Abstract The FtsZ protein is a central component of the bacterial cell division machinery. It polymerizes at mid-cell and recruits more than 30 proteins to assemble into a macromolecular complex to direct cell wall constriction. FtsZ polymers exhibit treadmilling dynamics, driving the processive movement of enzymes that synthesize septal peptidoglycan (sPG). Here, we combine theoretical modelling with single-molecule imaging of live bacterial cells to show that FtsZ’s treadmilling drives the directional movement of sPG enzymes via a Brownian ratchet mechanism. The processivity of the directional movement depends on the binding potential between FtsZ and the sPG enzyme, and on a balance between the enzyme’s diffusion and FtsZ’s treadmilling speed. We propose that this interplay may provide a mechanism to control the spatiotemporal distribution of active sPG enzymes, explaining the distinct roles of FtsZ treadmilling in modulating cell wall constriction rate observed in different bacteria.
Abstract Bacterial cell division is mediated by the tubulin-homolog FtsZ, which recruits peptidoglycan (PG) synthesis enzymes to the division site. Septal PG synthases promote inward growth of the division septum, but the mechanisms governing the spatiotemporal regulation of these enzymes are poorly understood. Recent studies on various organisms have proposed different models for the relationship between the movement and activity of septum-specific PG synthases and FtsZ treadmilling. Here, we studied the movement dynamics of conserved cell division proteins relative to the rates of septum constriction and FtsZ treadmilling in the Gram-positive pathogen Staphylococcus aureus . The septal PG synthesis enzyme complex FtsW/PBP1 and its putative activator protein, DivIB, moved processively, around the division site, with the same velocity. Impairing FtsZ treadmilling did not affect FtsW and DivIB velocities or septum constriction rates. Contrarily, inhibition of PG synthesis slowed down or completely stopped both septum constriction and the directional movement of FtsW/PBP1 and DivIB. Our findings support a model for S. aureus in which a single population of processively moving FtsW/PBP1 remains associated with DivIB to drive cell constriction independently of treadmilling FtsZ filaments.
ABSTRACT Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall (CW) assembly. In Escherichia coli , alterations in a CW synthase component of the system called PBP2 were identified that overcome other inactivating defects. Rod system activity was stimulated in the suppressors in vivo, and purified synthase complexes with these changes showed more robust CW synthesis in vitro. Polymerization of the actin-like MreB component of the Rod system was also found to be enhanced in cells with the activated synthase. The results suggest an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both CW glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient CW assembly. An analogous activation pathway involving similar enzymatic components is likely responsible for controlling CW synthesis by the division machinery.
2920-Pos Board B612 Quantitation of Cell Wall Growth Suggests Feedback Mechanisms that Robustly Build Rod-Like Bacteria Tristan Ursell, Kerwyn Casey Huang. Bioengineering, Stanford University, Stanford, CA, USA. In bacteria, a host of enzymes regulates the reproducible and robust construction of the cell wall, whose mechanical integrity is crucial for viability under osmotic stress. Antibiotics that target these enzymes disrupt cell wall construction, ultimately leading to mechanical failure of the cell. Our work explores the physical mechanisms of cell growth and death, as a guide to understanding antibiotic mechanisms that disrupt mechanical properties of the cell. We use a combination of cell wall fluorescent labeling, high resolution time-lapse microscopy, and computational image processing to characterize where, and with what dynamics, cell wall and outer membrane growth occurs. When cell-shape analysis is combined biophysical simulations of growth, our data strongly suggest that dynamic localization of the bacterial MreB cytoskeleton is part of a curvature sensing and growth feedback mechanism that orchestrates heterogeneous growth to maintain rod-like shape and regulate mechanical stress. Analysis of MreB and cell-surface marker fluorescence indicates that the cytoskeleton is present at sites of active growth and that chemical depolymerization of the cytoskeleton causes homogenous, unstructured growth and eventual cell death by rupture. Quantitative tracking of growth is an effective method for characterizing cell wall mechanical failure, and these techniques pave the way for studying the detailed dynamics of growth-associated proteins and their disturbance by antibiotics.
As biofilms grow, resident cells inevitably face the challenge of resource limitation. In the opportunistic pathogen Pseudomonas aeruginosa PA14, electron acceptor availability affects matrix production and, as a result, biofilm morphogenesis. The secreted matrix polysaccharide Pel is required for pellicle formation and for colony wrinkling, two activities that promote access to O2. We examined the exploitability and evolvability of Pel production at the air-liquid interface (during pellicle formation) and on solid surfaces (during colony formation). Although Pel contributes to the developmental response to electron acceptor limitation in both biofilm formation regimes, we found variation in the exploitability of its production and necessity for competitive fitness between the two systems. The wild type showed a competitive advantage against a non-Pel-producing mutant in pellicles but no advantage in colonies. Adaptation to the pellicle environment selected for mutants with a competitive advantage against the wild type in pellicles but also caused a severe disadvantage in colonies, even in wrinkled colony centers. Evolution in the colony center produced divergent phenotypes, while adaptation to the colony edge produced mutants with clear competitive advantages against the wild type in this O2-replete niche. In general, the structurally heterogeneous colony environment promoted more diversification than the more homogeneous pellicle. These results suggest that the role of Pel in community structure formation in response to electron acceptor limitation is unique to specific biofilm models and that the facultative control of Pel production is required for PA14 to maintain optimum benefit in different types of communities.
Abstract FtsZ, a highly conserved bacterial tubulin GTPase homolog, is a central component of the cell division machinery in nearly all walled bacteria. FtsZ polymerizes at the future division site and recruits greater than 30 proteins to assemble into a macromolecular complex termed the divisome. Many of these divisome proteins are involved in septal cell wall peptidoglycan (sPG) synthesis. Recent studies found that FtsZ polymers undergo GTP hydrolysis-coupled treadmilling dynamics along the circumference the division site, driving the processive movement of sPG synthesis enzymes. How FtsZ’s treadmilling drives the directional transport of sPG enzymes and what its precise role is in bacterial cell division are unknown. Combining theoretical modeling and experimental testing, we show that FtsZ’s treadmilling drives the directional movement of sPG-synthesis enzymes via a Brownian ratchet mechanism, where the shrinking end of FtsZ polymers introduces an asymmetry to rectify diffusions of single sPG enzymes into persistent end-tracking movement. Furthermore, we show that the processivity of this directional movement is dependent on the binding potential between FtsZ and the enzyme, and hinges on the balance between the enzyme’s diffusion and FtsZ’s treadmilling speed. This interplay could provide a mechanism to control the level of available enzymes for active sPG synthesis both in time and space, explaining the distinct roles of FtsZ treadmilling in modulating cell wall constriction rate observed in different bacterial species.
Abstract How bacteria produce a septum to divide in two is not well understood. This process is mediated by periplasmic cell-wall producing enzymes that are positioned by filaments of the cytoplasmic membrane-associated actin FtsA and the tubulin FtsZ (FtsAZ). To understand how these components act in concert to divide cells, we visualized their movements relative to the dynamics of cell wall synthesis during cytokinesis. We find that the division septum is built at discrete sites that move around the division plane. Furthermore, FtsAZ filaments treadmill in circumferential paths around the division ring, pulling along the associated cell-wall-synthesizing enzymes. We show that the rate of FtsZ treadmilling controls both the rate of cell wall synthesis and cell division. The coupling of both the position and activity of the cell wall synthases to FtsAZ treadmilling guides the progressive insertion of new cell wall, synthesizing increasingly small concentric rings to divide the cell. One-sentence summary Bacterial cytokinesis is controlled by circumferential treadmilling of FtsAZ filaments that drives the insertion of new cell wall.
Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall assembly by the glycan polymerase RodA and crosslinking enzyme PBP2. Using Escherichia coli as a model system, we identified a PBP2 variant that promotes Rod system function when essential accessory components of the machinery are inactivated. This PBP2 variant hyperactivates cell wall synthesis in vivo and stimulates the activity of RodA-PBP2 complexes in vitro. Cells with the activated synthase also exhibited enhanced polymerization of the actin-like MreB component of the Rod system. Our results define an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient cell wall assembly. In light of these results, previously isolated mutations that activate cytokinesis suggest that an analogous pathway may also control cell wall synthesis by the division machinery.
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