Mtundu watsopano wa mabakiteriya a Gram-negative, aerobic, mchere, wokangalika, wooneka ngati ndodo, komanso wolusa ASxL5T analekanitsidwa ndi dziwe la ndowe la ng'ombe ku Nottinghamshire, England, ndipo adagwiritsa ntchito Campylobacter ngati nyama yake. Pambuyo pake, mitundu ina ya Campylobacter ndi mamembala a banja la Enterobacteriaceae adapezeka ngati nyama. Pambuyo pa subculture yopanda ma cell olandila, kukula kofooka kwa aseptic kudakwaniritsidwa pa Brain Heart Infusion Agar. Kukula koyenera kwambiri ndi 37 °C ndipo pH ndi 7. Ma electron microscopy adawonetsa zinthu zachilendo kwambiri zokhudzana ndi kupezeka kwa nyama. Kusanthula kwa phylogenetic pogwiritsa ntchito 16S rRNA jini yotsatizana inasonyeza kuti kudzipatula kumagwirizana ndi membala wa banja la Marine Spirulina, koma sangathe kutchulidwa momveka bwino ngati membala wamtundu uliwonse wodziwika. Kutsata kwamtundu wonse wa ASxL5T kunatsimikizira ubale ndi mamembala a spirochetes apanyanja. Kusaka kwa database kudawonetsa kuti ma ASxL5T angapo amagawana ma jini a 16S rRNA ndi mabakiteriya angapo omwe sanapangidwe kuchokera kunyanja, pamtunda ndi pansi. Tikukulimbikitsani kuti mtundu wa ASxL5T ukuyimira mtundu watsopano wamtundu watsopano. Timalimbikitsa dzina la Venatorbacter cucullus gen. November, sp. Mu Novembala, ASxL5T idagwiritsidwa ntchito ngati mtundu wamavuto.
Mabakiteriya olusa ndi mabakiteriya omwe amawonetsa kuthekera kosaka ndi kupha mabakiteriya ena amoyo kuti apeze zida ndi mphamvu za biosynthetic. Izi ndizosiyana ndi kubwezeretsanso zakudya zambiri kuchokera ku tizilombo takufa, komanso zimasiyana ndi kuyanjana kwa tizilombo toyambitsa matenda, momwe mabakiteriya amapanga ubale wapamtima ndi wowalandira popanda kuwapha. Mabakiteriya olusa asintha machitidwe osiyanasiyana amoyo kuti atengere mwayi pazakudya zambiri zomwe zimapezeka m'malo omwe amapezeka (monga malo okhala m'madzi). Ndi gulu la mitundu yosiyanasiyana ya taxonomically, omwe amangolumikizidwa ndi moyo wawo wapadera wotsekereza1. Zitsanzo za mabakiteriya olusa zapezeka mu phyla zingapo zosiyanasiyana, kuphatikizapo: Proteobacteria, Bacteroides, ndi Chlorella.3. Komabe, mabakiteriya olusa omwe amaphunzitsidwa bwino kwambiri ndi Bdellovibrio ndi Bdellovibrio-and-like zamoyo (BALOs4). Mabakiteriya olusa ndi magwero odalirika a mankhwala atsopano komanso antibacterial agents5.
Tizilombo toyambitsa matenda timakhulupilira kuti timakulitsa kusiyanasiyana kwa tizilombo tating'onoting'ono komanso timakhudza thanzi, zokolola komanso kukhazikika kwa chilengedwe6. Ngakhale zili ndi makhalidwe abwinowa, pali maphunziro ochepa okhudza mabakiteriya atsopano omwe amadya chifukwa cha zovuta kulima mabakiteriya komanso kufunikira koyang'anitsitsa kuyanjana kwa maselo kuti amvetsetse zovuta za moyo wawo. Izi sizovuta kuzipeza kuchokera ku kafukufuku wamakompyuta.
M'nthawi ya kuwonjezeka kwa antimicrobial resistance, njira zatsopano zothandizira tizilombo toyambitsa matenda zikuphunziridwa, monga kugwiritsa ntchito bacteriophages ndi mabakiteriya owononga7,8. Mabakiteriya a ASxL5T adalekanitsidwa mu 2019 pogwiritsa ntchito ukadaulo wopatula ndowe za ng'ombe kuchokera ku Dairy Center ya University of Nottingham, Nottinghamshire. Cholinga cha kafukufukuyu ndikulekanitsa zamoyo zomwe zitha kukhala zowongolera zamoyo. Campylobacter hyointestinalis ndi zoonotic pathogen, yomwe imagwirizana kwambiri ndi matenda a m'mimba mwa anthu10. Imapezeka paliponse mu seramu ndipo imagwiritsidwa ntchito ngati chandamale.
Bakiteriya ya ASxL5T idalekanitsidwa ndi odzola a ng'ombe chifukwa idawonedwa kuti zolembera zomwe zidapanga pa udzu wa C. hyointestinalis zinali zofanana ndi zomwe zimapangidwa ndi bacteriophages. Izi ndizopezeka mosayembekezereka, chifukwa gawo la njira yodzipatula ya phage imaphatikizapo kusefa kudzera mu fyuluta ya 0.2 µm, yomwe imapangidwa kuti ichotse maselo a bakiteriya. Kuyang'ana kwapang'onopang'ono kwa zinthu zotengedwa pamwalawu kunawonetsa kuti mabakiteriya ang'onoang'ono opindika owoneka ngati ndodo osawundana ndi polyhydroxybutyrate (PHB). Chikhalidwe cha aseptic chodziyimira pawokha pama cell anyama chimazindikirika pazachuma cholimba (monga ubongo wamtima kulowetsedwa agar (BHI) ndi agar wamagazi (BA)), ndipo kukula kwake ndikofooka. Imapezedwa pambuyo pa subculture yokhala ndi inoculum yolemera. Imakula mofanana pansi pa microaerobic (7% v/v oxygen) ndi mpweya wa mumlengalenga, koma osati mumlengalenga. Pambuyo pa maola 72, m'mimba mwake m'mimba mwake munali ochepa kwambiri, kufika 2 mm, ndipo anali beige, translucent, kuzungulira, convex ndi chonyezimira. Kuyesa kwachilengedwe kwachilengedwe kumalephereka chifukwa ASxL5T singakulitsidwe modalirika muzamadzimadzi, zomwe zikuwonetsa kuti zitha kudalira zovuta zamoyo zomwe zimapangidwa ndi biofilm. Komabe, kuyimitsidwa kwa mbale kunasonyeza kuti ASxL5T ndi aerobic, yabwino kwa oxidase ndi catalase, ndipo imatha kulekerera 5% NaCl. ASxL5T imagonjetsedwa ndi 10 µg streptomycin, koma imamva maantibayotiki ena onse omwe ayesedwa. Maselo a bakiteriya a ASxL5T adawunikidwa ndi TEM (Chithunzi 1). Mukakula popanda ma cell a nyama pa BA, ma cell a ASxL5T ndi Campylobacter ang'onoang'ono, okhala ndi kutalika kwa 1.63 μm (± 0.4), m'lifupi mwake 0.37 μm (± 0.08), ndi mlongo umodzi wautali (mpaka 5 μm). Kugonana flagella. Pafupifupi 1.6% ya maselo amawoneka kuti ali ndi m'lifupi mwake osachepera 0.2 μm, zomwe zidzalola kudutsa mu chipangizo cha fyuluta. Kuwonjezedwa kwachilendo kwachilendo kunawonedwa pamwamba pa maselo ena, ofanana ndi fairing (Latin cucullus) (onani mivi mu 1D, E, G). Izi zikuwoneka kuti zimapangidwa ndi nembanemba yakunja yochulukirapo, yomwe ingakhale chifukwa cha kuchepa kwachangu kwa kukula kwa envelopu ya periplasmic, pomwe nembanemba yakunja imakhalabe, kuwonetsa mawonekedwe "otayirira". Kulima ASxL5T popanda zakudya (mu PBS) kwa nthawi yaitali pa 4 ° C kunapangitsa kuti maselo ambiri (koma osati onse) asonyeze coccal morphology (Chithunzi 1C). Pamene ASxL5T ikukula ndi Campylobacter jejuni ngati nyama kwa maola 48, kukula kwa maselo kumakhala kotalika kwambiri komanso kocheperapo kusiyana ndi maselo omwe amakula popanda khamu (Table 1 ndi Chithunzi 1E). Mosiyana ndi zimenezi, pamene ASxL5T ikukula ndi E. coli monga nyama kwa maola 48, kukula kwa maselo kumakhala kotalika komanso kokulirapo kuposa pamene ikukula popanda nyama (Table 1), ndipo kutalika kwa selo kumakhala kosiyana, kawirikawiri kumawonetsa filamentous (Chithunzi 1F). Pamene adakulungidwa ndi Campylobacter jejuni kapena E. coli ngati nyama kwa maola 48, maselo a ASxL5T sanawonetsere flagella konse. Table 1 ikufotokozera mwachidule zomwe zawona kusintha kwa kukula kwa selo kutengera kupezeka, kusapezeka, ndi mtundu wa ASxL5T.
TEM chiwonetsero cha ASx5LT: (A) ASx5LT chikuwonetsa chikwapu chachitali; (B) batire wamba ASx5LT; (C) cocci ASx5LT maselo pambuyo makulitsidwe yaitali popanda zakudya; (D) gulu la ma cell a ASx5LT limasonyeza zachilendo (E) ASx5LT gulu la selo lopangidwa ndi Campylobacter nyama linawonetsa kutalika kwa maselo poyerekeza ndi omwe alibe kukula kwa nyama (D) adawonetsanso mawonekedwe a apical; (F) Large Filamentous flagella, ASx5LT maselo, pambuyo pa kukulitsidwa ndi E. coli nyama; (G) Selo limodzi la ASx5LT pambuyo pa kukulitsidwa ndi E. coli, kusonyeza mawonekedwe apamwamba achilendo. The bar ikuyimira 1 μm.
Kuzindikira mndandanda wamtundu wa 16S rRNA (nambala yofikira MT636545.1) imathandizira kusaka kwa database kuti ikhazikitse zofananira zomwe zili m'gulu la Gammaproteobacteria, ndipo ali pafupi kwambiri ndi mabakiteriya am'madzi am'madzi am'madzi am'madzi a spirillum (Chithunzi 2), ndipo ndi mamembala amtundu wa Thalassolituus. Wachibale wapafupi kwambiri ndi Marine Bacillus. Mndandanda wa jini wa 16S rRNA ndi wosiyana kwambiri ndi mabakiteriya olusa a m'banja la Bdelvibrionaceae (Deltaproteobacteria). Kuyerekeza kwapawiri kwa B. bacteriovorus HD100T (mtundu wa zovuta, DSM 50701) ndi B. bacteriovorus DM11A kunali 48.4% ndi 47.7%, ndipo kwa B. exovorus JSS kunali 46.7%. Mabakiteriya a ASxL5T ali ndi makope a 3 a jini ya 16S rRNA, awiri omwe ali ofanana kwa wina ndi mzake, ndipo lachitatu ndi 3 maziko osiyana. Mabakiteriya ena awiri owopsa (ASx5S ndi ASx5O; 16S rRNA manambala olowa nawo jini ndi MT636546.1 ndi MT636547.1, motsatana) okhala ndi mawonekedwe ofanana a morphological ndi phenotypic ochokera kumalo omwewo sizofanana, koma ndi osiyana ndi ASxL5T ndi Bacterial uncultured Bact. Zotsatira za database zimaphatikizidwa pamodzi ndi magulu ena Oceanospirillaceae (Chithunzi 2). Mndandanda wonse wa genome wa ASxL5T watsimikiziridwa ndikusungidwa mu database ya NCBI, ndipo nambala yolowa ndi CP046056. Genome ya ASxL5T imakhala ndi chromosome yozungulira ya 2,831,152 bp yokhala ndi chiŵerengero cha G + C cha 56.1%. Mndandanda wa ma genome uli ndi 2653 CDS (yonse), yomwe 2567 imanenedweratu kuti idzalowetsa mapuloteni, omwe 1596 akhoza kuperekedwa ngati ntchito zopangira (60.2%). Genome ili ndi majini 67 a RNA-coding, kuphatikiza 9 rRNAs (3 iliyonse ya 5S, 16S, ndi 23S) ndi 57 tRNAs. Mawonekedwe a genomic a ASxL5T adafanizidwa ndi ma genome omwe alipo amtundu wamtundu wapafupi womwe umadziwika kuchokera ku 16S rRNA gene sequence (Table 2). Gwiritsani ntchito chidziwitso cha amino acid (AAI) kuyerekeza ma genome onse a Thalassolituus ndi ASxL5T. Njira yapafupi kwambiri (yosakwanira) yotsatiridwa ndi AAI ndi Thalassolituus sp. C2-1 (onjezani NZ_VNIL01000001). Vutoli linali lolekanitsidwa ndi matope akuzama a m'nyanja ya Mariana Trench, koma pakadali pano palibe chidziwitso chambiri chokhudza mtundu uwu poyerekeza. Poyerekeza ndi ASxL5T's 2.82 Mb, genome ya chamoyocho ndi yokulirapo pa 4.36 Mb. Kukula kwa genome kwa ma spirochetes am'madzi ndi pafupifupi 4.16 Mb (± 1.1; n = 92 ma genomes athunthu omwe amafufuzidwa kuchokera ku https://www.ncbi.nlm.nih.gov/assembly), kotero genome ya ASxL5T ikugwirizana ndi dongosolo Poyerekeza ndi mamembala ena, ndi ochepa. Gwiritsani ntchito GToTree 1.5.54 kuti mupange mtengo wa phylogenetic wotengera ma genome (Chithunzi 3A), pogwiritsa ntchito ma amino acid osakanikirana ndi ma gene 172 amtundu umodzi wa Gammaproteobacteria 11,12,13,14,15,16, 17, 18. Kusanthula kunasonyeza kuti ikugwirizana kwambiri ndi Thalassolituus, Bacterial Plane, ndi Marine Bacterium. Komabe, izi zikuwonetsa kuti ASxL5T ndi yosiyana ndi achibale ake ku Marine Spirulina ndipo deta yake yotsatizana ndi ma genome ilipo.
Mtengo wa phylogenetic wogwiritsa ntchito 16S rRNA gene sequence ikuwonetsa malo a ASxL5T, ASxO5, ndi ASxS5 strains (ndi matumbo) okhudzana ndi mabakiteriya osalimidwa ndi am'madzi a Marine Spirulinaceae. Nambala yolowa mu Genbank imatsatira dzina la zovuta m'makolo. Gwiritsani ntchito ClustalW kuti mugwirizanitse zotsatizana, ndikugwiritsa ntchito njira yothekera kwambiri ndi chitsanzo cha Tamura-Nei kuti mutsimikize maubale a phylogenetic, ndikuchitanso zobwereza motsogozedwa 1000 mu pulogalamu ya MEGA X. Nambala yomwe ili panthambi ikuwonetsa kuti kopi yowongoleredwa ndiyoposa 50%. Escherichia coli U/541T idagwiritsidwa ntchito ngati gulu.
(A) Mtengo wa phylogenetic wozikidwa pa genome, ukuwonetsa ubale pakati pa bakiteriya wa Marine Spirospiraceae ASxL5T ndi achibale ake apamtima, E. coli U 5/41T ngati gulu lakunja. (B) Poyerekeza ndi T. oleivorans MIL-1T, gawo logwira ntchito la kugawa kwa majini limanenedweratu kutengera gulu la orthologous (COG) la mapuloteni a ASx5LT. Chithunzi chakumanzere chikuwonetsa kuchuluka kwa majini mugulu lililonse la COG lomwe limagwira ntchito mumtundu uliwonse. Chithunzi chakumanja chikuwonetsa kuchuluka kwa ma genome omwe ali mugulu lililonse logwira ntchito la COG. (C) Poyerekeza ndi T. oleiverans MIL-1T, kusanthula kwa KEGG yonse (Kyoto Encyclopedia of Genes and Genomes) modular pathway ya ASxL5T.
Pogwiritsa ntchito nkhokwe ya KEGG kuti mufufuze zamitundu yomwe ilipo mu ASxL5T genome idawulula njira ya aerobic gamma Proteus. ASxL5T ili ndi majini okwana 75 omwe amaperekedwa ku mapuloteni amtundu wa bakiteriya, kuphatikizapo majini okhudzidwa ndi chemotaxis, flagella assembly, ndi type IV fimbriae system. M'gulu lomaliza, 9 mwa 10 majini ndi omwe amayang'anira kusuntha kwamitundu yosiyanasiyana ya zamoyo. Genome ya ASxL5T ili ndi njira yathunthu ya tetrahydropyrimidine biosynthetic yomwe imagwira nawo ntchito yoteteza ku osmotic stress20, monga momwe amayembekezera ma halophiles. Genome ilinso ndi njira zambiri zathunthu zopangira ma cofactors ndi mavitamini, kuphatikiza ma riboflavin synthesis pathways. Ngakhale jini ya alkane 1-monooxygenase (alkB2) ilipo mu ASxL5T, njira yogwiritsira ntchito hydrocarbon siinathe. M'magulu amtundu wa ASxL5T, ma homologue a majini omwe amadziwika kuti ndiwo omwe amachititsa kuwonongeka kwa ma hydrocarbons mu T. oleiverans MIL-1T21, monga TOL_2658 (alkB) ndi TOL_2772 (alcohol dehydrogenase) mwachiwonekere palibe. Chithunzi 3B chikuwonetsa kufananitsa kugawa kwa majini mugulu la COG pakati pa ASxL5T ndi Mafuta a Olive MIL-1T. Ponseponse, ma genome ang'onoang'ono a ASxL5T amakhala ndi majini ocheperako kuchokera mugulu lililonse la COG poyerekeza ndi ma genome akuluakulu. Pamene chiwerengero cha majini m'gulu lililonse logwira ntchito chikufotokozedwa ngati kuchuluka kwa ma genome, kusiyana kumazindikirika mu kuchuluka kwa majini mu kumasulira, kapangidwe ka ribosomal ndi magulu a biogenesis, ndi magulu opangira mphamvu ndi kutembenuka, omwe amapanga ASxL5T yaikulu. genome Peresenti imafananizidwa ndi gulu lomwelo lomwe likupezeka mu T. oleiverans MIL-1T genome. Mosiyana, poyerekeza ndi ASxL5T genome, T. oleivorans MIL-1T ili ndi chiwerengero chachikulu cha majini mumagulu obwerezabwereza, ophatikizana ndi okonzanso, ndi zolemba. Chochititsa chidwi n'chakuti, kusiyana kwakukulu pa zomwe zili m'gulu lililonse logwira ntchito la ma genome awiriwa ndi chiwerengero cha majini osadziwika omwe alipo mu ASxL5T (Chithunzi 3B). Kusanthula kopindulitsa kwa ma module a KEGG kunachitika, pomwe gawo lililonse la KEGG limayimira magawo omwe amafotokozedwa pamanja pazofotokozera komanso kutanthauzira kwachilengedwe kwa data yotsatizana. Kuyerekeza kwa kagawidwe ka majini munjira yonse ya KOG module ya ASxL5T ndi azitona MIL-1T ikuwonetsedwa pa Chithunzi 3C. Kusanthula uku kukuwonetsa kuti ngakhale ASxL5T ili ndi njira yathunthu ya sulfure ndi nitrogen metabolism, T. oleiverans MIL-1T alibe. Mosiyana ndi izi, T. oleiverans MIL-1T ili ndi njira yathunthu ya cysteine ​​​​ndi methionine, koma siyikwanira mu ASxL5T. Choncho, ASxL5T ili ndi gawo lodziwika bwino la sulfate assimilation (lotanthauzidwa ngati gulu la majini omwe angagwiritsidwe ntchito ngati zizindikiro za phenotypic, monga mphamvu ya metabolic kapena pathogenicity; https://www.genome.jp/kegg/module.html) Mu T. Zithunzi za MIL-1T. Kuyerekeza zomwe zili mu ASxL5T ndi mndandanda wa majini omwe akuwonetsa kuti moyo wolusa ndi wovuta. Ngakhale kuti waaL jini yomwe imalowetsa ligase yokhudzana ndi O antigen polysaccharide mpaka pachimake ilipo mu ASxL5T genome (koma imakhala yofala m'mabakiteriya ambiri a Gram-negative), tryptophan 2,3-dioxygenase (TDO ) Genes ingaphatikizepo 60 amino. zigawo za asidi zomwe zimapezeka m'mabakiteriya olusa omwe kulibe. Palibe majini ena olusa mumtundu wa ASxL5T, kuphatikiza ma enzymes omwe amaphatikizidwa mu isoprenoid biosynthesis munjira ya mevalonate. Dziwani kuti palibe jini yoyendetsera gntR pagulu lazilombo lomwe lawunikidwa, koma majini atatu onga gntR amatha kudziwika mu ASxL5T.
Makhalidwe a phenotypic a ASxL5T akufotokozedwa mwachidule mu Table 3 ndikuyerekeza ndi mawonekedwe a phenotypic amtundu wofananira 23, 24, 25, 26, ndi 27 wolembedwa m'mabuku. Zodzipatula kuchokera ku T. marinus, T. olevorans, B. sanyensis, ndi Oceanobacter kriegii ndizochita, zolekerera mchere, matupi opangidwa ndi ndodo ya okosijeni, koma alibe pafupifupi zizindikiro zina za phenotypic ndi ASxL5T. Avereji ya pH ya m’nyanja ndi 8.1 (https://ocean.si.edu/ocean-life/invertebrates/ocean-acidification#section_77), yomwe ikuwonetsedwa mu T. marinus, T. olevorans, B. sanyensis ndi O. kirigii. ASxL5T ndi yoyenera ku mitundu yokulirapo ya pH (4-9) yamitundu yomwe si ya m'madzi. Makhalidwe a Phenotypic a Thalassolituus sp. C2-1. Zosadziwika. Kutentha kwa kukula kwa ASxL5T nthawi zambiri kumakhala kokulirapo kuposa kunyanja (4-42 ° C), ngakhale kuti zina koma osati zonse za T. marinus zimalekerera kutentha. Kulephera kukula kwa ASxL5T mu media media kunalepheretsa mawonekedwe ena a phenotypic. Gwiritsani ntchito API 20E kuyesa zinthu zomwe zidachotsedwa mu mbale ya BA, ONPG, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, kugwiritsa ntchito citrate, urease, tryptophan deaminase, gelatin hydrolysis Enzyme, zotsatira zake zonse zinali zoipa, koma palibe indole, acetoin ndi H2S. zinapangidwa. Zakudya zopanda chotupitsa zimaphatikizapo: shuga, mannose, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin ndi arabinose. Poyerekeza ndi zovuta zofotokozera zomwe zimasindikizidwa, mawonekedwe amtundu wa mafuta amtundu wa ASxL5T akuwonetsedwa mu Table 4. Mafuta akuluakulu amtundu wa C16: 1ω6c ndi / kapena C16: 1ω7c, C16: 0 ndi C18: 1ω9. Ma hydroxy fatty acids C12:0 3-OH ndi C10:0 3-OH aliponso. Chiyerekezo cha C16:0 mu ASxL5T ndichokwera kuposa mtengo womwe wanenedwa wamtundu wofananira. Mosiyana, poyerekeza ndi T. marinus IMCC1826TT, chiwerengero cha C18: 1ω7c ndi / kapena C18: 1ω6c mu ASxL5T chachepetsedwa. oleivorans MIL-1T ndi O. kriegii DSM 6294T, koma sanapezeke mu B. sanyensis KCTC 32220T. Poyerekeza mbiri yamafuta a asidi a ASxL5T ndi ASxLS adawonetsa kusiyana kosawoneka bwino kwa kuchuluka kwamafuta amtundu wamafuta pakati pa mitundu iwiriyi, yomwe imagwirizana ndi ma genomic DNA amtundu womwewo. Palibe tinthu tating'ono ta poly-3-hydroxybutyrate (PHB) zomwe zidapezeka pogwiritsa ntchito mayeso akuda aku Sudan.
Zochita za predation za mabakiteriya a ASxL5T zidaphunziridwa kuti adziwe kuchuluka kwa nyama. Bakiteriyayu amatha kupanga zolembera pamitundu ya Campylobacter, kuphatikiza: 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 ndi C. upsaliensis NCTC 11541T. Gwiritsani ntchito zikhalidwe zomwe zalembedwa m'gawo lodziwikiratu za njirayo kuti muyese mabakiteriya ambiri a Gram-negative ndi gram-positive. Zotsatira zikuwonetsa kuti ASxL5T itha kugwiritsidwanso ntchito ku Escherichia coli NCTC 86 ndi Citrobacter freundii NCTC 9750T. Mapulani opangidwa pa Klebsiella oxytoca 11466. Kugwirizana kwa TEM ndi E. coli NCTC 86 kukuwonetsedwa mu Chithunzi 4A-D, ndipo kuyanjana ndi Campylobacter jejuni PT14 ndi Campylobacter suis S12 ikuwonetsedwa mu Chithunzi 4E-H pakati. Njira yowukirayo ikuwoneka kuti ndi yosiyana pakati pa mitundu ya nyama yomwe imayesedwa, yokhala ndi maselo amodzi kapena angapo a E. coli omwe amamangiriridwa ku selo lililonse la ASxL5T ndipo amaikidwa pambali pambali pa selo yowonjezera isanayambe kudsorption. Mosiyana ndi zimenezi, ASxL5T ikuwoneka kuti ikugwirizanitsa ndi Campylobacter kudzera pa malo amodzi okhudzana, nthawi zambiri amalumikizana ndi nsonga ya cell ya nyama yolusa komanso pafupi ndi nsonga ya Campylobacter cell (Chithunzi 4H).
TEM yosonyeza kuyanjana pakati pa ASx5LT ndi nyama: (AD) ndi E. coli nyama; (EH) ndi C. jejuni nyama. (A) Selo lodziwika bwino la ASx5LT lolumikizidwa ndi selo limodzi la E. coli (EC); (B) A filamentous ASx5LT yolumikizidwa ku selo limodzi la EC; (C) Selo la filamentous ASx5LT lolumikizidwa ndi ma cell angapo a EC; (D) Kumangirira Maselo Ang'onoang'ono a ASx5LT pa selo limodzi la E. coli (EC); (E) selo limodzi la ASx5LT lolumikizidwa ndi selo la Campylobacter jejuni (CJ); (F) ASx5LT imaukira C. hyointestinalis (CH) maselo; (G) awiri Amodzi ASx5LT cell anaukira CJ selo; (H) Kuyang'ana kwapafupi kwa malo olumikizirana ndi ASx5LT, pafupi ndi nsonga ya cell ya CJ (bar 0.2 μm). Malowa akuyimira 1 μm mu (A–G).
Mabakiteriya olusa asintha kuti agwiritse ntchito magwero ambiri a nyama. Mwachiwonekere, amapezeka kwambiri m'malo osiyanasiyana. Chifukwa cha kukula kochepa kwa anthu, ndizotheka kudzipatula mabakiteriya a ASxL5T kuchokera ku slurry pogwiritsa ntchito njira yolekanitsa phage. Kugwirizana kwa ma genomic a ASxL5T kwa mamembala a banja la oceanospirillaceae la mabakiteriya am'madzi ndi odabwitsa, ngakhale kuti chamoyocho sichimalekerera mchere ndipo chimatha kukula pa sing'anga yomwe ili ndi mchere wa 5%. Kusanthula kwamadzi kwa slurry kunawonetsa kuti sodium chloride inali yochepera 0.1%. Choncho, matope ali kutali kwambiri ndi chilengedwe cha m'nyanja - ponse pa malo ndi mankhwala. Kukhalapo kwa zigawenga zitatu zogwirizana koma zosiyana kuchokera ku gwero lomwelo kumapereka umboni wakuti zilombozi zikuyenda bwino m'malo omwe si a m'madzi. Kuphatikiza apo, kusanthula kwa ma microbiome (mafayilo a data omwe akupezeka kuchokera ku https://www.ebi.ac.uk/ena/browser/view/PRJEB38990) adawonetsa kuti mndandanda womwewo wa 16S rRNA gene uli pamitengo 50 yapamwamba kwambiri yogwira ntchito (OTU). ) Pazitsanzo zingapo za matope. Mabakiteriya angapo osakhazikika adapezeka mu database ya Genbank, yomwe ili ndi ma gene 16S rRNA ofanana ndi mabakiteriya a ASxL5T. Zotsatizanazi, pamodzi ndi machitidwe a ASxL5T, ASxS5, ndi ASxO5, zikuwoneka kuti zikuyimira magulu osiyanasiyana olekanitsidwa ndi Thalassolituus ndi Oceanobacter (Chithunzi 2). Mitundu itatu ya mabakiteriya omwe sanapangidwe (GQ921362, GQ921357 ndi GQ921396) adasiyanitsidwa ndi madzi ophwanyidwa pamtunda wa makilomita 1.3 mumgodi wa golidi waku South Africa mu 2009, ndipo ena awiri (DQ256320 ndi DQ337006) anali ochokera ku South Africa mu 2005). Mndandanda wa jini wa 16S rRNA womwe umagwirizana kwambiri ndi ASxL5T ndi gawo la 16S rRNA jini yotsatiridwa kuchokera ku chikhalidwe cholemeretsa cha mchenga chomwe chinapezedwa kuchokera ku magombe a kumpoto kwa France mu 2006 (chiwerengero cha AM29240828). Mtundu wina wofananira wa jini wa 16S rRNA wochokera ku bakiteriya wosabadwa HQ183822.1 unapezedwa kuchokera ku tanki yosonkhanitsira yotengedwa kuchokera kumalo otayirako matalala ku China. Mwachiwonekere, mabakiteriya a ASxL5T sali oimira kwambiri m'mabuku a taxonomic, koma mabakiteriya omwe alibe chikhalidwe amatha kuimira zamoyo zofanana ndi ASxL5T, zomwe zimagawidwa padziko lonse lapansi, nthawi zambiri m'madera ovuta. Kuchokera pakuwunika konse kwa genome phylogenetic, wachibale wapafupi kwambiri ndi ASxL5T ndi Thalassolituus sp. C2-1, T. marinus, T. oleivorans. Ndipo O. kriegii 23, 24, 25, 26, 27. Thalassolituus ndi membala wa marine obligate hydrocarbon fragmentation bacteria (OHCB), yomwe ili ponseponse m'madera a m'nyanja ndi padziko lapansi, ndipo nthawi zambiri imakhala yaikulu pambuyo pa zochitika zowonongeka kwa hydrocarbon30,31. Mabakiteriya a m'nyanja sali mamembala a gulu la OHCB, koma ali olekanitsidwa ndi chilengedwe cha m'nyanja.
Deta ya phenotypic ikuwonetsa kuti ASxL5T ndi mtundu watsopano komanso membala wamtundu womwe sunadziwike m'banja la marine spirospiraceae. Pakali pano palibe mulingo womveka bwino woyika mitundu yatsopano yodzipatula kukhala mtundu watsopano. Mayesero apangidwa kuti adziwe malire amtundu wapadziko lonse lapansi, mwachitsanzo, kutengera kuchuluka kwa genome ya protein yoteteza (POCP), tikulimbikitsidwa kuti mtengo wodulidwa ukhale 50% wofanana ndi mtundu33. Ena amati agwiritse ntchito mfundo za AAI, zomwe zili ndi zabwino kuposa POCP chifukwa zitha kupezeka kuchokera ku ma genomes34 osakwanira. Wolembayo amakhulupirira kuti ngati mtengo wa AAI uli wochepera 74% poyerekeza ndi mtundu wamtundu wamtundu wamtunduwu, zovutazo zimayimira mtundu wina. Mtundu wa chitsanzo mu marine spirillaceae ndi marine spirillum, ndipo mtundu wa chitsanzo ndi O. linum ATCC 11336T. Mtengo wa AAI pakati pa ASxL5T ndi O. linum ATCC 11336T ndi 54.34%, ndipo mtengo wa AAI pakati pa ASxL5T ndi T. oleivorans MIL-1T (mitundu yamtundu wamtundu) ndi 67.61%, kusonyeza kuti ASxL5T ikuyimira mtundu watsopano wosiyana ndi Thalassolituus. Pogwiritsa ntchito mndandanda wa jini wa 16S rRNA monga muyeso wamagulu, malire omwe akuperekedwa ndi 94.5% 35. ASxL5T ikhoza kuyikidwa mumtundu wa Thalassolituus, kusonyeza 95.03% 16S rRNA sequence identity ndi T. oleivorans MIL-1T ndi 96.17%. Mtengo wa IMCC1826T. Komabe, idzayikidwanso mu mtundu wa Bacteroides womwe uli ndi 94.64% 16S rRNA gene gene ndi B. sanyensis NV9, kusonyeza kuti kugwiritsa ntchito jini imodzi monga jini ya 16S rRNA kungayambitse kugawidwa mopanda malire ndi ntchito. Njira ina yomwe ikugwiritsidwa ntchito imagwiritsa ntchito ANI ndi Genome Alignment Score (AF) kuti iwunikire kusanjika kwa ma data kuchokera kumitundu yonse ndi mitundu yosakhala yamitundu yomwe ilipo. Wolemba amalimbikitsa kuphatikizira malire amtundu ndi malo osinthira amtundu womwe akuyerekezeredwa ndi msonkho womwe ukuwunikidwa. Komabe, ngati palibe ma genome okwanira okwanira kuchokera ku Thalassolituus isolates, ndizosatheka kudziwa ngati ASxL5T ndi ya mtundu wa Thalassolituus ndi njira iyi. Chifukwa cha kupezeka kochepa kwa mndandanda wathunthu wa ma genome kuti awunikenso, mtengo wonse wa genome phylogenetic uyenera kutanthauziridwa mosamala. Kachiwiri, njira zonse zofananira ma genome sizingawerengere kusiyana kwakukulu mu kukula kwa ma genome oyerekeza. Iwo anayeza kufanana kwa majini osungidwa amtundu umodzi wamtundu umodzi pakati pa mitundu yofananira, koma sanaganizire kuchuluka kwa majini omwe sapezeka mu genome yaying'ono kwambiri ya ASxL5T. Mwachiwonekere, ASxL5T ndi magulu kuphatikizapo Thalassolituus, Oceanobacter, ndi Bacterioplanes ali ndi kholo limodzi, koma chisinthiko chatenga njira yosiyana, zomwe zimabweretsa kuchepa kwa majeremusi, zomwe zingakhale zogwirizana ndi moyo wolusa. Izi zikusiyana ndi T. oleivorans MIL-1T, yomwe ndi 28% yokulirapo ndipo yasintha pansi pa zovuta zosiyanasiyana za chilengedwe kuti igwiritse ntchito ma hydrocarbons23,30. Kuyerekeza kosangalatsa kungapangidwe ndi tizirombo tomwe timayendera m'kati mwa maselo ndi ma symbionts, monga Rickettsia, Chlamydia, ndi Buchnera. Kukula kwawo kwa genome ndi pafupifupi 1 Mb. Kutha kugwiritsa ntchito ma cell cell metabolites kumabweretsa kutayika kwa majini, kotero Kuwonongeka kwakukulu kwa genomic. Kusintha kwachisinthiko kuchokera ku zamoyo zam'madzi zam'madzi kupita ku moyo wolusa kungapangitse kuchepetsedwa kofanana kwa kukula kwa ma genome. Kusanthula kwa COG ndi KEGG kukuwonetsa kuchuluka kwa majini omwe amagwiritsidwa ntchito pazinthu zinazake komanso kusiyana kwapadziko lonse lapansi panjira zama genomic pakati pa ASxL5T ndi T. oleivorans MIL-1T, zomwe sizili chifukwa cha kupezeka kwazinthu zamtundu wa mafoni. Kusiyana kwa chiŵerengero cha G + C cha genome yonse ya ASxL5T ndi 56.1%, ndipo ya T. oleivorans MIL-1T ndi 46.6%, zomwe zimasonyezanso kuti zimagawidwa.
Kuwunika kwazomwe zili mumtundu wa ASxL5T kumapereka chidziwitso chogwira ntchito pamakhalidwe a phenotypic. Kukhalapo kwa ma jini amtundu wa IV fimbriae (Tfp) ndikosangalatsa kwambiri chifukwa amalimbikitsa kuyenda kwa maselo, kotchedwa social gliding kapena convulsions, popanda flagella pamwamba. Malinga ndi malipoti, Tfp ili ndi ntchito zina, kuphatikiza predation, pathogenesis, biofilm mapangidwe, kutengeka kwachilengedwe kwa DNA, kuphatikiza ma cell ndi chitukuko38. Genome ya ASxL5T ili ndi majini 18 otsekera diguanylate cyclase (enzyme yomwe imathandizira kusintha kwa 2 guanosine triphosphate kukhala guanosine 2 phosphate ndi cyclic diGMP) ndi majini 6 omwe amasunga diguanylate cyclase phosphate diguanylate. Jini ya esterase (yoyambitsa kuwonongeka kwa cyclic di-GMP kukhala guanosine monophosphate) ndiyosangalatsa chifukwa cycl-di-GMP ndi mthenga wachiwiri wofunikira wokhudzidwa ndi chitukuko cha biofilm ndi kulekanitsa, kusuntha, kulumikizidwa kwa cell ndi virulence 39, 40 munjirayi. Tiyeneranso kukumbukira kuti mu Bdellovibrio bacteriovorus, cyclic double GMP yasonyezedwa kuti imayendetsa kusintha pakati pa moyo waufulu ndi moyo wolanda41.
Kafukufuku wambiri wokhudzana ndi mabakiteriya olusa ayang'ana kwambiri zamoyo za Bdellovibrio, Bdellovibrio, ndi mitundu ya Myxococcus. Izi ndi zitsanzo zina zodziwika za mabakiteriya olusa zimapanga magulu osiyanasiyana. Ngakhale kusiyanasiyana kumeneku, magulu amagulu a mapuloteni omwe amasonyeza ma phenotypes a 11 omwe amadziwika kuti ndi mabakiteriya owononga adadziwika3,22. Komabe, ma jini okha omwe amalemba O antigen ligase (waaL) adadziwika, omwe amapezeka makamaka mu mabakiteriya a Gram-negative. Kusanthula uku sikuthandiza posankha ASxL5T ngati nyama yolusa, mwina chifukwa imagwiritsa ntchito njira yowukira. Kupezeka kwa mitundu yosiyanasiyana ya mabakiteriya olusa kudzathandizira kupanga kusanthula kwabwinoko komwe kumatengera umboni wa kusiyana kwa magwiridwe antchito ndi chilengedwe pakati pa mamembala. Zitsanzo za mabakiteriya olusa omwe sanaphatikizidwe pakuwunikaku akuphatikizapo mamembala a Cupriavidus necator42 ndi Bradymonabacteria43, chifukwa ofufuza akamafufuza madera osiyanasiyana a tizilombo tating'onoting'ono, misonkho yambiri yolusa imakhazikitsidwa.
Chodziwika kwambiri cha mabakiteriya a ASxL5T ogwidwa ndi chithunzi cha TEM ndi mawonekedwe ake apadera komanso osinthika, omwe amatha kulimbikitsa kuyanjana ndi mabakiteriya odya nyama. Mtundu wa kuyanjana womwe umawonedwa ndi wosiyana ndi mabakiteriya ena olusa ndipo sanapezekepo kale kapena kunenedwa. Zomwe zikuyembekezeredwa za ASxL5T zamoyo zolusa zikuwonetsedwa mu Chithunzi 5. Pali zitsanzo zochepa m'mabuku omwe ali ndi mawonekedwe a apical ofanana monga tikunenera pano, koma zitsanzo izi zikuphatikiza Terasakiispira papahanaumokuakeensis, bakiteriya wa spirillum wam'madzi wokhala ndi kukula kwanthawi yayitali 44, ndi Alphaproteobacteria, pusillasa. , omwe kale anali a mtundu wa Oceanospirillum, kuwonetsa Zomwe zimatchedwa "filimu ya polar" 45. Mitundu ya Cocci nthawi zambiri imawonedwa m'zikhalidwe zakale, makamaka kwa mabakiteriya omwe ali ndi mawonekedwe opindika, monga Vibrio, Campylobacter, ndi Helicobacter 46, 47, 48, yomwe ingaimire dziko lowonongeka. Ntchito ina ikufunika kuti tifotokoze bwino momwe mabakiteriya a ASxL5T amakhalira. Kudziwa momwe imagwirira ndi kupha, komanso ngati ma genome ake amaphatikiza zinthu zomwe zimagwira ntchito pazamankhwala kapena zamankhwala.
Kufotokozera kwa Venatorbacter gen. November Venatorbacter (Ven.a.tor, ba'c.ter, L. amapangidwa ndi ma venator ochokera ku L. n. venator,'hunter' ndi Gr. n. bacter,'a rod'. Venatorbacter,'a hunting Rod' . Maselo ndi aerobic, kusanja mchere, zopindika zopindika, masewera olimbitsa thupi. kudziunjikira mu kutentha kwa 4 mpaka 42 °C Incrown Mtundu wa pH wa 4-9 ndi wachilendo Mu nkhono za m'madzi, zambiri zimakhala zosagwirizana ndi acidic acids C16: 1ω6c ndi / kapena C16: 1ω7c. C16:0 ndi C18:1ω9 ; 3-OH amapezeka ngati ma hydroxy fatty acids Sakula muzofalitsa za DNA G + C ndi 56.1 mol malo a mtundu uwu ali m'banja.
Kufotokozera kwa Venatorbacter cuculus sp. November Venatorbacter cucullus (cu'cull.us.; L. n. cucullus amatanthauza chilungamo).
Kuphatikiza apo, mawonekedwe ofotokozera amtunduwu ndikuti akakula pa BA kapena BHI, ma cell amakhala 1.63 µm kutalika ndi 0.37 µm m'lifupi. Magulu a BHI agar ndi ochepa kwambiri, amafika 2 mm m'mimba mwake pambuyo pa maola 72. Iwo ndi beige, translucent, kuzungulira, convex ndi chonyezimira. Mamembala amtunduwu amatha kugwiritsa ntchito Escherichia coli ndi Klebsiella. Campylobacter ndi mabakiteriya ena angapo a Gram-negative amakhala ngati nyama.
Mitundu yodziwika bwino ya ASxL5T idasiyanitsidwa ndi mkaka wa ng'ombe ku Nottinghamshire, UK, ndikuyikidwa mu National Type Culture Collection (UK): nambala yolowa NCTC 14397 ndi Netherlands Bacterial Culture Collection (NCCB) nambala yolowa NCCB 100775. Mndandanda wathunthu wa genome wa ASxL5T yaikidwa ku Genbank malinga ndi kuwonjezera kwa CP046056.
Mabakiteriya a ASxL5T adalekanitsidwa ndi mkaka wa ng'ombe pogwiritsa ntchito ukadaulo wodzipatula wa phage9,49. Slurry adasungunuka 1:9 (w/v) mu SM buffer (50 mM Tris-HCl [pH 7.5], 0.1 M NaCl, 8 mM MgSO4.7H2O ndi 0.01% gelatin; Sigma Aldrich, Gillingham, UK), Kenako pa 4°C kwa maola 24, kumazungulira pang'onopang'ono kuthamangitsa adani kuti alowe m'malo otetezedwa. Kuyimitsidwa kunali centrifuged pa 3000g kwa mphindi 3. The supernatant anasonkhanitsidwa ndi centrifuged pa 13,000g kachiwiri kwa mphindi 5. Chodabwitsacho chinadutsa mu sefa ya membrane ya 0.45 µm (Minisart; Sartorius, Gottingen, Germany) ndi 0.2 µm membrane fyuluta (Minisart) kuchotsa maselo a bakiteriya otsala. ASxL5T ikhoza kudutsa zosefera izi. Udzu wofewa wa agar wa Campylobacter enterosus S12 (NCBI accession number CP040464) kuchokera ku slurry womwewo unakonzedwa pogwiritsa ntchito njira zokhazikika. The slurry osefedwa anagawira aliyense wa mbale ma cell awa mu 10 µl madontho katatu ndi kuloledwa kuti ziume. Mbaleyi inayikidwa mu tanka ya microaerophilic pa 37 ° C kwa maola 48 pansi pa mikhalidwe ya microaerobic (5% O2, 5% H2, 10% CO2, ndi 80% N2). Zolemba zowonekera zomwe zidapezeka zidatulutsidwa mu SM buffer ndikusamutsira ku udzu watsopano wa C. hyointestinalis S12 kuti afalitse zamoyo zotchedwa lysed. Zikatsimikiziridwa kuti mabakiteriya ndi omwe amachititsa lytic plaque osati phage, yesetsani kukulitsa zamoyo popanda wolandirayo ndikuziwonetsanso. Chikhalidwe cha aerobic chinachitidwa pa 37 ° C ndi 5% v / v defibrinated horse blood (TCS Biosciences Lt, Buckingham, UK, supplement). Malinga ndi malangizo a National Clinical Standards Committee, njira ya diffusion ya disc imagwiritsidwa ntchito poyesa antibacterial susceptibility. BHI agar idalimidwa pa 37 °C pogwiritsa ntchito diski yomwe ili ndi maantibayotiki otsatirawa (Oxoid) ya chikhalidwe cha aerobic: amoxicillin ndi clavulanic acid 30 µg; cefotaxime 30 g; streptomycin 10 g; ciprofloxacin 5 g; Ceftazidime 30 µg Nalidixic asidi 30 µg; Imipenem 10 µg; Azithromycin 15 g; Chloramphenicol 30 g; Cefoxitin 30 g; Tetracycline 30 μg; Nitrofurantoin 300 g; Aztreonam 30 µg; Ampicillin 10 g; Cefpodoxime 10 g; Trimethoprim-Sulfamethoxazole 25 µg. Kulekerera kwa mchere kunakhazikitsidwa ndi aerobic incubation pa BHI agar mbale pa 37 °C. NaCl yowonjezera idawonjezedwa pama mbale a BHI agar kuti apereke kuchuluka kwa 10% w/v. Mtundu wa pH umatsimikiziridwa ndi chikhalidwe cha aerobic pa BHI agar mbale pa 37 ° C, pomwe pH yasintha kukhala pakati pa 4 ndi 9 ndi HCl wosabala kapena NaOH wosabala, ndipo pH yomwe mukufuna imatsimikiziridwa musanathire mbale. Pakuwunika kwamafuta amtundu wa cell, ASxL5T idakulitsidwa pa BHI agar kwa masiku atatu ndi aerobic pa 37 °C. Malinga ndi MIDI (Sherlock Microbial Identification System, version 6.10) standard protocol ya FERA Science Ltd, (York, UK), mafuta acids a cell adatulutsidwa, kukonzedwa ndikuwunikidwa.
Kwa TEM, ASxL5T inatukulidwa aerobic pofalitsa mofanana pa BA pa 37 ° C kwa maola 24, ndiyeno kukolola mu 1 ml ya 3% (v / v) glutaraldehyde mu 0.1 M cacodylate buffer kutentha kwa firiji Konzani kwa ola la 1, kenako centrifuge pa 10,000 g kwa mphindi zitatu. Kenako imitsaninso pang'onopang'ono pellet mu 600 μl 0.1 M cacodylate buffer. Tumizani kuyimitsidwa kwa ASxL5T kokhazikika ku Formvar/filimu ya kaboni pa gridi yamkuwa ya ma mesh 200. Mabakiteriyawo anali odetsedwa ndi 0.5% (w/v) uranyl acetate kwa mphindi imodzi ndipo amafufuzidwa ndi TEM pogwiritsa ntchito microscope ya TEI Tecnai G2 12 Biotwin. Monga tafotokozera pamwambapa, phatikizani chiwerengero chofanana cha nyama ndi nyama yodya nyama mu NZCYM msuzi (BD Difco™, Fisher Scientific UK Ltd, Loughborough) ndi kuumitsa kwa maola 48 pansi pa mikhalidwe ya microaerobic ya Campylobacter kapena Campylobacter pa 37 ° C , Kuyanjana kwa nyama zolusa ndi nyama. idawunikidwanso ndi TEM. Matenda a Aerobic a Escherichia coli. Unikani paokha mabakiteriya odya nyama ndi olanda kuti muwone kusintha kulikonse kwa ma cell morphology chifukwa chakupha. Njira yakuda ya ku Sudan idagwiritsidwa ntchito popanga ma microscope a PHB.
Kulitsani zikhalidwe za ASxL5T usiku umodzi popaka kukula pa BHI kapena BA mbale ndi swab wosabala. Sonkhanitsani ma cell a ASxL5T ndikuwayimitsa mu MRD (CM0733, Oxoid), ndiyeno muwaike pa 4 ° C kwa masiku 7 kuti asafe ndi njala. Chidziwitso cha NCTC kapena ma laboratory chikhalidwe cha bakiteriya chinalowetsedwa mu msuzi wa BHI kapena No. 2 msuzi wa zakudya (CM007, Oxoid), wopangidwa ndi usiku umodzi, centrifuged pa 13,000g ndipo anatsitsimutsidwa mu MRD mpaka OD600 inali 0.4. Chikhalidwe: Bacillus subtilis NCTC 3610T, Citrobacter freundii NCTC 9750T, Enterobacter aerogenes NCTC 10006T, Enterococcus faecalis NCTC 775T, Escherichia coli NCTC 86, Klebsiella 1uTC1co6 Oxysto6 10817. NCTC 10861, Staphylococcus aureus NCTC 8532T, Streptococcus pneumoniae NCTC 7465T, Yersinia enterocolitica NCTC 10460. The Campylobacter host host anali microaerobically incubated pa BA mbale pa 37 ° C ndi suspendedMNZCY suspended. Magulu oyesedwa a Campylobacter ndi: 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... Sungani ma cell mu MRD, centrifuge pa 13,000g ndikuyimitsanso mu MRD mpaka OD600 ndi 0.4. Onjezani aliquot ya kuyimitsidwa kwa 0.5 ml ku 5 ml yosungunuka NZCYM pamwamba agar (0.6% agar) ndikutsanulira pa 1.2% NZCYM pansi mbale. Pambuyo pochiritsa ndikuumitsa, ASxL5T yosungunuka idagawidwa ngati madontho 20 µl pa bolodi lililonse la udzu katatu. Kutentha kwa chikhalidwe ndi mlengalenga zimadalira zofunikira za mabakiteriya oyesa.
Gwiritsani ntchito GenElute ™ Bacterial Genomic DNA Kit (Sigma Aldridge) kuti mukonzekere DNA kuchokera kumagulu a mabakiteriya. Njira zokhazikika zidagwiritsidwa ntchito pakukulitsa kwa PCR kwa jini ya 16S rRNA ndikutsimikiza kutsata kwazinthu pogwiritsa ntchito chemistry yothetsa utoto (Eurofins Value Read Service, Germany). Gwiritsani ntchito pulogalamu ya BLAST-N kuti mufananize zotsatizanazi ndi ma gene 16S rRNA kuti muzindikire ndikusonkhanitsa mitundu yofananira. Izi zimalumikizidwa pogwiritsa ntchito ClustalW mu pulogalamu ya MEGA X. Mtengo wa phylogenetic unamangidwanso pogwiritsa ntchito MEGA X pogwiritsa ntchito njira yowonjezereka yotengera chitsanzo cha Tamura-Nei, ndi makope otsogolera 100054. Gwiritsani ntchito PureLink™ Genomic DNA Kit (Fisher Scientific, Loughborough, UK) kuti mutenge DNA kuti mutengere ma genome onse. Mayendedwe a genome a ASxL5T adatsimikiziridwa pogwiritsa ntchito kuphatikiza kwa Illumina MiSeq, komwe kumakhala ndi ma 250 bp owerengedwa kawiri opangidwa ndi laibulale yokonzedwa pogwiritsa ntchito zida zolembera za Nextera ndi 2 mpaka 20 kb yowerengera kutalika kuchokera papulatifomu ya PacBio. Genomics DNA Sequencing Research Facility ku Sembia University. Ma genome adasonkhanitsidwa pogwiritsa ntchito CLC Genomics Workbench 12.0.3 (Qiagen, Aarhus, Denmark). Zikhalidwe za ASxL5T zimayikidwa mu National Type Culture Collection (UK) ndi Netherlands Bacterial Culture Collection (NCCB). Ma genome a zamoyo zofananira zomwe zimagwiritsidwa ntchito poyerekezera ndi: Thalassolituus oleivorans MIL-1T (nambala yolumikizira HF680312, yokwanira); Bacterioplanes sanyensis KCTC 32220T (nambala yolowera BMYY01000001, yosakwanira); Oceanobacter kriegii DSM 6294T (nambala yolowera NZ_AUGV00000000, yosakwanira); Marinamonas community DSM 5604T (yowonjezera ASM436330v1, yosakwanira), Oceanospirullum linum ATCC 11336T (yowonjezera MTSD02000001, yosakwanira) ndi Thalassolituus sp. C2-1 (onjezani NZ_VNIL01000001, yosakwanira). Gwiritsani ntchito JGI Genome Portal36 pa https://img.jgi.doe.gov//cgi-bin/mer/main.cgi?section=ANI&page= kuti mudziwe kuchuluka kwa ma alignment (AF) ndi nucleic acid identity (ANI). Awiriawiri. Njira ya Rodriguez-R & Konstantinidis55 idagwiritsidwa ntchito pozindikira amino acid (AAI). Gwiritsani ntchito GToTree 1.5.5411,12,13,14,15,16,17,18 kuti mupange mtengo woyerekeza kwambiri wa phylogenetic. Ma genome oyimira ma genome omwe alipo amasankhidwa kuchokera kumitundu yodziwika kuti ikugwirizana ndi ASxL5T kuchokera ku 16S rRNA phylogeny. Tchulani mtengo pogwiritsa ntchito chida cha intaneti cha mtengo wa moyo (https://itol.embl.de/). Kufotokozera ndi kusanthula kwa ASxL5T genome kumachitika pogwiritsa ntchito chida chapaintaneti cha BlastKOALA KEGG pogwiritsa ntchito KEGG (Kyoto Encyclopedia of Genes and Genomes) yolemeretsa gawo. Kugawidwa kwa magulu a COG (magulu a orthologous) kumatsimikiziridwa pogwiritsa ntchito chida cha intaneti cha eggNOG-mapper.
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