University of Technology, Sydney. Faculty of Science.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
