Abstract
Enteropathogenic E. coli virulence genes are under the control of various regulators, one of which is PerA, an AraC/XylS-like regulator. PerA directly promotes its own expression and that of the bfp operon encoding the genes involved in the biogenesis of the bundle-forming pilus (BFP); it also activates PerC expression, which in turn stimulates locus of enterocyte effacement (LEE) activation through the LEE-encoded regulator Ler. Monomeric PerA directly binds to the per and bfp regulatory regions; however, it is not known whether interactions between PerA and the RNA polymerase (RNAP) are needed to activate gene transcription as has been observed for other AraC-like regulators. Results showed that PerA interacts with the alpha subunit of the RNAP polymerase and that it is necessary for the genetic and phenotypic expression of bfpA. Furthermore, an in silico analysis shows that PerA might be interacting with specific alpha subunit amino acids residues highlighting the direction of future experiments.
Highlights
Enteropathogenic Escherichia coli (EPEC) is an E. coli pathovar that causes diarrhea in in children under 5 years of age and elderly p eople[1,2]
Here we used the MBPPerA fusion to identify interacting proteins in two related experiments: pull downs and co-purifications with cellular extracts of an EPEC strain grown in bundle-forming pilus (BFP)-A/E inducing conditions
To corroborate the conditions used to induce the expression of BFP, E2348 WT and ΔperA mutant strains were grown in inducing and repressing conditions and the expression of BfpA was detected by Western blot
Summary
Enteropathogenic Escherichia coli (EPEC) is an E. coli pathovar that causes diarrhea in in children under 5 years of age and elderly p eople[1,2]. Transcriptional activation of bfp relays on PerA ( known as BfpT), a member of the AraC/XylS family of transcriptional activators encoded by the per (perABC) operon in the EAF plasmid, which regulates its own expression[11,12,13]. As an activator of the genes required for both virulence-related phenotypes, LA and A/E, PerA represents a central regulator of the infectious process caused by typical EPEC strains. One possibility is that these residues are involved in interactions with the RNA polymerase (RNAP) Such interactions have been documented in other members of the AraC/XylS family of transcriptional activators[22,23,24]. Results showed that these interactions occur with the alpha (α) subunit and confirm our previous hypothesis that PerA is a positive regulator that makes specific contacts with the transcriptional machinery and that these interactions are important for the expression of genes under PerA regulation
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