Understanding the dynamics of the parFGH segregation system at the cellular and molecular level

Antibiotic resistance is a growing global health concern, with the occurrences of multi-drug-resistant pathogens ever increasing. Genes that confer antibiotic resistance are often carried by low copy plasmids, making the understanding of how these plasmids are maintained within bacterial populations imperative. Low copy plasmids encode plasmid partition cassettes to ensure that plasmid copies are efficiently segregated prior to cell division.
This work examines the Type Ib parFGH plasmid partition system from the multidrug resistance plasmid TP228, first isolated from the pathogen Salmonella newport. This system is composed of the ATPase ParF, the centromere binding protein ParG and the centromeric site parH. To understand how this system segregates its plasmid copies efficiently we have probed the system at the cellular and molecular level, using both in vitro and in vivo techniques. Firstly, we developed a novel single plasmid construct which contained the entire parFGH cassette with parF fused to muGFP and parG fused to mScarlet-I. Using this construct, we visualised the parFGH system in live cells, leading us to elucidate how ParF separates plasmid copies, with ParF ensuring that plasmids occupy equidistant positions prior to cell division. The results of these investigations also revealed that ParF is distributed asymmetrically across the cell in early-stage plasmid partition and dynamically rearranges as plasmid partition progresses to become more symmetrical. Mutations to ParF residues affecting ATP binding and hydrolysis were also assessed to determine their impact on ParF dynamics in vivo. Finally, we have utilised atomic force microscopy to understand how ParF binding affects DNA topology. Additionally, we compared the topology of the ParF-DNA complex with the topology of the ParFGH complex, which allowed us to assess how ParG manipulates ParF-DNA binding. Based on these findings we have developed a new model of parFGH plasmid segregation.