Browsing School, Graduate by Title "Genetic analysis of BvgA activaton of virulence gene promoters in Bordetella pertussis"
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Genetic analysis of BvgA activaton of virulence gene promoters in Bordetella pertussisBordetella pertussis, the causative agent of whooping cough, regulates its virulence factors coordinately in response to external stimuli. The locus responsible for this regulation is the bvg locus which encodes a two component signal transduction system made up of a sensor protein, BvgS and a transcriptional activator protein, BvgA. The bvg locus regulates the expression of several virulence factors such as filamentous hemagglutinin (Fha), pertussis toxin (Ptx) and adenylate cyclase/hemolysin toxin (Cya). Previously it was hypothesized that BvgA directly activated the fha and bvg promoters and that a secondary activator was responsible for activation of the ptx promoter. This was based on the observations that bvg and fha promoter-lac fusions on the E. coli chromosome were activated in the presence of the bvg locus whereas a ptx promoter-lac fusion was inactive. Furthermore, BvgA was shown to bind directly upstream of the promoters of fha and bvg at heptanucleotide repeat sequences, but no such binding was observed upstream of the ptx promoter. However, attempts to identify a secondary activator for ptx were unsuccessful and mutants that had a Fha+Ptx- phenotype were found to possess mutations in either the rpoA gene, that encodes the alpha subunit of RNA polymerase (RNAP) or the bvgA gene itself In this study we determined that Bvg alone was responsible for the transcriptional activation of the ptx promoter and that compared to the fha promoter, a higher concentration of BvgA was necessary for ptx promoter activation. We also genetically analyzed the mechanism of ptx promoter activation and showed that a heptanucleotide inverted repeat sequence upstream of the ptx promoter that shared identity with the inverted repeat present upstream of the fha promoter, was important for promoter activation. This inverted repeat sequence has subsequently been shown to be within the primary binding site for BvgA at the ptx promoter in in vitro footprinting studies. The intervening sequence between the repeat region and the ptx promoter was analyzed. We determined in this study that this intervening region played an important role in ptx promoter activation. Replacement of this region as well as deletions of 10, 42 and 63 bp within this region resulted in the inactivation of the ptx promoter. Nevertheless, any of three adjacent 21 bp deletions from this region did not significantly reduce ptx promoter activity. These data support the cooperative-binding model of ptx promoter activation, in which this region provides weaker binding sites for cooperative binding of BvgA, probably as dimers, which places it in a location where it can assist in the binding of RNAP at the ptx promoter. The above result, as well as the fact that mutations with a Fha +Ptx- phenotype mapped to the alpha subunit of RNAP, suggests that there is some interaction between RNAP and BvgA. We tested this hypothesis by attempting to isolate positive control (pc) mutants of BvgA that were defective in their ability to activate the ptx and fha promoters but still retained wild type binding to DNA. One of the mutants obtained demonstrated pc mutant characteristics. This mutant will have to be characterized further but it strengthens the hypothesis that there is interaction between the transcriptional activator, BvgA and RNAP.