The mechanism by which bacteria divide is not well understood. Cell division is mediated by filaments of FtsZ and FtsA (FtsAZ) that recruit septal peptidoglycan synthesizing enzymes to the division site. To understand how these components coordinate to divide cells, we visualized their movements relative to the dynamics of cell wall synthesis during cytokinesis. We found that the division septum was built at discrete sites that moved around the division plane. FtsAZ filaments treadmilled circumferentially around the division ring, driving the motions of the peptidoglycan synthesizing enzymes. The FtsZ treadmilling rate controlled both the rate of peptidoglycan synthesis and cell division. Thus, FtsZ treadmilling guides the progressive insertion of new cell wall, building increasingly smaller concentric rings of peptidoglycan to divide the cell.

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.

One-sentence summary:Bacterial cytokinesis is controlled by circumferential treadmilling of FtsAZ filaments that drives the insertion of new cell wall. 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. Main Text:Cells from all domains of life must divide in order to proliferate. In bacteria, cell division involves the inward synthesis of the cell wall peptidoglycan (PG), creating a septum that cleaves the cell in two. Septation is directed by proteins that are highly conserved among bacteria. The location and activity of the septal PG synthesis enzymes are regulated by FtsZ, a tubulin homolog, which associates with the cytoplasmic side of the membrane via an actin-like protein FtsA. FtsZ can form filaments (1) and membrane-associated copolymers with FtsA (FtsAZ) (2). Together, the two proteins form a dynamic

cAs 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 O 2 . 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 O 2 -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. Most bacteria form multicellular communities called biofilms by producing and encasing themselves in matrices of secreted polymers (1). The National Institutes of Health has estimated that more than half of all bacterial infections involve such biofilm formation, a feature that complicates treatment due to a variety of associated mechanisms that confer increased antibiotic resistance and tolerance in these communities (2). Steep chemical gradients that form within cellular aggregates give rise to microenvironmental heterogeneity; consequently, cells in biofilms exist in diverse physiological states, at least some of which are unique to this lifestyle. For example, in the opportunistic pathogen Pseudomonas aeruginosa PA14, some genes expressed specifically in biofilms are critical for the establishment of lung infections in a mouse model (3). A better understanding of the physiology of biofilm development is required for rational approaches to new therapies for many types of bacterial infections.Using a colony biofilm model, we have found that a primary factor influencing P. aeruginosa PA14 community morphology is the availability of electron acceptors (4-6). As colonies increase in thickness, the formation of O 2 gradients renders the community anoxic at depth and leads to an increase in intr...

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.

How proteins in the bacterial cell division complex (the divisome) coordinate to divide bacteria remains unknown. To explore how these proteins collectively function, we conducted a complete dynamic characterization of the proteins involved, and then examined the function of FtsZ binding proteins (ZBPs) and their role in cytokinesis. We find that the divisome consists of two dynamically distinct subcomplexes: stationary ZBPs that transiently bind to treadmilling FtsZ filaments, and a directionally-moving complex that includes cell wall synthases. FtsZ filaments treadmill at steady state and the ZBPs have no effect on filament dynamics. Rather, ZBPs bundle FtsZ filaments, condensing them into Z rings. Z ring condensation increases the recruitment of cell wall synthesis enzymes to the division site, and this condensation is necessary for cytokinesis. Main Text:The mechanism by which bacteria divide remains poorly understood. In most bacteria, division begins when filaments of FtsZ, a tubulin homolog, form a "Z ring" at midcell (1). The Z ring then recruits other cell division proteins, collectively called the divisome (Fig 1A). The first group of these proteins (early proteins) arrives concurrently with FtsZ and includes the actin homolog FtsA and several other FtsZ binding proteins (ZBPs): EzrA, SepF, and ZapA. The second group of integral membrane proteins (late proteins) is then recruited, including DivIB, DivIC, and FtsL, and the cell wall synthesis enzymes Pbp2B and FtsW (2, 3). During cytokinesis, the Z ring constricts while the associated cell wall synthesis enzymes build a septum that divides the cell in half (4). Recent work has shown that FtsZ filaments treadmill around the division plane, moving at the same rate as the transpeptidase Pbp2B. (5, 6) (Movie S1). FtsZ treadmilling dynamics are critical for cell division: In Bacillus subtilis, the rate of treadmilling limits Pbp2B motion, the rate of septal cell wall synthesis, and the overall rate of septation (5).To understand how these proteins work to divide cells, we sought to build a dynamic characterization of how this multi-component machine functions in B. subtilis. We first worked to identify groups of divisome proteins that move together, then investigated how the FtsZ-.

AbstractHow proteins in the bacterial cell division complex (the divisome) coordinate to divide bacteria remains unknown. To explore how these proteins collectively function, we conducted a complete dynamic characterization of the proteins involved, and then examined the function of FtsZ binding proteins (ZBPs) and their role in cytokinesis. We find that the divisome consists of two dynamically distinct subcomplexes: stationary ZBPs that transiently bind to treadmilling FtsZ filaments, and a directionally-moving complex that includes cell wall synthases. FtsZ filaments treadmill at steady state and the ZBPs have no effect on filament dynamics. Rather, ZBPs bundle FtsZ filaments, condensing them into Z rings. Z ring condensation increases the recruitment of cell wall synthesis enzymes to the division site, and this condensation is necessary for cytokinesis.

Mycobacteriophages are a diverse group of viruses infecting Mycobacterium with substantial therapeutic potential. However, as this potential becomes realized, the molecular details of phage infection and mechanisms of resistance remain ill-defined. Here we use live-cell fluorescence microscopy to visualize the spatiotemporal dynamics of mycobacteriophage infection in single cells and populations, showing that infection is dependent on the host nucleoid-associated Lsr2 protein. Mycobacteriophages preferentially adsorb at Mycobacterium smegmatis sites of new cell wall synthesis and following DNA injection, Lsr2 reorganizes away from host replication foci to establish zones of phage DNA replication (ZOPR). Cells lacking Lsr2 proceed through to cell lysis when infected but fail to generate consecutive phage bursts that trigger epidemic spread of phage particles to neighbouring cells. Many mycobacteriophages code for their own Lsr2-related proteins, and although their roles are unknown, they do not rescue the loss of host Lsr2.