Bifidobacteria are the main component of the human microflora. We constructed a temperature-sensitive (Ts) plasmid by random mutagenesis of the Bifidobacterium-Escherichia coli shuttle vector pKKT427 using error-prone PCR. Mutant plasmids were introduced into Bifidobacterium longum 105-A and, after screening approximately 3,000 colonies, candidate clones that grew at 30 °C but not at 42 °C were selected. According to DNA sequence analysis of the Ts plasmid, five silent and one missense mutations were found in the repB region. The site-directed mutagenesis showed only the missense mutation to be relevant to the Ts phenotype. We designated this plasmid pKO403. The Ts phenotype was also observed in B. longum NCC2705 and Bifidobacterium adolescentis ATCC15703. Single-crossover homologous-recombination experiments were carried out to determine the relationship between the length of homologous sequences encoded on the plasmid and recombination frequency: fragments greater than 1 kb gave an efficiency of more than 10(3) integrations per cell. We performed gene knockout experiments using this Ts plasmid. We obtained gene knockout mutants of the pyrE region of B. longum 105-A, and determined that double-crossover homologous recombination occurred at an efficiency of 1.8 %. This knockout method also worked for the BL0033 gene in B. longum NCC2705.
ABSTRACTFor improvement of tolerance to oxidative stress inBifidobacterium longum105-A, we introduced theBacillus subtiliscatalase gene (katE) into it. The transformant showed catalase activity (39 U/mg crude protein) in the intracellular fraction, which increased survival by ∼100-fold after a 1-h exposure to 4.4 mM H2O2, decreasedde novoH2O2accumulation, and increased survival in aerated cultures by 105-fold at 24 h. The protection level was better than that conferred by exogenously added catalase.
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.
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