Investigating the cell division protein FtsZ and its regulation in Bacillus subtilis

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As with all organisms, bacterial cells divide with amazing precision. The first stage of this process is marked by the polymerisation of the essential tubulin-like FtsZ protein at midcell into a ring, the Z ring. Understanding its formation and regulation provides an insight into how the crucial event of cell division is controlled. However, despite intense investigation, these molecular mechanisms are not fully understood. One factor believed to play a role in midcell Z ring placement is the coordination between DNA replication and cell division. Previously it has been shown that when initiation of DNA replication is allowed, but DNA synthesis is inhibited by two different methods (thymine starvation or addition of HPUra), Z rings are able to form at midcell in one case, and not in the other. Both conditions block DNA synthesis at the same stage, the beginning of DNA chain elongation. In an attempt to understand these incongruous results, the possibility of the drug HPUra playing a nonreplicative role, leading to di placement of the Z ring, was examined. It was found that Z ring positioning in an HPUra-resistant strain was not significantly different to that of wild type. Z rings formed at midcell in both conditions. Thu in the wild type strain, the effect of HPUra on Z ring positioning is dependant on its ability to inhibit replication. Hence the block to the elongation stage of DNA replication mediated by the addition of HPUra is capable of misplacing the Z ring, strong evidence for a link between these essential processe of DNA replication and cell division. Ten years ago it was proposed that the Z ring forms by bidirectional growth from a midcell nucleation site. Work presented in this thesis now suggests this may not be the case. Using a modified immunofluorescence protocol it was discovered that, in addition to forming a Z ring, FtsZ forms a helical structure along the length of the cell in vegetatively growing wild type Bacillus subtilis cells. Time-lapse experiments in live cells using an inducible FtsZYFP fusion, showed that the helical FtsZ structure is highly dynamic and undergoes cell cycle-regulated changes in localization. The monitoring of a complete cell cycle revealed the early appearance of a pole-to-pole FtsZ helix, a subsequent short helix spatially restricted to midcell, and finally this redistributed to produce a sharp midcell Z ring. These observations led to the proposal of a novel assembly tnechanism for Z ring formation involving a cell-cycle mediated multi-step remodelling of FtsZ polymers. This was the first report of an FtsZ helix in B. subtilis during vegetative growth. The new model for Z ring formation predicts that in order for the cell to assemble a Z ring, FtsZ must go through long helix-to-short helix-to-Z ring polymerisation changes. How is the Min system, a known negative regulator of FtsZ responsible for inhibiting aberrant Z ring assembly at the cell poles, involved in regulating these FtsZ polymerisation transitions? To address this, FtsZ polymer remodelling was examined whilst modifying the effect of the Min system. Time-lapse studies of a strain carrying a deletion of the minCD genes showed FtsZ polymerising at the poles in the same fashion as wild type; that is going through a short helical intetmediate prior to Z ring formation. This indicated that the helical form of FtsZ is in fact a true intennediate, required for Z rings to form even at non-midcell locations. A minCD over-expression strain showed a marked decrease in Z ring formation and time-lapse imaging wa conducted to asses at which transitional stage FtsZ assembly was affected. Intere tingly, it wa found that both the long and hort helical polymerisation of FtsZ can actually form as wild type in the over-expression experiments. The excess of MinCO in the cell appeared to be able to severely impair division by hampering and prolonging the transition of the short midcell helix to a ring. It is proposed that this i mediated by the inhibition of lateral interactions of FtsZ protofilaments. Indeed a model is put forth etnphasizing the importance of lateral interactions in the helix -to-ring remodelling, and thus in stable Z ring formation. To examine the in vivo FtsZ helix with higher resolution, the advanced microscopic techniques of 4Pi and STED imaging were employed. Using alternative methods has the advantages of confirming the helical structure and extracts further information, for example is the helix continuous? STED microscopy breaks the diffraction barrier and lateral resolution is increased to ~ 100 ntn, ~2. 5 times that of normal confocal microscopy. Using STED, FtsZ localization showed a distinct periodicity, consistent with a helical conformation. Additionally FtsZ staining was revealed to be extremely punctate and discontinuous, suggesting that the helical structure of FtsZ may depend on a cellular track. Visualising cells and their sub-cellular structure in ever increasing detail ensures novel insights into the regulation of Z ring assembly in bacteria.
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