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Interaction of Ler at the LEE5 ( tir ) Operon of Enteropathogenic Escherichia coli

2002; American Society for Microbiology; Volume: 71; Issue: 1 Linguagem: Inglês

10.1128/iai.71.1.384-392.2003

1098-5522

Kenneth R. Haack, Christopher L. Robinson, Kristie J. Miller, Jonathan W. Fowlkes, Jay L. Mellies,

Viral gastroenteritis research and epidemiology

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.