Studies on expression, organization, and regulation of the gene cluster associated with the biosynthesis of Escherichia coli K99 adhesin
Lee, John Hwa
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https://hdl.handle.net/2142/21479
Description
Title
Studies on expression, organization, and regulation of the gene cluster associated with the biosynthesis of Escherichia coli K99 adhesin
Author(s)
Lee, John Hwa
Issue Date
1995
Doctoral Committee Chair(s)
Isaacson, Richard E.
Department of Study
Veterinary Medicine
Discipline
Veterinary Biosciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Microbiology
Language
eng
Abstract
The biogenesis of the pilus adhesin K99 is dependent upon the expression of eight contiguous genes: fanA-H. These genes are encoded on an 87.8 kb plasmid. To study gene expression, regulation, and transcriptional organization of the gene cluster associated with K99, transposon mutagenesis using TnphoA and TnlacZ was employed. Using these transposon mutants, expression of each K99 gene was measured. FanC was the most highly expressed and FanD was expressed at very low levels. The expression of TnlacZ insertions in fanA and B insertions was high. Deletion of fanA, B, and part of fanC abolished the transcription of fanD but had no effect on the distal genes fanE-H. To locate the regions regulating fanE-H, deletion mutations were prepared and the effects on expression of fanE-H was determined. The deletion of a segment between fanD and fanE abolished fanE and F expression but did not affect fanG and H expression. The deletion of a portion of fanF (approximately 1 kb proximal to fanG) abolished the expression of fanG and H. These results indicate the presence of regulatory units proximal to fanE and to fanG. Putative promoters were identified in these regions by DNA homology and by primer extension. A stem loop structure was also found at the beginning of fanF that may act as a transcriptional attenuator of fanF expression. Based on these data and our previous data, a model of the K99 gene cluster was constructed. The K99 genes are divided into 3 regions. Region I includes fanA-fanD, Region II includes fanE and fanF, and Region III includes fanG and fanH. The expression of these regions are independent of each other. Region I genes are dependent upon CRP-cAMP complex and LRP. The gene expression of Region II is dependent on CRP-cAMP complex. A promoter for the region II genes is located 17 bp upstream from the initiation codon for fanE. The region III genes may be expressed by multiple promoters within fanF. The expression is regulated by a repressor. The repressor may be regulated by CRP-cAMP.
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