The goal of this proposal is to understand the molecular basis of phase variation of the pyelonephritis-associated pili (pap) operon of uropathogenic Escherichia coli. The pap operon codes for a fimbrial-adhesin complex important in the pathogenesis of upper urinary tract infections. Pap phase variation is controlled by leucine-responsive regulatory protein (Lrp), PapI, and deoxyadenosine methylase (Dam). These proteins act within the pap regulatory region, which includes two Dam target sites designated GATC-I and GATC-II. Competitive interactions between Lrp and Dam at GATC-I and GATC-II result in pap DNA methylation patterns characteristic of phase ON and phase OFF cells. These methylation patterns, in turn, control Lrp binding to pap regulatory DNA. A key step in the phase OFF to ON switch is the movement of Lrp from the GATC-II region to GATC-I, located over 100 basepairs upstream. This Lrp translocation is facilitated by PapI, which specifically binds to the Lrp moiety of Lrp-pap DNA complexes.
One aim of this proposal is to identify the domains of PapI and Lrp that interact with each other to aid Lrp translocation. Both genetic and biochemical approaches will be used to identify the amino acids of Lrp and PapI that contact each other.
A second aim i s to determine how PapI and Dam affect the binding equilibria and stoichiometry of Lrp to pap DNA using gel shift, DNA footprinting, and fluorescence resonance energy transfer binding assays.
A third aim i s to isolate a recently identified factor that interacts with the pap regulatory region designated as Dam blocking factor (Dbf). Dbf will be purified by DNA affinity chromatography, and its role in regulating Pap phase variation will be assessed using a dbf null mutation for genetic analysis.
A fourth aim i s to determine the roles of Lrp, PapI, Dam, Dbf, and CAP in the activation and repression of pap transcription. In vitro transcription experiments will be carried out to measure the effects of these regulatory factors on the affinity of RNA polymerase for the papBAp promoter, the rate of open complex formation, and the rate of promoter clearance. The relevance of the in vitro data to Pap gene regulation in E. coli will be determined using in vivo footprinting. The studies proposed should provide a solid basis for understanding how DNA methylation patterns are formed and regulated by protein-DNA interactions and how methylation patterns, in turn, control gene expression.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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University of California Santa Barbara
Schools of Arts and Sciences
Santa Barbara
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Weyand, N J; Braaten, B A; van der Woude, M et al. (2001) The essential role of the promoter-proximal subunit of CAP in pap phase variation: Lrp- and helical phase-dependent activation of papBA transcription by CAP from -215. Mol Microbiol 39:1504-22
Hale, W B; van der Woude, M W; Braaten, B A et al. (1998) Regulation of uropathogenic Escherichia coli adhesin expression by DNA methylation. Mol Genet Metab 65:191-6
Kaltenbach, L; Braaten, B; Tucker, J et al. (1998) Use of a two-color genetic screen to identify a domain of the global regulator Lrp that is specifically required for pap phase variation. J Bacteriol 180:1224-31
van der Woude, M; Braaten, B; Low, D (1996) Epigenetic phase variation of the pap operon in Escherichia coli. Trends Microbiol 4:5-9
Kaltenbach, L S; Braaten, B A; Low, D A (1995) Specific binding of PapI to Lrp-pap DNA complexes. J Bacteriol 177:6449-55
Nou, X; Braaten, B; Kaltenbach, L et al. (1995) Differential binding of Lrp to two sets of pap DNA binding sites mediated by Pap I regulates Pap phase variation in Escherichia coli. EMBO J 14:5785-97
van der Woude, M W; Kaltenbach, L S; Low, D A (1995) Leucine-responsive regulatory protein plays dual roles as both an activator and a repressor of the Escherichia coli pap fimbrial operon. Mol Microbiol 17:303-12
Hale, W B; van der Woude, M W; Low, D A (1994) Analysis of nonmethylated GATC sites in the Escherichia coli chromosome and identification of sites that are differentially methylated in response to environmental stimuli. J Bacteriol 176:3438-41
van der Woude, M W; Low, D A (1994) Leucine-responsive regulatory protein and deoxyadenosine methylase control the phase variation and expression of the sfa and daa pili operons in Escherichia coli. Mol Microbiol 11:605-18
Braaten, B A; Nou, X; Kaltenbach, L S et al. (1994) Methylation patterns in pap regulatory DNA control pyelonephritis-associated pili phase variation in E. coli. Cell 76:577-88

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