Title page for ETD etd-09262012-161530


Type of Document Master's Thesis
Author Hartman, Andrea H
Author's Email Address ahhartman@vt.edu
URN etd-09262012-161530
Title Use of an Inducible Promoter to Characterize Type IV Pili Homologues in Clostridium perfringens
Degree Master of Science
Department Biology
Advisory Committee
Advisor Name Title
Melville, Stephen B. Committee Chair
Chen, Jiann-Shin Committee Member
Popham, David L. Committee Member
Schubot, Florian D. Committee Member
Keywords
  • Clostridium perfringens
  • Type II secretion
  • inducible promoter
  • Type IV pili
Date of Defense 2012-09-13
Availability unrestricted
Abstract
Researchers of Clostridium perfringens, a Gram-positive anaerobic pathogen, were lacking a tightlyregulated, inducible promoter system in their genetic toolbox. We constructed a lactose-inducible plasmid-based system utilizing the transcriptional regulator, BgaR. Using the E. coli reporter GusA, we characterized its induction in three different strains of C. perfringens. We then used a newly-developed mutation system to create in-frame deletion mutants in three genes with homology to Type IV pilins, and we used the promoter system described above to complement the mutants. We analyzed each pilin for localization and expression, as well as tested each of the mutants for various phenotypes frequently associated with type IV pili (TFP) and type II secretion systems. PilA2, PilA3, and PilA4 localized to the poles of the cells. PilA2 was expressed in the wildtype when C. perfringens was grown on agar plates, and the PilA3 mutant lacked a von Willebrand factor A domain-containing protein in its secretome. We used our promoter system to express GFP-tagged versions of the TFP ATPase homologues and view them in cells growing on surfaces. We saw that PilB1 and PilB2 co-localized nearly all of the time, while a portion of PilT was independent of the PilB proteins. PilT appeared necessary for the localization of PilB, and it localized independently of TFP proteins in Bacillus subtilis. PilT’s typical localization in Bacillus subtilis was disrupted when the GTPase and polymerization activity of cell division protein FtsZ was blocked, suggesting that PilT associates with cell division proteins.
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