Sharpea azabuensis KH1P5

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

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

Genome characteristics
Contigs	1
size (bp)	2,465,508
GC %	37.5
CDS	2364
rRNA	16
tRNA	66
CRISPR	2

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.