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The ability of eukaryotic cells to differentiate substrate stiffness is fundamental for many processes such as the development of stem cells into mature tissue. Here, we establish that bacteria feel their microenvironment in a similar manner. We show that Pseudomonas aeruginosa actively probes and measures substrate stiffness using type IV pili (TFP). The activity of the major virulence factor regulator Vfr is peaked with stiffness in a physiologically important range between 0.1 kPa (mucus) and 1000 kPa (cartilage). The local concentration of PilA at the base of dynamic TFP changes during extension and retraction in a surface dependent manner due to slow PilA diffusion in the cell membrane. Traction force measurements reveal that TFP retraction deforms even stiff substrates. Modeling of the measured substrate deformation and optical tweezers experiments suggest that TFP adhere at the tip only. Informed by these experimental results, we developed a model that describes substrate stiffness dependent dynamics of the polar PilA concentration which are quantitatively consistent with the transcriptional response to stiffness. Manipulating the ATPase activity of the TFP motors changes the TFP extension and retraction velocities and consequently the PilA concentration dynamics in a manner that is predictive of the experimental stiffness response. This work points to the use of a competition between PilA diffusion and TFP extension-retraction as a molecular shear rheometer. Our results highlight that stiffness sensing is a conserved property between the kingdoms of life.

Bacteria use extracellular appendages called type IV pili (T4P) for diverse behaviors including DNA uptake, surface sensing, virulence, protein secretion, and twitching motility. Dynamic extension and retraction of T4P is essential for their function, and T4P extension is thought to occur through the action of a single, highly conserved motor, PilB. Here, we develop Acinetobacter baylyi as a model to study T4P by employing a recently developed pilus labeling method. By contrast to previous studies of other bacterial species, we find that T4P synthesis in A. baylyi is dependent not only on PilB but also on an additional, phylogenetically distinct motor, TfpB. Furthermore, we identify a protein (CpiA) that inhibits T4P extension by specifically binding and inhibiting PilB but not TfpB. These results expand our understanding of T4P regulation and highlight how inhibitors might be exploited to disrupt T4P synthesis., Type IV pili (T4P) are retractile appendages used by bacteria for DNA uptake and other purposes. T4P extension is thought to occur through the action of a single motor protein, PilB. Here, Ellison et al. show that T4P synthesis in Acinetobacter baylyi depends not only on PilB but also on an additional, distinct motor, TfpB.

Bacteria use extracellular appendages called type IV pili (T4P) for diverse behaviors including DNA uptake, surface sensing, virulence, protein secretion, and twitching motility1. Dynamic extension and retraction of T4P is essential for their function, yet little is known about the mechanisms controlling these dynamics or the extent to which their regulation is conserved across bacterial species. Here, we develop Acinetobacter baylyi as a new model to study T4P by employing a recently developed pilus labeling method2,3. Our findings overturn the current dogma that T4P extension occurs through the action of a single, highly conserved motor, PilB, by showing that T4P synthesis in A. baylyi is dependent on an additional, phylogenetically distinct motor, TfpB. Furthermore, we uncover an inhibitor of T4P extension that specifically binds to and inhibits PilB but not TfpB. These results expand our understanding of T4P regulation and highlight how inhibitors might be exploited to inhibit T4P synthesis.

The predatory deltaproteobacterium Myxococcus xanthus uses a helically-trafficked motor at bacterial focal adhesion (bFA) sites to power gliding motility. Using TIRF and force microscopy, we herein identify the integrin αI-domain-like outer-membrane (OM) lipoprotein CglB as an essential substratum-coupling protein of the gliding motility complex. Similar to most known OM lipoproteins, CglB is anchored on the periplasmic side of the OM and thus a mechanism must exist to secrete it to the cell surface in order for it to interact with the underlying substratum. We reveal this process to be mediated by a predicted OM β-barrel structure of the gliding complex. This OM platform was found to regulate the conformational activation and secretion of CglB across the OM. These data suggest that the gliding complex promotes surface exposure of CglB at bFAs, thus explaining the manner by which forces exerted by inner-membrane motors are transduced across the cell envelope to the substratum; they also uncover a novel protein secretion mechanism, highlighting the ubiquitous connection between secretion and bacterial motility.

Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, γ-tubulin ring complex (γ-TuRC) has been identified, precisely how γ-TuRC nucleates a MT remains poorly understood. Here we developed a single molecule assay to directly visualize nucleation of a MT from purifiedXenopus laevisγ-TuRC. We reveal a high γ-/αβ-tubulin affinity, which facilitates assembly of a MT from γ-TuRC. Whereas spontaneous nucleation requires assembly of 8 αβ-tubulins, nucleation from γ-TuRC occurs efficiently with a cooperativity of 4 αβ-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally-associated αβ-tubulins drive γ-TuRC into a closed conformation. Putative activation domain of CDK5RAP2, NME7 and TPX2 do not enhance γ-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal γ-/αβ-tubulin interaction.

Mechanical properties of the extracellular matrix are important determinants of cellular migration in diverse processes, such as immune response, wound healing, and cancer metastasis. Moreover, recent studies indicate that even bacterial surface colonization can depend on the mechanics of the substrate. Here, we focus on physical mechanisms that can give rise to substrate-rigidity dependent migration. We study a "twitcher", a cell driven by extension-retraction cycles, to idealize bacteria and perhaps eukaryotic cells that employ a slip-stick mode of motion. The twitcher is asymmetric and always pulls itself forward at its front. Analytical calculations show that the migration speed of a twitcher depends non-linearly on substrate rigidity. For soft substrates, deformations do not lead to build-up of significant force and the migration speed is therefore determined by stochastic adhesion unbinding. For rigid substrates, forced adhesion rupture determines the migration speed. Depending on the force-sensitivity of front and rear adhesions, forced bond rupture implies an increase or a decrease of the migration speed. A requirement for the occurrence of rigidity-dependent stick-slip migration is a "sticky" substrate, with binding rates being an order of magnitude larger than unbinding rates in absence of force. Computer simulations show that small stall forces of the driving machinery lead to a reduced movement on high rigidities, regardless of force-sensitivities of bonds. The simulations also confirm the occurrence of rigidity-dependent migration speed in a generic model for slip-stick migration of cells on a sticky substrate.

Dynamic control of cell polarity is of critical importance for many aspects of cellular development and motility. In Myxococcus xanthus, MglA, a G protein, and MglB, its cognate GTPase-activating protein, establish a polarity axis that defines the direction of movement of the cell and that can be rapidly inverted by the Frz chemosensory system. Although vital for collective cell behaviours, how Frz triggers this switch has remained unknown. Here, we use genetics, imaging and mathematical modelling to show that Frz controls polarity reversals via a gated relaxation oscillator. FrzX, which we identify as a target of the Frz kinase, provides the gating and thus acts as the trigger for reversals. Slow relocalization of the polarity protein RomR then creates a refractory period during which another switch cannot be triggered. A secondary Frz output, FrzZ, decreases this delay, allowing rapid reversals when required. Thus, this architecture results in a highly tuneable switch that allows a wide range of reversal frequencies.

The actin-like protein MreB has been proposed to coordinate the synthesis of the cell wall to determine cell shape in bacteria. MreB is preferentially localized to areas of the cell with specific curved geometries, avoiding the cell poles. It remains unclear whether MreB’s curvature preference is regulated by additional factors, and which specific features of MreB promote specific features of rod shape growth. Here, we show that the transmembrane protein RodZ modulates MreB curvature preference and polymer number in E. coli, properties which are regulated independently. An unbiased machine learning analysis shows that MreB polymer number, the total length of MreB polymers, and MreB curvature preference are key correlates of cylindrical uniformity, the variability in radius within a single cell. Changes in the values of these parameters are highly predictive of the resulting changes in cell shape (r2 = 0.93). Our data thus suggest RodZ promotes the assembly of geometrically-localized MreB polymers that lead to the growth of uniform cylinders., The actin-like protein MreB coordinates the synthesis of the cell wall, which determines cell shape in bacteria. Here, Bratton et al. show that the transmembrane protein RodZ modulates MreB polymer number and curvature preference, contributing to the cylindrical uniform shape of E. coli cells.

Helical cell shape is necessary for efficient stomach colonization by Helicobacter pylori, but the molecular mechanisms for generating helical shape remain unclear. The helical centerline pitch and radius of wild-type H. pylori cells dictate surface curvatures of considerably higher positive and negative Gaussian curvatures than those present in straight- or curved-rod H. pylori. Quantitative 3D microscopy analysis of short pulses with either N-acetylmuramic acid or D-alanine metabolic probes showed that cell wall growth is enhanced at both sidewall curvature extremes. Immunofluorescence revealed MreB is most abundant at negative Gaussian curvature, while the bactofilin CcmA is most abundant at positive Gaussian curvature. Strains expressing CcmA variants with altered polymerization properties lose helical shape and associated positive Gaussian curvatures. We thus propose a model where CcmA and MreB promote PG synthesis at positive and negative Gaussian curvatures, respectively, and that this patterning is one mechanism necessary for maintaining helical shape., eLife digest Round spheres, straight rods, and twisting corkscrews, bacteria come in many different shapes. The shape of bacteria is dictated by their cell wall, the strong outer barrier of the cell. As bacteria grow and multiply, they must add to their cell wall while keeping the same basic shape. The cells walls are made from long chain-like molecules via processes that are guided by protein scaffolds within the cell. Many common antibiotics, including penicillin, stop bacterial infections by interrupting the growth of cell walls. Helicobacter pylori is a common bacterium that lives in the gut and, after many years, can cause stomach ulcers and stomach cancer. H. pylori are shaped in a twisting helix, much like a corkscrew. This shape helps H. pylori to take hold and colonize the stomach. It remains unclear how H. pylori creates and maintains its helical shape. The helix is much more curved than other bacteria, and H. pylori does not have the same helpful proteins that other curved bacteria do. If H. pylori grows asymmetrically, adding more material to the cell wall on its long outer side to create a twisting helix, what controls the process? To find out, Taylor et al. grew H. pylori cells and watched how the cell walls took shape. First, a fluorescent dye was attached to the building blocks of the cell wall or to underlying proteins that were thought to help direct its growth. The cells were then imaged in 3D, and images from hundreds of cells were reconstructed to analyze the growth patterns of the bacteria’s cell wall. A protein called CcmA was found most often on the long side of the twisting H. pylori. When the CcmA protein was isolated in a dish, it spontaneously formed sheets and helical bundles, confirming its role as a structural scaffold for the cell wall. When CcmA was absent from the cell of H. pylori, Taylor et al. observed that the pattern of cell growth changed substantially. This work identifies a key component directing the growth of the cell wall of H. pylori and therefore, a new target for antibiotics. Its helical shape is essential for H. pylori to infect the gut, so blocking the action of the CcmA protein may interrupt cell wall growth and prevent stomach infections.

Mechanical properties of the extracellular matrix are important determinants of cellular migration in diverse processes, such as immune response, wound healing, and cancer metastasis. Moreover, recent studies indicate that even bacterial surface colonization can depend on the mechanics of the substrate. Here, we focus on physical mechanisms that can give rise to substrate-rigidity dependent migration. We study a “twitcher”, a cell driven by extension-retraction cycles, to idealize bacteria and perhaps eukaryotic cells that employ a slip-stick mode of motion. The twitcher is asymmetric and always pulls itself forward at its front. Analytical calculations show that the migration speed of a twitcher depends non-linearly on substrate rigidity. For soft substrates, deformations do not lead to build-up of significant force and the migration speed is therefore determined by stochastic adhesion unbinding. For rigid substrates, forced adhesion rupture determines the migration speed. Depending on the force-sensitivity of front and rear adhesions, forced bond rupture implies an increase or a decrease of the migration speed. A requirement for the occurrence of rigidity-dependent stick-slip migration is a “sticky” substrate, with binding rates being an order of magnitude larger than unbinding rates in absence of force. Computer simulations show that small stall forces of the driving machinery lead to a reduced movement on high rigidities, regardless of force-sensitivities of bonds. The simulations also confirm the occurrence of rigidity-dependent migration speed in a generic model for slip-stick migration of cells.

To understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We useXenopusegg extracts to study the nucleation and dynamics of MTs in branched networks, a process that is critical for spindle assembly. Surprisingly, new branched MTs preferentially originate near the minus-ends of pre-existing MTs. A sequential reaction model, consisting of deposition of nucleation sites on an existing MT, followed by rate-limiting nucleation of branches, reproduces the measured spatial profile of nucleation, the distribution of MT plus-ends and tubulin intensity. By regulating the availability of the branching effectors TPX2, augmin and γ-TuRC, combined with single-molecule observations, we show that first TPX2 is deposited on pre-existing MTs, followed by binding of augmin/γ-TuRC to result in the nucleation of branched MTs. In sum, regulating the localization and kinetics of nucleation effectors governs the architecture of branched MT networks.Impact StatementA sequential reaction pathway involving TPX2, augmin and γ-TuRC governs the assembly and architecture of branched microtubule networks.

Directed surface motility of metazoan cells and protozoan parasites involves substratum engagement by surface-exposed integrin(-like) adhesins, directionally transported by molecular motors via coupling to the internal cytoskeleton. The predatory deltaproteobacterium Myxococcus xanthus uses a helically-trafficked motor at bacterial focal adhesions to power gliding motility. However, the mechanisms of gliding machinery-substratum coupling and force mechanotransduction between innermembrane motors and substratum are unknown. Herein, we use bead force spectroscopy and TIRF microscopy to characterize CglB as the essential substratum-coupling integrin αI-domain-like adhesin. Protease susceptibility reveals that CglB interacts with a globular-protein-accessorized β-barrel OM display platform, which regulates the cell-surface conformational state and accessibility of the adhesin. Surface retention of CglB is further regulated by a cell-surface metalloprotease, a phenomenon also modulated by the OM display platform. These data depict a complex mechanism for bacterial gliding adhesin secretion, cell-surface anchoring, and processing, with conserved themes between prokaryotic and eukaryotic cell motility.

Cell shape in bacteria is determined by the cell wall, which is synthesized by a variety of proteins whose actions are coordinated by the actin-like MreB protein. MreB uses local geometric cues of envelope curvature to avoid the cell poles and localize to specific regions of the cell body. However, it remains unclear whether MreB’s curvature preference is regulated by additional factors, and which features of MreB are essential for specific aspects of rod shape growth, such as cylindrical uniformity. Here we show that in addition to its previously-described role in mediating MreB motion, RodZ also modulates MreB polymer number and curvature preference. MreB polymer number and curvature localization can be regulated independently. Quantitative 3D measurements and a series of mutant strains show that among various properties of MreB, polymer number, total length of MreB polymers, and MreB curvature preference are the key determinants of cylindrical uniformity, a measure of the variability in radius within a single cell. Changes in the values of these parameters are highly predictive of the resulting changes in cell shape (r2=0.93). Our data suggest a model for rod shape in which RodZ promotes the assembly of multiple long MreB polymers that ensure the growth of a uniform cylinder.

SummaryDynamic control of cell polarity is of critical importance for many aspects of cellular development and motility. In Myxococcus xanthus, a G-protein and its cognate GTPase-activating protein establish a polarity axis that defines the direction of movement of the cell and which can be rapidly inverted by the Frz chemosensory system. Although vital for collective cell behaviours, how Frz triggers this switch has remained unknown. Here, we use genetics, imaging and mathematical modelling to show that Frz controls polarity reversals via a gated relaxation oscillator. FrzX, which we newly identify as the primary Frz output, provides the gating and thus acts as the trigger for reversals. Slow relocalisation of the polarity protein RomR then creates a refractory period during which another switch cannot be triggered. A secondary Frz output, FrzZ, decreases this delay allowing rapid reversals when required. This architecture thus results in a highly tunable switch that allows a wide range of motility responses.

Pathogenic Vibrio cholerae remains a major human health concern. V. cholerae has a characteristic curved rod morphology, with a longer outer face and a shorter inner face. Previously, the mechanism and function of this curvature were unknown. Here we identify and characterize CrvA, the first curvature determinant in V. cholerae. CrvA self-assembles into filaments at the inner face of cell curvature. Unlike traditional cytoskeletons, CrvA localizes to the periplasm, and thus could be considered a periskeletal element. To quantify how curvature forms, we developed QuASAR (Quantitative Analysis of Sacculus Architecture Remodeling), which measures subcellular peptidoglycan dynamics. QuASAR reveals that CrvA asymmetrically patterns peptidoglycan insertion rather than removal, causing more material insertion into the outer face than the inner face. Furthermore, crvA is quorum regulated and CrvA-dependent curvature increases at high cell density. Finally, we demonstrate that CrvA promotes motility in hydrogels and confers an advantage in host colonization and pathogenesis.

Bacteria derive and maintain a variety of shapes that carry selective benefits. The shapes are usually defined by a mechanically stiff exoskeletal cell wall -- a macro-molecular network of peptidoglycan. The growth of such a network is catalyzed by transglycosylases and transpeptidases, and various cell-wall remodeling enzymes further digest and process the network. To maintain the overall cell shape, the bacterial cytoskeleton coordinates cell wall synthesis on the cellular scale. Recent studies also suggest that the mechanical properties of the bacterial cytoskeleton are important for cell wall growth. Here, we review current experiments and theories on the structure, dynamics and interactions of the bacterial cell wall and cytoskeleton, and their contributions to cell shape maintenance. We also propose future research directions that will help clarify the mystery of bacterial cell morphogenesis.

A filamentous cytoskeleton largely governs the physical shape and mechanical properties of eukaryotic cells. In bacteria, proteins homologous to all three classes of eukaryotic cytoskeletal filaments have recently been discovered. These proteins are essential for the maintenance of bacterial cell shape and have been shown to guide the localization of key cell-wall-modifying enzymes. However, whether the bacterial cytoskeleton is stiff enough to affect the overall mechanical rigidity of a cell has not been probed. Here, we used an optical trap to measure the bending rigidity of live Escherichia coli cells. We find that the actin-homolog MreB contributes nearly as much to the stiffness of a cell as the peptidoglycan cell wall. By quantitatively modeling these measurements, our data indicate that the MreB is rigidly linked to the cell wall, increasing the mechanical stiffness of the overall system. These data are the first evidence that the bacterial cytoskeleton contributes to the mechanical integrity of a cell in much the same way as it does in eukaryotes.

Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable fidelity in vivo. Its low error rate may be achieved by means of a 'proofreading' mechanism comprised of two sequential events. The first event (backtracking) involves a transcriptionally upstream motion of RNAP through several base pairs, which carries the 3' end of the nascent RNA transcript away from the enzyme active site. The second event (endonucleolytic cleavage) occurs after a variable delay and results in the scission and release of the most recently incorporated ribonucleotides, freeing up the active site. Here, by combining ultrastable optical trapping apparatus with a novel two-bead assay to monitor transcriptional elongation with near-base-pair precision, we observed backtracking and recovery by single molecules of RNAP. Backtracking events ( approximately 5 bp) occurred infrequently at locations throughout the DNA template and were associated with pauses lasting 20 s to30 min. Inosine triphosphate increased the frequency of backtracking pauses, whereas the accessory proteins GreA and GreB, which stimulate the cleavage of nascent RNA, decreased the duration of such pauses.

Accumulating evidence suggests that molecular motors contribute to the apparent diffusion of molecules in cells. However, current literature lacks evidence for an active process that drives diffusive-like motion in the bacterial membrane. One possible mechanism is cell wall synthesis, which involves the movement of protein complexes in the cell membrane circumferentially around the cell envelope and may generate currents in the lipid bilayer that advectively transport other transmembrane proteins. We test this hypothesis in Escherichia coli using drug treatments that slow cell wall synthesis and measure their effect on the diffusion of the transmembrane protein mannitol permease using fluorescence recovery after photobleaching. We found no clear decrease in diffusion in response to vancomycin and no decrease in response to mecillinam treatment. These results suggest that cell wall synthesis is not an active contributor to mobility in the cytoplasmic membrane.

In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope, followed by an active recovery. After recovery, and if the external osmolality remains high, cells have been shown to grow more slowly, smaller, and at reduced turgor pressure. Despite the fact that the active recovery is a key stress response, the nature of these changes and how they relate to each other is not understood. Here, we use fluorescence imaging of single cells during hyperosmotic shocks, combined with custom made microfluidic devices, to show that cells fully recover their volume to the initial, preshock value and continue to grow at a slower rate immediately after the recovery. We show that the cell envelope material properties do not change after hyperosmotic shock, and that cell shape recovers along with cell volume. Taken together, these observations indicate that the turgor pressure recovers to its initial value so that reduced turgor is not responsible for the reduced growth rate observed immediately after recovery. To determine the point at which the reduction in cell size and turgor pressure occurs after shock, we measured the volume of E. coli cells at different stages of growth in bulk cultures. We show that cell volume reaches the same maximal level irrespective of the osmolality of the media. Based on these measurements, we propose that turgor pressure is used as a feedback variable for osmoregulatory pumps instead of being directly responsible for the reduction in growth rates. Reestablishment of turgor to its initial value might ensure correct attachment of the inner membrane and cell wall needed for cell wall biosynthesis.

Thermal variations can exert dramatic effects on the rates of enzymes. The influence of temperature on RNA polymerase is of particular interest because its transcriptional activity governs general levels of gene expression, and may therefore exhibit pleiotropic effects in cells. Using a custom-modified optical trapping apparatus, we used a tightly focused infrared laser to heat single molecules of Escherichia coli RNA polymerase while monitoring transcriptional activity. We found a significant change in rates of transcript elongation with temperature, consistent with a large enthalpic barrier to the condensation reaction associated with RNA polymerization (approximately 13 kcal/mol). In contrast, we found little change in either the frequency or the lifetime of off-pathway, paused states, indicating that the energetic barrier to transcriptional pausing is predominantly entropic.

We often take for granted the elegantly simple shapes adopted by bacteria. Spheres, crescents, helices, and other shapes fill the prokaryotic world (1). Despite the relative simplicity of these shapes compared to a scraggly Purkinje cell in the cerebellar cortex or even the crowned and tailed unicellular choanoflagellate, truly fundamental questions about bacterial growth remain unanswered. Nearly all bacteria use an exoskeletal, peptidoglycan cell wall to define their shape and structural properties. Cell growth can be incredibly fast, with doubling times on the order of 10 min, and involves the biogenesis of peptidoglycan material and its insertion into and modification of the existing cell wall. The generation of cellular-scale wall features such as micron lengths and widths are thought to be the result of spatial-temporal patterning of cell-wall insertion and modification. How molecular-scale enzymes collectively act to build a cell remains an open question in bacterial morphogenesis. This is largely due to the lack of tools that can probe scales between the molecular and the cellular regions—exactly those needed to understand cell shape and many other cellular-scale phenomena. On the smallest scales, structural and biochemical probes are able to provide atomistic details of protein form and function. Above the diffraction limit of visible light, light microscopy can provide vital information about biological processes. However, a dearth of techniques to probe biological dynamics on scales from 10 nm to 200 nm has hindered progress in understanding bacterial growth. Very recently, however, biophysical modeling has emerged as a tool to bridge these spatial scales (see, e.g., Huang et al. (2) and Gardner et al. (3)). In a recent issue of the Biophysical Journal, Misra et al. (4) use just this strategy to attack the problem of cell-wall thickness and the growth of Gram-positive bacteria. The Gram-positive Bacillus subtilis is a rod-shaped cell surrounded by a multilayered, thick cell wall. By combining a physical model of cell-wall growth and degradation with molecular-scale electron microscopy data and cellular-scale growth rate measurements, the authors find that a coupling between cell-wall synthesis and hydrolysis, a molecular-scale phenomenon, yields a fixed wall thickness and strain even when the rate of growth is increased substantially. Because the cell wall is under considerable turgor pressure, hydrolysis can generate as much strain in the material as insertion. This work also helps to explain seemingly confusing data from the 1970s, which reported that cell-wall hydrolysis itself can increase growth rate (5). This powerful combination of theory and experiment provides a guiding framework for dealing with scientific problems that lack direct experimental support. New techniques, such as superresolution light microscopy and high-resolution AFM imaging, may eventually serve to directly test these ideas. For now, though, a combination of new experimental designs and the development of more realistic and complex models of biological systems will continue to provide a window into the inner workings of the cell.

The regulation of cell shape is a common challenge faced by organisms across all biological kingdoms. In nearly all bacteria, cell shape is determined by the architecture of the peptidoglycan cell wall, a macromolecule consisting of glycan strands crosslinked by peptides. In addition to shape, cell growth must also maintain the wall structural integrity to prevent lysis due to large turgor pressures. Robustness can be accomplished by establishing a globally ordered cell-wall network, although how a bacterium generates and maintains peptidoglycan order on the micron scale using nanometer-sized proteins remains a mystery. Here, we demonstrate that left-handed chirality of the MreB cytoskeleton in the rod-shaped bacterium Escherichia coli gives rise to a global, right-handed chiral ordering of the cell wall. Local, MreB-guided insertion of material into the peptidoglycan network naturally orders the glycan strands and causes cells to twist left-handedly during elongational growth. Through comparison with the right-handed twisting of Bacillus subtilis cells, our work supports a common mechanism linking helical insertion and chiral cell-wall ordering in rod-shaped bacteria. These physical principles of cell growth link the molecular structure of the bacterial cytoskeleton, mechanisms of wall synthesis, and the coordination of cell-wall architecture.

The cell wall, a cross-linked 3D network of peptidoglycan, is the key structural element that dictates cell shape and counter-balances turgor forces in prokaryotes. During cell growth and division, the wall undergoes a coordinated series of addition and remodeling events that enable the cell shape to change in a stereotyped manner. Recent studies suggest that the spatial insertion pattern of new material into the peptidoglycan network, guided by the bacterial cytoskeleton, is critical for cell shape maintenance. In this work, we use a high-density of quantum dots (QDs) on the B. subtilis cell surface to track the local motions of the wall in three-dimensions during cell growth and division. From a 2D projection of the QD distribution onto the cylindrical cell surface, we obtain the local stretching and shearing rates on multiple spatial scales. The homogeneity of insertion and cell wall twist during elongation and division can then be quantified from these growth maps.

We study intact and bulging Escherichia coli cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the overall cell stiffness. We find strong evidence of power-law stress-stiffening in the E. coli cell wall, with an exponent of 1.22 \pm 0.12, such that the wall is significantly stiffer in intact cells (E = 23 \pm 8 MPa and 49 \pm 20 MPa in the axial and circumferential directions) than in unpressurized sacculi. These measurements also indicate that the turgor pressure in living cells E. coli is 29 \pm 3 kPa., Main Text: 4 pages, 4 figures. Supplemental Materials: 9 pages

The ability of bacteria to grow into specific shapes depends on the accurate self-organization of the cell wall growth machinery. Absent a global roadmap, these enzymes must rely on local cues to guide the location and orientation of new material inserted into the peptidoglycan (PG) network. I will discuss our recent experimental and computational efforts in understanding the strategy used by Escherichia coli cells to grow into a rod shape. By combining 3D microscopy, particle tracking and physical simulations, we show that helical insertion of PG material guided by the actin homolog MreB leads to constant radius elongation and a robust global ordering of the glycan strands in a helical pattern relative to the cell long axis.

The envelope of a gram-negative bacterium is composed of three layers: the inner and outer membranes and the periplasmic peptidoglycan (PG) cell wall. The PG layer is thought to be the stress bearing structure that determines and maintains cell shape, preventing cells from bursting due to a large inner turgor pressure. In vitro, PG mechanics have been measured with different techniques, yielding a high elastic modulus. However, in vivo such mechanical measurements are complicated by the turgor pressure. On the other hand, direct measurement of a cell's turgor pressure is difficult due to the presence of the PG layer. We are able to overcome these difficulties and simultaneously measure cell turgor pressure and PG mechanics in vivo with an atomic force microscope (AFM). Using a vancomycin sensitive strain of Escherichia coli, we locally induced small membrane blebs at sites of reduced PG integrity. AFM indentation of these membrane-bound blebs directly probes the turgor pressure. By comparing bleb indentation with indentation of the cell body, we can deduce the relative contributions of the PG layer and turgor pressure to the overall cell mechanics. Furthermore, drugs that inhibit bacterial cytoskeletal filaments can be used to evaluate the role of these proteins in modulating cell elasticity.

The growth of actin filament networks is a fundamental biological process that drives a variety of cellular and intracellular motions. During motility, eukaryotic cells and intracellular pathogens are propelled by actin networks organized by nucleation-promoting factors, which trigger the formation of nascent filaments off the side of existing filaments in the network. A Brownian ratchet (BR) mechanism has been proposed to couple actin polymerization to cellular movements, whereby thermal motions are rectified by the addition of actin monomers at the end of growing filaments. Here, by following actin--propelled microspheres using three--dimensional laser tracking, we find that beads adhered to the growing network move via an object--fluctuating BR. Velocity varies with the amplitude of thermal fluctuation and inversely with viscosity as predicted for a BR. In addition, motion is saltatory with a broad distribution of step sizes that is correlated in time. These data point to a model in which thermal fluctuations of the microsphere or entire actin network, and not individual filaments, govern motility. This conclusion is supported by Monte Carlo simulations of an adhesion--based BR and suggests an important role for membrane tension in the control of actin--based cellular protrusions., Comment: To be published in PNAS

During transcription, RNA polymerase (RNAP) moves processively along a DNA template, creating a complementary RNA. Here we present the development of an ultra-stable optical trapping system with angstrom-level resolution, which we used to monitor transcriptional elongation by single molecules of Escherichia coli RNAP. Records showed discrete steps averaging 3.7 ± 0.6 A, a distance equivalent to the mean rise per base found in B-DNA. By combining our results with quantitative gel analysis, we conclude that RNAP advances along DNA by a single base pair per nucleotide addition to the nascent RNA. We also determined the force–velocity relationship for transcription at both saturating and sub-saturating nucleotide concentrations; fits to these data returned a characteristic distance parameter equivalent to one base pair. Global fits were inconsistent with a model for movement incorporating a power stroke tightly coupled to pyrophosphate release, but consistent with a brownian ratchet model incorporating a secondary NTP binding site. In a paper building on over a decade of progress in single molecule biophysics, Abbondanzieri et al. report the first experiments capable of resolving base-pair steps for an individual enzyme moving along DNA. The enzyme is RNA polymerase, captured by an all-optical force clamp in the act of transcribing RNA from a DNA template. The ability to detect Angstrom-scale motions in a single enzyme opens up new areas of study of biomolecules including perhaps single-molecule DNA sequencing.

With every step it takes, the kinesin motor undergoes a mechanochemical reaction cycle that includes the hydrolysis of one ATP molecule, ADP/P i release, plus an unknown number of additional transitions. Kinesin velocity depends on both the magnitude and the direction of the applied load. Using specialized apparatus, we subjected single kinesin molecules to forces in differing directions. Sideways and forward loads up to 8 pN exert only a weak effect, whereas comparable forces applied in the backward direction lead to stall. This strong directional bias suggests that the primary working stroke is closely aligned with the microtubule axis. Sideways loads slow the motor asymmetrically, but only at higher ATP levels, revealing the presence of additional, load-dependent transitions late in the cycle. Fluctuation analysis shows that the cycle contains at least four transitions, and confirms that hydrolysis remains tightly coupled to stepping. Together, our findings pose challenges for models of kinesin motion.

The extreme concentrations of chemicals in a bacterium's cytoplasm generate a large osmotic pressure that inflates the cell. Using a number of interconnected systems, bacteria actively regulate their turgor pressure to resist changes in their local environment. In response to osmotic shock and changes in internal ion concentration, the osmosensory transporters ProP/U, BetT/U, TrkAH and KdpFABC transport external chemicals such as proline, choline and potassium into the cell, whereas the mechano-sensitive channels MscS and MscL export solutes from the cell in response to increased membrane strain. Although each has been shown to play a role in the regulation of turgor, details of how the different systems are coordinated by a cell is poorly understood. Previous measurements of osmoregulation in bacteria have been unable to directly probe the adaptation of turgor pressure, focusing instead on the activity of various transporters, or the change in cellular survival rates. Here we move beyond these limited measurements using AFM and fluorescence imaging to monitor turgor pressure and cell volume adaptation on a single cell level with a time resolution on the order of seconds. To explore different mechanisms used by bacteria, we moved exponentially growing cells from LB medium to an iso-osmotic buffered medium and allowed the cells to adapt. We subsequently challenged the cells with varying levels of sucrose (as an external osmolyte) and potassium or proline. We measured the dependence of the adaptation time and adaptation level on different amounts of extracellular potassium or proline and the magnitude of the osmotic shock. This type of measurement allows us to uncouple the different adaptation pathways and to study them individually and in small groups to quantify their function and interactions.

All living cells employ an array of different mechanisms to help them survive changes in extra cellular osmotic pressure. The difference in the concentration of chemicals in a bacterium's cytoplasm and the external environment generates an osmotic pressure that inflates the cell. It is thought that the bacterium Escherichia coli use a number of interconnected systems to adapt to changes in external pressure, allowing them to maintain turgor and live in surroundings that range more than two-hundred-fold in external osmolality. Here, we use fluorescence imaging to make the first measurements of cell volume changes over time during hyperosmotic shock and subsequent adaptation on a single cell level in vivo with a time resolution on the order of seconds. We directly observe two previously unseen phases of the cytoplasmic water efflux upon hyperosmotic shock. Furthermore, we monitor cell volume changes during the post-shock recovery and observe a two-phase response that depends on the shock magnitude. The initial phase of recovery is fast, on the order of 15-20 min and shows little cell-to-cell variation. For large sucrose shocks, a secondary phase that lasts several hours adds to the recovery. We find that cells are able to recover fully from shocks as high as 1 Osmol/kg using existing systems, but that for larger shocks, protein synthesis is required for full recovery.

Genetic and microscopy studies have shown that the newly discovered bacterial cytoskeleton is essential for the maintenance of cell shape in many prokaryotes. Moreover, the localization of different components of the cytoskeleton correlates with the localization of cell-wall-synthesis enzymes and of newly added cell-wall material. These correlations suggest that the cytoskeleton controls cell shape by regulating the location of cell-wall synthesis. However, whether the cytoskeleton also applies mechanical forces inside of cells is unknown. To address this issue, a quantitative measurement of the role of cytoskeletal proteins in cell mechanics is needed.We used an optical trap to measure the elasticity of live E. coli cells. Upon the addition of A22, a drug that promotes the disassembly of MreB filaments, cells become significantly weaker in their resistance to cell bending. This effect is reversible, and cells recover wild-type stiffness when A22 is removed from the environment. Our results show that MreB, a bacterial actin homologue, contributes as much to the stiffness of a cell as the peptidoglycan cell wall. This is the first direct evidence that the bacterial cytoskeleton contributes to the mechanical integrity of a cell in much the same way as it does in eukaryotes.

Fluctuations in biochemical processes can provide insights into the underlying kinetics beyond what can be gleaned from studies of average rates alone. Historically, analysis of fluctuating transmembrane currents supplied information about ion channel conductance states and lifetimes before single-channel recording techniques emerged. More recently, fluctuation analysis has helped to define mechanochemical pathways and coupling ratios for the motor protein kinesin as well as to probe the contributions of static and dynamic disorder to the kinetics of single enzymes. As growing numbers of assays are developed for enzymatic or folding behaviors of single macromolecules, the range of applications for fluctuation analysis increases. To evaluate specific biochemical models against experimental data, one needs to predict analytically the distribution of times required for completion of each reaction pathway. Unfortunately, using traditional methods, such calculations can be challenging for pathways of even modest complexity. Here, we derive an exact expression for the distribution of completion times for an arbitrary pathway with a finite number of states, using a recursive method to solve algebraically for the appropriate moment-generating function. To facilitate comparisons with experiments on processive motor proteins, we develop a theoretical formalism for the randomness parameter, a dimensionless measure of the variance in motor output. We derive the randomness for motors that take steps of variable sizes or that move on heterogeneous substrates, and then discuss possible applications to enzymes such as RNA polymerase, which transcribes varying DNA sequences, and to myosin V and cytoplasmic dynein, which may advance by variable increments.

The mechanical properties of gram-negative bacteria are governed by a rigid peptidoglycan (PG) cell wall and the turgor pressure generated by the large concentration of solutes in the cytoplasm. The elasticity of the PG has been measured in bulk and in isolated sacculi and shown to be very compliant compared to the overall stiffness of the cell itself. However, the stiffness of the cell wall in live cells has not been accurately measured. In particular, the effects that pressure-induced stress might have on the stiffness of the meshlike PG network have not been addressed even though polymeric materials often exhibit large amounts of stress-stiffening. In this study, we use two experimental systems to probe the effects of pressure on the wall stiffness. First, we study blebbing E. coli cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the overall cell stiffness. Second, we study the relationship between cell stiffness and external osmotic pressure which allows us to probe higher turgor pressures. Combining these two types of measurements, we find strong evidence of a power-law stress-stiffening in the E. coli cell wall such that the wall is significantly stiffer in live cells (E∼5 MPa) than the relatively compliant sacculus stiffness (E∼34 MPa).

A peptidoglycan cell wall determines the shape of nearly all bacteria. The cell wall, along with the shape that it adopts, is crucial to cellular physiology. The mechanical integrity and morphology of the cell wall is determined by the spatiotemporal patterning of cell wall synthesis; therefore a rod-shaped cell such as Escherichia coli faces the challenge of coordinating the nanoscale proteins responsible for peptidoglycan synthesis to construct a micron-scale sacculus. What are the principles that allow cell wall synthesis proteins to establish order over a range of length scales spanning nearly three orders of magnitude? We approached this question by examining the process of reversion in cell-wall-deficient ‘L-forms’ of E. coli, in which cell-wall synthesis has disrupted by beta-lactam antibiotics. An L-form undergoing reversion begins in a spherical shape without an intact cell wall. When cell wall synthesis inhibiting antibiotics are removed, the cell generates newrod-shaped protrusions, which eventually undergo septation and adopt the normal rod morphology of E. coli. The reversion of L-forms thus provides on opportunity to study morphogenesis in bacteria lacking an intact cell wall. We therefore investigated the morphological dynamics of the reversion process in L-forms of E. coli, and simultaneously imaged the localization of MreB, a bacterial actin required for the rod-like morphology of many bacteria. We found that MreB localizes to negatively curved regions of the cell (e.g. invaginations), and furthermore targets cell wall synthesis to those regions. Our results therefore suggest a model in which the localization of MreB in response to geometric cues is crucial to morphogenesis in E. coli.