Ler and H-NS, Regulators Controlling Expression of the Long Polar Fimbriae of Escherichia coli O157:H7

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Abstract
Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 colonizes the human intestine and is responsible for diarrheal outbreaks worldwide. Previously we showed that EHEC produces long polar fimbriae (LPF) and that maximum expression is observed during the exponential phase of growth at 37°C and pH 6.5. In this study, we analyzed the roles of several regulators in the expression of LPF using the β-galactosidase reporter system, and we found that H-NS functions as a transcriptional silencer while Ler functions as an antisilencer of LPF expression. Interestingly, deletion of the hns and ler genes in EHEC caused constitutive expression of the fusion reporter protein. Semiquantitative reverse transcription (RT)-PCR was also used to analyze LPF expression in the EHEC ler or hns mutant strain. The hns mutant exhibited an increase in lpf mRNA expression, while expression in the ler mutant was decreased, compared to that in 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 σ 70 -dependent promoters. Further, we determined whether H-NS and Ler interact directly with the lpf promoter region by using purified His-tagged proteins and electrophoretic mobility shift assays. Our data are the first to show direct binding interactions between the H-NS and Ler proteins within the regulatory sequence of the lpf operon. Based on the electrophoretic mobility shift assay, RT-PCR, primer extension, and β-galactosidase assay results, we concluded that the E. coli O157:H7 lpf operon possesses a promoter dependent on σ 70 , that H-NS binds to the regulatory sequence of lpfA and “silences” the transcription of lpf , and that Ler binds to the regulatory sequence and inhibits the action of the H-NS protein.