Characterization of plasmids from human infant Bifidobacterium strains: Sequence analysis and construction of E. coli–Bifidobacterium shuttle vectors

A survey of infant fecal Bifidobacterium isolates for plasmid DNA revealed that a significant portion of the strains, 17.6%, carry small plasmids. The majority of plasmid-harboring strains belonged to the Bifidobacterium longum/infantis group. Most of the plasmids could be assigned into two groups based on their sizes and the restriction profiles. Three plasmids, pB44 (3.6 kb) from B. longum, pB80 (4.9 kb) from Bifidobacterium bifidum, and pB21a (5.2 kb) from Bifidobacterium breve were sequenced. While the former two plasmids were found to be highly similar to previously characterized rolling-circle replicating pKJ36 and pKJ56, respectively, the third plasmid, pB21a, does not share significant nucleotide homology with known plasmids. However, it might be placed into the pCIBb1-like group of bifidobacterial rolling-plasmids based on the homology of its Rep protein and the overall molecular organization. Two sets of Escherichia coli–Bifidobacterium shuttle vectors constructed based on pB44 and pB80 replicons were capable of transforming B. bifidum and B. breve strains with efficiency up to 3 × 104 cfu/μg DNA. Additionally, an attempt was made to employ a broad host range conjugation element, RP4, in developing of E. coli–Bifidobacterium gene transfer system.