Use of an Inducible Promoter to Characterize Type IV Pili Homologues in Clostridium perfringens

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dc.contributor.authorHartman, Andrea H.en
dc.contributor.committeechairMelville, Stephen B.en
dc.contributor.committeememberSchubot, Florian D.en
dc.contributor.committeememberPopham, David L.en
dc.contributor.committeememberChen, Jiann-Shinen
dc.contributor.departmentBiologyen
dc.date.accessioned2017-04-04T19:49:54Zen
dc.date.adate2012-10-18en
dc.date.available2017-04-04T19:49:54Zen
dc.date.issued2012-09-13en
dc.date.rdate2016-09-30en
dc.date.sdate2012-09-26en
dc.description.abstractResearchers of <i>Clostridium perfringens</i>, 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 <i>E. coli</i> reporter GusA, we characterized its induction in three different strains of <i>C. perfringens</i>. 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 <i>C. perfringens</i> 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 <i>Bacillus subtilis</i>. PilT's typical localization in <i>Bacillus subtilis</i> was disrupted when the GTPase and polymerization activity of cell division protein FtsZ was blocked, suggesting that PilT associates with cell division proteins.en
dc.description.degreeMaster of Scienceen
dc.identifier.otheretd-09262012-161530en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-09262012-161530/en
dc.identifier.urihttp://hdl.handle.net/10919/76874en
dc.language.isoen_USen
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectClostridium perfringensen
dc.subjectType II secretionen
dc.subjectinducible promoteren
dc.subjectType IV pilien
dc.titleUse of an Inducible Promoter to Characterize Type IV Pili Homologues in Clostridium perfringensen
dc.typeThesisen
dc.type.dcmitypeTexten
thesis.degree.disciplineBiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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