The role of the FtsA protein in Bacillus subtilis cell division
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Bacterial cell division involves the invagination of the membrane and the cell wall to form a septum at midcell, between two replicated chromosomes. From a molecular perspective, the main event in cell division is the formation of a circumferential structure, the Z ring, formed by polymerisation of the tubulin-like FtsZ protein. The Z ring recruits a multi-protein complex to the division site, forming a division apparatus that eventually constricts as the septum forms. FtsA, a eukaryotic actin homologue, is another division protein, known to interact directly with FtsZ. It has been proposed that FtsA promotes Z ring formation; however its exact role has remained unknown. This thesis investigateshow FtsA affects the Z ring and cytokinesis in the Gram-positive model organism, Bacillus subtilis. Interestingly, FtsA is essential in Escherichia coli, the Gram-negative model organism, but not in Bacillus subtilis. Rather, deletion of the ftsA gene in vegetatively-growing B. subtilis cells causes a significant reduction in Z ring formation and cell division is severely diminished while cell growth is maintained, resulting in cell filamentation (long cells without septa). To confirm that this phenotype is due to the inability of FtsZ to efficiently form rings, Z ring formationwas examined in the absence of FtsA, during the first round of cell division following B. subtilis spore germination. Surprisingly the Z rings formed with wild-type efficiency. However, unlike wild-type cells that showed subsequent constriction of these Z rings leading to septum formation, Z rings did not constrict immediately in the ftsA mutant and persisted into the second cycle of division. These results reveal for the first time that, unlike E. coli, FtsA is not required for Z ring formation in B. subtilis. To understand the delay in Z ring constriction, further experiments were conducted to determine if the recruitment of downstream division proteins to the Z ring is affectedin the absence of FtsA. The live-cell microscopy data confirmed that the recruitment of DivIB, and presumably other downstream division proteins that are co-recruited with DivIB, is delayed in ftsA-mutant cells, but occurs with wild-type efficiency. However, after recruitment of DivIB, Z ring constriction and septation are still inefficient in the absence of FtsA. These observations indicate a primary role for FtsA in B. subtilis in the later stages of division, that is, after the division apparatus has assembled. This work reveals a novel perspective on the function of this protein. In an attempt to further explore how Z ring constriction is affected by FtsA, microscopy studies were designed to analyse this cell process. Different Z ring constriction defects were observed in ftsA-mutant cells. Importantly, it was shown that, in the absence of FtsA, constriction is either significantly delayed or never occurs, resulting in destabilisation of the Z ring, indicating that FtsA is required for efficient Z ring constriction in B. subtilis. This finding raised the possibility that FtsA may be affecting the dynamics of the Z ring during cytokinesis. To verify this, the rate of FtsZ turnover in Z rings of ftsA-mutant cells was investigated. The results demonstrated a decrease in the rate of the FtsZ turnover in the Z ring in the absence of FtsA, possibly enough to cause an effect on Z ring constriction.
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