FIG. 1.

Production of a cassette, flanked by neisserial uptake sequences, and deletion of the N. meningitidis-specific islands. (A) For production of the cassette, an inverted repeat containing two neisserial uptake sequences (arrows) and an internal BglII site (shaded) was constructed by ligating synthetic oligonucleotides T1 to T4 (Table 1). This molecule was cloned into pBluescript, and an Ω spectinomycin resistance cassette was cloned into the BglII site to yield a cassette that may be excised with BamHI. (B) Deletion of region 8. For replacement of the N. meningitidis-specific islands with the cassette, the flanking regions of the islands were PCR amplified by using the oligonucleotides in Table 1. These were ligated together at an internal restriction enzyme site (EcoRI), reamplified, and cloned into pBluescript. The construct was reopened at the EcoRI site, and the cassette was inserted between the flanking sequences. This plasmid was then used to transform N. meningitidis and replace the chromosomal island with the resistance cassette by homologous recombination.