Establishing how bacterial cells position the division site
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In virtually all bacteria cell division is essential and tightly regulated both temporally and spatially to ensure that cells divide precisely at the centre between segregated chromosomes. Failure to do so can lead to cell death. The earliest event in bacterial cell division is the polymerization of the highly conserved tubulin-like protein, FtsZ, to form a contractile structure called the Z ring, on the inner side of the cytoplasmic membrane at midcell and between chromosomes. The Z ring subsequently contracts causing the cell envelope to invaginate, generating two newborn cells. Thus the Z ring defines the position of the division site in bacterial cells. How the Z ring is positioned precisely at midcell is a controversial topic that remains unresolved. Division site positioning has long been believed to occur via the combined action of two factors: the Min system and nucleoid occlusion. Both factors have been proposed to prevent Z ring assembly along the length of the cell, allowing it to assemble only once chromosomes segregate and nucleoid occlusion is relieved specifically at midcell. The research described in this thesis challenges this paradigm, providing compelling evidence that other mechanisms in addition to nucleoid occlusion and the Min system act to position the Z ring at midcell in B. subtilis. Moreover, this work also shows that nucleoid occlusion and the Min system do not define the Z ring position at midcell but rather ensure that the midcell division site is utilized efficiently. A clue to an additional mechanism for positioning the Z ring has emerged from studies investigating the relationship between chromosome replication and Z ring position. The nature of this relationship has remained obscure for years. Part of this thesis involves a closer examination of this relationship. It was found that the ability to position the Z ring at midcell is linked specifically to the progress of the initiation stage of DNA replication, such that the frequency of Z rings at midcell increases as this stage of DNA replication is progressively completed. Moreover, this link was found to be nucleoid occlusion independent. Spatial and temporal control of Z ring assembly has been widely attributed to the Min system and nucleoid occlusion. While inactivating both systems substantially affects cell division, it is currently unknown whether their absence affects precise midcell Z ring positioning. This thesis deals with this question, and it was found that the combined effect of MinCD and Noc proteins actually affects the timing and efficiency of Z ring assembly, but not its spatial precision between nucleoids at midcell. If Noc and MinCD proteins do not position the Z ring at midcell, what other factores may play this role? Two hypotheses were proposed to help explain the precise Z ring positioning observed in absence of nae and minCD: 1) Noc-independent nucleoid occlusion or 2) factors completely independent of nucleoid occlusion position the Z ring at midcell. Experiments designed to discriminate between these hypotheses showed that they are actually both valid: while the data obtained suggests that factors completely independent of nucleoid occlusion (Noc inclusive) and the Min system position the Z ring at midcell, it also suggested that other Noc-independent nucleoid occlusion factors prevent the Z ring from assembling at midcell over unreplicated DNA.
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