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J. Bacteriol. doi:10.1128/JB.00245-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Ler and H-NS, regulators controlling the expression of the Long Polar Fimbriae of Escherichia coli O157:H7

Department of Microbiology and Immunology, Department of Pathology and Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas 77555-1070, and Centro de Investigaciones en Ciencias Microbiológicas, B. Universidad Autónoma de Puebla, Puebla, Puebla 72570 México

^* To whom correspondence should be addressed. Email: altorres{at}utmb.edu. igmatine{at}siu.buap.mx.

	Abstract

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 colonizes the human intestine and is responsible for diarrheal outbreaks worldwide. Previously, we showed that EHEC produce a Long Polar Fimbriae (LPF) and that maximum expression is observed during exponential phase of growth at 37°C and pH 6.5. In this study, we analyzed the role of several regulators on the expression of LPF using the -galactosidase reporter system, and we found that H-NS functions as a transcriptional silencer while Ler functions as an anti-silencer of LPF expression. Interestingly, deletion of hns and ler genes in EHEC caused constitutive expression of the fusion reporter protein. Semi-quantitative RT-PCR was also used to analyze LPF expression in the EHEC ler or hns mutant strains. The hns mutant exhibited an increase in lpf mRNA expression while expression in the ler mutant was decreased as compared to the wild type strain. Using primer extension analysis, we identified two potential transcriptional start sites within the regulatory region of lpf, and located consensus hexamers of -10 (CAAGAT) and -35 (TTCAAA), which are commonly found in sigma 70-dependent promoters. Further, we determined whether H-NS and Ler interacts directly with the lpf promoter region using purified His-tagged proteins and electrophoretic mobility shift assays. Our data is the first to show direct binding interactions between H-NS and Ler proteins within the regulatory sequence of the lpf operon. Based on the EMSA, RT-PCR, primer extension, and -galactosidase assays results, we concluded that the E. coli O157:H7 lpf operon possesses a promoter dependent on sigma 70, that H-NS binds to the regulatory sequence of lpfA and "silences" the transcription of lpf, while Ler binds to the regulatory sequence and inhibits the action of the H-NS protein.

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