Construction and characterization of Bordetella pertussis RecA- mutants

Antibiotic drug-resistance cassettes (DRCs) were used to insertionally inactivate the wild-type Bordetella pertussis recA gene cloned into a suicide vector. The mutant allele was mobilized by conjugal gene transfer from Escherichia coli strain SM10 into different genetic backgrounds of B. pertussis. Southern hybridization studies of one of these mutants showed that it contained a DRC integrated within a recA gene situated within a ClaI genomic DNA fragment. Selected mutants were assayed to quantify recombinational and DNA repair deficiencies. These mutants were shown to be highly sensitive to both chemically and physically induced DNA damage. Gene transfer studies of another RecA- mutant also indicated that it was defective in intergenic recombination. No difference in hemolytic activity or production of capsule was detected between the RecA- mutants and their corresponding wild-type strains. The results of this investigation corroborate previous studies with the cloned B. pertussis recA gene, and demonstrate that the expression of the B. pertussis recA gene in the original host promotes both DNA repair and recombination.