The Regulation of the icsP promoter of Shigella flexneri by the virulence factor VirB

Shigella flexneri is a pathogenic bacterium that causes severe dysentery in humans commonly known as shigellosis. Shigella encodes an outer membrane protease called IcsP. The regulation of icsP expression is under direct control of a transcriptional factor called VirB, which controls the expression of many virulence genes in Shigella. Previous work has shown through deletion analysis of the icsP promoter region that sequences as far as 1368 base pairs upstream of the transcription starting site are important for the regulation of the icsP gene by VirB. However, it is still unclear whether VirB activation requires sequences within the icsP promoter region or whether VirB activation is an artifact of the cloning vector. The aim of my project is to examine whether sequences located within the icsP promoter are necessary for the activation of the icsP promoter by VirB or whether VirB activation is dependent upon sequences located within the cloning vector. To determine whether VirB activation of the icsP promoter is dependent upon sequences located within the cloning vector, I will delete base pairs and introduce additional base pairs at the junction between the icsP promoter and the cloning vector. In addition, I will generate mutations on the sequence of a distal putative VirB binding site located far upstream of the icsP promoter through site-directed mutagenesis. To indirectly measure activity of the icsP promoter, the promoter has been fused with a reporter gene called lacZ. To measure the expression of the lacZ gene, and hence, the activity of the icsP promoter, a beta-galactosidase assay will be used. I will measure and compare the activity of the icsP promoter in the wild type and VirB mutant strains of Shigella. By using the beta-galactosidase assay, I will be able to determine if the icsP promoter activity is affected by addition, deletion and mutation of base pair sequences upstream of the promoter region