Tuesday, May 13, 2025
5/13/2025
Elucidating the spatiotemporal regulation of septal peptidoglycan synthases in E.coli - PROJECT SUMMARY In E. coli, cell division occurs at the midcell via initial assembly of the division apparatus into a septal ring and subsequent septal ring closure. The septal ring contains essential proteins including FtsZ (tubulin homolog an GTPase), FtsW (glycosyltransferase), FtsI (transpeptidase), and regulators including FtsN and the FtsBLQ complex. Together FtsW and FtsI form a septal peptidoglycan (sPG) synthase complex (FtsWI) that is essential for new septum synthesis. Recently, single molecules imaging studies in live E. coli cells revealed three different states of FtsWI along the septum: two processive moving states and one immobile state. The slow processive movement of FtsWI is driven by their own sPG synthesis activities by which PG-substrate (Lipid II) is continuously polymerized and crosslinked to the existing septal cell wall (termed on sPG track). The fast processive movement of FtsWI is driven by FtsZ’s treadmilling dynamics but not active in sPG synthesis (termed on the Z-track). Additionally, some FtsWI molecules can display an “immobile” state whereby they remain stationary at septum, but can transition into the fast- and slow-moving populations. These different mobility states of FtsWI complexes signal different states of their activities, and hence providing a new way to investigate the activity regulation pathway of FtsWI at the septum and the corresponding spatiotemporal coordination in septum synthesis. The broad goal of this work is to characterize the spatiotemporal regulation of FtsWI in live E. coli cells and investigate how such regulation impact septum cell wall synthesis and pole morphogenesis. In Aim 1, I will determine the kinetic pathway of FtsWI activation in live cells by developing a single-molecule imaging and analysis pipeline and measure transition frequencies between different states and the corresponding state lifetimes of FtsWI. Using this pipeline, I will investigate how lipid II levels or protein modulators of FtsWI differentially feed into the regulatory pathway. In Aim 2 I will investigate how the relative distributions of FtsWI on the sPG and Z-tracks are related to division progression and cell pole morphogenesis. Using a combination of single molecule tracking, Structured Illumination Microscopy (SIM), Scanning Electron Microscopy, and genetic and growth conditions known to unbalance the ratio of FtsWI on the two tracks, I will determine to what extent the two tracks are required to maintain balanced rates of septum closure and septum symmetry that give rise to cell pole shape. Elucidating the kinetic pathway for FtsWI regulation will reveal novel insights regarding how molecular inputs feed into this dynamic system.
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