B. pertussis infects the upper respiratory tract by adhering to ciliated epithelial cells and releasing toxins. The virulence genes that encode these proteins are regulated by the BvgS-BvgA two-component system. The response regulator BvgA binds to the promoter region of all known B. pertussis virulence genes to activate their transcription during infection. These genes include bipA, fha, ptx, prn, cya, bvgR, bvgA, and the fim genes. The fim2 and fim3 genes encode different surface proteins that facilitate adhesion of the bacterium to epithelial cells in the human respiratory tract. During transcription, the sigma subunit of RNA polymerase (RNAP) is the specificity factor that recognizes promoter elements. Primary sigmas like E. coli sigma70 and B. pertussis sigma A have specific regions that contact promoter sequences. Residues within Regions 2, 3, and 4 interact with sequences in the -35 element, the extended -10 sequence (positions -15, -14), and sequence in the -10 element, respectively. The fim3 promoter (Pfim3) has both the sigma70-dependent extended -10 sequence and a -10 element while the fim2 promoter (Pfim2) has only a canonical -10 sequence. However, neither promoter contains a recognizable -35 element. Instead each fim promoter contains a tract of cytosines (C) upstream of its -10 element and the length of this C-tract regulates BvgA activation through phase-variation. The mechanism of BvgA-dependent regulation is not understood. We are collaborating with the lab of S. Stibitz (FDA) to characterize the features of Pfim3 needed for its regulation. Using primer extension analyses, we determined or confirmed the in vivo start site for several BvgA-activated genes, including Pfim3 and Pfim2. Using RNA polymerase, BvgA protein, and Pfim3 DNA, we have established an in vitro transcription system that recapitulates BvgA regulation by the length of the C-tract. We have determined that both wild type Pfim3, which contains the TGn and -10 elements, and mutant fim3 promoters, which lack the extended -10 sequence, are similarly activated by BvgA. Fe-BABE footprinting has located two BvgA binding sites within Pfim3. The proximal site lies within the C-tract, which includes the -35 region of Pfim3. Sigma70 Region 4 normally interacts with the -35 sequences, and many activators are known to bind upstream of the -35 element and to promote sigma70 binding to a non-canonical -35 sequence. However, it is atypical to find an activator site within the core promoter DNA. To determine the importance of sigma Region 4 for BvgA activation at Pfim3, we have performed in vitro transcription experiments using mutant sigma70 and sigma A proteins. Our results indicate that Pfim3 does not require interactions with Region 4. Sigma Region 4 mutations that eliminate the interaction of sigma70 with the -35 element or addition of AsiA, which prevents the Region 4/-35 DNA interaction, does not significantly affect BvgA activation of wild type Pfim3. Furthermore, BvgA activation of wild type Pfim3 is still seen even if Region 4 of sigma70 or sigma A is deleted. However, a mutant Pfim3 that lacks the TGn element is dependent on Region 4. These results suggest an unusual flexibility of Region 4 in the BvgA/polymerase/Pfim3 complex, in which Region 4 contacts can be used with BvgA when needed. We speculate that activation of Pfim3 may involve a novel mechanism of gene activation.
