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. B. pertussis adhesin and adhesin-related genes, such as fim2, fim3, and fimX, are among the earliest genes activated by BvgA upon its induction. The Fim proteins 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 4, 3, and 2 interact with the -35 element, the extended -10 sequence (positions -15, -14), and 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 Pfim2 nor Pfim3 contains a recognizable -35 element. Instead each fim promoter contains a tract of cytosines (C), and the length of this C-tract regulates BvgA activation through phase-variation. The mechanism of BvgA-dependent regulation is not understood. In a collaboration with the lab of S. Stibitz (FDA) we have examined in detail BvgA activation of transcription from Pfim3 and PfhaB. At PfhaB, a BvgA binding site lies immediately upstream of the -35 promoter element recognized by Region 4 of the sigma subunit of RNA polymerase. We demonstrate that sigma Region 4 is required for BvgA-activation of PfhaB, a hallmark of Class II activation. In contrast, the promoter proximal BvgA binding site at Pfim3 includes the -35 region and includes the tract of cytosines that lacks specific sequence information. We demonstrate that sigma Region 4 is not required for BvgA activation at Pfim3. Nonetheless, Region 4 mutations that impair its typical interactions with core and with the -35 DNA affect Pfim3 transcription. Hydroxyl radical cleavage using RNA polymerase with sigmaD581C-FeBABE positions Region 4 near the -35 region of Pfim3;cleavage using RNA polymerase with alpha276C−FeBABE or alpha302C−FeBABE also positions an alphaCTD within the -35 region, on a different helical face from the promoter proximal BvgAP. Our results suggest that the -35 region of Pfim3 accommodates BvgAP, an alphaCTD, and sigma Region 4. Molecular modeling suggests how BvgA, sigma Region 4, and alpha might coexist within this DNA in a conformation that suggests a novel mechanism of activation.
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