Abstract

The focus of this proposal is on the role of DNA methylation in the regulation of pyelonephritis-associated pili (pap) gene expression in uropathogenic Escherichia coli. Pap pili expression is subject to a phase variation regulatory mechanism which involves DNA methylation and specific methylation inhibition at two GATC sites (GATC1028 and GATC1130) within pap regulatory DNA sequences. This mechanism appears to be different from other phase variation systems previously studied. The overall objective of this proposal is to determine the mechanism by which the methylation of the GATC1028 and GATC1130 sites is inhibited by the papI and methylation blocking factor (mbf) gene products, and how these gene products regulate pilin transcription. These proteins will be used to analyze protein-DNA interactions in vitro using DNaseI and methylation protection assays. To determine the protein-DNA interactions occurring in transcriptionally inactive (off) and active (on) cells, we will use in vivo DNA footprinting. An abortive transcriptional initiation assay will be developed to determine if MBF, PapI and cAMP-CAP are sufficient to stimulate pap transcription by RNA polymerase. If they are not, we will use this assay to purify additional factors from crude E. coli extracts. The role(s) of Dam methylase in pap gene regulation will be examined by determining the effects of in vitro methylation on protein-DNA interactions and transcription initiation. In addition to the above proposed biochemical approach, pap regulatory mutants will be isolated after random mutagenesis. In these studies, pilin transcription will be analyzed using single copy pap-lac gene fusion constructs. A search for both pap-linked and unlinked second site revertants will be carried out as well. Regulatory mutants will be characterized by DNA sequence analysis and by DNA-protein interaction analysis as described above. Site-directed mutagenesis will be used to further probe the functions of specific pap regulatory sequences that are identified by random mutagenesis. The experiments proposed are important and significant because they explore a new area in bacterial gene regulation; the epigenetic modulation of gene expression by DNA methylation inhibition.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI023348-07
Application #
2062151
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1987-04-01
Project End
1996-07-31
Budget Start
1994-08-01
Budget End
1995-07-31
Support Year
7
Fiscal Year
1994
Total Cost
Indirect Cost

  Institution

Name
University of Utah
Department
Pathology
Type
Schools of Medicine
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112

  Publications

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
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
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
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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