Intrinsic and extrinsic factors regulate FtsZ function in Caulobacter crescentus

Bacterial cell division is crucial for replication and requires careful coordination via a complex set of proteins collectively known as the divisome. The tubulin-like GTPase FtsZ is the master regulator of this process and serves to recruit downstream divisome proteins and regulate their activities. Upon arrival at mid-cell, FtsZ associates with the membrane via anchoring proteins and exhibits treadmilling motion, driven by its GTP binding and hydrolysis activities. Treadmilling is proposed to play a role in Z-ring condensation, as well as in distribution and regulation of peptidoglycan (PG) cell wall remodeling enzymes. FtsZ polymer superstructure and dynamics are central to its function, yet their regulation is incompletely understood. We sought to address these gaps in knowledge by modulating intrinsic and extrinsic regulators of FtsZ and evaluating their effects in vitro and in cells, alone and in combination. To do this, we leveraged the cell cycle control features of Caulobacter crescentus. We observed that Caulobacter FtsZ variants that abrogate GTP hydrolysis impact FtsZ dynamics and Z-ring positioning, with little to no effect on Z-ring structure or constriction. Production of an FtsZ variant lacking its disordered C-terminal linker ({Delta}CTL) resulted in aberrant Z-ring dynamics and morphology, misregulated PG metabolism, and cell lysis. Combining {Delta}CTL and GTPase mutations was additive, suggesting they each act independently to control the Z-ring. Modulating levels of FtsA resulted in formation of multiple Z-rings that failed to constrict, suggesting roles in regulating both FtsZ superstructure and the activity of downstream divisome components. Collectively, our results indicate that GTP hydrolysis serves primarily to position the Z-ring at mid-cell, the CTL regulates both Z-ring structure and downstream signaling, and FtsA contributes to all aspects of FtsZ assembly and function. The additive effects of these elements are required to support robust and efficient cell division.