Mechanistic studies of bacterial chromosome segregation using single-molecule microscopy

In all forms of life, DNA must be properly segregated to each daughter cell prior to cell division to ensure genetic material is inherited. Prokaryotes encode their own systems which facilitate the process of segregation of low-copy number plasmids and chromosomes, including active partitioning (Par) systems. Recent studies have used fluorescence microscopy to reveal the highly organised structure of bacterial chromosomes and their distinct localisation patterns which occur within the cell. The aim of this project was to gain new insight into the molecular mechanisms of chromosome partition systems. A bespoke TIRF microscope was used to study the interplay between the partition proteins of V. cholerae Chromosome II. The non-specific interaction between ParA and DNA was characterised, furthering our understanding of the role of the nucleoid during the segregation process. The partitioning system of V. cholerae Chromosome II was reconstituted in vitro and revealed the formation of chromosomal ParA depletion zones, akin to those seen during the reconstitutions of plasmid partition systems. The TIRF microscope was also adapted to allow the detection of freely diffusing single molecules using a sCMOS detector to multiplex detection channels.