H-081. Genetic Studies on the Fimbrial Subunit Promoters of Bordetella pertussis

Q. Chen1, P. Boucher1, K. Baxter2, D. Hinton2, S. Stibitz1;
1CBER/FDA, Bethesda, MD, 2NIDDK/NIH, Bethesda, MD.

Bordetella pertussis, the causative agent of whooping cough, can express fimbriae of two different serotypes. Accessory fimbrial genes are encoded together at one locus, while the genes for the different serotype fimbrial subunits, fim2, and fim3, are unlinked. A third unlinked gene, fimX, encodes an intact subunit gene that is not expressed. Willems et al. (EMBO J. 1990, 9:2803-9) demonstrated that expression of the fim subunit genes is phase-variable through changes in the lengths of monotonic runs of 13 or more G:C base-pairs within the fim subunit promoters. Interestingly, this “C-stretch” overlaps the position expected for a -35 region in each promoter. We have begun a number of genetic analyses of these promoters. Each was subjected to site-directed mutagenesis, creating a series of derivatives, differing in the number of residues in the C-stretch. BvgA-dependent expression was assessed in vivo, in B. pertussis using fim-lux transcriptional fusions. Aligning the three promoters based on partial DNA sequence conservation and transcriptional start-sites verified by primer extension, it could be seen that optimal activity in each series corresponded to the same spacing between features upstream and downstream of the C-string. Although it has been known for years that fim gene expression is dependent on the bvgASR locus, it has not been known if BvgA interacts directly with the fim promoters, as obvious BvgA consensus binding sites were not recognized. We have shown that activation of transcription from Pfim3 is dependent on BvgA~P in vitro, and that BvgA~P binds strongly to positions in Pfim3, in part at locations of partial DNA sequence conservation among the three promoters. Interestingly, the region bound by BvgA~P at Pfim3 extends into the C-stretch. The C-stretch is thus in a position to affect BvgA-binding, RNA polymerase-binding, and the spacing of regulatory and promoter elements. We have initiated mutagenesis studies to determine the degree to which the DNA sequence of the C-stretch contributes to fim promoter activity.