Chromosome Translocation Inflates Bacillus Forespores and Impacts Cellular Morphology.

The means by which the physicochemical properties of different cellular components together determine bacterial cell shape remain poorly understood. Here, we investigate a programmed cell-shape change during Bacillus subtilis sporulation, when a rod-shaped vegetative cell is transformed to an ovoid spore. Asymmetric cell division generates a bigger mother cell and a smaller, hemispherical forespore. The septum traps the forespore chromosome, which is translocated to the forespore by SpoIIIE. Simultaneously, forespore size increases as it is reshaped into an ovoid. Using genetics, timelapse microscopy, cryo-electron tomography, and mathematical modeling, we demonstrate that forespore growth relies on membrane synthesis and SpoIIIE-mediated chromosome translocation, but not on peptidoglycan or protein synthesis. Our data suggest that the hydrated nucleoid swells and inflates the forespore, displacing ribosomes to the cell periphery, stretching septal peptidoglycan, and reshaping the forespore. Our results illustrate how simple biophysical interactions between core cellular components contribute to cellular morphology.

Phase separation in the outer membrane of Escherichia coli.

Gram-negative bacteria are surrounded by a protective outer membrane (OM) with phospholipids in its inner leaflet and lipopolysaccharides (LPS) in its outer leaflet. The OM is also populated with many ß-barrel outer-membrane proteins (OMPs), some of which have been shown to cluster into supramolecular assemblies. However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane. Here, we reveal how the OM is organized from molecular to cellular length scales, using atomic force microscopy to visualize the OM of live bacteria, including engineered Escherichia coli strains and complemented by specific labeling of abundant OMPs. We find that a predominant OMP in the E. coli OM, the porin OmpF, forms a near-static network across the surface, which is interspersed with barren patches of LPS that grow and merge with other patches during cell elongation. Embedded within the porin network is OmpA, which forms noncovalent interactions to the underlying cell wall. When the OM is destabilized by mislocalization of phospholipids to the outer leaflet, a new phase appears, correlating with bacterial sensitivity to harsh environments. We conclude that the OM is a mosaic of phase-separated LPS-rich and OMP-rich regions, the maintenance of which is essential to the integrity of the membrane and hence to the lifestyle of a gram-negative bacterium.

Bacterial cell shape control by nutrient-dependent synthesis of cell division inhibitors.

By analyzing cell size and shapes of the bacterium Bacillus subtilis under nutrient perturbations, protein depletion, and antibiotic treatments, we find that cell geometry is extremely robust, reflected in a well-conserved scaling relation between surface area (S) and volume (V), S∼Vγ, with γ=0.85. We develop a molecular model supported by single-cell simulations to predict that the surface-to-volume scaling exponent γ is regulated by nutrient-dependent production of metabolic enzymes that act as cell division inhibitors in bacteria. Using theory that is supported by experimental data, we predict the modes of cell shape transformations in different bacterial species and propose a mechanism of cell shape adaptation to different nutrient perturbations. For organisms with high surface-to-volume scaling exponent γ, such as B. subtilis, cell width is not sensitive to growth-rate changes, whereas organisms with low γ, such as Acinetobacter baumannii, cell shape adapts readily to growth-rate changes.

A Roadblock-and-Kill Mechanism of Action Model for the DNA-Targeting Antibiotic Ciprofloxacin.

Fluoroquinolones, antibiotics that cause DNA damage by inhibiting DNA topoisomerases, are clinically important, but their mechanism of action is not yet fully understood. In particular, the dynamical response of bacterial cells to fluoroquinolone exposure has hardly been investigated, although the SOS response, triggered by DNA damage, is often thought to play a key role. Here, we investigated the growth inhibition of the bacterium Escherichia coli by the fluoroquinolone ciprofloxacin at low concentrations. We measured the long-term and short-term dynamical response of the growth rate and DNA production rate to ciprofloxacin at both the population and single-cell levels. We show that, despite the molecular complexity of DNA metabolism, a simple roadblock-and-kill model focusing on replication fork blockage and DNA damage by ciprofloxacin-poisoned DNA topoisomerase II (gyrase) quantitatively reproduces long-term growth rates in the presence of ciprofloxacin. The model also predicts dynamical changes in the DNA production rate in wild-type E. coli and in a recombination-deficient mutant following a step-up of ciprofloxacin. Our work highlights that bacterial cells show a delayed growth rate response following fluoroquinolone exposure. Most importantly, our model explains why the response is delayed: it takes many doubling times to fragment the DNA sufficiently to inhibit gene expression. We also show that the dynamical response is controlled by the timescale of DNA replication and gyrase binding/unbinding to the DNA rather than by the SOS response, challenging the accepted view. Our work highlights the importance of including detailed biophysical processes in biochemical-systems models to quantitatively predict the bacterial response to antibiotics.

Bistable forespore engulfment in Bacillus subtilis by a zipper mechanism in absence of the cell wall.

To survive starvation, the bacterium Bacillus subtilis forms durable spores. The initial step of sporulation is asymmetric cell division, leading to a large mother-cell and a small forespore compartment. After division is completed and the dividing septum is thinned, the mother cell engulfs the forespore in a slow process based on cell-wall degradation and synthesis. However, recently a new cell-wall independent mechanism was shown to significantly contribute, which can even lead to fast engulfment in [Formula: see text] 60 [Formula: see text] of the cases when the cell wall is completely removed. In this backup mechanism, strong ligand-receptor binding between mother-cell protein SpoIIIAH and forespore-protein SpoIIQ leads to zipper-like engulfment, but quantitative understanding is missing. In our work, we combined fluorescence image analysis and stochastic Langevin simulations of the fluctuating membrane to investigate the origin of fast bistable engulfment in absence of the cell wall. Our cell morphologies compare favorably with experimental time-lapse microscopy, with engulfment sensitive to the number of SpoIIQ-SpoIIIAH bonds in a threshold-like manner. By systematic exploration of model parameters, we predict regions of osmotic pressure and membrane-surface tension that produce successful engulfment. Indeed, decreasing the medium osmolarity in experiments prevents engulfment in line with our predictions. Forespore engulfment may thus not only be an ideal model system to study decision-making in single cells, but its biophysical principles are likely applicable to engulfment in other cell types, e.g. during phagocytosis in eukaryotes.

Differential growth regulates asymmetric size partitioning in Caulobacter crescentus.

Cell size regulation has been extensively studied in symmetrically dividing cells, but the mechanisms underlying the control of size asymmetry in asymmetrically dividing bacteria remain elusive. Here, we examine the control of asymmetric division in Caulobacter crescentus, a bacterium that produces daughter cells with distinct fates and morphologies upon division. Through comprehensive analysis of multi-generational growth and shape data, we uncover a tightly regulated cell size partitioning mechanism. We find that errors in division site positioning are promptly corrected early in the division cycle through differential growth. Our analysis reveals a negative feedback between the size of daughter cell compartments and their growth rates, wherein the larger compartment grows slower to achieve a homeostatic size partitioning ratio at division. To explain these observations, we propose a mechanistic model of differential growth, in which equal amounts of growth regulators are partitioned into daughter cell compartments of unequal sizes and maintained over time via size-independent synthesis.

Antibiotic Resistance via Bacterial Cell Shape-Shifting.

Bacteria have evolved to develop multiple strategies for antibiotic resistance by effectively reducing intracellular antibiotic concentrations or antibiotic binding affinities, but the role of cell morphology in antibiotic resistance remains poorly understood. By analyzing cell morphological data for different bacterial species under antibiotic stress, we find that bacteria increase or decrease the cell surface-to-volume ratio depending on the antibiotic target. Using quantitative modeling, we show that by reducing the surface-to-volume ratio, bacteria can effectively reduce the intracellular antibiotic concentration by decreasing antibiotic influx. The model further predicts that bacteria can increase the surface-to-volume ratio to induce the dilution of membrane-targeting antibiotics, in agreement with experimental data. Using a whole-cell model for the regulation of cell shape and growth by antibiotics, we predict shape transformations that bacteria can utilize to increase their fitness in the presence of antibiotics. We conclude by discussing additional pathways for antibiotic resistance that may act in synergy with shape-induced resistance.

Cellular resource allocation strategies for cell size and shape control in bacteria.

Bacteria are highly adaptive microorganisms that thrive in a wide range of growth conditions via changes in cell morphologies and macromolecular composition. How bacterial morphologies are regulated in diverse environmental conditions is a long-standing question. Regulation of cell size and shape implies control mechanisms that couple the growth and division of bacteria to their cellular environment and macromolecular composition. In the past decade, simple quantitative laws have emerged that connect cell growth to proteomic composition and the nutrient availability. However, the relationships between cell size, shape, and growth physiology remain challenging to disentangle and unifying models are lacking. In this review, we focus on regulatory models of cell size control that reveal the connections between bacterial cell morphology and growth physiology. In particular, we discuss how changes in nutrient conditions and translational perturbations regulate the cell size, growth rate, and proteome composition. Integrating quantitative models with experimental data, we identify the physiological principles of bacterial size regulation, and discuss the optimization strategies of cellular resource allocation for size control.

Kinetics of myosin node aggregation into a contractile ring.

We study a stochastic aggregation model for the assembly of the contractile ring from a broad band of nodes during cytokinesis in fission yeast. We found that bands of nodes condense into rings when the range of node interactions is larger than the width of the band. Wide bands are unstable to clump formation due to Poisson density fluctuations. We derive expressions for node kinetics and times for ring vs clump formation and test them using numerical simulations. These results suggest clump formation mechanisms in mutant cells.

Nutrient-Dependent Trade-Offs between Ribosomes and Division Protein Synthesis Control Bacterial Cell Size and Growth.

Cell size control emerges from a regulated balance between the rates of cell growth and division. In bacteria, simple quantitative laws connect cellular growth rate to ribosome abundance. However, it remains poorly understood how translation regulates bacterial cell size and shape under growth perturbations. Here, we develop a whole-cell model for growth dynamics of rod-shaped bacteria that links ribosomal abundance with cell geometry, division control, and the extracellular environment. Our study reveals that cell size maintenance under nutrient perturbations requires a balanced trade-off between ribosomes and division protein synthesis. Deviations from this trade-off relationship are predicted under translation inhibition, leading to distinct modes of cell morphological changes, in agreement with single-cell experimental data on Escherichia coli. Furthermore, by calibrating our model with experimental data, we predict how combinations of nutrient-, translational-, and shape perturbations can be chosen to optimize bacterial growth fitness and antibiotic resistance.

Surface-to-volume scaling and aspect ratio preservation in rod-shaped bacteria.

Rod-shaped bacterial cells can readily adapt their lengths and widths in response to environmental changes. While many recent studies have focused on the mechanisms underlying bacterial cell size control, it remains largely unknown how the coupling between cell length and width results in robust control of rod-like bacterial shapes. In this study we uncover a conserved surface-to-volume scaling relation in Escherichia coli and other rod-shaped bacteria, resulting from the preservation of cell aspect ratio. To explain the mechanistic origin of aspect-ratio control, we propose a quantitative model for the coupling between bacterial cell elongation and the accumulation of an essential division protein, FtsZ. This model reveals a mechanism for why bacterial aspect ratio is independent of cell size and growth conditions, and predicts cell morphological changes in response to nutrient perturbations, antibiotics, MreB or FtsZ depletion, in quantitative agreement with experimental data.

Cell-wall remodeling drives engulfment during Bacillus subtilis sporulation.

When starved, the Gram-positive bacterium Bacillus subtilis forms durable spores for survival. Sporulation initiates with an asymmetric cell division, creating a large mother cell and a small forespore. Subsequently, the mother cell membrane engulfs the forespore in a phagocytosis-like process. However, the force generation mechanism for forward membrane movement remains unknown. Here, we show that membrane migration is driven by cell wall remodeling at the leading edge of the engulfing membrane, with peptidoglycan synthesis and degradation mediated by penicillin binding proteins in the forespore and a cell wall degradation protein complex in the mother cell. We propose a simple model for engulfment in which the junction between the septum and the lateral cell wall moves around the forespore by a mechanism resembling the 'template model'. Hence, we establish a biophysical mechanism for the creation of a force for engulfment based on the coordination between cell wall synthesis and degradation.

α-Actinin and fimbrin cooperate with myosin II to organize actomyosin bundles during contractile-ring assembly.

The actomyosin contractile ring assembles through the condensation of a broad band of nodes that forms at the cell equator in fission yeast cytokinesis. The condensation process depends on actin filaments that interconnect nodes. By mutating or titrating actin cross-linkers α-actinin Ain1 and fimbrin Fim1 in live cells, we reveal that both proteins are involved in node condensation. Ain1 and Fim1 stabilize the actin cytoskeleton and modulate node movement, which prevents nodes and linear structures from aggregating into clumps and allows normal ring formation. Our computer simulations modeling actin filaments as semiflexible polymers reproduce the experimental observations and provide a model of how actin cross-linkers work with other proteins to regulate actin-filament orientations inside actin bundles and organize the actin network. As predicted by the simulations, doubling myosin II Myo2 level rescues the node condensation defects caused by Ain1 overexpression. Taken together, our work supports a cooperative process of ring self-organization driven by the interaction between actin filaments and myosin II, which is progressively stabilized by the cross-linking proteins.

Model of myosin node aggregation into a contractile ring: the effect of local alignment.

Actomyosin bundles frequently form through aggregation of membrane-bound myosin clusters. One such example is the formation of the contractile ring in fission yeast from a broad band of cortical nodes. Nodes are macromolecular complexes containing several dozens of myosin-II molecules and a few formin dimers. The condensation of a broad band of nodes into the contractile ring has been previously described by a search, capture, pull and release (SCPR) model. In SCPR, a random search process mediated by actin filaments nucleated by formins leads to transient actomyosin connections among nodes that pull one another into a ring. The SCPR model reproduces the transport of nodes over long distances and predicts observed clump-formation instabilities in mutants. However, the model does not generate transient linear elements and meshwork structures as observed in some wild-type and mutant cells during ring assembly. As a minimal model of node alignment, we added short-range aligning forces to the SCPR model representing currently unresolved mechanisms that may involve structural components, cross-linking and bundling proteins. We studied the effect of the local node alignment mechanism on ring formation numerically. We varied the new parameters and found viable rings for a realistic range of values. Morphologically, transient structures that form during ring assembly resemble those observed in experiments with wild-type and cdc25-22 cells. Our work supports a hierarchical process of ring self-organization involving components drawn together from distant parts of the cell followed by progressive stabilization.

EXTRACTION AND ANALYSIS OF ACTIN NETWORKS BASED ON OPEN ACTIVE CONTOUR MODELS.

Network structures formed by actin filaments are present in many kinds of fluorescence microscopy images. In order to quantify the conformations and dynamics of such actin filaments, we propose a fully automated method to extract actin networks from images and analyze network topology. The method handles well intersecting filaments and, to some extent, overlapping filaments. First we automatically initialize a large number of Stretching Open Active Contours (SOACs) from ridge points detected by searching for plus-to-minus sign changes in the gradient map of the image. These initial SOACs then elongate simultaneously along the bright center-lines of filaments by minimizing an energy function. During their evolution, they may merge or stop growing, thus forming a network that represents the topology of the filament ensemble. We further detect junction points in the network and break the SOACs at junctions to obtain "SOAC segments". These segments are then re-grouped using a graph-cut spectral clustering method to represent the configuration of actin filaments. The proposed approach is generally applicable to extracting intersecting curvilinear structures in noisy images. We demonstrate its potential using two kinds of data: (1) actin filaments imaged by Total Internal Reflection Fluorescence Microscopy (TIRFM) in vitro; (2) actin cytoskeleton networks in fission yeast imaged by spinning disk confocal microscopy.