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Permanent link (DOI): https://doi.org/10.7939/R3J38KR75

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Insights into the regulation of F and ColE1 plasmid transfer Open Access

Descriptions

Other title
Subject/Keyword
TraD
F plasmid
bacterial conjugation
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Pedrycz, Barbara J
Supervisor and department
Glover, Mark (Biochemistry)
Examining committee member and department
Lemieux, Joanne (Biochemistry)
James, Michael (Biochemistry)
Raivio, Tracy (Biological Sciences)
Department
Department of Biochemistry
Specialization

Date accepted
2014-06-25T15:37:02Z
Graduation date
2014-11
Degree
Master of Science
Degree level
Master's
Abstract
Multidrug resistant bacteria pose a threat to human health and economic burden for the healthcare system. Transmission of antibiotic resistance genes can occur by the conjugative transfer of the F plasmid between bacteria. The transferosome forms a channel linking the two cells together, while formation of the relaxosome at the oriT nicks and unwinds DNA for transfer. This thesis explores the interaction of the hexameric coupling protein and transferosome component, TraD, with TraM, the DNA binding protein of the relaxosome. Via mating assays, we determined that TraD-TraM interaction does not depend on specific sequences upstream of the terminal 8 residues within the TraD C-terminal domain (CTD). Rather, this region of the TraD CTD functions as a flexible unstructured tether linking the ATPase domain to the terminal 8 residues. These tethers must be more than 44 residues long in order to make multiple simultaneous contacts with TraM and form a stable TraD-TraM complex through avidity effects. Mobilizable plasmids, such as ColE1 encode the relaxase, but lack genes encoding coupling proteins and components for mating pair formation. For this reason, their transmission relies on the presence of conjugative plasmids to encode the necessary conjugation machinery. Expression constructs were made to further structurally and functionally characterize the ColE1-encoded relaxase, MbeA and accessory protein MbeC, which together with MbeB form the relaxosome. EMSA indicated that MbeC binds double-stranded oriT ColE1 DNA in a non-specific manner.
Language
English
DOI
doi:10.7939/R3J38KR75
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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