Cell cycle-dependent recruitment of FtsN to the divisome in Escherichia coli

Abstract Cell division in Escherichia coli starts with the formation of an FtsZ protofilament network in the middle of the cell, the Z ring. However, only after a considerable lag period do the cells start to form a midcell constriction. The basis of this cell cycle checkpoint is yet unclear. The onset of constriction is dependent upon the arrival of so-called late divisome proteins, among which, FtsN is the last arriving essential one. The timing and dependency of FtsN arrival to the divisome, along with genetic evidence, suggests it triggers cell division. In this study, we used high throughput fluorescence microscopy to quantitatively determine the arrival of FtsN and the early divisome protein ZapA to midcell at a single-cell level during the cell cycle. Our data show that recruitment of FtsN coincides with the initiation of constriction within experimental uncertainties and that the relative fraction of ZapA/FtsZ reaches its highest value at this event. We also find that FtsN is recruited to midcell in two distinct temporal stages with septal peptidoglycan synthesis starting in the first stage and accelerating in the second stage, during which the amount of ZapA/FtsZ in the midcell decreases. In the presence of FtsA*, recruitment of FtsN becomes concurrent with the formation of the Z-ring, but constriction is still delayed indicating FtsN recruitment is not rate limiting, at least under these conditions. Finally, our data support the recently proposed idea that ZapA/FtsZ and FtsN are part of physically separate complexes in midcell throughout the whole septation process. Importance In E. coli , FtsN has been considered a trigger for septal wall synthesis and the onset of constriction. While FtsN is critical for cell division, its recruitment kinetics to midcell has not been characterized. Using quantitative high throughput microscopy, we find that FtsN is recruited to midcell in two temporal stages. The septal cell wall synthesis starts at the first stage and accelerates in the second stage. In the presence of an FtsA mutant defective in self-interaction, recruitment of FtsN to midcell is enhanced, but constriction is still delayed. Our results shed new light on an essential but not rate-limiting role of FtsN in E. coli cell division and also support the view that ZapA/FtsZ and FtsN are part of physically separate complexes in midcell throughout the division process.