Hfr mapping of mutations in Bordetella pertussis that define a genetic locus involved in virulence gene regulation.

We report the development of techniques for the genetic mapping of point mutations in the bacterial pathogen Bordetella pertussis. A plasmid vector which is self-transmissible by conjugation and which, by insertion into the B. pertussis chromosome, can mobilize chromosomal sequences during conjugation with a recipient B. pertussis bacterium has been constructed. This vector is used in conjunction with a set of strains containing kanamycin resistance gene insertions at defined physical locations in the B. pertussis genome. In crosses between these donor strains and a mutant recipient strain, transfer of a chromosomal segment flanking the kanamycin resistance gene insertion is selected for, and the percentage of exconjugants which reacquire the wild-type trait is scored. In this way the linkage of the mutant allele to these markers, and thus the approximate chromosomal position of the mutant allele, is determined. We have used this genetic system to map a newly described locus in B. pertussis involved in the regulation of the virulence genes ptx (pertussis toxin) and cya (adenylate cyclase toxin).