Abstract
During bacterial cell division, synthesis of new septal peptidoglycan (sPG) is crucial for successful cytokinesis and cell pole morphogenesis. FtsW, a SEDS (Shape, Elongation, Division and Sporulation) family protein and an indispensable component of the cell division machinery in all walled bacterial species, was recently identified in vitro as a new monofunctional peptidoglycan glycosyltransferase (PGTase). FtsW and its cognate monofunctional transpeptidase (TPase) class B penicillin binding protein (PBP3 or FtsI in E. coli) may constitute the essential, bifunctional sPG synthase specific for new sPG synthesis. Despite its importance, the septal PGTase activity of FtsW has not been documented in vivo. How its activity is spatiotemporally regulated in vivo has also remained unknown. Here we investigated the septal PGTase activity and dynamics of FtsW in E. coli cells using a combination of single-molecule imaging and genetic manipulations. We show that FtsW exhibits robust activity to incorporate an N-acetylmuramic acid analog at septa in the absence of other known PGTases, confirming FtsW as the essential septum-specific PGTase in vivo. Notably, we identified two populations of processive moving FtsW molecules at septa. A fast-moving population is driven by the treadmilling dynamics of FtsZ and independent of sPG synthesis. A slow-moving population is driven by active sPG synthesis and independent of FtsZ’s treadmilling dynamics. We further identified that FtsN, a potential sPG synthesis activator, plays an important role in promoting the slow-moving, sPG synthesis-dependent population. Our results support a two-track model, in which inactive sPG synthase molecules follow the fast treadmilling “Z-track” to be distributed along the septum; FtsN promotes their release from the “Z-track” to become active in sPG synthesis on the slow “sPG-track”. This model explains how the spatial information is integrated into the regulation of sPG synthesis activity and suggests a new mechanistic framework for the spatiotemporal coordination of bacterial cell wall constriction.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
1.We performed dwell time analysis on the processive moving FtsW molecules to validate the two distinct populations of FtsW (Fig.3). The dwell time of stationary FtsW molecules indicated the nature of the stationary FtsW molecules are either bound to the middle of FtsZ filaments or trapped in sPG synthesis sites.
2. We monitored the changes in the slow-moving population of FtsW in cells with different levels of cell wall precursors using different growth media and overexpression of a precursor synthesis enzyme(UppS) (Fig.4).
3. We updated our segmentation algorithm to avoid human bias.
4. We performed additional new experiments to show that PGTase activities from other enzymes such as PBP1A/B also contribute to the slow-moving dynamics of FtsW molecules. In our previous submission, we observed that when FtsW is inhibited by MTSES in the ftsWI302C background, there was still a ∼20% slow-moving population of FtsW. Referee #2 questioned this observation. In the revised manuscript, we show that the slow-moving population of FtsW is completely abolished and the entire population is shifted to fast-moving in the del3, ponBS247C, ftsWI302C strain in the presence of MTSES, where all known PGTase activity is inhibited. This result further confirmed that the slow-moving population of FtsW is active in sPG synthesis and that other PGTase activity could also drive the slow, processive movement of FtsW.
5. We measured FtsZ treadmilling speed in most of the conditions to compare with the fast-moving population.
6. We monitored FtsI in several conditions to confirm it also showed two motions as FtsW.