The pyrE Gene as a Bidirectional Selection Marker in Bifidobacterium Longum 105-A

Bioscience of Microbiota, Food and Health

Aya HOBO¹  Kouta SAKAGUCHI²  Tohru SUZUKI²  Tohru MITSUNAGA¹  Yasunobu KANO³  Saori TANI¹  Nobutaka FUNAOKA¹ 

[1] Faculty of Applied Biological Sciences, Gifu University, Japan;The United Graduate School of Agricultural Science, Gifu University, Japan;Department of Molecular Genetics, Kyoto Pharmaceutical University, Japan

关键词: pyrimidine metabolism;    gene knockout;    homologous recombination;    5-FOA;    B. longum 105-A;    bifidobacterial minimal medium (BMM);    gene inactivation;   

DOI  :  10.12938/bmfh.32.59

学科分类：生物科学（综合）

来源: Nihon Bifizusukin Senta / Japan Bifidus Foundation

【 摘 要 】

References(29)Cited-By(2)We constructed a deletion mutant of the pyrE gene in Bifidobacterium longum 105-A. A pyrE knockout cassette was cloned into pKKT427, a Bifidobacterium-Escherichia coli shuttle vector, and then introduced into B. longum 105-A by electroporation. The transformants were propagated and spread onto MRS plates containing 5-fluoroorotic acid (5-FOA) and uracil. 5-FOA-resistant mutants were obtained at a frequency of 4.7 × 10�?5 integrations per cell. To perform pyrE gene complementation, the pyrE gene was amplified by PCR and used to construct a complementation plasmid (pKKT427-pyrE+). B. longum 105-A ∆pyrE harboring this plasmid could not grow on MRS plates containing 5-FOA, uracil and spectinomycin. We also developed a chemically defined medium (bifidobacterial minimal medium; BMM) containing inorganic salts, glucose, vitamins, isoleucine and tyrosine for positive selection of pyrE transformants. B. longum 105-A ∆pyrE could not grow on BMM agar, but the same strain harboring pKKT427-pyrE+ could. Thus, pyrE can be used as a counterselection marker in B. longum 105-A and potentially other Bifidobacterium species as well. We demonstrated the effectiveness of this system by constructing a knockout mutant of the xynF gene in B. longum 105-A by using the pyrE gene as a counterselection marker. This pyrE-based selection system will contribute to genetic studies of bifidobacteria.

【 授权许可】

Unknown