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.