Regulation of the Shigella flexneri icsP gene and H-NS dependent repression

The gram negative bacterium Shigella flexneri is known to cause dysentery in humans and primates. In order to help prevent the spread of shigellosis, gene regulation must be understood. Studies show that the virulence genes in S .flexneri are thermo regulated. At 30°C histone-like nucleoid structuring protein (H-NS) represses transcription of virulence genes and at 37°C VirB derepresses virulence genes. One of the genes that contribute to the virulence of S. flexneri is icsP. My project focuses on the regulation of the S. flexneri icsP gene and has two main goals. The first is to identify the sequence of H-NS dependent repression. Previous studies suggest that the DNA sequence necessary for the H-NS dependent repression is located between -1058 and -523 upstream of the icsP promoter. We are going to create a series of promoter truncations to positions -637, -601,-550, and -436 relative to the icsP promoter. We anticipate this will pin-point which sequences are required for H-NS dependent repression. My second goal is to further characterize the PicsP-lacZ reporter plasmid. Specifically we want to know if icsP promoter activity is being affected by an upstream chloramphenicol cassette. Our hypothesis is that the chloramphenicol cassette is not affecting icsP promoter activity. To test this we will insert a transcription terminator after the chloramphenicol cassette stopping all transcription before the icsP promoter. Promoter activity will be measured by beta-galactosidase assays. These studies will improve our understanding of the regulation of the icsP promoter, and this knowledge may be helpful when studying the regulation of other virulence genes in S. flexneri