Role of PapI in pyelonephritis-associated pili phase variation in Escherichia coli

Update item information
Publication Type dissertation
School or College School of Medicine
Department Pathology
Author Kaltenbach, Linda S.
Title Role of PapI in pyelonephritis-associated pili phase variation in Escherichia coli
Date 1997-06
Description The expression of Pap pili alternates between ON (pili+) and OFF (pili-) states, a process called phase variation. Pap phase variation is controlled by Leucine responsive regulatory protein (Lrp), PapI, and Deoxyadenosine methylase (Dam). Methylation patterns of two GATC sites in the pap regulatory region are established by competition between Dam methylation and Lrp binding. In the phase OFF state, Lrp binds cooperatively to promoter proximal binding sites (1,2,3) and protects GATC-II from methylation whereas GATC-I is methylated. In the phase ON state Lrp binds to distal binding sites (4,5) protecting GATC-I from methylation whereas GATC-II is methylated. Lrp binding to GATC-I required PapI, suggesting that PapI is required for switching to the phase ON state. PapI bound specifically to Lrp-pap DNA complexes, but not Lrp-ilvIH, Lrp in solution or pap DNA. PapI reduced the affinity of Lrp for sites (1,2,3) and increased the affinity for sites (4,5) resulting in translocation of Lrp from the pap GATC-II region to the pap GATC-I region. These observations suggested that Lrp and PapI may directly interact in switching to phase ON. To identify PapI-Lrp binding sites, mutagenesis of papI and lrp was performed. Mutagenesis of papI yielded mutations that resulted in unstable proteins. A novel two color genetic screen was performed to identify pap-specific mutations in lrp. papBA-phoA (blue indicator) and ilvIH-lacZYA (red indicator) fusions generated purple colonies in the presence of wild type lrp. Red colonies were chosen because they contained lrp mutations that could activate ilvIH but not papBA transcription. One mutant, designated Lrp[E133G failed to translocate to GATC-I but was competent for pap and ilvIH DNA binding and PapI-pap DNA binding. Another pap-specific mutation, LrpY115C] may be defective in PapI binding. Furthermore, radiolabeled PapI bound to an Lrp peptide spanning amino acids 105-118. Together, these data indicate that Lrp contains a PapI binding site and a region required for translocation to GATC-I. A model for PapI mediated translocation of Lrp in the phase OFF to phase ON switch is presented.
Type Text
Publisher University of Utah
Subject Genetics; DNA; Urinary Tract Infection
Subject MESH Escherichia coli; Fimbriae, Bacterial
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "The role of PapI in pyelonephritis-associated pili phase variation in Escherichia coli". Spencer S. Eccles Health Sciences Library. Print version of "The role of PapI in pyelonephritis-associated pili phase variation in Escherichia coli". available at J. Willard Marriott Library Special Collection. QR6.5 1997 .K35.
Rights Management © Linda S. Kaltenbach
Format Medium application/pdf
Format Extent 2,780,791 bytes
Identifier undthes,4553
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available).
Master File Extent 2,780,829 bytes
ARK ark:/87278/s6cj8gdn
Setname ir_etd
Date Created 2012-04-24
Date Modified 2012-04-24
ID 191943
Reference URL https://collections.lib.utah.edu/ark:/87278/s6cj8gdn