogenetic, kamano e haufi haholo le ASxL5T ke Thalassolituus sp. C2-1, T. marinus, T. oleivorans. Le O. kriegii 23, 24, 25, 26, 27. Thalassolituus ke setho sa marine obligate hydrocarbon fragmentation bacteria (OHCB), e atileng tikolohong ea leoatle le lefats'e, 'me hangata e ba eona e hlahelletseng kamora liketsahalo tsa tšilafalo ea hydrocarbon30,31. Libaktheria tsa metsing ha se litho tsa sehlopha sa OHCB, empa li arohane le tikoloho ea leoatle.
Lintlha tsa phenotypic li bonts'a hore ASxL5T ke mofuta o mocha ebile ke setho sa mofuta o neng o sa tsejoe pele lelapeng la marine spirospiraceae. Hajoale ha ho na maemo a hlakileng a ho arola mefuta e sa tsoa aroloa hore e be mofuta o mocha. Boiteko bo entsoe ho khetholla meeli ea kakaretso ea kakaretso, mohlala, ho ipapisitsoe le peresente ea genome ea protheine e bolokang bophelo (POCP), ho khothaletsoa hore boleng bo khaotsoeng bo ts'oana ka 50% le mofuta oa litšupiso33. Ba bang ba fana ka maikutlo a ho sebelisa litekanyetso tsa AAI, tse nang le melemo ho feta POCP hobane li ka fumanoa ho tsoa ho genomes34 e sa fellang. Sengoli se lumela hore haeba boleng ba AAI bo ka tlase ho 74% ha bo bapisoa le mofuta oa mofuta oa mofuta oa mofuta, khatello ke moemeli oa mofuta o fapaneng. Mofuta oa mofuta oa marine spirillaceae ke marine spirillum, 'me mofuta oa mofuta ke O. linum ATCC 11336T. Boleng ba AAI pakeng tsa ASxL5T le O. linum ATCC 11336T ke 54.34%, 'me boleng ba AAI pakeng tsa ASxL5T le T. oleivorans MIL-1T (mofuta oa mofuta oa genus) ke 67.61%, e leng se bontšang hore ASxL5T e emela mofuta o mocha o fapaneng le Thalassolitu. Ka ho sebelisa tatellano ea liphatsa tsa lefutso tsa 16S rRNA e le maemo a ho arola, moeli oa moeli oa mefuta ke 94.5%35. ASxL5T e kanna ea beoa mofuteng oa Thalassolituus, e bonts'a 95.03% 16S rRNA boitsebiso ba tatelano le T. oleivorans MIL-1T le 96.17%. Marinus IMCC1826T. Leha ho le joalo, e tla boela e behoe lelokong la Bacteroides le nang le 94.64% 16S rRNA gene identity le B. sanyensis NV9, e leng se bontšang hore tšebeliso ea gene e le 'ngoe e kang ea 16S rRNA gene e ka lebisa ho khethollo le kabelo e sa reroang. Mokhoa o mong o khothalelitsoeng o sebelisa ANI le Genome Alignment Score (AF) ho hlahloba ho bokellana ha lintlha tsa data ho tsoa mefuteng eohle le mefuta e seng ea mofuta oa mofuta o teng. Mongodi o kgothalletsa ho kopanya moedi wa leloko le ntlha ya ho fetofetoha ha leloko le hakanyetswang le tobane le lekgetho le hlahlojwang. Leha ho le joalo, haeba ho se na tatellano e felletseng ea liphatsa tsa lefutso tse tsoang ho Thalassolituus isolate, ha ho khonehe ho tseba hore na ASxL5T ke ea mofuta oa Thalassolituus ka mokhoa ona. Ka lebaka la ho fumaneha ho fokolang ha tatellano e feletseng ea genome bakeng sa tlhahlobo, sefate sohle sa genome phylogenetic se lokela ho hlalosoa ka hloko. Taba ea bobeli, mekhoa ea ho bapisa liphatsa tsa lefutso kaofela e ke ke ea ikarabella bakeng sa liphapang tse kholo ka boholo ba liphatsa tsa lefutso tse bapisoang. Ba ile ba lekanya ho tšoana ha liphatsa tsa lefutso tse bolokiloeng tsa kopi e le 'ngoe pakeng tsa lihlopha tse amanang, empa ha lia ka tsa ela hloko palo e kholo ea liphatsa tsa lefutso tse sieo ho genome e nyenyane haholo ea ASxL5T. Ho hlakile hore ASxL5T le lihlopha tse kenyeletsang Thalassolituus, Oceanobacter, le Bacterioplanes li na le moholo-holo a le mong, empa ho iphetola ha lintho ho nkile tsela e fapaneng, e lebisang ho fokotseha ha genome, e ka 'nang ea e-ba ho ikamahanya le mokhoa oa bophelo oa ho tsoma. Sena se fapane le T. oleivorans MIL-1T, e kholoanyane ka 28% mme e fetohile tlas'a likhatello tse fapaneng tsa tikoloho ho sebelisa hydrocarbons23,30. Papiso e thahasellisang e ka etsoa ka likokoana-hloko tse tlamang le li-symbionts, tse kang Rickettsia, Chlamydia le Buchnera. Boholo ba bona ba genome bo ka ba 1 Mb. Bokhoni ba ho sebelisa li-metabolite tsa lisele tse amohelang bo lebisa tahlehelong ea liphatsa tsa lefutso, ka hona, ho ile ha e-ba le tšenyo e kholo ea phetoho ea genomic. Liphetoho tsa ho iphetola ha lintho ho tloha ho likokoana-hloko tsa limatlafatsi tsa lik'hemik'hale tsa leoatleng ho ea ho bophelo ba libatana li ka baka phokotso e tšoanang ea boholo ba genome. Tlhahlobo ea COG le KEGG e totobatsa palo ea liphatsa tsa lefutso tse sebelisetsoang mesebetsi e itseng le liphapang tsa lefats'e ka litsela tsa genomic pakeng tsa ASxL5T le T. oleivorans MIL-1T, e seng ka lebaka la ho fumaneha hohle ha likarolo tsa liphatsa tsa lefutso tsa mobile. Phapang ka karolelano ea G + C ea genome eohle ea ASxL5T ke 56.1%, 'me ea T. oleivorans MIL-1T ke 46.6%, e bontšang hape hore e arohane.
Tlhahlobo ea dikahare tsa likhoutu tsa genome ea ASxL5T e fana ka lintlha tse sebetsang mabapi le litšobotsi tsa phenotypic. Ho ba teng ha liphatsa tsa lefutso tsa mofuta oa IV fimbriae (Tfp) ho bohlokoa haholo hobane li khothalletsa ho tsamaea ha lisele, ho bitsoang social gliding kapa convulsions, ntle le flagella holim'a metsi. Ho ea ka litlaleho, Tfp e na le mesebetsi e meng, ho kenyelletsa le predation, pathogenesis, biofilm formation, tlhaho ea DNA ea tlhaho, ho kopanya lisele le nts'etsopele38. Genome ea ASxL5T e na le liphatsa tsa lefutso tse 18 tse khoutong diguanylate cyclase (enzyme e susumetsang phetoho ea 2 guanosine triphosphate ho guanosine 2 phosphate le cyclic diGMP) le liphatsa tsa lefutso tse 6 tse kenyelletsang diguanylate cyclase phosphate diguanylate. Mofuta oa esterase (o susumetsang ho senyeha ha cyclic di-GMP ho guanosine monophosphate) oa thahasellisa hobane cycl-di-GMP ke len messengerosa la bohlokoa la bobeli le amehang ho nts'etsopele ea biofilm le karohano, motsamao, ho ikopanya le lisele le virulence 39, 40 ts'ebetsong. Hape hoa lokela ho hlokomeloa hore ho Bdellovibrio bacteriovorus, cyclic double GMP e bontšitsoe ho laola phetoho pakeng tsa bophelo bo sa lefelloeng le mokhoa oa bophelo oa ho ja liphoofolo41.
Lipatlisiso tse ngata ka libaktheria tse jang liphoofolo li tsepamisitse maikutlo ho Bdellovibrio, likokoana-hloko tse kang Bdellovibrio, le mefuta ea Myxococcus. Mehlala ena le tse ling tse tsejoang tsa libaktheria tse jang li etsa sehlopha se fapaneng. Ho sa tsotellehe mefuta ena ea mefuta-futa, lihlopha tsa malapa a liprotheine tse nang le litšobotsi tse bontšang phenotypes tsa libaktheria tse 11 tse tsejoang tse hlaselang li khethiloe3,22. Leha ho le joalo, ke liphatsa tsa lefutso tse kenyelletsang O antigen ligase (waaL) feela tse fumanoeng, tse tloaelehileng haholo ho libaktheria tsa Gram-negative. Mofuta ona oa tlhahlobo ha o thuse ho khetha ASxL5T joalo ka sebatana, mohlomong hobane e sebelisa leano le lecha la tlhaselo. Ho ba teng ha liphatsa tsa lefutso tsa baktheria tse fapaneng ho tla thusa ho nts'etsapele litlhahlobo tse ntle tse nkang bopaki ba phapang ea tšebetso le tikoloho lipakeng tsa litho tsa sehlopha. Mehlala ea libaktheria tse jang liphoofolo tse sa kenyelletsoeng tlhahlobisong ena e kenyelletsa litho tsa Cupriavidus necator42 le Bradymonabacteria43, hobane ha bafuputsi ba ntse ba etsa lipatlisiso ka lichaba tse fapaneng tsa likokoana-hloko, ho thehoa lekhetho le eketsehileng la liphoofolo.
Ntho e ikhethang ka ho fetisisa ea libaktheria tsa ASxL5T tse nkiloeng ke setšoantšo sa TEM ke morphology ea eona e ikhethang le e feto-fetohang, e ka khothalletsang ho sebelisana le libaktheria tsa phofu. Mofuta oa tšebelisano-'moho o bonoang o fapane le libaktheria tse ling 'me ha o so ka o fumanoa kapa ho tlalehoa. Potoloho e reriloeng ea bophelo ba libatana tsa ASxL5T e bonts'itsoe ho Setšoantšo sa 5. Ho na le mehlala e fokolang libukeng tse nang le meaho e tšoanang ea apical joalo ka ha re tlaleha mona, empa mehlala ena e kenyelletsa Terasakiispira papahanaumokuakeensis, baktheria ea marine spirillum e nang le keketseho ea 44 ka linako tse ling, le Alphaproteobacteria, Terasillasa. , eo pele e neng e le setho sa mofuta oa Oceanospirillum, ho bonts'a seo ho thoeng ke "filimi ea polar" 45. Mefuta ea cocci e atisa ho hlokomeloa litsong tsa khale, haholo-holo bakeng sa libaktheria tse nang le mefuta e kobehileng, tse kang Vibrio, Campylobacter, le Helicobacter 46, 47, 48, e ka 'nang ea emela boemo bo senyehileng. Ho hlokahala mosebetsi o mong ho hlakisa potoloho e nepahetseng ea bophelo ba libaktheria tsa ASxL5T. Ho fumana hore na e hapa joang le hore na e tšoasa joang, le hore na genome ea eona e kenyelletsa metsoako ea bioloji e ka sebelisetsoang merero ea bongaka kapa ea biotechnological.
Tlhaloso ea mofuta oa Venatorbacter. November Venatorbacter (Ven.a.tor, ba'c.ter, L. e entsoe ka li-venator tse tsoang L. n. venator,'hunter' and Gr. n. bacter,'a rod'. Venatorbacter,'a hunting Rod' . Lisele ke aerobic, mamellehe, bokellela Boemong ba mocheso oa 4 ho isa ho 42 °C Incrown The pH ea 4-9 e sa tloaeleha Likhofung tsa leoatleng, tse ngata ha li mamelle pH ea mafura ke C16:1ω6c le/kapa C16:1ω7c. C16:0 le C18:1ω9 ; 3-OH li fumanoa e le hydroxy fatty acids Ha li hōle ka har'a moro oa DNA Likahare tsa 56.1 mol boemo ba leloko lena bo lelapeng.
Tlhaloso ea Venatorbacter cuculus sp. Pulungoana Venatorbacter cucullus (cu'cull.us.; L. n. cucullus e bolela ho loka).
Ho feta moo, tšobotsi e hlalosang ea mofuta ona ke hore ha e holile ho BA kapa BHI, lisele li bolelele ba 1.63 µm le bophara ba 0.37 µm. Likolone tsa BHI agar li nyane haholo, li fihla ho 2 mm ka bophara kamora lihora tse 72. Ke beige, translucent, chitja, convex le benyang. Litho tsa mofuta ona li ka sebelisa Escherichia coli le Klebsiella. Campylobacter le libaktheria tse ling tse 'maloa tsa Gram-negative li sebetsa e le phofu.
Mofuta o tloaelehileng oa ASxL5T o ne o arohane le lebese la nama ea khomo Nottinghamshire, UK, 'me o kenngoa ho National Type Culture Collection (UK): nomoro ea ho kena NCTC 14397 le Netherlands Bacterial Culture Collection (NCCB) nomoro ea NCCB 100775. Tatelano e feletseng ea genome ea ASxL5T e kentsoe Genbank ho latela tlatsetso ea CP046056.
Libaktheria tsa ASxL5T li ne li arohane le lebese la nama ea khomo li sebelisa theknoloji ea ho itšehla thajana ea phage9,49. The slurry e hlakotsoe 1:9 (w/v) ka SM buffer (50 mM Tris-HCl [pH 7.5], 0.1 M NaCl, 8 mM MgSO4.7H2O le 0.01% gelatin; Sigma Aldrich, Gillingham, UK), Ebe e kenya ka 4°C bakeng sa lihora tse 24, e potoloha butle ho leleka libatana ka har'a buffer. Ho emisoa ho ne ho le centrifuged ho 3000g bakeng sa metsotso e 3. The supernatant e ile ea bokelloa 'me ea centrifuged ho 13,000g lekhetlo la bobeli ka metsotso e 5. Ka mor'a moo, supernatant e ile ea fetisoa ka 0.45 µm membrane filter (Minisart; Sartorius, Gottingen, Germany) le 0.2 µm membrane filter (Minisart) ho tlosa lisele tse setseng tsa baktheria. ASxL5T e ka fetisa lihloela tsena. Mohloa o bonolo oa agar oa Campylobacter enterosus S12 (nomoro ea ho kena NCBI CP040464) ho tloha slurry e tšoanang e ile ea lokisoa ho sebelisoa mekhoa e tloaelehileng. The slurry e tlhotliloeng e ile ea ajoa ho e 'ngoe le e' ngoe ea li-cell plate tsena ka marotholi a 10 µl ka makhetlo a mararo 'me a lumelloa hore a ome. Letlapa le ne le kenngoa ka tanka ea microaerophilic ka 37 ° C bakeng sa lihora tse 48 tlas'a maemo a microaerobic (5% O2, 5% H2, 10% CO2, le 80% N2). Letlapa le bonahalang le fumanoeng le ile la ntšoa ka har'a SM buffer 'me la fetisetsoa mohloeng o mocha oa C. hyointestinalis S12 ho tsoela pele ho phatlalatsa likokoana-hloko tse nang le lysed. Hang ha ho etsoa qeto ea hore libaktheria ke sesosa sa letlapa la lytic eseng phage, leka ho hōlisa ntho e phelang ntle le moeti le ho e khetholla ka ho eketsehileng. Tloaelo ea aerobic e ne e etsoa ka 37 ° C le 5% v / v defibrinated pere ea mali (TCS Biosciences Lt, Buckingham, UK, supplement). Ho ea ka litataiso tsa National Clinical Standards Committee, mokhoa oa ho jala disc o sebelisoa bakeng sa tlhahlobo ea ts'oaetso ea antibacterial. BHI agar e ne e lengoa ka 37 ° C e sebelisa disc e nang le lithibela-mafu tse latelang (Oxoid) bakeng sa setso sa aerobic: amoxicillin le 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; Chloramphenicol 30 μg; Cefoxitin 30 μg; Tetracycline 30 µg; Nitrofurantoin 300 μg; Aztreonam 30 µg; Ampicillin 10 µg; Cefpodoxime 10 μg; Trimethoprim-Sulfamethoxazole 25 µg. Mamello ea letsoai e thehiloe ke aerobic incubation holim'a lipoleiti tsa agar tsa BHI ka 37 °C. NaCl e eketsehileng e ile ea kenngoa lipoleiting tsa agar tsa BHI ho fana ka mefuta e mengata ea mahloriso ho fihlela ho 10% w/v. Boemo ba pH bo khethoa ke setso sa aerobic lipoleiting tsa BHI agar tse 37°C, moo pH e fetotsoeng ho ba lipakeng tsa 4 le 9 ka HCl e sa hloekang kapa NaOH e hloekisitsoeng, 'me boleng ba pH bo tiisitsoeng pele u tšela poleiti . Bakeng sa tlhahlobo ea cellular fatty acid, ASxL5T e hlahisitsoe BHI agar ka matsatsi a 3 le aerobic ho 37 °C. Ho ea ka MIDI (Sherlock Microbial Identification System, version 6.10) protocol e tloaelehileng ea FERA Science Ltd, (York, UK), li-cell fatty acids li ile tsa ntšoa, tsa lokisoa le ho hlahlojoa.
Bakeng sa TEM, ASxL5T e ile ea etsoa aerobic ka ho hasana ka mokhoa o ts'oanang ho BA ho 37 ° C bakeng sa lihora tse 24, 'me ea kotuloa ka 1 ml ea 3% (v/v) glutaraldehyde ka 0.1 M cacodylate buffer mocheso oa kamore Lokisa hora ea 1, ebe centrifuge. ka 10,000 g bakeng sa metsotso e 3. Ebe o emisa pellet ka bonolo ho 600 μl 0.1 M cacodylate buffer. Fetisetsa ho emisoa ho tsitsitseng ha ASxL5T ho filimi ea Formvar/carbon ka marang-rang a koporo a 200 mesh. Libaktheria li ne li silafalitsoe ke 0.5% (w/v) uranyl acetate bakeng sa motsotso oa 1 'me tsa hlahlojoa ke TEM ho sebelisa microscope ea TEI Tecnai G2 12 Biotwin. Joalokaha ho boletsoe ka holimo, kopanya palo e tšoanang ea liphofu le sebata ho NZCYM moro (BD Difco™, Fisher Scientific UK Ltd, Loughborough) 'me u emise lihora tse 48 tlas'a maemo a microaerobic a Campylobacter kapa Campylobacter ho 37 ° C , Tšebelisano ea sebata le phofu. e ile ea hlahlojoa hape ke TEM. Maemo a Aerobic bakeng sa Escherichia coli. Lekola liphofu le libaktheria tse jang liphoofolo ka boikemelo ho fumana hore na ho na le liphetoho life ho morphology ea sele ka lebaka la ho hlaseloa. Mokhoa o motšo oa Sudan o ne o sebelisoa bakeng sa microscope ea optical ea ho bokella PHB.
Holisa litloaelo tsa ASxL5T ka bosiu bo le bong ka ho tlotsa kholo holim'a lipoleiti tsa BHI kapa BA ka swab e sa hloekang. Bokella lisele tsa ASxL5T ebe u li emisa ho MRD (CM0733, Oxoid), ebe u li beha ho 4 ° C matsatsi a 7 ho bolaea lisele. Tlhaloso ea NCTC kapa setso sa baktheria sa laboratori se ile sa kenngoa ka har'a moro oa BHI kapa No. 2 moro oa limatlafatsi (CM007, Oxoid), o kenngoa ka bosiu bo le bong, o kenngoa ka 13,000g mme o khutlisetsoa MRD ho fihlela OD600 e le 0.4. Setso: Bacillus subtilis NCTC 3610T, Citrobacter freundii NCTC 9750T, Enterobacter aerogenes NCTC 10006T, Enterococcus faecalis NCTC 775T, Escherichia coli NCTC 86, Klebsiella 1uTC1co4 oxyto6 10817, Listeria Libaktheria tse khethehileng NCTC 4885, Bacillus macerans NCTC 6355T, Providencia stuartsii NCTC 10318, Pseudomonas fluorescens SMDL, Rhodococcus submarine hamburger NCTC 1621T, TC7 Salmonella 5 bacteria ea Mokokotlo NCTC 10861, Staphylococcus aureus NCTC 8532T, Streptococcus pneumoniae NCTC 7465T, Yersinia enterocolitica NCTC 10460. Motlatsi oa Campylobacter o ne a kenngoa ka microaerobically ka lipoleiti tsa BA ka 37 ° C le suspendedMNZCYMNZ e emisitsoe. Lihlopha tse lekiloeng tsa Campylobacter ke: C. coli 12667 NCTC, C. jejuni 12662, C. jejuni PT14, C. jejuni NCTC 11168T, C. helveticus NCTC 12472, C. lari NCTC 114548, C. lari NCTC 114548, C. PT14, C... Bokella lisele ho MRD, centrifuge ho 13,000g le ho emisa hape ho MRD ho fihlela OD600 e le 0.4. Eketsa aliquot ea 0.5 ml ho fanyeha ho 5 ml e qhibilihisitsoeng holimo agar ea NZCYM (0.6% agar) 'me ue tšollele holim'a poleiti e ka tlase ea 1.2% NZCYM. Kamora ho folisa le ho omisoa, ASxL5T e hlapolotsoeng e ile ea ajoa joalo ka marotholi a 20 µl botong ka 'ngoe ea mohloa ka makhetlo a mararo. Mocheso oa setso le sepakapaka li itšetlehile ka litlhoko tsa libaktheria tsa teko.
Sebelisa GenElute ™ Bacterial Genomic DNA Kit (Sigma Aldridge) ho lokisa DNA ho tsoa libakeng tse arohaneng le baktheria. Mekhoa e tloaelehileng e ile ea sebelisoa bakeng sa ho hōlisa PCR ea 16S rRNA gene le boikemisetso ba tatellano ea lihlahisoa ho sebelisoa k'hemistri ea ho felisa dae (Eurofins Value Read Service, Jeremane). Sebelisa lenaneo la BLAST-N ho bapisa tatellano ena le liphatsa tse ling tsa 16S rRNA ho khetholla le ho bokella mefuta e amanang haufi-ufi. Tsena li hokahantsoe ho sebelisoa ClustalW lenaneong la MEGA X. Sefate sa phylogenetic se ile sa tsosolosoa ho sebelisoa MEGA X ho sebelisoa mokhoa o phahameng oa monyetla o thehiloeng ho mohlala oa Tamura-Nei, ka likopi tse tataisoang tse 100054. Sebelisa PureLink™ Genomic DNA Kit (Fisher Scientific, Loughborough, UK) ho ntša DNA bakeng sa tatellano ea liphatsa tsa lefutso. Tatelano ea genome ea ASxL5T e ne e khethiloe ho sebelisoa motsoako oa Illumina MiSeq, o nang le 250 bp e phethiloeng habeli e entsoeng ka laebrari e lokiselitsoeng ho sebelisa Nextera labeling kit le 2 ho 20 kb nako e telele ho tloha sethaleng sa PacBio. Genomics DNA Sequencing Research Facility Univesithing ea Sembia. Genome e ile ea bokelloa ho sebelisoa CLC Genomics Workbench 12.0.3 (Qiagen, Aarhus, Denmark). Litloaelo tsa ASxL5T li behiloe ho National Type Culture Collection (UK) le Netherlands Bacterial Culture Collection (NCCB). Liphatsa tsa lefutso tsa lintho tse phelang tse amanang tse sebelisoang ho bapisa ke: Thalassolituus oleivorans MIL-1T (nomoro ea tumello ea HF680312, e feletse); Bacterioplanes sanyensis KCTC 32220T (nomoro ea tumello BMYY01000001, e sa fellang); Oceanobacter kriegii DSM 6294T (nomoro ea tumello NZ_AUGV00000000, e sa fellang); Sechaba sa Marinamonas DSM 5604T (e kenyelelitsoe ASM436330v1, e sa fellang), Oceanospirullum linum ATCC 11336T (e kenyelelitsoe MTSD02000001, e sa fellang) le Thalassolituus sp. C2-1 (eketsa NZ_VNIL01000001, e sa fellang). Sebelisa JGI Genome Portal36 ho https://img.jgi.doe.gov//cgi-bin/mer/main.cgi?section=ANI&page= ho fumana lintlha tsa alignment (AF) le karolelano ea nucleic acid identity (ANI). Ka bobeli. Mokhoa oa Rodriguez-R & Konstantinidis55 o ne o sebelisetsoa ho fumana boitsebiso ba amino acid (AAI). Sebelisa GToTree 1.5.5411,12,13,14,15,16,17,18 ho hlahisa palo e hakanyetsoang ea monyetla oa sefate sa phylogenetic. Genome e kentsoeng e emelang genome e fumanehang ea litšupiso e khethiloe ho mofuta oa litšupiso o tsejoang e le o amanang le ASxL5T ho tsoa ho 16S rRNA phylogeny. Hlalosa sefate ka ho sebelisa sefate sa bophelo se sebelisanang inthaneteng (https://itol.embl.de/). Tlhaloso e sebetsang le tlhahlobo ea genome ea ASxL5T e etsoa ho sebelisoa sesebelisoa sa marang-rang sa BlastKOALA KEGG ho sebelisoa KEGG (Kyoto Encyclopedia of Genes and Genomes) kabo ea ntlafatso ea module. Kabo ea lihlopha tsa COG (lihlopha tsa orthologous) li ikemiselitse ho sebelisa sesebelisoa sa marang-rang sa eggNOG-mapper.
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