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    Characterization of the genes required for the expression of a type IV pilus in enteropathogenic Escherichia coli

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    Author
    Anantha, Ravi Prakash
    Advisor
    Donnenberg, Michael S.
    Date
    2000
    Type
    dissertation
    
    Metadata
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    Abstract
    Enteropathogenic Escherichia coli (EPEC) express a type IV fimbria known as the bundle-forming pilus (BFP). This pilus is required for bacterial autoaggregation and adherence to epithelial cells in a distinctive pattern called localized adherence (LA). EPEC strains that express BFP have a large plasmid. A cluster of fourteen genes on this plasmid is sufficient to reconstitute pilus biogenesis and localized adherence in a laboratory strain of E. coli. Type IV pilus biogenesis is a poorly understood process, and the identification of all the genes required for BFP expression in EPEC makes this an attractive system to study. The first gene in the cluster, bfpA, encodes bundlin, the major structural subunit of BFP. Bundlin is expressed as a pre-protein with a characteristic type IV pilin leader sequence. We have undertaken a systematic mutagenesis of the individual genes of the bfp cluster to determine which genes are required for BFP biogenesis, LA, and autoaggregation. Here we report the construction and analysis of nonpolar mutations in seven bfp genes, bfpG, bfpB, bfpC, bfpD, bfpF, bfpP, and bfpH, as well as the further analysis of a previously described bfpA mutant that is unable to express bundlin. The mutation in bfpP, the gene encoding the pre-pilin peptidase, does not affect pre-bundlin expression, but blocks pre-bundlin processing, BFP biogenesis, LA, and autoaggregation. The mutations in bfpG, bfpB, bfpC, and bfpD do not affect pre-bundlin expression or processing, but block BFP biogenesis, LA, and autoaggregation. The mutation in bfpF does not block any of these events, and in fact bfpF mutant strains adhere to epithelial cells in greater numbers than do wild-type EPEC. The mutation in bfpH has no discernable effect on BFP expression or function. We also show by sucrose density floatation gradient analysis that the association of prebundlin or bundlin with sucrose density floatation gradient fractions containing both inner and outer membrane proteins does not require any other Bfp proteins. Finally, we show that BfpC is a bitopic inner membrane protein. These results show that BfpP is the only prepilin peptidase in EPEC capable of processing prebundlin, BfpG, BfpB, BfpC, and BfpD are all required for BFP expression, BfpF is not required for BFP expression but does play a role in BFP function, and the bfpH gene is not required for any of the phenotypes examined here.
    Description
    University of Maryland, Baltimore. Molecular and Cellular Biology. Ph.D. 2000
    Keyword
    Biology, Molecular
    Biology, Microbiology
    bundle-forming pilus biogenesis
    type IV pilus
    Enteropathogenic Escherichia coli--genetics
    Fimbriae, Bacterial--genetics
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/1243
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    Theses and Dissertations School of Medicine
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