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Infection and Immunity, January 2003, p. 384-392, Vol. 71, No. 1
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.1.384-392.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Kenneth R. Haack, Christopher L. Robinson, Kristie J. Miller,{dagger} Jonathan W. Fowlkes, and Jay L. Mellies*

Biology Department, Reed College, Portland, Oregon 97202

Received 1 March 2002/ Returned for modification 7 May 2002/ Accepted 18 September 2002

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.

* Corresponding author. Mailing address: Biology Department, Reed College, 3203 S.E. Woodstock Blvd., Portland, OR 97202. Phone: (503) 771-1112, ext. 7964. Fax: (503) 777-7773. E-mail: jay.mellies{at}reed.edu.

Editor: V. J. DiRita

{dagger} Present address: Molecular Microbiology and Immunology Department, Oregon Health and Science University, Portland, OR 97201.

Infection and Immunity, January 2003, p. 384-392, Vol. 71, No. 1
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.1.384-392.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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