Work in our laboratory has been concerned with the vir locus of Bordetella pertussis. This locus is responsible for the regulation of B. pertussis virulence determinants (pertussis toxin, filamentous hemagglutinin, etc.) in response to environmental stimuli. We have recently developed powerful new physical and genetic methods for the analysis of bacterial genomes. The physical methods have allowed us to compare the genomes of five B. pertussis and one B. parapertussis strain. Surprisingly, our results have shown that there can be a great deal of variation in genomic organization both between strains of B. pertussis and between the species B. pertussis and B. parapertussis. The new genetic tools (specifically, a set of donor strains that allow us to perform Hfr mapping) that we have developed have allowed us, in collaboration with Dr. Nick Carbonetti at the University of Maryland at Baltimore, to map a new gene involved in the regulation of pertussis toxin and adenylate cyclase, but not filamentous hemagglutinin. Such a finding lends support to the concept of a regulatory cascade in B. pertussis, where all virulence determinants are ultimately regulated by the vir locus, but where there may be other regulatory loci involved only in the regulation of one or a few virulence determinants. Using our new genetic tools we have begun a systematic search for other genes which affect the regulation of virulence determinants in a differential fashion with the hopes of defining all of the factors involved in the regulation of virulence in B. pertussis.