Sharpea azabuensis KH1P5: Difference between revisions
Rodriguezcrj (talk | contribs) →Introduction: Added context for the EPS formation in Sharpea on commercial RCM and linked the page with more context on RCM. |
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== Global Methane Hub Metadata == | == Global Methane Hub Metadata == | ||
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Use a strain of ''E. coli'' that lacks dam, dcm, and hsdRMS, such as AG5589. | Use a strain of ''E. coli'' that lacks dam, dcm, and hsdRMS, such as AG5589. | ||
== Culturing Conditions == | == Culturing Conditions and Growth Characteristics == | ||
''S. azabuensis'' KH1P5 grows well in stock [[RCM]] at 37 °C. | ''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. | ||
''S. azabuensis KH1P5'' also grows well in M2G medium at 39 °C, reaching stationary phase after approximately 6-7 h. Cells grow as long poorly-defined chains (to insert image), preventing the use of cell counting methods to calculate plating efficiencies. | |||
== Antibiotic MIC Tests == | == Antibiotic MIC Tests == | ||
Revision as of 21:06, 22 October 2026
Introduction
Sharpea azabuensis KH1P5
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.
Sharpea and Kandleria were identified as key bacteria associated with the S-type (Sharpea-enriched) bacterial community, one of the two low-CH4 community types in rumen samples from sheep that differed in the amount of CH4 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-CH4 emitting sheep than in their high-CH4 emitting flock mates. It was hypothesized that lactate produced by these bacteria is further converted to butyrate by Megasphaera spp., resulting in lower CH4 emissions.
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.
LPSN: Sharpea azabuensis Morita et al. 2008
NCBI: Sharpea azabuensis.
GTDB: Sharpea azabuensis. KH1P5 groups with the type strain (ST18, DSM 18934) [5]
Risk group: 1
Global Methane Hub Metadata
| Origin: | New Zealand, Kelly Lab |
| Sequenced by: | Sam Mahoney Kurpe |
| Assembled by: | William G. Alexander |
| Methylome called by: | William G. Alexander |
| Methyltransferases cloned by: | Roy Rodriguez Carrero |
| MIC and Culturing Conditions by: | Roy Rodriguez Carrero |
| Transformation by: | Roy Rodriguez Carrero |
Genome
DefenseFinder Results
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.
Methylating E. coli Strains
Use a strain of E. coli that lacks dam, dcm, and hsdRMS, such as AG5589.
Culturing Conditions and Growth Characteristics
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 extracellular polymeric substance (EPS) that requires 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.
S. azabuensis KH1P5 also grows well in M2G medium at 39 °C, reaching stationary phase after approximately 6-7 h. Cells grow as long poorly-defined chains (to insert image), preventing the use of cell counting methods to calculate plating efficiencies.
Antibiotic MIC Tests
| antibiotic | concentration (ug/mL) |
|---|---|
| chloramphenicol | 0.38 |
| erythromycin | 1 |
| kanamycin | insensitive |
| spectinomycin | 96 |
| tetracycline | 0.23 |
Protocols
Transformation Results
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.
[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.
[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.
[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.
[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.
