title: Interaction of Ler at the LEE5 (tir) operon of enteropathogenic Escherichia coli.
abstract: The genome of enteropathogenic Escherichia coli (EPEC) encodes a global regulator, Ler (locus of enterocyte effacement [LEE]-encoded regulator), which activates expression of several polycistronic operons within the 35.6-kb LEE pathogenicity island, including the LEE2-LEE3 divergent operon pair containing overlapping -10 regions and the LEE5 (tir) operon. Ler is a predicted 15-kDa protein that exhibits amino acid similarity with the nucleoid protein H-NS. In order to study Ler-mediated activation of virulence operons in EPEC, we used a molecular approach to characterize the interactions of purified Ler protein with the upstream regulatory sequences of the LEE5 operon. We determined the cis-acting DNA sequences necessary for Ler binding at LEE5 by mobility shift and DNase I protection assays, demonstrating that Ler acts directly at LEE5 by binding sequences between positions -190 and -73 in relation to the transcriptional start site. Based on the molecular weight of Ler, the similarity to H-NS, and the extended region of protection observed in a DNase I footprint at LEE5, we hypothesized that multiple Ler proteins bind upstream of the LEE5 promoter to increase transcriptional activity from a distance. Using an hns deletion strain, we demonstrated that like the LEE2-LEE3 operon pair, H-NS represses LEE5 transcription. We describe a model in which Ler activates transcription at both divergent overlapping paired and single promoters by displacing H-NS, which results in the disruption of a repressing nucleoprotein complex.
authors.affiliation: Biology Department, Reed College, Portland, Oregon 97202, USA.
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Haack
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Robinson
authors.forename: Kristie J
authors.initials: KJ
authors.lastname: Miller
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Fowlkes
authors.forename: Jay L
authors.initials: JL
authors.lastname: Mellies
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D009876:Operon; D011401:Promoter Regions, Genetic; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C031908:H-NS protein, bacteria; C465556:Ler protein, E coli; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; D015534:Trans-Activators