Vectors for the insertion of a second copy of the ptx operon into the B. pertussis chromosome. A: The insertion site for a second copy of the ptx operon was selected between two abandoned genes, each carrying two frameshift mutations. B: Allelic-exchange elements used to insert a chloramphenicol marker into the selected site. C: Schematic structure of the ptx operon with its original promoter. The ptx-ptl terminator was cloned and inserted downstream of the S3 gene. This cluster was finally integrated into the SS4245 derivative to replace the chloramphenicol marker and generate the second allelic-exchange event to insert the second copy of the PT structural genes.