Streptococcus bovis JB1

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

No methylation motifs were detected in Streptococcus bovis JB1.

Methylating E. coli Strains

No E. coli methylation strain has been constructed for S. bovis JB1 strain

Culturing Conditions

Streptococcus bovis JB1 grows on both M2GSC and SDM (Semi Defined medium). Cells grow overnight at 37 °C in SDM or M2GSC culture medium with head space composed of 95% CO₂ and 5% H₂. Similarly, Streptococcus bovis JB1 grows overnight on SDM agar plates. Agar plates were placed in anaerobic Jar and incubated in anaerobic chamber under gas mixture that is composed of 95% CO₂ and 5% H₂.

Antibiotic MIC Tests

MIC tests of Streptococcus bovis JB1

antibiotic	concentration (ug/mL)
Ampicillin	0.25
Kanamycin	Resistant (>200 ug/mL)
Chloramphenicol	0.50
Tetracycline	0.23
Gentamicin	1.0
Streptomycin	Resistant (>200 ug/mL)
Erythromycin	1

Protocols

Preparation of SDM culture medium:

• Transformation protocol

Culture media:

1.     For overnight culture (SGM17):

Beef extract ………………..    5.00 g/l

Polypeptone ………………     5.00 g/l

Phytone-peptone ……………  5.00g/l

Yeast extract ………………..  2.50 g/l

Disodium glycerol phosphate 19.00 g/l

Ascorbic acid ………………...0.50 g/l

Glucose ………………………5.00 g/l

MgSO4.7H2O ………………. 0.25 g/l

+

0.5 M Sucrose (171.15 g/l)

-     Filter-sterilize                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            

2.     For growing the cells to make electrocompetent cells:

SGM17 (above) + 0.5% glycine

3.     For recovery after electroporation:

SGM17 containing 20 mM MgCl2 and 2 mM CaCl2

4.     For plating:

SDM culture medium + 1.5% agar

Method:

Day1. Grow Streptococcus bovis JB1 in 10 ml of GM17+Sucrose culture medium overnight aerobically.

Day2. Preparation of competent cells

-       Inoculate prewarmed 250 ml of GM17+Sucrose +0.5% glycine culture medium with 5 ml of the overnight culture.

-       Grow aerobically at 37 °C to an OD600 of 0.2 (approximately 5 to 7 h).

-       Place the culture on ice for 30 minutes and centrifuge at 5000 g, 4 °C, for 15 min.

-       Wash the cell pellets three times in 25 ml of ice-cold 0.5 M sucrose/10% glycerol, and centrifuge as before.

-       Resuspend the cell pellet in 0.5 ml of 0.5 M sucrose/10% glycerol and either use immediately for electroporation or store at -80 °C.

Electroporation:

-       Mix 100 ml of the cell suspension was mixed with up to 5 ml of DNA and place in ice-cold 0.2-cm electroporation cuvette (Bio-Rad Laboratories, Richmond, CA).

-       Electroporate at 2.5 kV, 200 W, 25 mF using the Bio-Rad Gene Pulser.

-       Immediately after electroporation, add 0.9 ml of SGM17 containing 20 mM MgCl2 and 2 mM CaCl2 to the cell suspension.

-       Transfer to a tube and incubate the mixture at 37 °C for 1 h.

-       Dilutions of the cells were plated on SR and SDM agar plates supplemented with antibiotic (5 mg/ml erythromycin).

-       Incubate in the anaerobic chamber and incubate at 37 °C for 1-2 days.

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.