Molecular basis of pyelonephritis-associated pili phase variation in Escherichia coli

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Title Molecular basis of pyelonephritis-associated pili phase variation in Escherichia coli
Publication Type dissertation
School or College School of Medicine
Department Pathology
Author Nou, Xiangwu
Date 1996-06
Description The expression of pyelonephritis-associated pili (Pap) in Escherichia coli is under a phase variation control mechanism in which individual cells alternate between pili+ (ON) and pili- (OFF) states. This occurs through a process involving DNA methylation by deoxyadenosine methylase (Dam). Methylation of two GATC sites (GATC-I and GATC-II) within the pap regulatory region is differentially inhibited in phase ON and phase OFF cells. The GATC-I site of phase ON cells is nonmethylated and GATC-II site is fully methylated. Conversely, in phase OFF cells the GATC-I site is fully methylated whereas the GATC-II site is nonmethylated. Two transcription activators, Lrp and PapI, are required for this specific methylation inhibition. Low resolution DNA footprint analyses using nonmethylated pap DNA indicated that Lrp binds near the GATC-II, whereas PapI does not bind specifically to pap regulatory region. However, the addition of Lrp and PapI together resulted in an additional footprint around the GATC-I site, indicating that both Lrp and PapI are required for binding to the GATC-I region. To define the role of Dam methylation in pap gene regulation, the GATC-I and GATC-II sites were mutated so that they could not be methylated, and the effects of these mutations on Pap phase variation were examined. The results indicated that methylation of GATC-I blocks formation of the phase ON state by inhibiting PapI-dependent Lrp binding to this DNA region. In contrast, methylation of GATC-II is required for the phase OFF to ON transition. Evidence suggests that this occurs by the inhibition of Lrp to sites overlapping the papBA promoter, which may occlude RNA polymerase. The Lrp binding sites in the pap regulatory region were further defined by methylation protection analysis. Six Lrp binding sites were found, each separated by about three helical turns of DNA. Lrp bound with highest affinity to three sites (1, 2, and 3) proximal to the papBA promoter. A mutational analysis indicated that the binding of Lrp to sites 2 and 3 inhibits pap transcription, which is consistent with the fact that Lrp binding site 3 is located between the -35 and -10 RNA polymerase binding region of papBA promoter. The addition of PapI decreased the affinity of Lrp for sites 1, 2, and 3 and increased its affinity for the distal Lrp binding sites 4 and 5. Mutations within Lrp binding sites 4 and 5 shut off pap transcription, indicating that the binding of Lrp to this pap region activated transcription. The pap GATC-I and GATC-II sites are located within Lrp binding sites 5 and 2, respectively, providing a mechanism by which Dam controls Lrp binding and Pap phase variation. A model for Pap phase variation is presented based on these results..
Type Text
Publisher University of Utah
Subject Nonmethylated pap DNA; Gene Regulation; Etiology
Subject MESH Escherichia coli Infections; Pyelonephritis; Urinary Tract Infections
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "The molecular basis of pyelonephritis-associated pili phase variation in Escherichia coli". Spencer S. Eccles Health Sciences Library. Print version of "The molecular basis of pyelonephritis-associated pili phase variation in Escherichia coli". available at J. Willard Marriott Library Special Collection. QR6.5 1996 .N68.
Rights Management © Xiangwu Nou.
Format application/pdf
Format Medium application/pdf
Format Extent 2,895,400 bytes
Identifier undthes,4449
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available).
Master File Extent 2,895,470 bytes
ARK ark:/87278/s6jd4zkb
Setname ir_etd
ID 190951
Reference URL https://collections.lib.utah.edu/ark:/87278/s6jd4zkb
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