Title page for ETD etd-12142004-185557


Type of Document Master's Thesis
Author Horton, William Henry Clay
Author's Email Address whorton@vt.edu
URN etd-12142004-185557
Title Characterization of the Components of Carbon Catabolite Repression in Clostridium perfringens
Degree Master of Science
Department Biology
Advisory Committee
Advisor Name Title
Melville, Stephen B. Committee Chair
Chen, Jiann-Shin Committee Member
Stevens, Ann M. Committee Member
Keywords
  • CRE-site
  • carbon catabolite repression
  • HPr kinase/phosphatase
  • Clostridium perfringens
  • sporulation
  • CcpA
Date of Defense 2004-12-08
Availability unrestricted
Abstract
Clostridium perfringens is a versatile pathogen capable of causing a wide array of diseases, ranging from clostridial food poisoning to tissue infections such as gas gangrene. An important factor in virulence as well as in the distribution of C. perfringens is its ability to form an endospore. The symptoms of C. perfringens food poisoning are directly correlated to the release of an enterotoxin at the end of the sporulation process. The sporulation process in C. perfringens is subject to carbon catabolite repression (CCR) by sugars, especially glucose. CCR is a regulatory pathway that alters transcription based on carbon source availability. In Gram-positive bacteria, the HPr kinase/phosphatase is responsible for this nutritional sensing by phosphorylating or dephosphorylating the serine-46 residue of HPr. HPr-Ser-P then forms a complex with the transcriptional regulator CcpA to regulate transcription. We were able to show here that purified recombinant C. perfringens HPr kinase/phosphatase was able to phosphorylate the serine-46 residue of HPr. When the codon for this serine residue is mutated through PCR mutagenesis to encode alanine, phosphorylation could not take place. We have also shown that in gel retardation assays, CcpA and HPr-Ser-P were able to bind to two DNA fragments containing putative C. perfringens CRE-sites, sequences where CcpA binds to regulate transcription. The genome sequence of a food poisoning strain of C. perfringens was searched for potential CRE-sites using degenerate sequences designed to match those CRE-sites CcpA was shown to bind. DNA fragments containing these newly identified CRE-sites were then used in gel retardation assays to determine whether CcpA binds to these CRE-sites, making them candidates for CCR regulation. These results, combined with comparisons of metabolic characteristics of a ccpA- strain versus wild-type C. perfringens, provide evidence that CcpA participates in the regulation of carbon catabolite repression in the pathogenic bacterium C. perfringens
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