FIG. 7.

Construction of an integrative vector for S. pneumoniae. (A) Schematic representation of the pIAPU1 integrative vector. Ap, ampicillin; Ln, lincomycin. (B) Site-specific integration of a single copy of pIAPU1 into the S. pneumoniae chromosome. Strain 708 was transformed with pIAPU1. DNAs extracted from the parental strain 708 (lane 1) and from two transformants, PM11 and PM12 (lanes 2 and 3), were cut with NcoI, run on an agarose gel, Southern blotted, and hybridized with the int-attP cassette as a probe. The sizes of hybridizing fragments are indicated. (C) Attachment site detection using PCR. The positions of the different attachment sites are indicated. The DNAs used as templates were from strain 708 (lanes 1, 4, 7, and 10), from transformant PM11 (lanes 2, 5, 8, and 11), and from lysogenic strain 949 (lanes 3, 6, 9, and 12). The oligonucleotides used were EGP4 and EGP9 for attP, EGP14 and EGP15 for attB, EGP9 and EGP14 for attL, and EGP4 and EGP15 for attR.