Analysis of BvgA activation of the pertactin gene promoter in Bordetella pertussis

Bordetella pertussis, the causative agent of whooping cough, regulates expr ession of its virulence factors via a two-component signal transduction sys tem encoded by the bvg regulatory locus. It has been shown by activation ki netics that several of the virulence factors are differentially regulated. fha is transcribed at 10 min following an inducing signal, while ptx is not transcribed until 2 to 4 h after the inducing signal. We present data indi cating that prn is transcribed at 1 h, an intermediate time compared to tho se of fha and ptx. We have identified cis-acting sequences necessary for ex pression of pm in B. pertussis by using prn-lac fusions containing alterati ons in the sequence upstream of the prn open reading frame. In vitro transc ription and DNase I footprinting analyses provided evidence to support our hypothesis that BvgA binds to this sequence upstream of prn to activate tra nscription from the promoter. Our genetic data indicate that the region cri tical for prn activation extends upstream to position -84. However, these d ata do not support the location of the prn transcription start site as prev iously published. We used a number of methods, including prn-lac fusions, r everse transcriptase PCR, and 5' rapid amplification of cDNA ends, to local ize and identify the bvg-dependent 5' end of the prn transcript to the cyto sine at -125 with respect to the published start site.