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| [[File:Sabu_tube.png|thumb|346x346px|''S. azabuensis'' KH1P5 in liquid culture exhibiting its dense layer of EPS. Source: Dr. Roy Rodriguez Carrero]]
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| == Introduction == | | == Introduction == |
| '''Sharpea azabuensis KH1P5'''
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| Sharpea azabuensis KH1P5 was isolated from bovine rumen contents in NZ using KH medium which was supplemented with haemin and vitamin K [1]. KH1P5 ferments glucose, fructose, galactose, cellobiose, sucrose, lactose and raffinose with D-lactate as the main fermentation product together with small amounts of acetate, formate and ethanol [2]. It is a member of the family Coprobacillaceae.
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| Sharpea and Kandleria were identified as key bacteria associated with the S-type (Sharpea-enriched) bacterial community, one of the two low-CH<sub>4</sub> community types in rumen samples from sheep that differed in the amount of CH<sub>4</sub> formed per unit of feed consumed when these sheep were fed pelletized lucerne [3]. A metagenomic and metatranscriptomic study conducted by Kamke et al. [4] confirmed that the relative abundance of Sharpea spp. was greater in a group of low-CH<sub>4</sub>emitting sheep than in their high-CH<sub>4</sub> emitting flock mates. It was hypothesized that lactate produced by these bacteria is further converted to butyrate by Megasphaera spp., resulting in lower CH<sub>4</sub> emissions.
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| The draft genome for KH1P5 (2,329,465 bp) was generated through the Hungate1000 project with JGI [1]. Resequencing gave a single chromosome of 2,465,508 bp.
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| LPSN: ''Sharpea azabuensis'' Morita ''et al.'' 2008
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| NCBI: Sharpea azabuensis.
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| GTDB: Sharpea azabuensis. KH1P5 groups with the type strain (ST18, DSM 18934) [5]
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| Risk group: 1
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| == Global Methane Hub Metadata == | | == Global Methane Hub Metadata == |
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| |Origin: | | |Origin: |
| |New Zealand, Kelly Lab | | | |
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| |Sequenced by: | | |Sequenced by: |
| |Sam Mahoney Kurpe | | | |
| |- | | |- |
| |Assembled by: | | |Assembled by: |
| |William G. Alexander | | | |
| |- | | |- |
| |Methylome called by: | | |Methylome called by: |
| |William G. Alexander | | | |
| |- | | |- |
| |Methyltransferases cloned by: | | |Methyltransferases cloned by: |
| |Roy Rodriguez Carrero | | | |
| |- | | |- |
| |MIC and Culturing Conditions by: | | |MIC and Culturing Conditions by: |
| |Roy Rodriguez Carrero | | | |
| |- | | |- |
| |Transformation by: | | |Transformation by: |
| |Roy Rodriguez Carrero | | | |
| |} | | |} |
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| == Genome == | | == Genome == |
| [[File:S_azabuensis_circos.png|center|thumb|Circos plot of ''S. azabuensis'' KH1P5. Source: Dr. William G. Alexander]]
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| == DefenseFinder Results == | | == DefenseFinder Results == |
| == Methylome == | | == Methylome == |
| While ''S. azabuensis'' KH1P5 possesses two putative Type II restriction endonucleases, no methylation is detectable in the isolate. Also, these two genes do not methylate in ''E. coli'' strains, suggesting that they're vestigial defense systems.
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| == Methylating E. coli Strains == | | == Methylating E. coli Strains == |
| Use a strain of ''E. coli'' that lacks dam, dcm, and hsdRMS, such as AG5589.
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| == Culturing Conditions == | | == Culturing Conditions == |
| ''S. azabuensis'' KH1P5 grows well in stock [[RCM]] at 37 °C. However, the inclusion of agar in commercially made RCM causes the strain to produce a thick, mucoidal layer of [[wikipedia:Extracellular_polymeric_substance|extracellular polymeric substance (EPS)]] that requires [[Sharpea azabuensis KH1P5#Protocols|removal via washing]] with chelating agents and detergent before work with the culture. Growing the organism in lab made RCM without agar eliminates the dense EPS layer.
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| KH1P5 also grows well on M2G medium at 39 °C, with 10 ml cultures reaching stationary phase after 6-7 h at optical densities of ~4-5. Cells of KH1P5 form long chains.
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| [[File:KH1P5_micrograph.jpg|thumb|Phase-contrast micrograph of ''S. azabuensis'' KH1P5 grown in M2G medium. Source: Dr. Sam Mahoney-Kurpe.]]
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| == Antibiotic MIC Tests == | | == Antibiotic MIC Tests == |
| [[File:Sabu_MIC.png|left|thumb|MIC tests of ''S. azabuensis''. Source: Dr. Roy Rodriguez Carrero]]
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| {| class="wikitable"
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| !antibiotic
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| !concentration (ug/mL)
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| |-
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| |carbenicillin
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| |5
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| |-
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| |chloramphenicol
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| |0.38
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| |-
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| |clarithromycin
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| |25
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| |-
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| |erythromycin
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| |1
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| |-
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| |kanamycin
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| |insensitive
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| |-
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| |spectinomycin
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| |96
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| |-
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| |tetracycline
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| |0.23
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| |-
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| |thiamphenicol
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| |5
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| |}
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| == Protocols == | | == Protocols == |
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| * [[Removal of extracellular material via chelation washing]]
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| == Transformation Results == | | == Transformation Results == |
| == External Links == | | == External Links == |
| [1] Seshadri R, Leahy SC, Attwood GT, Teh KH, Lambie SC, Cookson AL, Eloe-Fadrosh EA, Pavlopoulos GA, Hadjithomas M, Varghese NJ, Paez-Espino D; Hungate1000 project collaborators; Perry R, Henderson G, Creevey CJ, Terrapon N, Lapebie P, Drula E, Lombard V, Rubin E, Kyrpides NC, Henrissat B, Woyke T, Ivanova NN, Kelly WJ. 2019. Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection. Nature Biotechnol 36: 359-367. doi: 10.1038/nbt.4110.
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| [2] Kumar S, Treloar BP, Teh KH, McKenzie CM, Henderson G, Attwood GT, Waters SM, Patchett ML, Janssen PH. 2018. Sharpea and Kandleria are lactic acid producing rumen bacteria that do not change their fermentation products when co-cultured with a methanogen. Anaerobe 54: 31-38. doi: 10.1016/j.anaerobe.2018.07.008.
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| [3] Kittelmann S, Pinares-Patiño CS, Seedorf H, Kirk MR, Ganesh S, McEwan JC, Janssen PH. 2014. Two different bacterial community types are linked with the low-methane emission trait in sheep. PLoS One 9: e103171. doi: 10.1371/journal.pone.0103171.
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| [4] Kamke J, Kittelmann S, Soni P, Li Y, Tavendale M, Ganesh S, Janssen PH, Shi W, Froula J, Rubin EM, Attwood GT. 2016. Rumen metagenome and metatranscriptome analyses of low methane yield sheep reveals a Sharpea-enriched microbiome characterised by lactic acid formation and utilisation. Microbiome 4: 56. doi: 10.1186/s40168-016-0201-2.
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| [5] Morita H, Shiratori C, Murakami M, Takami H, Toh H, Kato Y, Nakajima F, Takagi M, Akita H, Masaoka T, Hattori M. 2008. Sharpea azabuensis gen. nov., sp. nov., a Gram-positive, strictly anaerobic bacterium isolated from the faeces of thoroughbred horses. Int J Syst Evol Microbiol 58: 2682-2686 doi: 10.1099/ijs.0.65543-0.
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