The cis requirements for transcriptional activation by HilA, a virulence determinant encoded on SPI-1

In several models of pathogenesis, Salmonella requires genes encoded on Salmonella pathogenicity island 1 (SPI-1) for virulence. In Salmonella enterica serovar Typhimurium (S. typhimurium), most SPI-1 genes are arranged in operons and are co-ordinately regulated in response to environmental signals via the SPI-1-encoded protein HilA. In order to understand how HilA controls the transcription of SPI-1 genes, we have analysed the invF and prgH promoters. We have reconstituted HilA-dependent activation of both promoters in Escherichia coli by supplying hilA on a plasmid, strongly suggesting that HilA acts directly on the promoters. By analysing the HilA-dependent activity of deletions and mutations in PinvF, we identified cis elements necessary for HilA-dependent activation. Through biochemical studies, we have defined a probable HilA-binding sequence in the invF promoter. This 'HilA box' is intact in the minimal promoter identified through deletion analysis, and it is disrupted in one class of PinvF mutants that has reduced activation by HilA. The prgH promoter also contains a HilA box in the same position relative to the +1 of transcription. This work is the first to connect HilA-dependent environmental regulation with a specific sequence in a SPI-1 virulence gene promoter.