MreB Cytoskeleton Research Articles

A molecular switch controls assembly of bacterial focal adhesions.

Cell motility universally relies on spatial regulation of focal adhesion complexes (FAs) connecting the substrate to cellular motors. In bacterial FAs, the Adventurous gliding motility machinery (Agl-Glt) assembles at the leading cell pole following a Mutual gliding-motility protein (MglA)-guanosine 5'-triphosphate (GTP) gradient along the cell axis. Here, we show that GltJ, a machinery membrane protein, contains cytosolic motifs binding MglA-GTP and AglZ and recruiting the MreB cytoskeleton to initiate movement toward the lagging cell pole. In addition, MglA-GTP binding triggers a conformational shift in an adjacent GltJ zinc-finger domain, facilitating MglB recruitment near the lagging pole. This prompts GTP hydrolysis by MglA, leading to complex disassembly. The GltJ switch thus serves as a sensor for the MglA-GTP gradient, controlling FA activity spatially.

Interacting bactofilins impact cell shape of the MreB-less multicellular Rhodomicrobium vannielii.

Most non-spherical bacteria rely on the actin-like MreB cytoskeleton to control synthesis of a cell-shaping and primarily rod-like cell wall. Diverging from simple rod shape generally requires accessory cytoskeletal elements, which locally interfere with the MreB-guided cell wall synthesis. Conserved and widespread representatives of this accessory cytoskeleton are bactofilins that polymerize into static, non-polar bundles of filaments. Intriguingly, many species of the Actinobacteria and Rhizobiales manage to grow rod-like without MreB by tip extension, yet some of them still possess bactofilin genes, whose function in cell morphogenesis is unknown. An intricate representative of these tip-growing bacteria is Rhodomicrobium vannielii; a member of the hitherto genetically not tractable and poorly studied Hyphomicrobiaceae within the MreB-less Rhizobiales order. R. vannielii displays complex asymmetric cell shapes and differentiation patterns including filamentous hyphae to produce offspring and to build dendritic multicellular arrays. Here, we introduce techniques to genetically access R. vannielii, and we elucidate the role of bactofilins in its sophisticated morphogenesis. By targeted mutagenesis and fluorescence microscopy, protein interaction studies and peptidoglycan incorporation analysis we show that the R. vannielii bactofilins are associated with the hyphal growth zones and that one of them is essential to form proper hyphae. Another paralog is suggested to represent a novel hybrid and co-polymerizing bactofilin. Notably, we present R. vannielii as a powerful new model to understand prokaryotic cell development and control of multipolar cell growth in the absence of the conserved cytoskeletal element, MreB.

Importance of being cross-linked for the bacterial cell wall.

The bacterial cell wall is primarily composed of a mesh of glycan strands cross-linked by peptide bridges and is essential for safeguarding the cell. The structure of the cell wall has to be stiff enough to bear the high turgor pressure and sufficiently tough to ensure protection against failure. Here we explore the role of various design features of the cell in enhancing the toughness of the cell wall. We explain how the glycan strand length distribution, the degree of cross-linking and the placement of the cross-links on the glycan strands can act in tandem to ensure that the cell wall offers sufficient resistance to propagation of cracks. Further, we suggest a possible mechanism by which peptide bond hydrolysis, via judicious cleaving of peptide cross-links, can act to mitigate this risk of failure. We also study the reinforcing effect of MreB cytoskeleton, which can offer a degree of safety to the cell wall. However, we show that the cross-linked structure of the cell wall is its primary line of defense against mechanical failure.

The bacterial Sec system is required for the organization and function of the MreB cytoskeleton.

The Sec system is responsible for protein insertion, translocation and secretion across membranes in all cells. The bacterial actin homolog MreB controls various processes, including cell wall synthesis, membrane organization and polarity establishment. Here we show that the two systems genetically interact and that components of the Sec system, especially the SecA motor protein, are essential for spatiotemporal organization of MreB in E. coli, as evidenced by the accumulation of MreB at irregular sites in Sec-impaired cells. MreB mislocalization in SecA-defective cells significantly affects MreB-coordinated processes, such as cell wall synthesis, and induce formation of membrane invaginations enriched in high fluidity domains. Additionally, MreB is not recruited to the FtsZ ring in secA mutant cells, contributing to division arrest and cell filamentation. Our results show that all these faults are due to improper targeting of MreB to the membrane in the absence of SecA. Thus, when we reroute RodZ, MreB membrane-anchor, by fusing it to a SecA-independent integral membrane protein and overproducing it, MreB localization is restored and the defect in cell division is corrected. Notably, the RodZ moiety is not properly inserted into the membrane, strongly suggesting that it only serves as a bait for placing MreB around the cell circumference. Finally, we show that MreB localization depends on SecA also in C. crescentus, suggesting that regulation of MreB by the Sec system is conserved in bacteria. Taken together, our data reveal that the secretion system plays an important role in determining the organization and functioning of the cytoskeletal system in bacteria.

Confined Mobility of TonB and FepA in Escherichia coli Membranes.

The important process of nutrient uptake in Escherichia coli, in many cases, involves transit of the nutrient through a class of beta-barrel proteins in the outer membrane known as TonB-dependent transporters (TBDTs) and requires interaction with the inner membrane protein TonB. Here we have imaged the mobility of the ferric enterobactin transporter FepA and TonB by tracking them in the membranes of live E. coli with single-molecule resolution at time-scales ranging from milliseconds to seconds. We employed simple simulations to model/analyze the lateral diffusion in the membranes of E.coli, to take into account both the highly curved geometry of the cell and artifactual effects expected due to finite exposure time imaging. We find that both molecules perform confined lateral diffusion in their respective membranes in the absence of ligand with FepA confined to a region μm in radius in the outer membrane and TonB confined to a region μm in radius in the inner membrane. The diffusion coefficient of these molecules on millisecond time-scales was estimated to be μm2/s and μm2/s for FepA and TonB, respectively, implying that each molecule is free to diffuse within its domain. Disruption of the inner membrane potential, deletion of ExbB/D from the inner membrane, presence of ligand or antibody to FepA and disruption of the MreB cytoskeleton was all found to further restrict the mobility of both molecules. Results are analyzed in terms of changes in confinement size and interactions between the two proteins.

RodZ and PgsA Play Intertwined Roles in Membrane Homeostasis of Bacillus subtilis and Resistance to Weak Organic Acid Stress.

Weak organic acids like sorbic and acetic acid are widely used to prevent growth of spoilage organisms such as Bacilli. To identify genes involved in weak acid stress tolerance we screened a transposon mutant library of Bacillus subtilis for sorbic acid sensitivity. Mutants of the rodZ (ymfM) gene were found to be hypersensitive to the lipophilic weak organic acid. RodZ is involved in determining the cell’s rod-shape and believed to interact with the bacterial actin-like MreB cytoskeleton. Since rodZ lies upstream in the genome of the essential gene pgsA (phosphatidylglycerol phosphate synthase) we hypothesized that expression of the latter might also be affected in rodZ mutants and hence contribute to the phenotype observed. We show that both genes are co-transcribed and that both the rodZ::mini-Tn10 mutant and a conditional pgsA mutant, under conditions of minimal pgsA expression, were sensitive to sorbic and acetic acid. Both strains displayed a severely altered membrane composition. Compared to the wild-type strain, phosphatidylglycerol and cardiolipin levels were lowered and the average acyl chain length was elongated. Induction of rodZ expression from a plasmid in our transposon mutant led to no recovery of weak acid susceptibility comparable to wild-type levels. However, pgsA overexpression in the same mutant partly restored sorbic acid susceptibility and fully restored acetic acid sensitivity. A construct containing both rodZ and pgsA as on the genome led to some restored growth as well. We propose that RodZ and PgsA play intertwined roles in membrane homeostasis and tolerance to weak organic acid stress.

MreB-Dependent Organization of the E. coli Cytoplasmic Membrane Controls Membrane Protein Diffusion

The functional organization of prokaryotic cell membranes, which is essential for many cellular processes, has been challenging to analyze due to the small size and nonflat geometry of bacterial cells. Here, we use single-molecule fluorescence microscopy and three-dimensional quantitative analyses in live Escherichia coli to demonstrate that its cytoplasmic membrane contains microdomains with distinct physical properties. We show that the stability of these microdomains depends on the integrity of the MreB cytoskeletal network underneath the membrane. We explore how the interplay between cytoskeleton and membrane affects trans-membrane protein (TMP) diffusion and reveal that the mobility of the TMPs tested is subdiffusive, most likely caused by confinement of TMP mobility by the submembranous MreB network. Our findings demonstrate that the dynamic architecture of prokaryotic cell membranes is controlled by the MreB cytoskeleton and regulates the mobility of TMPs.

Natural transformation occurs independently of the essential actin-like MreB cytoskeleton in Legionella pneumophila.

Natural transformation is the process by which bacteria can actively take up and integrate exogenous DNA thereby providing a source of genetic diversity. Under specific growth conditions the coordinated expression of several genes – a situation referred to as “competence” – allows bacteria to assemble a highly processive and dedicated system that can import high molecular weight DNA. Within the cell these large imported DNA molecules are protected from degradation and brought to the chromosome for recombination. Here, we report elevated expression of mreB during competence in the Gram-negative pathogen Legionella pneumophila. Interestingly a similar observation had previously been reported in the distantly-related Gram-positive organism Bacillus subtilis. MreB is often viewed as the bacterial actin homolog contributing to bacterial morphogenesis by coordinating peptidoglycan-synthesising complexes. In addition MreB is increasingly found to be involved in a growing number of processes including chromosome segregation and motor-driven motility. Using genetic and pharmacological approaches, we examined the possible role of MreB during natural transformation in L. pneumophila. Our data show that natural transformation does not require MreB dynamics and exclude a direct role of MreB filaments in the transport of foreign DNA and its recombination in the chromosome.

The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions.

In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate-bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA-MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein-cytoskeleton interactions are a universally conserved feature.

Principles of Bacterial Cell-Size Determination Revealed by Cell-Wall Synthesis Perturbations

Although bacterial cell morphology is tightly controlled, the principles of size regulation remain elusive. In Escherichia coli, perturbation of cell-wall synthesis often results in similar morphologies, making it difficult to deconvolve the complex genotype-phenotype relationships underlying morphogenesis. Here we modulated cell width through heterologous expression of sequences encoding the essential enzyme PBP2 and through sublethal treatments with drugs that inhibit PBP2 and the MreB cytoskeleton. We quantified the biochemical and biophysical properties of the cell wall across a wide range of cell sizes. We find that, although cell-wall chemical composition is unaltered, MreB dynamics, cell twisting, and cellular mechanics exhibit systematic large-scale changes consistent with altered chirality and a more isotropic cell wall. This multiscale analysis enabled identification of distinct roles for MreB and PBP2, despite having similar morphological effects when depleted. Altogether, our results highlight the robustness of cell-wall synthesis and physical principles dictating cell-size control.

Curvature-dependent localization of MreB during reversion of Escherichia coli L-forms to rod-like morphology, visualized through kymographs of curvature (left) and localization of the MreB cytoskeleton (right). For further details readers are referred to

Molecular MicrobiologyVolume 93, Issue 5 p. i-i Front CoverFree Access Curvature-dependent localization of MreB during reversion of Escherichia coli L-forms to rod-like morphology, visualized through kymographs of curvature (left) and localization of the MreB cytoskeleton (right). For further details readers are referred to the article by Billings et al. on pp. 883–896 of this issue First published: 29 August 2014 https://doi.org/10.1111/mmi.12754AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume93, Issue5September 2014Pages i-i RelatedInformation

Organization of Ribosomes and Nucleoids in Escherichia coli Cells during Growth and in Quiescence

We have examined the distribution of ribosomes and nucleoids in live Escherichia coli cells under conditions of growth, division, and in quiescence. In exponentially growing cells translating ribosomes are interspersed among and around the nucleoid lobes, appearing as alternative bands under a fluorescence microscope. In contrast, inactive ribosomes either in stationary phase or after treatment with translation inhibitors such as chloramphenicol, tetracycline, and streptomycin gather predominantly at the cell poles and boundaries with concomitant compaction of the nucleoid. However, under all conditions, spatial segregation of the ribosomes and the nucleoids is well maintained. In dividing cells, ribosomes accumulate on both sides of the FtsZ ring at the mid cell. However, the distribution of the ribosomes among the new daughter cells is often unequal. Both the shape of the nucleoid and the pattern of ribosome distribution are also modified when the cells are exposed to rifampicin (transcription inhibitor), nalidixic acid (gyrase inhibitor), or A22 (MreB-cytoskeleton disruptor). Thus we conclude that the intracellular organization of the ribosomes and the nucleoids in bacteria are dynamic and critically dependent on cellular growth processes (replication, transcription, and translation) as well as on the integrity of the MreB cytoskeleton.

The actin homologue MreB organizes the bacterial cell membrane

The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate bacterial cell wall synthesis. We noticed that the MreB cytoskeleton influences fluorescent staining of the cytoplasmic membrane. Detailed analyses combining an array of mutants, using specific lipid staining techniques and spectroscopic methods, revealed that MreB filaments create specific membrane regions with increased fluidity (RIFs). Interference with these fluid lipid domains (RIFs) perturbs overall lipid homeostasis and affects membrane protein localization. The influence of MreB on membrane organization and fluidity may explain why the active movement of MreB stimulates membrane protein diffusion. These novel MreB activities add additional complexity to bacterial cell membrane organization and have implications for many membrane-associated processes.

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.

Quantitation of Cell Wall Growth Suggests Feedback Mechanisms that Robustly Build Rod-Like Bacteria

In bacteria, a host of enzymes regulates the reproducible and robust construction of the cell wall, whose mechanical integrity is crucial for viability under osmotic stress. Antibiotics that target these enzymes disrupt cell wall construction, ultimately leading to mechanical failure of the cell. Our work explores the physical mechanisms of cell growth and death, as a guide to understanding antibiotic mechanisms that disrupt mechanical properties of the cell. We use a combination of cell wall fluorescent labeling, high resolution time-lapse microscopy, and computational image processing to characterize where, and with what dynamics, cell wall and outer membrane growth occurs. When cell-shape analysis is combined biophysical simulations of growth, our data strongly suggest that dynamic localization of the bacterial MreB cytoskeleton is part of a curvature sensing and growth feedback mechanism that orchestrates heterogeneous growth to maintain rod-like shape and regulate mechanical stress. Analysis of MreB and cell-surface marker fluorescence indicates that the cytoskeleton is present at sites of active growth and that chemical depolymerization of the cytoskeleton causes homogenous, unstructured growth and eventual cell death by rupture. Quantitative tracking of growth is an effective method for characterizing cell wall mechanical failure, and these techniques pave the way for studying the detailed dynamics of growth-associated proteins and their disturbance by antibiotics.

The deletion of bacterial dynamin and flotillin genes results in pleiotrophic effects on cell division, cell growth and in cell shape maintenance

BackgroundIn eukaryotic cells, dynamin and flotillin are involved in processes such as endocytosis and lipid raft formation, respectively. Dynamin is a GTPase that exerts motor-like activity during the pinching off of vesicles, while flotillins are coiled coil rich membrane proteins with no known enzymatic activity. Bacteria also possess orthologs of both classes of proteins, but their function has been unclear.ResultsWe show that deletion of the single dynA or floT genes lead to no phenotype or a mild defect in septum formation in the case of the dynA gene, while dynA floT double mutant cells were highly elongated and irregularly shaped, although the MreB cytoskeleton appeared to be normal. DynA colocalizes with FtsZ, and the dynA deletion strain shows aberrant FtsZ rings in a subpopulation of cells. The mild division defect of the dynA deletion is exacerbated by an additional deletion in ezrA, which affects FtsZ ring formation, and also by the deletion of a late division gene (divIB), indicating that DynA affects several steps in cell division. DynA and mreB deletions generated a synthetic defect in cell shape maintenance, showing that MreB and DynA play non-epistatic functions in cell shape maintenance. TIRF microscopy revealed that FloT forms many dynamic membrane assemblies that frequently colocalize with the division septum. The deletion of dynA did not change the pattern of localization of FloT, and vice versa, showing that the two proteins play non redundant roles in a variety of cellular processes. Expression of dynamin or flotillin T in eukaryotic S2 cells revealed that both proteins assemble at the cell membrane. While FloT formed patch structures, DynA built up tubulated structures extending away from the cells.ConclusionsBacillus subtilis dynamin ortholog DynA plays a role during cell division and in cell shape maintenance. It shows a genetic link with flotillin T, with both proteins playing non-redundant functions at the cell membrane, where they assemble even in the absence of any bacterial cofactor.

Helical insertion of peptidoglycan produces chiral ordering of the bacterial cell wall

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 actin-like MreB proteins in Bacillus subtilis a new turn

A decade ago, two breakthrough descriptions were reported: 1) the first helix-like protein localization pattern of MreB and its paralog Mbl in Bacillus subtilis and 2) the crystal structure of Thermotoga maritima MreB1, which was remarkably similar to that of actin. These discoveries strongly stimulated the field of bacterial development, leading to the identification of many new cytoskeletal proteins (1) and the publication of many studies describing the helical patterns of protein, DNA and even lipid domains. However, today, new breakthroughs are shaking up what had become a dogma. Instead of helical structures, MreBs appear to form discrete patches that move circumferentially around the cell, questioning the idea of MreB cables forming an actin-like cytoskeleton. Furthermore, increasing evidence of biochemical properties that are unlike the properties of actin suggest that the molecular behavior of MreB proteins may be different. The aim of this review is to summarize the current knowledge of the so-called "actin-like" MreB cytoskeleton through a discussion of the model Gram-positive bacterium B. subtilis and the most recent findings in this rapidly evolving research field.

Rotate into shape: MreB and bacterial morphogenesis

Rotate into shape: MreB and bacterial morphogenesis

Assembly of MreB Filaments on Liposome Membranes: A Synthetic Biology Approach

The physical interaction between the cytoskeleton and the cell membrane is essential in defining the morphology of living organisms. In this study, we use a synthetic approach to polymerize bacterial MreB filaments inside phospholipid vesicles. When the proteins MreB and MreC are expressed inside the liposomes, the MreB cytoskeleton structure develops at the inner membrane. Furthermore, when purified MreB is used inside the liposomes, MreB filaments form a 4-10 μm rigid bundle structure and deform the lipid vesicles in physical contact with the vesicle inner membrane. These results indicate that the fibrillation of MreB filaments can take place either in close proximity of deformable lipid membrane or in the presence of associated protein. Our finding might be relevant for the self-assembly of cytoskeleton filaments toward the construction of artificial cell systems.