Assembly Of MreB Research Articles

Localized cardiolipin synthesis is required for the assembly of MreB during the polarized cell division of Chlamydia trachomatis.

Pathogenic Chlamydia species are coccoid bacteria that use the rod-shape determining protein MreB to direct septal peptidoglycan synthesis during their polarized cell division process. How the site of polarized budding is determined in this bacterium, where contextual features like membrane curvature are seemingly identical, is unclear. We hypothesized that the accumulation of the phospholipid, cardiolipin (CL), in specific regions of the cell membrane induces localized membrane changes that trigger the recruitment of MreB to the site where the bud will arise. To test this, we ectopically expressed cardiolipin synthase (Cls) and observed a polar distribution for this enzyme in Chlamydia trachomatis. In early division intermediates, Cls was restricted to the bud site where MreB is localized and peptidoglycan synthesis is initiated. The localization profile of 6xHis tagged Cls (Cls_6xH) throughout division mimicked the distribution of lipids that stain with NAO, a dye that labels CL. Treatment of Chlamydia with 3’,6-dinonylneamine (diNN), an antibiotic targeting CL-containing membrane domains, resulted in redistribution of Cls_6xH and NAO-staining phospholipids. In addition, 6xHis tagged MreB localization was altered by diNN treatment, suggesting an upstream regulatory role for CL-containing membranes in directing the assembly of MreB. This hypothesis is consistent with the observation that the clustered localization of Cls_6xH is not dependent upon MreB function or peptidoglycan synthesis. Furthermore, expression of a CL-binding protein at the inner membrane of C. trachomatis dramatically inhibited bacterial growth supporting the importance of CL in the division process. Our findings implicate a critical role for localized CL synthesis in driving MreB assembly at the bud site during the polarized cell division of Chlamydia.

AimB Is a Small Protein Regulator of Cell Size and MreB Assembly

The MreB actin-like cytoskeleton assembles into dynamic polymers that coordinate cell shape in many bacteria. In contrast to most other cytoskeleton systems, few MreB-interacting proteins have been well characterized. Here, we identify a small protein from Caulobacter crescentus, an assembly inhibitor of MreB (AimB). AimB overexpression mimics inhibition of MreB polymerization, leading to increased cell width and MreB delocalization. Furthermore, aimB appears to be essential, and its depletion results in decreased cell width and increased resistance to A22, a small-molecule inhibitor of MreB assembly. Molecular dynamics simulations suggest that AimB binds MreB at its monomer-monomer protofilament interaction cleft and that this interaction is favored for C. crescentus MreB over Escherichia coli MreB because of a closer match in the degree of opening with AimB size, suggesting coevolution of AimB with MreB conformational dynamics in C. crescentus. We support this model through functional analysis of point mutants in both AimB and MreB, photo-cross-linking studies with site-specific unnatural amino acids, and species-specific activity of AimB. Together, our findings are consistent with AimB promoting MreB dynamics by inhibiting monomer-monomer assembly interactions, representing a new mechanism for regulating actin-like polymers and the first identification of a non-toxin MreB assembly inhibitor. Because AimB has only 104 amino acids and small proteins are often poorly characterized, our work suggests the possibility of more bacterial cytoskeletal regulators to be found in this class. Thus, like FtsZ and eukaryotic actin, MreB may have a rich repertoire of regulators to tune its precise assembly and dynamics.

Division-site localization of RodZ is required for efficient Z ring formation in Escherichia coli.

Bacteria such as Escherichia coli must coordinate cell elongation and cell division. Elongation is regulated by an elongasome complex containing MreB actin and the transmembrane protein RodZ, which regulates assembly of MreB, whereas division is regulated by a divisome complex containing FtsZ tubulin. These complexes were previously thought to function separately. However, MreB has been shown to directly interact with FtsZ to switch to cell division from cell elongation, indicating that these complexes collaborate to regulate both processes. Here, we investigated the role of RodZ in the regulation of cell division. RodZ localized to the division site in an FtsZ-dependent manner. We also found that division-site localization of MreB was dependent on RodZ. Formation of a Z ring was delayed by deletion of rodZ, suggesting that division-site localization of RodZ facilitated the formation or stabilization of the Z ring during early cell division. Thus, RodZ functions to regulate MreB assembly during cell elongation and facilitates the formation of the Z ring during cell division in E. coli.

The periplasmic disordered domain of RodZ promotes its self-interaction in Escherichia coli.

Rod shape of bacterial cells such as Escherichia coli is mainly regulated by a supramolecular complex called elongasome including MreB actin. Deletion of the mreB gene in rod-shaped bacterium E.coli results in round-shaped cells. RodZ was isolated as a determinant of rod shape in E.coli, Caulobacter crescentus and Bacillus subtilis and it has been shown to be an interaction partner and a regulator of assembly of MreB through its cytoplasmic domain. As opposed to functions of the N-terminal cytoplasmic domain of RodZ, functions of the C-terminal periplasmic domain including a disordered region are still unclear. To understand it, we adopted an in vivo photo-cross-linking assay to analyze interaction partners to identify proteins which interact with RodZ via its periplasmic domain, finding that the RodZ self-interacts in the periplasmic disordered domain. Self-interaction of RodZ was affected by MreB actin. Deletion of this region resulted in aberrant cell shape. Our results suggest that MreB binding to the cytoplasmic domain of RodZ causes structural changes in the disordered periplasmic domain of RodZ. We also found that the disordered domain of RodZ contributes to fine-tune rod shape in E.coli.

Large-scale purification and in vitro characterization of the assembly of MreB from Leptospira interrogans

BackgroundWeil's syndrome is caused by Leptospira interrogans infections, a Gram negative bacterium with a distinct thin corkscrew cell shape. The molecular basis for this unusual morphology is unknown. In many bacteria, cell wall synthesis is orchestrated by the actin homolog, MreB. MethodsHere we have identified the MreB within the L.interrogans genome and expressed the His-tagged protein product of the synthesized gene (Li-MreB) in Escherichia coli. Li-MreB did not purify under standard nucleotide-free conditions used for MreBs from other species, requiring the continual presence of ATP to remain soluble. Covalent modification of Li-MreB free thiols with Alexa488 produced a fluorescent version of Li-MreB. ResultsWe developed native and denaturing/refolding purification schemes for Li-MreB. The purified product was shown to assemble and disassemble in MgCl2 and KCl dependent manners, as monitored by light scattering and sedimentation studies. The fluorescence spectrum of labeled Li-MreB-Alexa488 showed cation-induced changes in line with an activation process followed by a polymerization phase. The resulting filaments appeared as bundles and sheets under the fluorescence microscope. Finally, since the Li-MreB polymerization was cation dependent, we developed a simple method to measure monovalent cation concentrations within a test case prokaryote, E. coli. ConclusionsWe have identified and initially characterized the cation-dependent polymerization properties of a novel MreB from a non-rod shaped bacterium and developed a method to measure cation concentrations within prokaryotes.General significance.This initial characterization of Li-MreB will enable future structural determination of the MreB filament from this corkscrew-shaped bacterium.

Mutations in cell elongation genes mreB, mrdA and mrdB suppress the shape defect of RodZ‐deficient cells

RodZ interacts with MreB and both factors are required to maintain the rod shape of Escherichia coli. The assembly of MreB into filaments regulates the subcellular arrangement of a group of enzymes that synthesizes the peptidoglycan (PG) layer. However, it is still unknown how polymerization of MreB determines the rod shape of bacterial cells. Regulatory factor(s) are likely to be involved in controlling the function and dynamics of MreB. We isolated suppressor mutations to partially recover the rod shape in rodZ deletion mutants and found that some of the suppressor mutations occurred in mreB. All of the mreB mutations were in or in the vicinity of domain IA of MreB. Those mreB mutations changed the property of MreB filaments in vivo. In addition, suppressor mutations were found in the periplasmic regions in PBP2 and RodA, encoded by mrdA and mrdB genes. Similar to MreB and RodZ, PBP2 and RodA are pivotal to the cell wall elongation process. Thus, we found that mutations in domain IA of MreB and in the periplasmic domain of PBP2 and RodA can restore growth and rod shape to ΔrodZ cells, possibly by changing the requirements of MreB in the process.

A22 Disrupts the Bacterial Actin Cytoskeleton by Directly Binding and Inducing a Low-Affinity State in MreB

S-(3,4-Dichlorobenzyl)isothiourea (A22) disrupts the actin cytoskeleton of bacteria, causing defects of morphology and chromosome segregation. Previous studies have suggested that the actin homologue MreB itself is the target of A22, but there has been no direct observation of A22 binding to MreB and no mechanistic explanation of its mode of action. We show that A22 binds MreB with at least micromolar affinity in its nucleotide-binding pocket in a manner that is sterically incompatible with simultaneous ATP binding. A22 negatively affects both the time course and extent of MreB polymerization in vitro in the presence of ATP. A22 prevents assembly of MreB into long, rigid polymers, as determined by both fluorescence microscopy and sedimentation assays. A22 increases the critical concentration of ATP-bound MreB assembly from 500 nM to approximately 2000 nM. We therefore conclude that A22 is a competitive inhibitor of ATP binding to MreB. A22-bound MreB is capable of polymerization, but with assembly properties that more closely resemble those of the ADP-bound state. Because the cellular concentration of MreB is in the low micromolar range, this mechanism explains the ability of A22 to largely disassemble the actin cytoskeleton in bacterial cells. It also represents a novel mode of action for a cytoskeletal drug and the first biochemical characterization of the interaction between a small molecule inhibitor of the bacterial cytoskeleton and its target.

Assembly of the MreB‐associated cytoskeletal ring of Escherichia coli

The Escherichia coli actin homologue MreB is part of a helical cytoskeletal structure that winds around the cell between the two poles. It has been shown that MreB redistributes during the cell cycle to form circumferential ring structures that flank the cytokinetic FtsZ ring and appear to be associated with division and segregation of the helical cytoskeleton. We show here that the MreB cytoskeletal ring also contains the MreC, MreD, Pbp2 and RodA proteins. Assembly of MreB, MreC, MreD and Pbp2 into the ring structure required the FtsZ ring but no other known components of the cell division machinery, whereas assembly of RodA into the cytoskeletal ring required one or more additional septasomal components. Strikingly, MreB, MreC, MreD and RodA were each able to independently assemble into the cytoskeletal ring and coiled cytoskeletal structures in the absence of any of the other ring components. This excludes the possibility that one or more of these proteins acts as a scaffold for incorporation of the other proteins into these structures. In contrast, incorporation of Pbp2 required the presence of MreC, which may provide a docking site for Pbp2 entry.

Polymerization properties of the Thermotoga maritima actin MreB: roles of temperature, nucleotides, and ions.

MreB is a bacterial orthologue of actin that affects cell shape, polarity, and chromosome segregation. Although a significant body of work has explored its cellular functions, we know very little about the biochemical behavior of MreB. We have cloned, overexpressed in Escherichia coli, and purified untagged MreB1 from Thermotoga maritima. We have characterized the conditions that regulate its monomer-to-polymer assembly reaction, the critical concentrations of that reaction, the manner in which MreB uses nucleotides, its stability, and the structure of the assembled polymer. MreB requires a bound purine nucleotide for polymerization and rapidly hydrolyzes it following assembly. MreB assembly contains two distinct components, one that does not require divalent cations and one that does, which may comprise the nucleation and elongation phases of assembly, respectively. MreB assembly is strongly favored by increasing temperature or protein concentration but inhibited differentially by high concentrations of monovalent salts. The polymerization rate increases and the bulk critical concentration decreases with increasing temperature, but in contrast to previous reports, MreB is capable of polymerizing across a broad range of temperatures. MreB polymers are shorter and stiffer and scatter more light than eukaryotic actin filaments. Due to rapid ATP hydrolysis and phosphate release, we suggest that most assembled MreB in cells is in the ADP-bound state. Because of only moderate differences between the ATP and ADP critical concentrations, treadmilling may occur, but we do not predict dynamic instability in cells. Because of the relatively low cellular concentration of MreB and the observed structural properties of the polymer, a single MreB assembly may exist in cells.

GTPase Activity, Structure, and Mechanical Properties of Filaments Assembled from Bacterial Cytoskeleton Protein MreB

MreB, a major component of the recently discovered bacterial cytoskeleton, displays a structure homologous to its eukaryotic counterpart actin. Here, we study the assembly and mechanical properties of Thermotoga maritima MreB in the presence of different nucleotides in vitro. We found that GTP, not ADP or GDP, can mediate MreB assembly into filamentous structures as effectively as ATP. Upon MreB assembly, both GTP and ATP release the gamma phosphate at similar rates. Therefore, MreB is an equally effective ATPase and GTPase. Electron microscopy and quantitative rheology suggest that the morphologies and micromechanical properties of filamentous ATP-MreB and GTP-MreB are similar. In contrast, mammalian actin assembly is favored in the presence of ATP over GTP. These results indicate that, despite high structural homology of their monomers, T. maritima MreB and actin filaments display different assembly, morphology, micromechanics, and nucleotide-binding specificity. Furthermore, the biophysical properties of T. maritima MreB filaments, including high rigidity and propensity to form bundles, suggest a mechanism by which MreB helical structure may be involved in imposing a cylindrical architecture on rod-shaped bacterial cells.

The Assembly of MreB, a Prokaryotic Homolog of Actin

MreB, a major component of the bacterial cytoskeleton, exhibits high structural homology to its eukaryotic counterpart actin. Live cell microscopy studies suggest that MreB molecules organize into large filamentous spirals that support the cell membrane and play a key shape-determining function. However, the basic properties of MreB filament assembly remain unknown. Here, we studied the assembly of Thermotoga maritima MreB triggered by ATP in vitro and compared it to the well-studied assembly of actin. These studies show that MreB filament ultrastructure and polymerization depend crucially on temperature as well as the ions present on solution. At the optimal growth temperature of T. maritima, MreB assembly proceeded much faster than that of actin, without nucleation (or nucleation is highly favorable and fast) and with little or no contribution from filament end-to-end annealing. MreB exhibited rates of ATP hydrolysis and phosphate release similar to that of F-actin, however, with a critical concentration of approximately 3 nm, which is approximately 100-fold lower than that of actin. Furthermore, MreB assembled into filamentous bundles that have the ability to spontaneously form ring-like structures without auxiliary proteins. These findings suggest that despite high structural homology, MreB and actin display significantly different assembly properties.