Characterization of the type III secretion locus of Bordetella pertussis

Multiple sequence comparisons of proteins of the LcrD/FlbF family allowed t he design of primers that specifically amplify sequences coding for type II I secretion components. Amplification of Bordetella pertussis DNA with thes e primers yielded a fragment that was further used as a probe for screening a genomic library. The nucleotide sequence of a positive clone revealed a 2100-bp gene, called bcrD, which specifies a 75-kDa polypeptide homologous to the Yersinia LcrD protein. Chromosome walking allowed the characterizati on of a 35-kb DNA segment that contains the entire locus and flanking house keeping genes. The B. pertussis type III secretion locus consists of more t han 30 open reading frames (ORFs), most of which are identical to annotated genes of Bordetella spp and share similarities with known type III secreti on genes of related bacteria. In order to assess the function of this locus , we engineered a bcrD null mutant. However, none of the tested phenotypes, such as protein secretion, cellular invasion, cytotoxicity or mouse lung c olonization, differentiated the mutant from its parental strain. Studies of bcrD and bscN expressions indicated that, under our experimental condition s, these genes are not expressed in vitro. Restriction analyses on pulsed-f ield gel electrophoresis allowed the type III locus mapping at coordinate p osition 1,590 kb on the Tohama I strain chromosome.