Assembling the ParFGH segrosome, the nucleoprotein machine for proper segregation of plasmid TP228

Abstract

TP228 is a low copy number, multidrug-resistance plasmid that uses a partition system to ensure its accurate distribution during Escherichia coli cell division. The segregation cassette of TP228 consists of the parF, parG and parH loci. The first gene encodes the ParF protein which is a member of the Walker-type ATPase superfamily. The second gene, parG, encodes a site-specific DNA binding protein that autoregulates parFG expression by binding to the OF operator that overlaps the parFG promoter. OF comprises an array of variant 5'-ACTC-3' boxes separated by 4-bp AT-rich spacers. ParG also is predicted to be a centromere binding protein at the parH site which is located 5' of the parFG cassette. Analysis of the sequences of parH, and the parF-parG intergenic region revealed different numbers and arrangements of variant 5'-ACTC-3' motifs compared to OF. Numerous important questions for the parFGH system remained unanswered at the beginning of this work. For instance, the roles of parH and the intergenic region and the interaction of ParG with these sites during segregation had not been investigated. Using DNase I footprinting and gel retardation assays in this thesis, it was shown that (i) ParG binds specifically to the parH and parF-parG intergenic regions, the latter at a novel operator site, OG, that is implicated in parG transcriptional regulation. ParG-parH complexes were also observed in AFM. (ii) ParG binds to subsites from parH and OG with different binding affinities. These results were confirmed by competition assays. (iii) The N-terminal mobile tail of ParG plays important roles in parH and OG binding and in assembly of the ParFGH segrosome. Moreover, an assay was developed which showed that parH possesses centromere function. The OF and OG sites also independently possess centromere activity. Furthermore, segrosome formation by ParF, ParG and parH was demonstrated in gel retardation and footprinting experiments. The work provides new information on the formation of the ParG-parH complex and how this interacts with ParF subsequently to move plasmid DNA molecules apart during partitioning. In summary, the work presented in this thesis highlights the importance of the three major components of the parFGH system and provides evidence for segrosome assembly, an essential aspect of plasmid segregation in prokaryotic cells.

Date of Award	1 Aug 2011
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Finbarr Hayes (Supervisor)