The toxigenic element of Clostridium difficile strain 10463 and its transcriptional analysis in strains which differ in toxigenicity

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dc.contributor.authorHammond, Georgia Annen
dc.contributor.committeechairJohnson, John L.en
dc.contributor.committeememberDean, Dennis R.en
dc.contributor.committeememberKrieg, Noel R.en
dc.contributor.committeememberWilkins, Tracy D.en
dc.contributor.committeememberLarson, Timothy J.en
dc.contributor.departmentAnaerobic Microbiologyen
dc.date.accessioned2014-03-14T21:09:54Zen
dc.date.adate2006-03-02en
dc.date.available2014-03-14T21:09:54Zen
dc.date.issued1994-03-05en
dc.date.rdate2006-03-02en
dc.date.sdate2006-03-02en
dc.description.abstractClostridium difficile is a Gram positive, anaerobic bacterium which produces two potent protein toxins, A and B. The genes for toxins A and B have been previously cloned and sequenced and lie within 1.4 kb of each other. Upstream and downstream boundaries between sequences shared by both toxigenic and nontoxigenic strains and those sequences which are unique to toxigenic strains were established. A toxigenic element was defined in C. difficile strain 10463 which is 19.6 kb in length and is comprised of five open reading frames, including the toxin A and B genes. One of these open reading frames is previously unidentified and is located upstream of toxin B. Products of Polymerase Chain Reaction (PCR) amplification of three regions in the toxigenic element: the upstream boundary, the downstream boundary, and the region between the toxin A and B genes, were all identical in length in six toxigenic strains, indicating that the toxigenic element is conserved among these strains. A short fragment unique to nontoxigenic strains and occupying the same position on the chromosome as the toxigenic element was identified. peR products of this region were identical in length in three nontoxigenic strains. Transcriptional analyses were undertaken using probes to each of the five open reading frames in the toxigenic element. Transcripts were detected for four of the open reading frames which are contiguous and transcribed in the same direction. In addition, a very large transcript, corresponding to the length of the four open reading frames and processing intermediates were detected, indicating that the toxin genes are cotranscribed. A promoter region and processing sites were identified. Sizes were determined for each of the individual transcripts which correspond well with the sizes of the open reading frames. Six toxigenic strains which vary considerably in toxin production were selected for analysis to determine whether DNA sequence variation could account for the observed differences in toxin production. DNA restriction fragment length polymorphisms were examined, toxin-specific transcripts were analyzed, and sequences of regulatory regions were determined and compared. Whereas quantitative differences in toxin-specific transcripts were found among the toxigenic strains, the remaining analyses showed that DNA sequences were conserved among these strains.en
dc.description.degreePh. D.en
dc.format.extentx, 124 leavesen
dc.format.mediumBTDen
dc.format.mimetypeapplication/pdfen
dc.identifier.otheretd-03022006-093403en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-03022006-093403/en
dc.identifier.urihttp://hdl.handle.net/10919/37447en
dc.language.isoenen
dc.publisherVirginia Techen
dc.relation.haspartLD5655.V856_1994.H366.pdfen
dc.relation.isformatofOCLC# 30909659en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subject.lccLD5655.V856 1994.H366en
dc.subject.lcshClostridium difficileen
dc.subject.lcshToxinsen
dc.subject.lcshTranscription factorsen
dc.titleThe toxigenic element of Clostridium difficile strain 10463 and its transcriptional analysis in strains which differ in toxigenicityen
dc.typeDissertationen
dc.type.dcmitypeTexten
thesis.degree.disciplineAnaerobic Microbiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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