P1 prophage is stably maintained in E. coli as a low-copy-number plasmid. Stable maintenance of P1 is dependent on the function of the plasmid encoded partition system, parABS. ParA is the partition ATPase, ParB is the partition-site binding protein, and parS is the partition site. The concerted action of these proteins results in dynamic movement of the plasmid over the bacterial nucleoid, which results in its stable maintenance. Plasmid movement has been proposed to be caused by interactions between parS bound ParB and nucleoid bound
ParA. In this thesis, I have identified a complex of ParA, ParB, and DNA that is capable of promoting plasmid stability. ParA, ParB, DNA interactions required the ATP bound conformation of ParA. The ParA-ParB-DNA complex was dynamically regulated by nucleotide hydrolysis, which promoted complex disassembly. Complex formation resulted from the cooperative binding of ParA and ParB to DNA. ParA-ParB and ParB-DNA interactions were both necessary for complex formation. ParA-ParB-DNA complex size was regulated by ParB stimulation of ParA-ATP hydrolysis. Microscopy demonstrated that complexes resulted in the association of multiple DNA molecules due to protein binding. The properties of complex assembly, dynamics, and DNA grouping lead me to propose a model where associations between ParA bound to the bacterial nucleoid and the partition complex mediated plasmid movement and localization.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/32730 |
| Date | 21 August 2012 |
| Creators | Havey, James C. |
| Contributors | Funnell, Barbara E. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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