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J Biol Chem, Vol. 275, Issue 5, 3192-3200, February 4, 2000

Regulation of Pap Phase Variation
Lrp IS SUFFICIENT FOR THE ESTABLISHMENT OF THE PHASE OFF pap DNA METHYLATION PATTERN AND REPRESSION OF pap TRANSCRIPTION IN VITRO^*

Nathan J. Weyand and David A. Low

From the Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106

The pyelonephritis-associated pili (pap) operon in Escherichia coli is regulated by an epigenetic mechanism involving the formation of specific DNA methylation patterns characteristic of transcriptionally active (phase ON) and inactive (phase OFF) cells. The formation of pap DNA methylation patterns in vivo was previously shown to require the leucine-responsive regulatory protein (Lrp) and DNA adenine methylase (Dam). To monitor the binding of Lrp to pap DNA, an in vitro methylation protection assay was developed. Binding of Lrp to a Dam target site proximal to the papBA promoter (designated GATC^prox) blocked methylation of this site and specifically repressed transcription. The DNA methylation pattern and transcription state are identical to those observed in vivo in phase OFF cells. To determine if binding of Lrp at GATC^prox was necessary for repression of papBA transcription, we analyzed a pap mutation (pap-13) that reduced the affinity of Lrp for the GATC^prox region. Binding of Lrp to pap-13 DNA was shifted to a promoter distal Dam target site (designated GATC^dist). Lrp blocked methylation of GATC^dist in the pap-13 mutant, but did not repress papBA transcription. Together, these results show that binding of Lrp to the GATC^prox region is sufficient for the establishment of the phase OFF DNA methylation pattern and repression of papBA transcription.

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^* This work was supported by National Institutes of Health Grant AI23348 (to D.A.L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Molecular, Cellular, and Developmental Biology, Rm. 3129, Bioscience II Bldg., University of California, Santa Barbara, CA 93106. Tel.: 805-893-5597; Fax: 805-893-7558; E-mail: low@lifesci.ucsb.edu.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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