Ua hoʻokaʻawale ʻia kekahi ʻano hou o ka Gram-negative, aerobic, paʻakai-tolerant, active, rod-shaped, and predatory bacteria ASxL5T mai kahi loko wai lepo bipi ma Nottinghamshire, ʻEnelani, a ua hoʻohana ʻo Campylobacter i kāna mea pio. Ma hope mai, ua ʻike ʻia nā ʻano ʻano Campylobacter ʻē aʻe a me nā lālā o ka ʻohana Enterobacteriaceae i mea pio. Ma hope o ka subculture me ka ʻole o nā cell host, ua loaʻa ka ulu aseptic nāwaliwali ma Brain Heart Infusion Agar. ʻO nā kūlana ulu maikaʻi loa he 37 °C a ʻo ka pH he 7. Hōʻike ʻia ka microscopy electron transmission i kekahi mau hiʻohiʻona morphological maʻamau e pili ana i ka loaʻa ʻana o ka mea pio. Hōʻike ʻia ka loiloi phylogenetic me ka 16S rRNA gene sequence e pili ana ka mea hoʻokaʻawale i kahi lālā o ka ʻohana Marine Spirulina, akā ʻaʻole hiki ke hoʻokaʻawale ʻia ma ke ʻano he lālā o kekahi ʻano genus i ʻike ʻia. Ua hōʻoia ka holoʻokoʻa-genome sequencing o ASxL5T i ka pilina me nā lālā o nā spirochetes moana. Ua hōʻike ʻia kahi hulina waihona ʻikepili he nui nā ASxL5T e kaʻana like ana i nā kaʻina gene 16S rRNA me kekahi mau bacteria i kanu ʻole ʻia mai ke kai, ka ʻāina a me ka wai lepo. Manaʻo mākou e hōʻike ana ke kānana ASxL5T i kahi ʻano hou i loko o kahi genus hou. Paipai mākou i ka inoa Venatorbacter cucullus gen. Nowemapa, sp. I Nowemapa, ua hoʻohana ʻia ʻo ASxL5T e like me ke ʻano ʻano.
ʻO ka bacteria predatory he bacteria e hōʻike ana i ka hiki ke hahai a pepehi i nā hua bacteria ola ʻē aʻe e loaʻa ai nā mea biosynthetic a me ka ikehu. He ʻokoʻa kēia mai ka hoʻihoʻi maʻamau o nā meaʻai mai nā microorganism make, a he ʻokoʻa hoʻi ia me nā pilina parasitic, kahi i hana ai ka bacteria i kahi pilina pili me kā lākou mea hoʻokipa me ka pepehi ʻole iā lākou. Ua hoʻololi ka bacteria predatory i nā pōʻai ola like ʻole e hoʻohana pono i nā kumu meaʻai nui ma nā niches kahi i loaʻa ai (e like me nā wahi noho kai). He hui ʻokoʻa ʻauhau lākou, kahi i hoʻopili wale ʻia e kā lākou ʻokoʻa ola sterilization kūikawā1. Ua loaʻa nā laʻana o ka bacteria predatory i loko o nā phyla like ʻole, e like me: Proteobacteria, Bacteroides, a me Chlorella.3. Eia nō naʻe, ʻo ka bacteria predatory maikaʻi loa i aʻo ʻia ʻo ia ka Bdelovibrio a me Bdellovibrio-and-like organisms (BALOs4). ʻO ka bacteria predatory kahi kumu hoʻohiki o nā pūhui hana olaola hou a me nā mea antibacterial5.
Manaʻo ʻia nā bacteria predatory e hoʻonui i ka ʻokoʻa microbial a loaʻa i kahi hopena maikaʻi i ke olakino kaiaola, huahana a me ke kūpaʻa6. ʻOiai kēia mau hiʻohiʻona maikaʻi, he liʻiliʻi nā haʻawina e pili ana i ka bacteria predatory hou ma muli o ka paʻakikī o ka hoʻoulu ʻana i ka bacteria a me ka pono e nānā pono i nā pilina cell e hoʻomaopopo i ko lākou mau pōʻai ola paʻakikī. ʻAʻole maʻalahi ka loaʻa ʻana o kēia ʻike mai ka loiloi kamepiula.
I ka wā o ka hoʻonui ʻana i ka pale antimicrobial, ke aʻo ʻia nei nā hoʻolālā hou no ka huli ʻana i nā pathogen bacteria, e like me ka hoʻohana ʻana i nā bacteriophages a me nā bacteria predatory7,8. Ua hoʻokaʻawale ʻia ka bacteria ASxL5T i ka makahiki 2019 me ka hoʻohana ʻana i ka ʻenehana hoʻokaʻawale phage mai ka lepo bipi i hōʻiliʻili ʻia mai ka Dairy Center o ke Kulanui o Nottingham, Nottinghamshire. ʻO ke kumu o ka hoʻokolokolo ʻana, ʻo ia ka hoʻokaʻawale ʻana i nā mea ola i hiki ke lilo i mau mea hoʻomalu olaola. ʻO Campylobacter hyointestinalis kahi pathogen zoonotic, e pili nui ana me nā maʻi ʻōpū kanaka10. Loaʻa ia ma nā wahi āpau i ka serum a hoʻohana ʻia e like me ka host host.
Ua hoʻokaʻawale ʻia ka maʻi maʻi ASxL5T mai ka meli pipi no ka mea ua ʻike ʻia ua like nā plaques i hana ʻia ma ka lau o C. hyointestinalis me nā mea i hana ʻia e nā bacteriophages. He mea i manaʻo ʻole ʻia kēia, no ka mea, ʻo kahi ʻāpana o ke kaʻina kaʻawale phage e pili ana i ka kānana ma o kahi kānana 0.2 µm, i hoʻolālā ʻia e wehe i nā cell bacteria. ʻO ka nānā ʻana i ka microscopic o nā mea i unuhi ʻia mai ka plaque i hōʻike ʻia ʻaʻole i hōʻuluʻulu ka polyhydroxybutyrate (PHB) ka hua bacteria liʻiliʻi i ke ʻano o ke koʻokoʻo curved gram-negative. Hoʻomaopopo ʻia ka moʻomeheu aseptic kūʻokoʻa i nā cell prey ma ke ʻano paʻa paʻa (e like me ka brain heart infusion agar (BHI) a me ke koko agar (BA)), a nāwaliwali kona ulu ʻana. Loaʻa ia ma hope o ka subculture me ka hoʻomaikaʻi ʻana i ka inoculum kaumaha. Ua ulu like ia ma lalo o ka microaerobic (7% v/v oxygen) a me nā kūlana oxygen o ka lewa, akā ʻaʻole i loko o kahi ea anaerobic. Ma hope o 72 mau hola, ua liʻiliʻi loa ke anawaena o ke kolo, hiki i ka 2 mm, a he beige, translucent, poepoe, convex a ʻālohilohi. Hoʻopilikia ʻia ka hoʻāʻo biochemical maʻamau no ka mea ʻaʻole hiki ke hoʻomaʻamaʻa hilinaʻi ʻia ʻo ASxL5T i loko o ka media wai, e hōʻike ana e hilinaʻi paha ia i ka pōʻai ola paʻakikī o ka hoʻokumu ʻana i ka biofilm. Eia naʻe, ua hōʻike ʻia ka hoʻokuʻu ʻana o ka pā i ka ASxL5T he aerobic, maikaʻi no ka oxidase a me ka catalase, a hiki ke ʻae i ka 5% NaCl. Kūpaʻa ʻo ASxL5T i ka 10 µg streptomycin, akā paʻakikī i nā lāʻau ʻokoʻa ʻē aʻe i hoʻāʻo ʻia. Ua nānā ʻia nā cell bacteria ASxL5T e TEM (Figure 1). Ke ulu me ka ʻole o nā pūnaewele pio ma ka BA, he Campylobacter liʻiliʻi nā pūnaewele ASxL5T, me ka lōʻihi o 1.63 μm (± 0.4), ka laulā o 0.37 μm (± 0.08), a me ka lōʻihi hoʻokahi (a hiki i ka 5 μm) pole. Ka hae moe kolohe. Ma kahi o 1.6% o nā pūnaewele e ʻike ʻia he liʻiliʻi ka laulā ma mua o 0.2 μm, kahi e hiki ai ke hele i loko o ka mea kānana. Ua ʻike ʻia kahi hoʻonui ʻokoʻa ma luna o kekahi mau cell, e like me ka fairing (Latin cucullus) (e ʻike i nā pua ma 1D, E, G). Me he mea lā ua hoʻokumu ʻia kēia me ka membrane o waho, ʻo ia paha ma muli o ka emi wikiwiki ʻana o ka nui o ka envelop periplasmic, ʻoiai e paʻa mau ana ka membrane o waho, e hōʻike ana i kahi hiʻohiʻona "loose". ʻO ka moʻomeheu ASxL5T me ka loaʻa ʻole o nā meaʻai (ma PBS) no ka manawa lōʻihi ma 4 ° C ka hopena i ka hapa nui (akā ʻaʻole nā ​​​​mea āpau) e hōʻike ana i kahi morphology coccal (Figure 1C). Ke ulu ʻo ASxL5T me Campylobacter jejuni ma ke ʻano he pio no 48 mau hola, ʻoi aku ka lōʻihi a ʻoi aku ka lōʻihi o ka nui o ka cell cell ma mua o nā cell i ulu me ka ʻole o ka host (Papa 1 a me Figure 1E). ʻO ka ʻokoʻa, ke ulu ʻo ASxL5T me E. coli ma ke ʻano he 48 hola, ʻoi aku ka lōʻihi o ka nui o ka cell cell ma mua o ka ulu ʻana me ka ʻole o ka mea pio (Papa 1), a ʻokoʻa ka lōʻihi o ka cell, ma ke ʻano he filamentous (Figure 1F). Ke hoʻokomo ʻia me Campylobacter jejuni a i ʻole E. coli ma ke ʻano he pio no 48 mau hola, ʻaʻole hōʻike ʻia nā pūnaewele ASxL5T ʻaʻohe flagella. Hōʻuluʻulu ka Papa 1 i nā ʻike o ka hoʻololi ʻana i ka nui o ke kelepona ma muli o ke ʻano, ka ʻole, a me ke ʻano pio o ASxL5T.
Hōʻike TEM o ASx5LT: (A) Hōʻike ka ASx5LT i ka hahau lōʻihi; (B) pākaukau maʻamau ASx5LT; (C) cocci ASx5LT pūnaewele ma hope o ka lōʻihi incubation me ka meaʻai ʻole; (D) hōʻike ka pūʻulu o nā pūnaewele ASx5LT i nā mea ʻino (E) hōʻike pū ʻia ka pūʻulu pūnaewele ASx5LT i hoʻomoʻa ʻia me Campylobacter pio i hoʻonui ʻia ka lōʻihi o ke kelepona i hoʻohālikelike ʻia me ka poʻe ʻaʻole ulu pio (D) hōʻike pū i ke ʻano apical; (F) Large Filamentous flagella, ASx5LT cell, after incubation with E. coli pio; (G) Hoʻokahi pūnaewele ASx5LT ma hope o ka hoʻoulu ʻana me E. coli, e hōʻike ana i kahi ʻano maʻamau o luna. He 1 μm ka pā.
ʻO ka hoʻoholo ʻana i ke kaʻina 16S rRNA gene sequence (helu komo MT636545.1) hiki i ka huli ʻikepili ke hoʻokumu i nā ʻano like me nā mea i loko o ka papa Gammaproteobacteria, a kokoke loa i ka bacteria moana i loko o ka ʻohana spirillum moana (Figure 2), a he lālā ia o ka genus Thalassolituus. ʻO ka pili pili loa iā Marine Bacillus. He ʻokoʻa loa ke kaʻina gene 16S rRNA mai ka bacteria predatory no ka ʻohana Bdelvibrionaceae (Deltaproteobacteria). ʻO nā hoʻohālikelike pālua o B. bacteriovorus HD100T (type strain, DSM 50701) a me B. bacteriovorus DM11A he 48.4% a me 47.7%, a no B. exovorus JSS he 46.7%. Loaʻa i ka bacteria ASxL5T he 3 kope o ka gene 16S rRNA, ʻelua o ia mau mea like kekahi me kekahi, a ʻo ke kolu he 3 kumu kaʻawale. ʻElua mau mea hoʻokaʻawale bacteria predatory (ASx5S a me ASx5O; 16S rRNA gene accession numbers he MT636546.1 a me MT636547.1, kēlā me kēia) me nā ʻano morphological a me nā phenotypic like ʻole mai ka wahi hoʻokahi, akā ʻokoʻa lākou mai ka ASxL5T a me ka Bacterial uncultured. Hoʻopili pū ʻia nā ʻōkuhi waihona me nā ʻano ʻē aʻe ma Oceanospirillaceae (Figure 2). Ua hoʻoholo ʻia a mālama ʻia ke kaʻina genome holoʻokoʻa o ASxL5T i ka waihona NCBI, a ʻo ka helu komo ʻana ʻo CP046056. Aia ka genome o ASxL5T i kahi chromosome circular o 2,831,152 bp me ka lakio G + C o 56.1%. Aia ka genome sequence he 2653 CDS (total), a he 2567 ka mea i wānana ʻia e hoʻopili i nā protein, kahi o 1596 hiki ke hoʻonoho ʻia ma ke ʻano he hana putative (60.2%). Aia i loko o ka genome he 67 mau genes RNA-coding, me 9 rRNAs (3 kēlā me kēia no 5S, 16S, a me 23S) a me 57 tRNAs. Hoʻohālikelike ʻia nā hiʻohiʻona genomic o ASxL5T me nā genomes i loaʻa o nā ʻano o ke ʻano pili kokoke loa i ʻike ʻia mai ke kaʻina 16S rRNA gene sequence (Table 2). E hoʻohana i ka ʻike amino acid (AAI) e hoʻohālikelike i nā genomes Thalassolituus i loaʻa iā ASxL5T. ʻO ke kaʻina genome kokoke loa i loaʻa (incomplete) i hoʻoholo ʻia e AAI ʻo Thalassolituus sp. C2-1 (hoʻohui NZ_VNIL01000001). Ua hoʻokaʻawale ʻia kēia ʻāʻī mai nā sediment hohonu o ke kahawai ʻo Mariana, akā i kēia manawa ʻaʻohe ʻike phenotypic e pili ana i kēia kānana no ka hoʻohālikelike ʻana. Hoʻohālikelike ʻia me ASxL5T 2.82 Mb, ʻoi aku ka nui o ka genome o ke kino ma 4.36 Mb. ʻO ka awelika genome nui o nā spirochetes moana ma kahi o 4.16 Mb (± 1.1; n = 92 mau genomes kuhikuhi piha i noiʻi ʻia mai https://www.ncbi.nlm.nih.gov/assembly), no laila ua like ka genome o ASxL5T me ka kauoha Hoʻohālikelike ʻia me nā lālā ʻē aʻe, he liʻiliʻi loa ia. E hoʻohana i ka GToTree 1.5.54 no ka hoʻoulu ʻana i kahi kumulāʻau phylogenetic likelihood ma muli o ka genome (Figure 3A), me ka hoʻohana ʻana i nā kaʻina amino acid i hoʻopili ʻia o 172 mau ʻano kope kope hoʻokahi e pili ana iā Gammaproteobacteria 11,12,13,14,15,16, 17 ,18. Ua hōʻike ka hōʻike ʻana he pili pili loa ia me Thalassolituus, Bacterial Plane, a me Marine Bacterium. Eia naʻe, hōʻike kēia mau ʻikepili he ʻokoʻa ʻo ASxL5T mai kona ʻohana ma Marine Spirulina a loaʻa kāna ʻikepili kaʻina genome.
ʻO ka lāʻau phylogenetic e hoʻohana ana i ke kaʻina gene 16S rRNA e hōʻike ana i ke kūlana o nā kānana ASxL5T, ASxO5, a me ASxS5 (me nā ʻōpū) e pili ana i nā maʻi bacteria i kanu ʻole ʻia a me ka moana i loko o ka Marine Spirulinaceae. ʻO ka helu komo ʻana o Genbank e hahai i ka inoa strain ma nā pale. E hoʻohana iā ClustalW e hoʻoponopono i nā kaʻina, a e hoʻohana i ke ʻano likelihood kiʻekiʻe loa a me ke kumu hoʻohālike ʻo Tamura-Nei no ka hoʻomaopopo ʻana i nā pilina pili phylogenetic, a hana i nā replication alakaʻi 1000 i ka papahana MEGA X. Hōʻike ka helu ma ka lālā ua ʻoi aku ka waiwai o ke kope alakaʻi ma mua o 50%. Ua hoʻohana ʻia ʻo Escherichia coli U/541T ma ke ʻano he outgroup.
(A) He lāʻau phylogenetic e pili ana i ka genome, e hōʻike ana i ka pilina ma waena o ke kai Spirospiraceae bacterium ASxL5T a me kona mau hoahānau pili, E. coli U 5/41T ma ke ʻano he outgroup. (B) Hoʻohālikelike ʻia me T. oleivorans MIL-1T, ua wānana ʻia ka māhele hana o nā genes ma muli o ka hui orthologous (COG) pūʻulu o ka protein ASx5LT. Hōʻike ka helu ma ka hema i ka helu o nā genes i kēlā me kēia māhele COG hana i kēlā me kēia genome. Hōʻike ka pakuhi ma ka ʻākau i ka pākēneka o nā genome i loko o kēlā me kēia hui COG hana. (C) Hoʻohālikelike ʻia me T. oleiverans MIL-1T, ka ʻike ʻana o ke ala modular KEGG (Kyoto Encyclopedia of Genes and Genomes) holoʻokoʻa o ASxL5T.
Ke hoʻohana nei i ka ʻikepili KEGG e nānā i nā ʻano genes i loaʻa i ka genome ASxL5T i hōʻike i ke ala metabolic maʻamau o ka aerobic gamma Proteus. Loaʻa i ka ASxL5T ka huina o 75 mau genes i hāʻawi ʻia i nā protein motor bacterial, me nā genes i komo i ka chemotaxis, ka hui flagella, a me ke ʻano IV fimbriae system. Ma ka māhele hope loa, he 9 o 10 mau genes ke kuleana no ka neʻe ʻana o nā ʻano mea ola ʻē aʻe. Aia i loko o ka genome o ASxL5T kahi alahele biosynthetic tetrahydropyrimidine piha e komo ana i ka pane pale i ka osmotic stress20, e like me ka mea i manaʻo ʻia no nā halophiles. Loaʻa i ka genome nā ala piha he nui no nā cofactors a me nā huaora, me nā ala ala riboflavin synthesis. ʻOiai aia ka gene alkane 1-monooxygenase (alkB2) ma ASxL5T, ʻaʻole i pau ke ala hoʻohana hydrocarbon. Ma ke kaʻina genome o ASxL5T, ʻaʻole ʻike ʻia nā homologues o nā genes i ʻike ʻia ʻo ia ke kumu nui o ka hoʻohaʻahaʻa ʻana i nā hydrocarbons ma T. oleiverans MIL-1T21, e like me TOL_2658 (alkB) a me TOL_2772 (dehydrogenase waiʻona). Hōʻike ka Figure 3B i ka hoʻohālikelike ʻana o ka hāʻawi ʻana i nā gene i ka māhele COG ma waena o ASxL5T a me ka ʻaila ʻoliva MIL-1T. ʻO ka holoʻokoʻa, ʻo ka genome ASxL5T liʻiliʻi he liʻiliʻi liʻiliʻi nā genes mai kēlā me kēia māhele COG i hoʻohālikelike ʻia me ka genome pili nui. Ke hōʻike ʻia ka helu o nā genes i kēlā me kēia ʻāpana hana ma ke ʻano he pākēneka o ka genome, ʻike ʻia nā ʻokoʻa i ka pākēneka o nā genes i ka unuhi, ribosomal structure a me biogenesis categories, a me ka hana ikehu a me ka hoʻololi ʻana i nā kāwai, ʻo ia ka ASxL5T nui aʻe. genome Hoʻohālikelike ʻia ka pākēneka me ka hui like i loko o ka genome T. oleiverans MIL-1T. I ka hoʻohālikelike ʻia me ka genome ASxL5T, ʻoi aku ka nui o ka T. oleivorans MIL-1T i ka pākēneka o nā genes i ka replication, recombination a me ka hoʻoponopono ʻana, a me nā ʻano unuhi. ʻO ka mea e mahalo ai, ʻo ka ʻokoʻa nui loa o ka ʻike o kēlā me kēia māhele hana o nā genome ʻelua, ʻo ia ka helu o nā genes ʻike ʻole i loaʻa ma ASxL5T (Figure 3B). Ua hana ʻia kahi loiloi hoʻonui ʻana i nā modula KEGG, kahi i hōʻike ʻia ai kēlā me kēia module KEGG i kahi pūʻulu o nā ʻāpana hana i wehewehe lima ʻia no ka hōʻike ʻana a me ka wehewehe biological o ka ʻikepili sequence genome. Hōʻike ʻia ka hoʻohālikelike ʻana o ka hāʻawi ʻana i ke ʻano ma ke ala holoʻokoʻa KOG module o ASxL5T a me ka ʻoliva MIL-1T ma ke Kiʻi 3C. Hōʻike kēia hōʻike ʻana ʻoiai aia ka ASxL5T i ka sulfur a me ka naikokene metabolic alahele, ʻaʻole ʻo T. oleiverans MIL-1T. ʻO ka ʻokoʻa, loaʻa iā T. oleiverans MIL-1T kahi ala cysteine ​​​​a me methionine metabolic holoʻokoʻa, akā ʻaʻole i piha i ka ASxL5T. No laila, loaʻa i ka ASxL5T kahi module hiʻohiʻona no ka assimilation sulfate (i wehewehe ʻia ma ke ʻano he pūʻulu o nā genes hiki ke hoʻohana ʻia ma ke ʻano he phenotypic markers, e like me ka metabolic capacity a pathogenicity; https://www.genome.jp/kegg/module.html) I T . ʻO ka hoʻohālikelike ʻana i ka maʻiʻo o ka ASxL5T me ka papa inoa o nā genes e hōʻike ana i kahi nohona predatory ʻaʻole maopopo. ʻOiai ʻo ka gene waaL e hoʻopili ana i ka ligase e pili ana me ka O antigen polysaccharide i ke kumu i loko o ka genome ASxL5T (akā, he mea maʻamau ia i nā bacteria Gram-negative he nui), tryptophan 2,3-dioxygenase (TDO) hiki ke hoʻokomo i ka 60 amino. ʻāpana ʻakika i loaʻa pinepine ʻia i loko o nā bacteria predatory ʻaʻole i loaʻa. ʻAʻohe ʻano genes predatory ʻē aʻe i loko o ka genome ASxL5T, me kēlā mau enzyme e hoʻopili ai i ka biosynthesis isoprenoid ma ke ala mevalonate. E hoʻomaopopo ʻaʻole ʻaʻohe transcriptional regulatory gene gntR i ka pūʻulu predator i nānā ʻia, akā hiki ke ʻike ʻia ʻekolu mau genes like gntR ma ASxL5T.
Hōʻuluʻulu ʻia nā hiʻohiʻona phenotypic o ASxL5T ma ka Papa 3 a hoʻohālikelike ʻia me nā hiʻohiʻona phenotypic o nā genera pili 23, 24, 25, 26, a me 27 i hōʻike ʻia ma ka palapala. ʻO nā mea hoʻokaʻawale mai T. marinus, T. olevorans, B. sanyensis, a me Oceanobacter kriegii he mau kino koʻikoʻi koʻikoʻi, hoʻomanawanui i ka paʻakai, akā ʻaneʻane ʻaʻohe ʻano phenotypic ʻē aʻe me ASxL5T. ʻO ka awelika pH o ka moana he 8.1 (https://ocean.si.edu/ocean-life/invertebrates/ocean-acidification#section_77), i ʻike ʻia ma T. marinus, T. olevorans, B. sanyensis a me O. kriegii. Ua kūpono ʻo ASxL5T no ka pae pH nui (4-9) maʻamau o nā ʻano ʻano kai ʻole. Nā hiʻohiʻona phenotypic o Thalassolituus sp. C2-1. ʻAʻole ʻike ʻia. ʻOi aku ka laulā o ka pae wela o ka ulu ʻana o ASxL5T ma mua o ko ke kai moana (4–42 °C), ʻoiai ʻo kekahi akā ʻaʻole nā ​​​​mea hoʻokaʻawale T. marinus a pau i ka wela. ʻO ka hiki ʻole ke ulu i ka ASxL5T i loko o ka pāpaʻi broth i pale aku i ke ʻano phenotypic hou aʻe. E hoʻohana i ka API 20E no ka hoʻāʻo ʻana i nā mea i ʻoki ʻia mai ka pā BA, ONPG, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate hoʻohana, urease, tryptophan deaminase, gelatin hydrolysis Enzyme, ʻaʻohe hopena indole, acetoin a me H2S. i hanaia. ʻO nā kaʻa kaʻa ʻole i hoʻomoʻa ʻia: glucose, mannose, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin a me arabinose. Ke hoʻohālikelike ʻia me nā ʻano kikoʻī e pili ana i paʻi ʻia, hōʻike ʻia ka profile fatty acid cellular o ka ASxL5T strain ma ka Papa 4. ʻO nā ʻakika momona cellular nui ʻo C16: 1ω6c a / a i ʻole C16: 1ω7c, C16: 0 a me C18: 1ω9. ʻO nā waikawa momona hydroxy C12: 0 3-OH a me C10: 0 3-OH pū kekahi. ʻOi aku ka lākiō o C16:0 ma ASxL5T ma mua o ka waiwai i hōʻike ʻia o nā genera pili. I ka hoʻohālikelike ʻia me ka T. marinus IMCC1826TT i hōʻike ʻia, ua hoʻemi ʻia ka ratio o C18: 1ω7c a me / a i ʻole C18: 1ω6c ma ASxL5T. oleivorans MIL-1T a me O. kriegii DSM 6294T, aka, aole i ikeia ma B. sanyensis KCTC 32220T. ʻO ka hoʻohālikelike ʻana i nā kiʻina momona momona o ASxL5T a me ASxLS ua hōʻike ʻia nā ʻokoʻa liʻiliʻi o ka nui o nā ʻakika momona pākahi ma waena o nā kānana ʻelua, i kūlike me ke kaʻina DNA genomic o ka ʻano like. ʻAʻole i ʻike ʻia nā ʻāpana poly-3-hydroxybutyrate (PHB) me ka hoʻohana ʻana i ka hoʻāʻo ʻeleʻele Sudan.
Ua aʻo ʻia ka hana predation o ASxL5T bacteria e hoʻoholo i ka laulā o nā mea pio. Hiki i kēia hua bacteria ke hana i nā pāpaʻi ma nā ʻano Campylobacter, e like me: 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 a me C. upsaliensis NCTC 11541T. E hoʻohana i nā moʻomeheu i helu ʻia ma ka ʻāpana hoʻoholo hoʻokipa hoʻokipa o ke ʻano e hoʻāʻo ai i kahi ākea ākea o nā hua bacteria Gram-negative a me Gram-positive. Hōʻike nā hopena i hiki ke hoʻohana ʻia ka ASxL5T ma Escherichia coli NCTC 86 a me Citrobacter freundii NCTC 9750T. ʻO nā plaques i hanaʻia ma Klebsiella oxytoca 11466. Hōʻikeʻia ka pilina TEM me E. coli NCTC 86 ma ka Figure 4A-D, aʻo ka pilina me Campylobacter jejuni PT14 a me Campylobacter suis S12 i hōʻikeʻia ma ka Figure 4E-H waena. He ʻokoʻa ke ʻano hoʻouka kaua ma waena o nā ʻano mea pio i hoʻāʻo ʻia, me hoʻokahi a ʻoi aʻe paha nā pūnaewele E. coli i hoʻopili ʻia i kēlā me kēia cell ASxL5T a hoʻonoho ʻia ma ka ʻaoʻao o ka cell extended ma mua o ka adsorption. ʻO ka ʻokoʻa, ʻike ʻia ʻo ASxL5T e hoʻopili iā Campylobacter ma kahi o ka hoʻopili ʻana, ma ke ʻano maʻamau me ka piko o ka cell predator a kokoke i ka piko o ka cell Campylobacter (Figure 4H).
TEM e hōʻike ana i ka pilina ma waena o ASx5LT a me nā mea pio: (AD) a me E. coli pio; (EH) a me C. jejuni pio. (A) He kelepona maʻamau ASx5LT i hoʻopili ʻia i hoʻokahi cell E. coli (EC); (B) He filamentous ASx5LT i hoʻopili ʻia i hoʻokahi cell EC; (C) He pūnaewele ASx5LT filamentous i hoʻopili ʻia i nā pūnana EC lehulehu; (D) Hoʻopili i nā pūnaewele ASx5LT liʻiliʻi ma kahi pūnaewele E. coli (EC); (E) hoʻokahi pūnaewele ASx5LT i hoʻopili ʻia i kahi pūnaewele Campylobacter jejuni (CJ); (F) ASx5LT hoʻouka C. hyointestinalis (CH); (G) ʻelua Hoʻokahi pahu ASx5LT i hoʻouka i kahi pūnaewele CJ; (H) ʻO kahi hiʻohiʻona kokoke i ka helu hoʻopili ASx5LT, kokoke i ka piko o ka cell CJ (bar 0.2 μm). Hōʻike ka pā i 1 μm i (A–G).
Ua ulu ka bacteria predatory e hoʻohana i nā kumu waiwai pio. ʻIke loa, aia lākou ma nā wahi like ʻole he nui. Ma muli o ka haiki o nā lālā o ka heluna kanaka, hiki ke hoʻokaʻawale i ka bacteria ASxL5T mai ka slurry me ka hoʻohana ʻana i ke ʻano hoʻokaʻawale phage. ʻO ka pilina genomic o ASxL5T i nā lālā o ka ʻohana oceanospirillaceae o nā maʻi bacteria ma ka moana he mea kupanaha, ʻoiai ʻo ke kino ka mea hoʻomanawanui i ka paʻakai a hiki ke ulu ma kahi ʻano me 5% paʻakai. Ua hōʻike ʻia ka nānā ʻana i ka maikaʻi o ka wai o ka slurry i ka liʻiliʻi o ka sodium chloride ma lalo o 0.1%. No laila, mamao loa ka lepo mai ke kai-ma ka ʻāina a me ke kemika. ʻO ka loaʻa ʻana o ʻekolu mau mea pili akā ʻokoʻa mai kahi kumu hoʻokahi e hōʻike ana i ka ulu ʻana o kēia mau mea ʻaihue i kēia ʻano kai ʻole. Eia kekahi, hōʻike ʻia ka ʻikepili microbiome (nā waihona ʻikepili mai https://www.ebi.ac.uk/ena/browser/view/PRJEB38990) i hōʻike ʻia ʻo ka 16S rRNA gene sequence i loaʻa i ka 50 ʻauhau hana nui loa (OTU). ) Ma kekahi mau la'ana o ka lepo. Ua loaʻa kekahi mau maʻi bacteria i hoʻomaʻamaʻa ʻole ʻia i loko o ka waihona Genbank, nona nā ʻano gene 16S rRNA e like me ASxL5T bacteria. ʻO kēia mau kaʻina, me nā kaʻina o ASxL5T, ASxS5, a me ASxO5, me he mea lā e hōʻike ana i nā papa like ʻole i hoʻokaʻawale ʻia mai Thalassolituus a me Oceanobacter (Figure 2). Ua hoʻokaʻawale ʻia ʻekolu mau ʻano bacteria i hoʻomaʻamaʻa ʻole ʻia (GQ921362, GQ921357 a me GQ921396) mai ka wai māwae ma kahi hohonu o 1.3 mau kilomita i ka lua gula o ʻApelika Hema i ka makahiki 2009, a ʻo nā mea ʻelua ʻē aʻe (DQ256320 a me DQ337006) ma ʻApelika Hema. i ka makahiki 2005). ʻO ka 16S rRNA gene sequence pili loa i ka ASxL5T he māhele ia o ka 16S rRNA gene sequence i loaʻa mai ka moʻomeheu hoʻonui o nā sediments one i loaʻa mai nā kahakai o Farani akau i ka makahiki 2006 (helu helu AM29240828). Ua loaʻa mai kekahi pūnaʻi gene 16S rRNA pili pili loa mai ka bacteria uncultured HQ183822.1 mai kahi pahu hōʻiliʻili i hoʻoheheʻe ʻia mai kahi pae ʻāina kūloko ma Kina. ʻOiaʻiʻo, ʻaʻole i hōʻike nui ʻia ka bacteria ASxL5T i ka ʻikepili taxonomic, akā ʻo kēia mau kaʻina mai ka bacteria uncultured e hōʻike ana paha i nā meaola e like me ASxL5T, i laha ʻia ma ka honua holoʻokoʻa, ma nā wahi paʻakikī. Mai ka hōʻike phylogenetic genome holoʻokoʻa, ʻo ka pili kokoke loa iā ASxL5T ʻo Thalassolituus sp. C2-1, T. marinus, T. oleivorans. A ʻo O. kriegii 23, 24, 25, 26, 27. ʻO Thalassolituus kekahi lālā o ka marine obligate hydrocarbon fragmentation bacteria (OHCB), ka mea i laha ma ka moana a me ka honua, a lilo i mea nui ma hope o nā hanana pollution hydrocarbon30,31. ʻAʻole nā ​​lālā o ka pūʻulu OHCB ka bacteria moana, akā ua hoʻokaʻawale ʻia mai ke kai moana.
Hōʻike ka ʻikepili phenotypic he ʻano hou ʻo ASxL5T a he lālā o ka genus i ʻike ʻole ʻia ma mua o ka ʻohana spirospiraceae moana. I kēia manawa ʻaʻohe maʻamau maʻamau no ka hoʻokaʻawale ʻana i nā kānana i hoʻokaʻawale ʻia i loko o kahi genus hou. Ua ho'āʻo ʻia e hoʻoholo i nā palena genera āpau, no ka laʻana, e pili ana i ka pākēneka o ka genome o kahi protein conservative (POCP), ua ʻōlelo ʻia ʻo 50% ka waiwai o ka ʻoki ʻia me ka strain reference33. Manaʻo ka poʻe ʻē aʻe e hoʻohana i nā waiwai AAI, ʻoi aku ka maikaʻi ma mua o POCP no ka mea hiki ke loaʻa iā lākou mai nā genomes34. Manaʻo ka mea kākau inā emi ka waiwai AAI ma mua o 74% i ka hoʻohālikelike ʻia me ke kumu hoʻohālike o nā ʻano kumu hoʻohālike, ʻo ke kānana he hōʻailona o nā ʻano genera ʻokoʻa. ʻO ke kumu hoʻohālike i loko o ka marine spirillaceae he marine spirillum, a ʻo ke kumu hoʻohālike ʻo O. linum ATCC 11336T. ʻO ka waiwai AAI ma waena o ASxL5T a me O. linum ATCC 11336T he 54.34%, a he 67.61% ka waiwai AAI ma waena o ASxL5T a me T. oleivorans MIL-1T (genus type strains) he 67.61%, e hōʻike ana ʻo ASxL5T ke hōʻike nei i kahi moʻo hou ʻokoʻa mai Thalassolituus. Ke hoʻohana nei i ke kaʻina gene 16S rRNA ma ke ʻano he maʻamau hoʻohālikelike, ʻo 94.5%35 ka palena palena ʻāina i manaʻo ʻia. Hiki ke waiho ʻia ʻo ASxL5T i loko o ka genus Thalassolituus, e hōʻike ana i ka 95.03% 16S rRNA sequence identity me T. oleivorans MIL-1T a me 96.17%. marinus IMCC1826T. Eia nō naʻe, e hoʻokomo pū ʻia i loko o ka genus Bacteroides nona ka 94.64% 16S rRNA gene identity me B. sanyensis NV9, e hōʻike ana i ka hoʻohana ʻana i hoʻokahi gene e like me ka 16S rRNA gene hiki ke alakaʻi i ka hoʻokaʻawale ʻana a me ka hāʻawi ʻana. Hoʻohana ʻia kekahi ala ʻē aʻe i ka ANI a me Genome Alignment Score (AF) no ka nānā ʻana i ka hui ʻana o nā helu ʻikepili mai nā ʻano a me nā ʻano ʻano ʻole o nā genera e kū nei. Paipai ka mea kākau i ka hoʻohui ʻana i ka palena ʻano me ka helu inflection o ka genus i manaʻo ʻia e pili ana i ka ʻauhau e nānā ʻia nei. Eia naʻe, inā ʻaʻole lawa nā kaʻina genome piha mai nā Thalassolituus isolates, ʻaʻole hiki ke hoʻoholo inā pili ʻo ASxL5T i ka genus Thalassolituus ma kēia ʻano. Ma muli o ka liʻiliʻi o ka loaʻa ʻana o nā kaʻina genome piha no ka nānā ʻana, pono e unuhi ʻia ka lāʻau phylogenetic genome holoʻokoʻa me ka akahele. ʻO ka lua, ʻaʻole hiki i nā ʻano hoʻohālikelike genome holoʻokoʻa ke helu i nā ʻokoʻa nui o ka nui o nā genome i hoʻohālikelike ʻia. Ua ana lākou i ka like o nā genes kope hoʻokahi i mālama ʻia ma waena o nā genera pili, akā ʻaʻole i noʻonoʻo i ka nui o nā genes i loaʻa ʻole i ka genome liʻiliʻi loa o ASxL5T. ʻIke loa, ʻo ASxL5T a me nā hui pū me Thalassolituus, Oceanobacter, a me Bacterioplanes he kupuna maʻamau, akā ua hele ka evolution i kahi ala ʻē aʻe, e alakaʻi ana i ka hōʻemi ʻana i ka genome, ʻo ia paha ke hoʻololi ʻana i kahi nohona predatory. He ʻokoʻa kēia me T. oleivorans MIL-1T, ʻo ia ka 28% ka nui a ua ulu aʻe ma lalo o nā kaomi kaiapuni like ʻole e hoʻohana i nā hydrocarbons23,30. Hiki ke hoʻohālikelike ʻia me nā parasites intracellular obligate a me nā hōʻailona, ​​​​e like me Rickettsia, Chlamydia, a me Buchnera. ʻO ko lākou nui genome ma kahi o 1 Mb. ʻO ka hiki ke hoʻohana i nā metabolites cell host e alakaʻi i ka nalowale o ke ʻano, no laila ua hoʻemi nui ʻia ka genomic evolutionary. Hiki ke ho'ēmi like ka nui o ka genome i nā hoʻololi hoʻololi ʻana mai nā mea ʻai meaʻai kemika o ka moana a i ka nohona predatory. Hōʻike ka loiloi COG a me KEGG i ka helu o nā genes i hoʻohana ʻia no nā hana kikoʻī a me nā ʻokoʻa honua i nā ala genomic ma waena o ASxL5T a me T. oleivorans MIL-1T, ʻaʻole ia ma muli o ka loaʻa ākea o nā mea genetic mobile. ʻO ka ʻokoʻa o ka ratio G + C o ka genome holoʻokoʻa o ASxL5T he 56.1%, a ʻo ka T. oleivorans MIL-1T he 46.6%, e hōʻike ana hoʻi ua hoʻokaʻawale ʻia.
Hāʻawi ka nānā ʻana i ka ʻike coding o ka genome ASxL5T i nā ʻike hana i nā hiʻohiʻona phenotypic. ʻO ka loaʻa ʻana o nā genes encoding type IV fimbriae (Tfp) he mea hoihoi nui ia no ka mea e hoʻoikaika lākou i ka neʻe ʻana o ke kelepona, i kapa ʻia ʻo social gliding a i ʻole convulsions, me ka ʻole o ka hae ma ka ʻili. Wahi a nā hōʻike, he mau hana ʻē aʻe ʻo Tfp, me ka predation, pathogenesis, biofilm formation, natural DNA uptake, automatic cell aggregation and development38. Aia i loko o ka genome ASxL5T he 18 mau genes e ho'opili ana i ka diguanylate cyclase (he enzyme e ho'ololi ana i ka ho'ololi 'ana o 2 guanosine triphosphate i guanosine 2 phosphate a me cyclic diGMP) a me 6 genes e ho'opili ana i ka diguanylate cyclase phosphate diguanylate. ʻO ka gene for esterase (catalyzing the degradation of cyclic di-GMP to guanosine monophosphate) he mea hoihoi ia no ka mea he mea nui ka cycl-di-GMP i ka lua o ka ʻelele e pili ana i ka hoʻomohala ʻana i ka biofilm a me ka hoʻokaʻawale ʻana, ka neʻe ʻana, ka hoʻopili cell a me ka virulence 39, 40 i ke kaʻina hana. Pono e hoʻomaopopo ʻia i loko o Bdellovibrio bacteriovorus, ua hōʻike ʻia ka GMP pālua cyclic e hoʻomalu i ka hoʻololi ʻana ma waena o ke ola manuahi a me ka nohona predatory41.
ʻO ka hapa nui o nā noiʻi e pili ana i ka bacteria predatory i kālele ʻia ma Bdelovibrio, nā mea ola like me Bdelovibrio, a me nā ʻano Myxococcus. ʻO kēia a me nā hiʻohiʻona ʻē aʻe i ʻike ʻia o ka bacteria predatory he hui like ʻole. ʻOiai ʻo kēia ʻokoʻa, ua ʻike ʻia kahi pūʻulu o nā ʻohana protein e hōʻike ana i nā phenotypes o 11 i ʻike ʻia i ka bacteria predatory3,22. Eia nō naʻe, ʻo nā genes e hoʻopili ana i ka O antigen ligase (waaL) i ʻike ʻia, ʻo ia ka mea maʻamau i ka maʻi bacteria Gram-negative. ʻAʻole kōkua kēia ʻano loiloi i ka koho ʻana iā ASxL5T ma ke ʻano he predator, no ka mea hoʻohana ia i kahi hoʻolālā hoʻouka kaua hou. ʻO ka loaʻa ʻana o nā genomes predatory bacterial e kōkua i ka hoʻomohala ʻana i nā loiloi hoʻonā maikaʻi e noʻonoʻo ana i nā hōʻike o ka ʻokoʻa hana a me ke kaiapuni ma waena o nā lālā o ka hui. ʻO nā laʻana o ka bacteria predatory ʻaʻole i hoʻokomo ʻia i loko o kēia loiloi ʻo nā lālā o Cupriavidus necator42 a me Bradymonabacteria43, no ka mea i ka noiʻi ʻana o nā mea noiʻi i nā kaiāulu microbial like ʻole, ua hoʻokumu ʻia nā ʻauhau predatory.
ʻO ka hiʻohiʻona kaulana loa o ka bacteria ASxL5T i hopu ʻia e ke kiʻi TEM, ʻo ia kona ʻano morphology kū hoʻokahi a maʻalahi, hiki ke hoʻoikaika i ka launa pū ʻana me nā bacteria prey. He ʻokoʻa ke ʻano o ka launa pū ʻana me nā bacteria predatory ʻē aʻe a ʻaʻole i ʻike mua ʻia a hōʻike ʻia paha. Hōʻike ʻia ka pōʻai ola predatory ASxL5T i manaʻo ʻia ma ke Kiʻi 5. He liʻiliʻi nā hiʻohiʻona i loko o ka puke me nā hale apical like e like me kā mākou e hōʻike nei ma ʻaneʻi, akā ʻo kēia mau hiʻohiʻona ʻo Terasakiispira kohoumokuakeensis, he marine spirillum bacterium me ka apex enlargement 44, a me Alphaproteobacteria, Terasakiella pusilla , nona mua i ka genus Oceanospirillum, e hōʻike ana i ka mea i kapa ʻia "kiʻi polar" 45. ʻIke pinepine ʻia nā ʻano Cocci i nā moʻomeheu kahiko, ʻoi aku hoʻi no nā hua bacteria me nā ʻano curved, e like me Vibrio, Campylobacter, a me Helicobacter 46, 47, 48. Pono nā hana hou aʻe no ka hoʻomaʻamaʻa ʻana i ka pōʻaiapuni o ke ola o ka bacteria ASxL5T. No ka hoʻoholo ʻana i ke ʻano o ka hopu ʻana a me ka hopu ʻana, a inā paha e hoʻopili ana kāna genome i nā pūhui hana biologically i hiki ke hoʻohana ʻia no nā kumu lāʻau lapaʻau a biotechnological paha.
ʻO ka wehewehe ʻana o Venatorbacter gen. ʻO Nowemapa Venatorbacter (Ven.a.tor, ba'c.ter, L. i haku ʻia e nā venator mai L. n. venator, 'hunter' a me Gr. n. bacter, 'he koʻokoʻo'. Venatorbacter, 'he koʻokoʻo hahai holoholona' . ^ E Ha yM.ʻO nā cells he aerobic, salk-tolerant, curd gram e hoʻoikaika maikaʻi,ʻaʻole e hōʻiliʻili i ke koʻokoʻo. ka laulā o 4 a 42 °C Ingrown ʻAʻole maʻamau ka pae pH o 4-9 I loko o nā ʻeke kai, ʻaʻole ʻae ʻia ka nui o ka pH acidic ʻo C16:1ω6c a / a i ʻole C16:1ω7c, C16:0 a me C18. :1ω9 ; ʻAʻole ulu lākou i loko o ka media broth He 56.1 mol% nā lālā o kēia ʻano a me ka hana predation o nā lālā o ka ʻohana Enterobacteriaceae .
ʻO ka wehewehe ʻana o Venatorbacter cucullus sp. ʻO Nowemapa Venatorbacter cucullus (cu'cull.us.
Eia kekahi, ʻo ka hiʻohiʻona wehewehe o kēia ʻano ʻo ia ke ulu ʻia ma BA a i ʻole BHI, ʻo nā cell he 1.63 µm ka lōʻihi a me 0.37 µm ākea. He liʻiliʻi loa nā kolo ma BHI agar, hiki i 2 mm ke anawaena ma hope o 72 mau hola. He beige lākou, translucent, poepoe, convex a ʻālohilohi. Hiki i nā lālā o kēia ʻano ke hoʻohana iā Escherichia coli a me Klebsiella. ʻO Campylobacter a me kekahi mau hua bacteria Gram-negative ʻē aʻe i mea pio.
Ua hoʻokaʻawale ʻia ke kānana maʻamau ASxL5T mai ka waiū pipi ma Nottinghamshire, UK, a waiho ʻia i loko o ka National Type Culture Collection (UK): helu komo NCTC 14397 a me ka Netherlands Bacterial Culture Collection (NCCB) helu komo NCCB 100775. ʻO ke kaʻina genome piha o ASxL5T ua waiho ʻia ma Genbank e like me ka hoʻohui o CP046056.
Ua hoʻokaʻawale ʻia ka bacteria ASxL5T mai ka waiū pipi me ka hoʻohana ʻana i ka ʻenehana hoʻokaʻawale phage9,49. Ua hoʻoheheʻe ʻia ka slurry 1: 9 (w / v) i ka SM buffer (50 mM Tris-HCl [pH 7.5], 0.1 M NaCl, 8 mM MgSO4.7H2O a me 0.01% gelatin; Sigma Aldrich, Gillingham, UK), A laila hoʻomoʻa. ma 4°C no 24 mau hola, e hoʻololi mālie e hoʻoheheʻe i nā mea ʻaihue i loko o ka pahu. Ua centrifuged ka hoʻokuʻu ʻia ma 3000g no 3 mau minuke. Ua hōʻiliʻili ʻia ka supernatant a centrifuged ma 13,000g no ka lua o ka manawa no 5 mau minuke. Lawe ʻia ka supernatant i loko o kahi kānana membrane 0.45 µm (Minisart; Sartorius, Gottingen, Kelemānia) a me kahi kānana membrane 0.2 µm (Minisart) no ka wehe ʻana i nā cell bacteria i koe. Hiki iā ASxL5T ke hala i kēia mau kānana. Ua hoʻomākaukau ʻia kahi lau nahele agar o Campylobacter enterosus S12 (NCBI accession number CP040464) mai ka slurry like me ka hoʻohana ʻana i nā ʻenehana maʻamau. Ua māhele ʻia ka slurry kānana ma kēlā me kēia o kēia mau pā hale hoʻokipa ma 10 µl droplets i ʻekolu a ʻae ʻia e maloʻo. Hoʻokomo ʻia ka pā i loko o kahi pahu microaerophilic ma 37 ° C no 48 mau hola ma lalo o nā kūlana microaerobic (5% O2, 5% H2, 10% CO2, a me 80% N2). Lawe ʻia ka plaque i ʻike ʻia i loko o ka SM buffer a hoʻoneʻe ʻia i ka lau hou o C. hyointestinalis S12 e hoʻolaha hou aku i nā mea ola lysed. Ke hoʻoholo ʻia ʻo ka bacteria ke kumu o ka lytic plaque a ʻaʻole ka phage, e hoʻāʻo e ulu i ke kino kūʻokoʻa mai ka mea hoʻokipa a hoʻohālikelike hou iā ia. Hana ʻia ka moʻomeheu aerobic ma 37 ° C me 5% v / v defibrinated koko lio lio (TCS Biosciences Lt, Buckingham, UK, supplement). Wahi a nā alakaʻi o ke Kōmike Kūʻai Kūlohelohe National, hoʻohana ʻia ke ʻano diffusion disc no ka hoʻāʻo ʻana i ka antibacterial susceptibility. Ua kanu ʻia ka BHI agar ma 37 °C me ka hoʻohana ʻana i kahi diski i loaʻa nā lāʻau antibiotic (Oxoid) no ka moʻomeheu aerobic: amoxicillin a me 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. Ua hoʻokumu ʻia ka ʻae ʻana o ka paʻakai e ka incubation aerobic ma nā pā BHI agar ma 37 °C. Ua hoʻohui ʻia ʻo NaCl hou i nā pā BHI agar e hāʻawi i kahi pae hoʻonaʻauao a hiki i ka 10% w/v. Hoʻoholo ʻia ka pae pH e ka moʻomeheu aerobic ma nā pā BHI agar ma 37 ° C, kahi i hoʻoponopono ʻia ka pae pH ma waena o 4 a me 9 me ka sterile HCl a i ʻole sterile NaOH, a ua hōʻoia ʻia ka waiwai pH ma mua o ka ninini ʻana i ka pā. No ka nānā ʻana i ka waika momona cellular, ua hoʻoulu ʻia ʻo ASxL5T ma BHI agar no 3 mau lā a me ka aerobic ma 37 °C. Wahi a ka MIDI (Sherlock Microbial Identification System, version 6.10) protocol maʻamau o FERA Science Ltd, (York, UK), ua unuhi ʻia nā ʻakika momona cell, hoʻomākaukau a nānā ʻia.
No TEM, ASxL5T i moʻomeheu aerobic ma ka laha like 'ana ma luna o BA ma 37°C no 24 hola, a laila ohi i loko o 1 ml o 3% (v/v) glutaraldehyde i 0.1 M cacodylate buffer ma ka lumi wela Hoʻoponopono no 1 hola, a laila centrifuge. ma 10,000 g no 3 mau minuke. A laila hoʻomaha mālie i ka pellet ma 600 μl 0.1 M cacodylate buffer. E hoʻololi i ka hoʻokuʻu ʻia ʻana o ASxL5T i ke kiʻiʻoniʻoni Formvar/carbon ma kahi pahu keleawe 200 mesh. Ua hoʻopaʻa ʻia ka bacteria me ka 0.5% (w/v) uranyl acetate no 1 minuke a nānā ʻia e TEM me ka microscope TEI Tecnai G2 12 Biotwin. E like me ka mea i ʻōlelo ʻia ma luna, e hoʻohui i ka helu like o ka mea pio a me ka predator ma NZCYM broth (BD Difco™, Fisher Scientific UK Ltd, Loughborough) a incubate no 48 mau hola ma lalo o nā kūlana microaerobic o Campylobacter a i ʻole Campylobacter ma 37 ° C , ʻO ka pilina o ka predator a me ka pio. ua nānā pū ʻia e TEM. Nā kūlana aerobic no Escherichia coli. E noʻonoʻo kūʻokoʻa i nā mea pio a me nā bacteria predatory e hoʻoholo ai i nā loli i ka morphology cell ma muli o ka predation. Ua hoʻohana ʻia ke ʻano ʻeleʻele Sudan no ka microscopy optical o ka hōʻiliʻili PHB.
E hoʻoulu i ka moʻomeheu ASxL5T i ka pō ma o ka hamo ʻana i ka ulu ʻana ma luna o nā pā BHI a i ʻole BA me kahi swab sterile. E hōʻiliʻili i nā pūnaewele ASxL5T a hoʻopaneʻe iā lākou i MRD (CM0733, Oxoid), a laila e kau iā lākou ma 4°C no 7 mau lā e pōloli i nā cell. Hoʻokomo ʻia ka moʻomeheu bacterial NCTC a i ʻole ka moʻomeheu bacteria i loko o ka broth BHI a i ʻole No. 2 nutrient broth (CM007, Oxoid), incubated i ka pō, centrifuged ma 13,000g a hoʻokuʻu hou ʻia i MRD a hiki i ka OD600 he 0.4. Moʻomeheu: Bacillus subtilis NCTC 3610T, Citrobacter freundii NCTC 9750T, Enterobacter aerogenes NCTC 10006T, Enterococcus faecalis NCTC 775T, Escherichia coli NCTC 86, Klebsiella oxytoca NCTC 11466, Klebsiella oxytoca NCTC11466 Listeria Bakteria kūikawā NCTC 4885, Bacillus macerans NCTC 6355T, Providencia stuartsii NCTC 10318, Pseudomonas fluorescens SMDL, Rhodococcus submarine hamburger NCTC 1621T, Salmonella intestinal bacteria Mondeville NC6TC1 ʻO Staphylococcus aureus NCTC 8532T, Streptococcus pneumoniae NCTC 7465T, Yersinia enterocolitica NCTC 10460. Ua hoʻokomoʻia ka pūʻali Campylobacter i ka microaerobic ma nā papa BA ma 37 ° C a ua hoʻokuʻuʻia i ka broth NZCYM. ʻO nā pūʻali Campylobacter i ho'āʻoʻia: C. coli 12667 NCTC, C. jejuni 12662, C. jejuni PT14, C. jejuni NCTC 11168T, C. helveticus NCTC 12472, C. lari NCTC 11458, C. jejuni NCTC 11458, C. jejuni NCTC 11458, C. jejuni NC8TC , C... E hōʻiliʻili i nā pūnaewele i MRD, centrifuge ma 13,000g a hoʻomaha hou i MRD a hiki i ka OD600 he 0.4. E hoʻohui i kahi aliquot o 0.5 ml hoʻokuʻu i ka 5 ml NZCYM i hoʻoheheʻe ʻia i luna agar (0.6% agar) a ninini ʻia ma luna o kahi pā 1.2% NZCYM lalo. Ma hope o ka ho'ōla ʻana a me ka hoʻomaloʻo ʻana, ua puʻunaue ʻia ka ASxL5T i hoʻoheheʻe ʻia ma ke ʻano he 20 µl droplets ma kēlā me kēia papa lawn i ʻekolu. ʻO ka mahana moʻomeheu a me ka lewa e pili ana i nā koi o ka bacteria hoʻāʻo.
E hoʻohana i ka GenElute ™ Bacterial Genomic DNA Kit (Sigma Aldridge) e hoʻomākaukau ai i ka DNA mai nā kaʻawale bacteria. Ua hoʻohana ʻia nā ʻano hana maʻamau no ka hoʻonui ʻana i ka PCR o ka gene 16S rRNA a me ka hoʻoholo ʻana i ke kaʻina huahana me ka hoʻohana ʻana i ka kemika hoʻopau dye (Eurofins Value Read Service, Germany). E hoʻohana i ka polokalamu BLAST-N e hoʻohālikelike i kēia mau kaʻina me nā kaʻina gene 16S rRNA ʻē aʻe e ʻike a hōʻiliʻili i nā ʻano pili pili. Hoʻopili ʻia kēia me ka hoʻohana ʻana iā ClustalW i ka papahana MEGA X. Ua kūkulu hou ʻia ka lāʻau phylogenetic me ka hoʻohana ʻana iā MEGA X me ka hoʻohana ʻana i ke ʻano likelihood kiʻekiʻe ma muli o ke kumu hoʻohālike Tamura-Nei, me 1000 mau kope alakaʻi54. E hoʻohana i ka PureLink™ Genomic DNA Kit (Fisher Scientific, Loughborough, UK) e unuhi i ka DNA no ke kaʻina o ka genome holoʻokoʻa. Ua hoʻoholo ʻia ke kaʻina genome o ASxL5T me ka hui pū ʻana o Illumina MiSeq, ʻo ia hoʻi he 250 bp helu ʻelua-hopena i haku ʻia me kahi waihona i hoʻomākaukau ʻia me ka pahu lepili Nextera a me 2 a 20 kb heluhelu lōʻihi mai ka paepae PacBio. ʻO Genomics DNA Sequencing Research Facility ma Sembia University. Ua hōʻuluʻulu ʻia ka genome me ka hoʻohana ʻana iā CLC Genomics Workbench 12.0.3 (Qiagen, Aarhus, Denmark). Waiho ʻia nā moʻomeheu ASxL5T i ka National Type Culture Collection (UK) a me ka Netherlands Bacterial Culture Collection (NCCB). ʻO nā genomes o nā meaola pili i hoʻohana ʻia no ka hoʻohālikelike ʻana: Thalassolituus oleivorans MIL-1T (helu komo HF680312, piha); Bacterioplanes sanyensis KCTC 32220T (helu komo BMYY01000001, pau ole); Oceanobacter kriegii DSM 6294T (helu komo NZ_AUGV00000000, ʻaʻole i piha); ʻO ke kaiāulu Marinamonas DSM 5604T (hoʻohui ʻia ʻo ASM436330v1, ʻaʻole piha), Oceanospirullum linum ATCC 11336T (hoʻohui ʻia ʻo MTSD02000001, piha ʻole) a me Thalassolituus sp. C2-1 (hoʻohui NZ_VNIL01000001, ʻaʻole i piha). E hoʻohana iā JGI Genome Portal36 ma https://img.jgi.doe.gov//cgi-bin/mer/main.cgi?section=ANI&page= no ka hoʻoholo ʻana i ka helu alignment (AF) a me ka awelika nucleic acid identity (ANI). ʻElua. Ua hoʻohana ʻia ke ʻano o Rodriguez-R & Konstantinidis55 e hoʻoholo ai i ka ʻike amino acid (AAI). E hoʻohana i ka GToTree 1.5.5411,12,13,14,15,16,17,18 no ka hoʻoulu ʻana i ka lāʻau phylogenetic. Ua koho ʻia ka genome hoʻokomo e hōʻike ana i ka genome kuhikuhi i loaʻa mai ka genera reference i ʻike ʻia e pili ana me ASxL5T mai ka phylogeny 16S rRNA. Hōʻike i ka lāʻau me ka hoʻohana ʻana i ka lāʻau pili o ke ola ma ka pūnaewele (https://itol.embl.de/). Hoʻokō ʻia ka hōʻike a me ka nānā ʻana o ka genome ASxL5T me ka hoʻohana ʻana i ka BlastKOALA KEGG mea paahana pūnaewele me ka hoʻohana ʻana i ka hoʻolaha hoʻonui module KEGG (Kyoto Encyclopedia of Genes and Genomes). Hoʻoholo ʻia ka hāʻawi ʻana i nā kāʻei COG (hui orthologous) me ka hoʻohana ʻana i ka mea hana pūnaewele eggNOG-mapper.
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