Genetic analysis of phase change in Bordetella pertussis

Avirulent-phase derivatives of Bordetella pertussis (those which have simultaneously lost the ability to synthesize several virulence-associated factors) and the genetic mechanism of the phase change were studied. Increased tolerance to erythromycin was shown to be an avirulent-phase marker. By the use of efficiency of plating on erythromycin, the proportion of avirulent-phase (Vir) variants in a virulent-phase (Vir+) population was determined to be between 10(-3) and 10(-6), depending on the strain. We showed that the phase shift is reversible and detected a complete Vir- to Vir+ to Vir- to cycle. In other experiments, hybridization studies with avirulent-phase mutants obtained by Tn5 mutagenesis suggested that a single region located at a unique site in the B. pertussis chromosome controls the phase change. One of the avirulent Tn5 mutants was used as a recipient in a conjugative cross with a virulent-phase donor. All recombinants which had reacquired the virulence-associated factors also lost Tn5, indicating the loss of Tn5 was required to restore the Vir+ phenotype. The Tn5 avirulent-phase mutants behave as if the insertion interrupted the function of a transacting gene product which is required for the expression of the other virulent-phase genes. A model of the molecular basis of the phase regulation is presented.