High frequency of independent IS50 transposition during Tn5 mutagenesis of Bordetella pertussis virulence-associated genes.

Transposon Tn5-generated mutants of Bordetella pertussis were selected on the basis of their inability to bind the dye Congo red (Crb-). No mutants which were solely Crb- were found. Ten mutants were phenotypically equivalent to previously described strains with mutations in the virulence regulatory (bvg) locus and failed to express a range of virulence-associated factors. Two of these mutants were shown to have Tn5 insertions within the bvg locus, while another two mutants showed deletions in this regulatory region. Complementation studies indicated that the other six mutants had spontaneous mutations in the bvg locus, but with Tn5 inserted elsewhere in the chromosome. Several of the mutants, besides having a single Tn5 insertion, also showed additional IS50 insertions, indicating that the IS50 element contained within Tn5 had transposed independently. Such additional insertion events, which themselves would have the potential to cause mutation, could complicate the interpretation of mutant phenotypes which could thus arise from the insertional inactivation of more than one gene.