| Popis: |
The plasmid-encoded Per regulatory locus of enteropathogenic Escherichia coli (EPEC) is generally considered to consist of three genes, perA, perB and perC. PerA, a member of the AraC-like family of transcriptional regulators, is known to be an activator of its own promoter (autoactivation) as well as of the plasmid-located bfp operon encoding bundle-forming pili, but its role in activation of the chromosomal locus of enterocyte effacement (LEE) pathogenicity island, which confers the property of intimate adherence on EPEC, requires clarification. Here, we show that PerA is also required for activation of the master regulatory LEE operon, LEE1, but that this activation is indirect, being achieved via autoactivation of the per promoter which ensures sufficient production of the PerC protein to activate LEE1. In contrast, PerA-dependent activation of the per and bfp promoters is direct and does not require the other Per proteins, but is modulated by the nucleoid-associated protein H-NS. The closely related VirF regulator from Shigella flexneri cannot substitute for PerA to activate these promoters, despite being able to bind their upstream regions in vitro. PerA can bind the per and bfp promoter fragments to form multiple complexes, while VirF forms only a single complex. Site-directed mutagenesis of the PerA protein suggests that, like VirF, it may use both of its carboxy-terminal helix-turn-helix motifs for DNA interaction, and may also make direct contacts with RNA polymerase. In addition, we have isolated mutations in the poorly characterized amino-terminal domain of PerA which affect its ability to activate gene expression. |
Plný text