Biology subjects

Whitley, K. D.

Publications and source records attributed to Whitley, K. D..

5 recordsLinked to original sources

bioRxiv2023

A one-track model for spatiotemporal coordination of Bacillus subtilis septal cell wall synthesis

Bacterial cell division requires synthesis of a septal peptidoglycan (sPG) wall across the middle of the cell. This is accomplished by the divisome synthesis complex in coordination with numerous other division proteins--such as the essential tubulin homolog FtsZ--but the molecular mechanism of its spatiotemporal regulation remains unclear. Here, we investigate the dynamics of sPG synthesis in the model Gram-positive bacterium Bacillus subtilis using live-cell single-molecule imaging of the divisome transpeptidase PBP2B. In contrast to previous models for division, we show that there is a single population of processively-moving PBP2B molecules whose motion is driven by peptidoglycan synthesis and is not associated with FtsZ treadmilling. However, although the motions of PBP2B and FtsZ are asynchronous, we demonstrate that processive PBP2B motion is partially dependent on FtsZ treadmilling. Additionally, we provide evidence that the divisome synthesis complex is multimeric. Our results support a new model for division in B. subtilis where a multimeric synthesis complex follows a single track dependent on sPG synthesis whose activity and dynamics are asynchronous with FtsZ treadmilling.

microbiology↗

bioRxiv2023

Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics in Bacillus subtilis

Most rod-shaped bacteria elongate by inserting new cell wall material into the inner surface of the cell sidewall. This is performed by class A penicillin binding proteins (PBPs) and a highly conserved protein complex, the elongasome, which moves processively around the cell circumference and inserts long glycan strands that act as barrel-hoop-like reinforcing structures, thereby giving rise to a rod-shaped cell. However, it remains unclear how elongasome synthesis dynamics and termination events are regulated to determine the length of these critical cell-reinforcing structures. To address this, we developed a method to track individual elongasome complexes around the entire circumference of Bacillus subtilis cells for minutes-long periods using single molecule fluorescence microscopy. We found that the B. subtilis elongasome is highly processive and that processive synthesis events are frequently terminated by rapid reversal or extended pauses. We found that cellular levels of RodA regulate elongasome processivity, reversal and pausing. Our single molecule data, together with stochastic simulations, show that elongasome dynamics and processivity are regulated by molecular motor tug-of-war competition between several, likely two, oppositely oriented peptidoglycan synthesis complexes associated with the MreB filament. Our data, thus, demonstrate that molecular motor tug-of-war is a key regulator of elongasome dynamics in B. subtilis, which likely also regulates the cell shape via modulation of elongasome processivity.

microbiology↗

bioRxiv2023

Self-organisation of mortal filaments: the role of FtsZ treadmilling in bacterial division ring formation

Protein filaments in the cell commonly treadmill - they grow on one end while shrinking on the other, driven by energy consumption. Treadmilling filaments appear to be moving, even though individual proteins remain static. Here, we investigate the role of treadmilling, implemented as dynamic turnover, in the collective filament self-organisation. On the example of the bacterial FtsZ protein, a highly conserved tubulin homologue, we show, in computer simulations and in vitro experiments, that treadmilling drives filament nematic ordering by dissolving misaligned filaments. We demonstrate that ordering via local dissolution allows the system to quickly respond to chemical and geometrical biases in the cell, and is necessary for the formation of the FtsZ ring required for bacterial cell division in living Bacillus subtilis cells. We finally use simulations to quantitatively explain the characteristic dynamics of FtsZ ring formation in vivo. Beyond FtsZ and other cytoskeletal filaments, our study identifies a novel mechanism for nematic ordering via constant birth and death of energy-consuming filaments.

biophysics↗

bioRxiv2021

Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

biophysics↗

bioRxiv2020

FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in bacterial cell division

Despite the central role of division in bacterial physiology, how division proteins work together as a nanoscale machine to divide the cell remains poorly understood. Cell division by cell wall synthesis proteins is guided by the cytoskeleton protein FtsZ, which assembles at mid-cell as a dense Z-ring formed of treadmilling filaments1,2. However, although FtsZ treadmilling is essential for cell division, the function of FtsZ treadmilling remains unclear2-5. Here, we systematically resolve the function of FtsZ treadmilling across each stage of division in the Gram-positive model organism Bacillus subtilis using a novel combination of nanofabrication, advanced microscopy, and microfluidics to measure the division-protein dynamics in live cells with ultrahigh sensitivity. We find that FtsZ treadmilling has two essential functions: mediating condensation of diffuse FtsZ filaments into a dense Z-ring, and initiating constriction by guiding septal cell wall synthesis. After constriction initiation, FtsZ treadmilling has a dispensable function in accelerating septal constriction rate. Our results show that FtsZ treadmilling is critical for assembling and initiating the bacterial cell division machine.

microbiology↗