Cell division in Bacillus subtilis : new insights from an old mutant
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In bacteria, cell division is mediated by a macromolecular complex consisting of numerous proteins that act together to split the cell into two. The earliest event in this process is the formation of a polymeric ring, composed of the tubulin-like protein FtsZ, at the future site of division. This so-called ‘Z ring’ plays a pivotal role in the cell division mechanism, at least in part because it serves as a scaffold for the assembly of the division apparatus. Importantly, Z ring formation establishes both when and where the cell will divide, and is therefore subject to stringent spatiotemporal control. This thesis is concerned with the molecular mechanism of Z ring assembly and its regulation in the gram-positive model organism Bacillus subtilis. It involves the use of powerful fluorescence microscopy techniques in combination with molecular biological and genetic methods to examine the specific effects of a temperature sensitive FtsZ mutation on Z ring formation in vivo. The B. subtilis strain harbouring this mutation is known as tsl, while the mutant protein itself has been designated FtsZ(Tsl). Initial experiments examined the intracellular localisation pattern of the FtsZ(Tsl) protein in live cells growing at 49°C (the non-permissive temperature for tsl). This work revealed that while FtsZ(Tsl) is unable to form Z rings under non-permissive conditions, it retains the capacity to polymerise in vivo and instead assembles into short helical-like structures. Interestingly, these helices were observed to reorganise into fully functional Z rings following a shift from 49°C down to permissive temperatures. These and other observations suggest an exciting new model for Z ring assembly in wild-type bacterial cells, involving a regulated helix-to-ring remodelling of FtsZ polymers. The work also suggests that at non- permissive temperatures, the FtsZ(Tsl) protein is unable to complete the final stages of this remodelling process, and becomes trapped as a short helical intermediate of Z ring formation in vivo. To explore how the FtsZ helix-to-ring assembly mechanism is orchestrated within the cell, further experiments aimed to identify exactly why FtsZ(Tsl) is unable to complete this process at 49°C. During this work, it was discovered that in the presence of elevated levels of the FtsZ-binding protein ZapA, FtsZ(Tsl) regains the capacity to form functional Z rings at 49°C via the normal assembly pathway. The ZapA protein has previously been shown to promote Z ring assembly in the cell, and to stimulate the association of simple FtsZ polymers (protofilaments) into higher-order polymeric structures in vitro. These and other results suggest that FtsZ(Tsl) is specifically defective in its ability to support higher-order polymer association in vivo under non-permissive conditions. This enables FtsZ(Tsl) to polymerise into a helix, while preventing the helix from undergoing the structural changes required for it to reorganise into a stable ring. These findings have important implications regarding the molecular mechanism of the FtsZ helix-to-ring transition in wild-type cells. Other work presented in this thesis involved a genetic screen for extragenic suppressors of tsl thermosensitivity. Using insertional mutagenesis, a total of four unique genes were identified that rescue tsl to temperature resistance when inactivated. Given that the tsl strain is specifically defective in FtsZ function, and taking into account other findings in the literature, this strongly suggests that these genes act in some capacity to control FtsZ activity in vivo. Further characterisation of the gene products promises to uncover novel insights into the regulation of Z ring assembly in bacteria.
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