Cell-wall Insertion Research Articles

Cell envelope growth of Gram‐negative bacteria proceeds independently of cell wall synthesis

All bacterial cells must expand their envelopes during growth. The main load‐bearing and shape‐determining component of the bacterial envelope is the peptidoglycan cell wall. Bacterial envelope growth and shape changes are often thought to be controlled through enzymatic cell wall insertion. We investigated the role of cell wall insertion for cell shape changes during cell elongation in Gram‐negative bacteria. We found that both global and local rates of envelope growth of Escherichia coli remain nearly unperturbed upon arrest of cell wall insertion—up to the point of sudden cell lysis. Specifically, cells continue to expand their surface areas in proportion to biomass growth rate, even if the rate of mass growth changes. Other Gram‐negative bacteria behave similarly. Furthermore, cells plastically change cell shape in response to differential mechanical forces. Overall, we conclude that cell wall‐cleaving enzymes can control envelope growth independently of synthesis. Accordingly, the strong overexpression of an endopeptidase leads to transiently accelerated bacterial cell elongation. Our study demonstrates that biomass growth and envelope forces can guide cell envelope expansion through mechanisms that are independent of cell wall insertion.

Polar protein Wag31 both activates and inhibits cell wall metabolism at the poles and septum.

Mycobacterial cell elongation occurs at the cell poles; however, it is not clear how cell wall insertion is restricted to the pole or how it is organized. Wag31 is a pole-localized cytoplasmic protein that is essential for polar growth, but its molecular function has not been described. In this study we used alanine scanning mutagenesis to identify Wag31 residues involved in cell morphogenesis. Our data show that Wag31 helps to control proper septation as well as new and old pole elongation. We have identified key amino acid residues involved in these essential functions. Enzyme assays revealed that Wag31 interacts with lipid metabolism by modulating acyl-CoA carboxylase (ACCase) activity. We show that Wag31 does not control polar growth by regulating the localization of cell wall precursor enzymes to the Intracellular Membrane Domain, and we also demonstrate that phosphorylation of Wag31 does not substantively regulate peptidoglycan metabolism. This work establishes new regulatory functions of Wag31 in the mycobacterial cell cycle and clarifies the need for new molecular models of Wag31 function.

FtsZ-mediated fission of a cuboid bacterial symbiont

SummaryLess than a handful of cuboid and squared cells have been described in nature, which makes them a rarity. Here, we show how Candidatus Thiosymbion cuboideus, a cube-like gammaproteobacterium, reproduces on the surface of marine free-living nematodes. Immunostaining of symbiont cells with an anti-fimbriae antibody revealed that they are host-polarized, as these appendages exclusively localized at the host-proximal (animal-attached) pole. Moreover, by applying a fluorescently labeled metabolic probe to track new cell wall insertion in vivo, we observed that the host-attached pole started septation before the distal one. Similarly, Ca. T. cuboideus cells immunostained with an anti-FtsZ antibody revealed a proximal-to-distal localization pattern of this tubulin homolog. Although FtsZ has been shown to arrange into squares in synthetically remodeled cuboid cells, here we show that FtsZ may also mediate the division of naturally occurring ones. This implies that, even in natural settings, membrane roundness is not required for FtsZ function.

The Stress-Active Cell Division Protein ZapE Alters FtsZ Filament Architecture to Facilitate Division in Escherichia coli.

During pathogenic infections, bacterial cells experience environmental stress conditions, including low oxygen and thermal stress. Bacterial cells proliferate during infection and divide by a mechanism characterized by the assembly of a large cytoskeletal structure at the division site called the Z-ring. The major protein constituting the Z-ring is FtsZ, a tubulin homolog and GTPase that utilizes the nucleotide to assemble into dynamic polymers. In Escherichia coli, many cell division proteins interact with FtsZ and modulate Z-ring assembly, while others direct cell wall insertion and peptidoglycan remodeling. Here, we show that ZapE, an ATPase that accumulates during late constriction, directly interacts with FtsZ and phospholipids in vitro. In the presence of adenosine triphosphate (ATP), ZapE induces bundling of GTP-induced FtsZ polymers; however, ZapE also binds FtsZ in the absence of GTP. The ZapE mutant protein ZapE(K84A), which is defective for ATP hydrolysis, also interacts with FtsZ and induces FtsZ filament bundling. In vivo, cultures of zapE deletion cells contain a low percentage of filamentous cells, suggesting that they have a modest division defect; however, they are able to grow when exposed to stress, such as high temperature and limited oxygen. When combined with the chromosomal deletion of minC, which encodes an FtsZ disassembly factor, ΔzapE ΔminC cells experience growth delays that slow proliferation at high temperature and prevent recovery. This synthetic slow growth phenotype after exposure to stress suggests that ZapE may function to ensure proliferation during and after stress, and this is exacerbated when cells are also deleted for minC. Expression of either ZapE or ZapE(K84A) complements the aberrant growth phenotypes in vivo suggesting that the division-associated role of ZapE does not require ZapE ATP hydrolysis. These results support that ZapE is a stress-regulated cell division protein that interacts directly with FtsZ and phospholipids, promoting growth and division after exposure to environmental stress.

Ftsz-Mediated Fission of a Cuboid Bacterial Symbiont

Less than a handful of cuboid and squared cells have been described in nature which makes them a rarity. Here, we show how Candidatus Thiosymbion cuboideus, a cube-like gammaproteobacterium, reproduces on the surface of marine free-living nematodes. Morphometric analysis showed that most non-dividing Ca . T. cuboideus cells are squared and 1.3 μm-thick on average. Immunostaining of symbiont cells with an anti-fimbriae antibody revealed that they are host-polarized, as these appendages exclusively localized at the host-proximal (animal-attached) pole. Moreover, by applying a fluorescently labelled metabolic probe to track new cell wall insertion in vivo , we observed that the host-attached pole started to septate before the distal one. Proximal-to-distal insertion of new cell wall material at the septum displayed a similar localization pattern as the tubulin homolog FtsZ. Although this has been shown to arrange into squares in synthetically remodelled cuboid cells, here we show that FtsZ may also mediate the division of naturally occurring ones. This implies that, even in natural settings, membrane roundness is not required for FtsZ function.

Chiral twisting in a bacterial cytoskeletal polymer affects filament size and orientation

In many rod-shaped bacteria, the actin homolog MreB directs cell-wall insertion and maintains cell shape, but it remains unclear how structural changes to MreB affect its organization in vivo. Here, we perform molecular dynamics simulations for Caulobacter crescentus MreB to extract mechanical parameters for inputs into a coarse-grained biophysical polymer model that successfully predicts MreB filament properties in vivo. Our analyses indicate that MreB double protofilaments can exhibit left-handed twisting that is dependent on the bound nucleotide and membrane binding; the degree of twisting correlates with the length and orientation of MreB filaments observed in vitro and in vivo. Our molecular dynamics simulations also suggest that membrane binding of MreB double protofilaments induces a stable membrane curvature of similar magnitude to that observed in vivo. Thus, our multiscale modeling correlates cytoskeletal filament size with conformational changes inferred from molecular dynamics simulations, providing a paradigm for connecting protein filament structure and mechanics to cellular organization and function.

Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects.

Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell: the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that aPBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level, we then present evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.

Conformational Dynamics of a Bacterial Actin Filament Predict In Vivo Filament Length

While cytoskeletal proteins in the actin family are structurally similar, as filaments they act as critical components of diverse cellular processes across all kingdoms of life. In many rod-shaped bacteria, the actin homolog MreB directs cell-wall insertion and maintains cell shape, but it remains unclear how structural changes to MreB affect its physiological function. To bridge this gap, we performed molecular dynamics simulations for Caulobacter crescentus MreB and then utilized a coarse-grained biophysical model to successfully predict MreB filament properties in vivo. We discovered that MreB double protofilaments exhibit left-handed twisting that is dependent on the bound nucleotide and membrane binding; the degree of twisting determines the limit length and orientation of MreB filaments in vivo. Membrane binding of MreB also induces a stable membrane curvature that is physiologically relevant. Together, our data empower the prediction of cytoskeletal filament size from molecular dynamics simulations, providing a paradigm for connecting protein filament structure and mechanics to cellular functions.

RodZ modulates geometric localization of the bacterial actin MreB to regulate cell shape

In the rod-shaped bacterium Escherichia coli, the actin-like protein MreB localizes in a curvature-dependent manner and spatially coordinates cell-wall insertion to maintain cell shape, although the molecular mechanism by which cell width is regulated remains unknown. Here we demonstrate that the membrane protein RodZ regulates the biophysical properties of MreB and alters the spatial organization of E. coli cell-wall growth. The relative expression levels of MreB and RodZ change in a manner commensurate with variations in growth rate and cell width, and RodZ systematically alters the curvature-based localization of MreB and cell width in a concentration-dependent manner. We identify MreB mutants that alter the bending properties of MreB filaments in molecular dynamics simulations similar to RodZ binding, and show that these mutants rescue rod-like shape in the absence of RodZ alone or in combination with wild-type MreB. Thus, E. coli can control its shape and dimensions by differentially regulating RodZ and MreB to alter the patterning of cell-wall insertion, highlighting the rich regulatory landscape of cytoskeletal molecular biophysics.

Cell Size Regulation Through Tunable Geometric Localization of the Bacterial Actin Cytoskeleton

Virtually all cells have the ability to adjust their size in response to environmental cues, yet relatively little is known about the molecular factors responsible for tuning cell size or the mechanisms through which they reconfigure cellular dimensions. In the rod-shaped bacterium Escherichia coli, the actin-like protein MreB localizes in a curvaturedependent manner and spatially coordinates cell-wall insertion to maintain cell shape across changing environments. Here, we demonstrate that the MreB-binding protein RodZ regulates the biophysical properties of MreB filaments and thereby alters the spatial patterning of cell-wall growth to modulate cell size. The relative expression levels of MreB and RodZ changed in a manner commensurate with variations in growth rate and cell width, and single-cell analyses demonstrated that RodZ systematically alters the curvature-based localization of MreB and cell width in a manner dependent on the concentration of RodZ. We identified MreB mutants that we predict using molecular dynamics simulations to alter the bending properties of MreB filaments at the molecular scale in a manner similar to RodZ binding, and showed that these mutants rescue rod-like shape in the absence of RodZ alone or in combination with wild-type MreB. Together, our results show that E. coli controls its shape and dimensions by differentially regulating RodZ and MreB expression, highlighting the rich capacity for precise coordination of cell-shape changes with environmental conditions by exploiting cytoskeletal molecular biophysics.

Remodeling of the Z-Ring Nanostructure during the Streptococcus pneumoniae Cell Cycle Revealed by Photoactivated Localization Microscopy.

ABSTRACTOvococci form a morphological group that includes several human pathogens (enterococci and streptococci). Their shape results from two modes of cell wall insertion, one allowing division and one allowing elongation. Both cell wall synthesis modes rely on a single cytoskeletal protein, FtsZ. Despite the central role of FtsZ in ovococci, a detailed view of the in vivo nanostructure of ovococcal Z-rings has been lacking thus far, limiting our understanding of their assembly and architecture. We have developed the use of photoactivated localization microscopy (PALM) in the ovococcus human pathogen Streptococcus pneumoniae by engineering spDendra2, a photoconvertible fluorescent protein optimized for this bacterium. Labeling of endogenously expressed FtsZ with spDendra2 revealed the remodeling of the Z-ring’s morphology during the division cycle at the nanoscale level. We show that changes in the ring’s axial thickness and in the clustering propensity of FtsZ correlate with the advancement of the cell cycle. In addition, we observe double-ring substructures suggestive of short-lived intermediates that may form upon initiation of septal cell wall synthesis. These data are integrated into a model describing the architecture and the remodeling of the Z-ring during the cell cycle of ovococci.

Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization

Cells typically maintain characteristic shapes, but the mechanisms of self-organization for robust morphological maintenance remain unclear in most systems. Precise regulation of rod-like shape in Escherichia coli cells requires the MreB actin-like cytoskeleton, but the mechanism by which MreB maintains rod-like shape is unknown. Here, we use time-lapse and 3D imaging coupled with computational analysis to map the growth, geometry, and cytoskeletal organization of single bacterial cells at subcellular resolution. Our results demonstrate that feedback between cell geometry and MreB localization maintains rod-like cell shape by targeting cell wall growth to regions of negative cell wall curvature. Pulse-chase labeling indicates that growth is heterogeneous and correlates spatially and temporally with MreB localization, whereas MreB inhibition results in more homogeneous growth, including growth in polar regions previously thought to be inert. Biophysical simulations establish that curvature feedback on the localization of cell wall growth is an effective mechanism for cell straightening and suggest that surface deformations caused by cell wall insertion could direct circumferential motion of MreB. Our work shows that MreB orchestrates persistent, heterogeneous growth at the subcellular scale, enabling robust, uniform growth at the cellular scale without requiring global organization.

Various scenarios of the cell pattern formation in Arabidopsis lateral root

During lateral root (LR) development a coordinate sequence of cell divisions, accompanied by a change of the organ form takes place. Both the order of anatomical events and morphological features may vary for individual primordia. At early stages of LR primordia development oblique division walls are inserted in cells that are symmetrically located on both sides of the axis of the developing LR primordium, and thereby allow for the protrusion of the LR. We hypothesize that both oblique cell wall insertion and continuous changes in primordium form could be a consequence of a local change in stress distribution in the region of the LR initiation.

Combining Modeling and Experiment to Understand Bacterial Growth

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.

Stability and Assembly of Pilus Subunits of Streptococcus pneumoniae

Pili are surface-exposed virulence factors involved in bacterial adhesion to host cells. The Streptococcus pneumoniae pilus is composed of three structural proteins, RrgA, RrgB, and RrgC and three transpeptidase enzymes, sortases SrtC-1, SrtC-2, and SrtC-3. To gain insights into the mechanism of pilus formation we have exploited biochemical approaches using recombinant proteins expressed in Escherichia coli. Using site-directed mutagenesis, mass spectrometry, limited proteolysis, and thermal stability measurements, we have identified isopeptide bonds in RrgB and RrgC and demonstrate their role in protein stabilization. Co-expression in E. coli of RrgB together with RrgC and SrtC-1 leads to the formation of a covalent RrgB-RrgC complex. Inactivation of SrtC-1 by mutation of the active site cysteine impairs RrgB-RrgC complex formation, indicating that the association between RrgB and RrgC is specifically catalyzed by SrtC-1. Mass spectrometry analyses performed on purified samples of the RrgB-RrgC complex show that the complex has 1:1 stoichiometry. The deletion of the IPQTG RrgB sorting signal, but not the corresponding sequence in RrgC, abolishes complex formation, indicating that SrtC-1 recognizes exclusively the sorting motif of RrgB. Finally, we show that the intramolecular bonds that stabilize RrgB may play a role in its efficient recognition by SrtC-1. The development of a methodology to generate covalent pilin complexes in vitro, facilitating the study of sortase specificity and the importance of isopeptide bond formation for pilus biogenesis, provide key information toward the understanding of this complex macromolecular process.

Bacterial intermediate filaments: in vivo assembly, organization, and dynamics of crescentin

Crescentin, which is the founding member of a rapidly growing family of bacterial cytoskeletal proteins, was previously proposed to resemble eukaryotic intermediate filament (IF) proteins based on structural prediction and in vitro polymerization properties. Here, we demonstrate that crescentin also shares in vivo properties of assembly and dynamics with IF proteins by forming stable filamentous structures that continuously incorporate subunits along their length and that grow in a nonpolar fashion. De novo assembly of crescentin is biphasic and involves a cell size-dependent mechanism that controls the length of the structure by favoring lateral insertion of crescentin subunits over bipolar longitudinal extension when the structure ends reach the cell poles. The crescentin structure is stably anchored to the cell envelope, and this cellular organization requires MreB function, identifying a new function for MreB and providing a parallel to the role of actin in IF assembly and organization in metazoan cells. Additionally, analysis of an MreB localization mutant suggests that cell wall insertion during cell elongation normally occurs along two helices of opposite handedness, each counterbalancing the other's torque.

Bacterial cell curvature through mechanical control of cell growth

The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament-like protein, is required for the curved shape of Caulobacter crescentus and localizes to the inner cell curvature. Here, we show that crescentin forms a single filamentous structure that collapses into a helix when detached from the cell membrane, suggesting that it is normally maintained in a stretched configuration. Crescentin causes an elongation rate gradient around the circumference of the sidewall, creating a longitudinal cell length differential and hence curvature. Such curvature can be produced by physical force alone when cells are grown in circular microchambers. Production of crescentin in Escherichia coli is sufficient to generate cell curvature. Our data argue for a model in which physical strain borne by the crescentin structure anisotropically alters the kinetics of cell wall insertion to produce curved growth. Our study suggests that bacteria may use the cytoskeleton for mechanical control of growth to alter morphology.

Actin and actin‐binding proteins in yeast

Recently it has been shown that the amoeboid form of Dictyosteliurn discoideurn can chemotax in the absence of either myosin, a-actinin or severin without immediately obvious defects [reviewed by Bray and Vasiliev, 19891. These observations lead one to wonder what contractile proteins do in nonmuscle cells. A number of investigators have begun in the past few years to study the actin cytoskeleton in the yeast Saccharornyces cerevisiae with the hope of associating the biochemical activities of contractile proteins observed in vitro with in vivo functions. Although yeast do not exhibit the full range of biological behaviors of higher organisms such as Dictyosteliurn, the simplicity of yeast can be an advantage in elucidating functional interactions. Moreover, S. cerevisiae offers the most sophisticated genetics available in a eukaryote (see the accompanying review by Stearns). A number of components of the yeast actin cytoskeleton have now been identified, and, although this work is still in its infancy, genetic studies on several of these proteins are already shedding light on their in vivo functions. Since yeast are nonmotile and have not been shown to exhibit actin-based organelle transport or cytoplasmic streaming, possible roles for yeast actin are not immediately obvious. Clues about what actin might be doing in yeast were first provided by studies on the organization of the actin cytoskeleton [Kilmartin and Adams, 1984; Adams and Pringle, 19841. S. cerevisiae cells replicate by growing in a highly polarized manner to form buds, which ultimately become daughter cells. Budding occurs when a spatially restricted region of the cell surface enlarges by the incorporation of new membrane and cell wall material. Fluorescence microscopy has shown that the yeast actin cytoskeleton is disposed in a manner that reflects the asymmetry of growth [Kilmartin and Adams, 1984; Adams and Pringle, 19841. The figure shows schematically how actin (encoded by the ACT1 gene) is organized in a budding yeast cell. Cables are aligned along the growth axis and cortical patches of actin are concentrated at the growing cell surface in the bud. These patches first appear clustered in a ring at the cell cortex early in the cell cycle (not shown); this ring predicts the precise site of bud emergence. Late in the cell cycle, the patches cluster in the region of the neck separating the daughter from the mother cell. Localized cell wall growth in this region results in the separation of the mother and daughter cells. The patches often seem to be located at the ends of actin filaments. Staining of the patches with rhodamine-phalloidin suggests that they contain filamentous actin [Adams and Pringle, 19841. The tight association of the actin patches with regions of surface growth suggests that they might be parts of the machinery responsible for localized membrane and cell wall insertion. The cables are well positioned to mediate directed transport of organelles synthesized in the mother cell to the bud. Understanding the function of actin in yeast will require an understanding of its organization on an ultrastructural level. The actin cables seen by fluorescence microscopy are likely to be filament bundles. Bundles of filaments have been observed in electron micrographs of yeast [Adams and Pringle, 19841; immunogold-labeling of yeast with antiactin antisera is needed to determine whether these filaments are composed of actin. Labeling actin filaments with myosin heads will determine if the actin filaments in a yeast cell have a uniform polarity. A uniform filament polarity with the barbed ends attached to the bud surface would make it possible for myosin-like motors to move organelles toward the bud surface. No structures corresponding to the cortical actin patches have been observed in the electron microscope. The patches might be transmembrane linkages between the

Yeast actin-binding proteins: evidence for a role in morphogenesis.

Three yeast actin-binding proteins were identified using yeast actin filaments as an affinity matrix. One protein appears to be a yeast myosin heavy chain; it is dissociated from actin filaments by ATP, it is similar in size (200 kD) to other myosins, and antibodies directed against Dictyostelium myosin heavy chain bind to it. Immunofluorescence experiments show that a second actin-binding protein (67 kD) colocalizes in vivo with both cytoplasmic actin cables and cortical actin patches, the only identifiable actin structures in yeast. The cortical actin patches are concentrated at growing surfaces of the yeast cell where they might play a role in membrane and cell wall insertion, and the third actin-binding protein (85 kD) is only detected in association with these structures. This 85-kD protein is therefore a candidate for a determinant of growth sites. The in vivo role of this protein was tested by overproduction; this overproduction causes a reorganization of the actin cytoskeleton which in turn dramatically affects the budding pattern and spatial growth organization of the yeast cell.