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.