title: The pyrE Gene as a Bidirectional Selection Marker in Bifidobacterium Longum 105-A.
abstract: 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.
authors.affiliation: The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Kouta
authors.initials: K
authors.lastname: Sakaguchi
authors.affiliation: Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Funaoka
authors.affiliation: Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Saori
authors.initials: S
authors.lastname: Tani
authors.affiliation: Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Hobo
authors.affiliation: Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Mitsunaga
authors.affiliation: Department of Molecular Genetics, Kyoto Pharmaceutical University, 1 Shichono-cho, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan.
authors.forename: Yasunobu
authors.initials: Y
authors.lastname: Kano
authors.affiliation: The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Suzuki
keywords: 5-FOA; B. longum 105-A; bifidobacterial minimal medium (BMM); gene inactivation; gene knockout; homologous recombination; pyrimidine metabolism