Strain template

S. azabuensis KH1P5 in liquid culture exhibiting its dense layer of EPS. Source: Dr. Roy Rodriguez Carrero

Introduction

Streptococcus bovis JB1 was isolated from the rumen of alfalfa-fed cow. It is a major amylolytic bacterium that depolymerize starch to glucose, fermenting it to large amounts of lactic acid that can lead to lactic acidosis in ruminants.

Global Methane Hub Metadata

Origin:	Illinois, Mackie Lab
Sequenced by:	Ahmed Hetta and Yuqi Li
Assembled by:	William G. Alexander
Methylome called by:	William G. Alexander
Methyltransferases cloned by:	No methylation motifs have been detected
MIC and Culturing Conditions by:	Ahmed Hetta and Yuqi Li
Transformation by:	Ahmed Hetta

Genome

Circos plot of S. azabuensis KH1P5. Source: Dr. William G. Alexander

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

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.

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.

Phase-contrast micrograph of S. azabuensis KH1P5 grown in M2G medium. Source: Dr. Sam Mahoney-Kurpe.

Antibiotic MIC Tests

MIC tests of S. azabuensis. Source: Dr. Roy Rodriguez Carrero

antibiotic	concentration (ug/mL)
carbenicillin	5
chloramphenicol	0.38
clarithromycin	25
erythromycin	1
kanamycin	insensitive
spectinomycin	96
tetracycline	0.23
thiamphenicol	5

Protocols

• Removal of extracellular material via chelation washing

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.