Dysfunctional MreB inhibits chromosome segregation in Escherichia coli

The mechanism of prokaryotic chromosome segregation is not known. MreB, an actin homolog, is a shape-determining factor in rod-shaped prokaryotic cells. Using immunofluorescence microscopy we found that MreB of Escherichia coli formed helical filaments located beneath the cell surface. Flow cytometric and cytological analyses indicated that MreB-depleted cells segregated their chromosomes in pairs, consistent with chromosome cohesion. Overexpression of wild-type MreB inhibited cell division but did not perturb chromosome segregation. Overexpression of mutant forms of MreB inhibited cell division, caused abnormal MreB filament morphology and induced severe localization defects of the nucleoid and of the oriC and terC chromosomal regions. The chromosomal terminus regions appeared cohered in both MreB-depleted cells and in cells overexpressing mutant forms of MreB. Our observations indicate that MreB filaments participate in directional chromosome movement and segregation.     

Dynamic localization and interaction with other Bacillus subtilis actin-like proteins are important for the function of MreB

Bacterial actin-like proteins play a key role in cell morphology and in chromosome segregation. Many bacteria, like Bacillus subtilis, contain three genes encoding actin-like proteins, called mreB, mbl and mreBH in B. subtilis. We show that MreB and Mbl colocalize extensively within live cells, and that all three B. subtilis actin paralogues interact with each other underneath the cell membrane. A mutation in the phosphate 2 motif of MreB had a dominant negative effect on cell morphology and on chromosome segregation. Expression of this mutant allele of MreB interfered with the dynamic localization of Mbl. These experiments show that the interaction between MreB and Mbl has physiological significance. An mreB deletion strain can grow under special media conditions, however, depletion of Mbl in this mutant background abolished growth, indicating that actin paralogues can partially complement each other. The membrane protein MreC was found to interact with Mbl, but not with MreB, revealing a clear distinction between the function of the two paralogues. The phosphate 2 mutant MreB protein allowed for filament formation of mutant or wild-type MreB, but abolished the dynamic reorganization of the filaments. The latter mutation led to a strong reduction, but not complete loss, of function of MreB, both in terms of chromosome segregation and of cell morphology. Our work shows that that the dynamic localization of MreB is essential for the proper activity of the actin-like protein and that the interactions between MreB paralogues have important physiological significance.     

DNA and origin region segregation are not affected by the transition from rod to sphere after inhibition of Escherichia coli MreB by A22

The bacterial actin homologue MreB forms a helix underneath the cytoplasmic membrane and was shown to be essential in the morphogenesis of the rod-shaped cells. Additionally, MreB was implicated to be involved in DNA segregation. However, in our hands the mreBCD deletion strain (PA340-678) grew without apparent DNA segregation defect, suggesting that the reported chromosome segregation inhibition could be caused by a temporarily effect of MreB inhibition or depletion. To assess the involvement of MreB in DNA segregation during the transition from rod to sphere, we compared the effect of A22 and the PBP2 inhibitor mecillinam on the percentage of cells with segregated nucleoids and the number of oriC foci in wild-type Escherichia coli cells. Cells became spherical in the same time window during both treatments and we could not detect any difference in the chromosome or oriC segregation between these two treatments. Additionally, flow cytometric analyses showed that A22 and mecillinam treatment gave essentially the same chromosome segregation pattern. We conclude that MreB is not directly involved in DNA segregation of E. coli.     

Actin-like proteins MreB and Mbl from Bacillus subtilis are required for bipolar positioning of replication origins

Actin-like proteins MreB and Mbl are required for proper cell shape and for viability in B. subtilis and form dynamic helical filaments underneath the cell membrane. We have found that depletion of MreB and Mbl proteins leads to a rapid defect in chromosome segregation before a defect in cell shape becomes detectable. Under these conditions, the SMC chromosome segregation complex that is essential for proper chromosome arrangement and segregation loses its specific subcellular localization, and replication origins fail to localize in a regular bipolar manner as in wild type cells. Time-lapse microscopy showed that during depletion of MreB, origin regions can move towards the same cell pole, showing that bipolar orientation of origin separation is lost. Contrarily, depletion of three other cell shape determinants, MreC, MreD, or MreBH (the third B. subtilis actin homolog) had no effect on chromosome segregation but varying effects on cell morphology. Depletion of MreC and MreD resulted in formation of round cells, while depletion of MreBH led to formation of vibrio-shaped cells. The data show that actin proteins Mbl and MreB are required for proper chromosome segregation and that Mre proteins affect different aspects in cell shape.     

MreB actin-mediated segregation of a specific region of a bacterial chromosome

Faithful chromosome segregation is an essential component of cell division in all organisms. The eukaryotic mitotic machinery uses the cytoskeleton to move specific chromosomal regions. To investigate the potential role of the actin-like MreB protein in bacterial chromosome segregation, we first demonstrate that MreB is the direct target of the small molecule A22. We then demonstrate that A22 completely blocks the movement of newly replicated loci near the origin of replication but has no qualitative or quantitative effect on the segregation of other loci if added after origin segregation. MreB selectively interacts, directly or indirectly, with origin-proximal regions of the chromosome, arguing that the origin-proximal region segregates via an MreB-dependent mechanism not used by the rest of the chromosome.     

Bacterial DNA segregation by the actin-like MreB protein

Faithful chromosome segregation is vital to all organisms. Eukaryotic cells use the tubulin-based cytoskeleton to segregate their chromosomes during mitosis. A handful of papers have provided convincing evidence that, in bacteria, this task is accomplished by the actin homolog MreB. In particular, a recent study by Gitai et al. demonstrates that MreB specifically binds to and segregates the replication origin of the bacterial chromosome.     

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.     

Changes in nucleoid morphology and origin localization upon inhibition or alteration of the actin homolog, MreB, of Vibrio cholerae

MreB is an actin homolog required for the morphogenesis of most rod-shaped bacteria and for other functions, including chromosome segregation. In Caulobacter crescentus and Escherichia coli, the protein seems to play a role in the segregation of sister origins, but its role in Bacillus subtilis chromosome segregation is less clear. To help clarify its role in segregation, we have here studied the protein in Vibrio cholerae, whose chromosome I segregates like the one in C. crescentus and whose chromosome II like the one in E. coli or B. subtilis. The properties of Vibrio MreB were similar to those of its homologs in other bacteria in that it formed dynamic helical filaments, was essential for viability, and was inhibited by the drug A22. Wild-type (WT) cells exposed to A22 became spherical and larger. The nucleoids enlarged correspondingly, and the origin positions for both the chromosomes no longer followed any fixed pattern. However, the sister origins separated, unlike the situation in other bacteria. In mutants isolated as A22 resistant, the nucleoids in some cases appeared compacted even when the cell shape was nearly normal. In these cells, the origins of chromosome I were at the distal edges of the nucleoid but not all the way to the poles where they normally reside. The sister origins of chromosome II also separated less. Thus, it appears that the inhibition or alteration of Vibrio MreB can affect both the nucleoid morphology and origin localization.     

MreB is important for cell shape but not for chromosome segregation of the filamentous cyanobacterium Anabaena sp. PCC 7120

MreB is a bacterial actin that plays important roles in determination of cell shape and chromosome partitioning in Escherichia coli and Caulobacter crescentus. In this study, the mreB from the filamentous cyanobacterium Anabaena sp. PCC 7120 was inactivated. Although the mreB null mutant showed a drastic change in cell shape, its growth rate, cell division and the filament length were unaltered. Thus, MreB in Anabaena maintains cell shape but is not required for chromosome partitioning. The wild type and the mutant had eight and 10 copies of chromosomes per cell respectively. We demonstrated that DNA content in two daughter cells after cell division in both strains was not always identical. The ratios of DNA content in two daughter cells had a Gaussian distribution with a standard deviation much larger than a value expected if the DNA content in two daughter cells were identical, suggesting that chromosome partitioning is a random process. The multiple copies of chromosomes in cyanobacteria are likely required for chromosome random partitioning in cell division.     

MreB of Streptomyces coelicolor is not essential for vegetative growth but is required for the integrity of aerial hyphae and spores

MreB forms a cytoskeleton in many rod-shaped bacteria which is involved in cell shape determination and chromosome segregation. PCR-based and Southern analysis of various actinomycetes, supported by analysis of genome sequences, revealed mreB homologues only in genera that form an aerial mycelium and sporulate. We analysed MreB in one such organism, Streptomyces coelicolor. Ectopic overexpression of mreB impaired growth, and caused swellings and lysis of hyphae. A null mutant with apparently normal vegetative growth was generated. However, aerial hyphae of this mutant were swelling and lysing; spores doubled their volume and lost their characteristic resistance to stress conditions. Loss of cell wall consistency was observed in MreB-depleted spores by transmission electron microscopy. An MreB-EGFP fusion was constructed to localize MreB in the mycelium. No clearly localized signal was seen in vegetative mycelium. However, strong fluorescence was observed at the septa of sporulating aerial hyphae, then as bipolar foci in young spores, and finally in a ring- or shell-like pattern inside the spores. Immunogold electron microscopy using MreB-specific antibodies revealed that MreB is located immediately underneath the internal spore wall. Thus, MreB is not essential for vegetative growth of S. coelicolor, but exerts its function in the formation of environmentally stable spores, and appears to primarily influence the assembly of the spore cell wall.     

A magnesium-dependent mreB null mutant: implications for the role of mreB in Bacillus subtilis

MreB shares a common prokaryotic ancestor with actin and is present in almost all rod-shaped bacteria. MreB proteins have been implicated in a range of important cell processes, including cell morphogenesis, chromosome segregation and cell polarity. The mreB gene frequently lies at the beginning of a cluster of genes, immediately upstream of the conserved mreC and mreD genes. RNA analysis showed that in Bacillus subtilis mreB is co-transcribed with mreC and that these genes form part of an operon under the control of a promoter(s) upstream of mreB. Construction of an in-frame deletion of mreB and its complementation by mreB(+) only, in trans, established that the gene is important for maintenance of cell width and cell viability under normal growth conditions, independent of polar effects on downstream genes. Remarkably, virtually normal growth was restored to the mreB null mutant in the presence of high concentrations of magnesium, especially when high concentrations of the osmoprotectant, sucrose were also present. Under these conditions, cells could be maintained in the complete absence of an mreB gene, with almost normal morphology. No detectable effect on chromosome segregation was evident in the mutant, nor was there an effect on the topology of nascent peptidoglycan insertion. A GFP-MreB fusion was used to look at the localization of MreB in live cells. The pattern of localization was similar to that previously described, but no tight linkage to nucleoid positioning was evident. Propagation of the mreB null mutant in the absence of magnesium and sucrose led to a progressive increase in cell width, culminating in cell lysis. Cell division was also perturbed but this effect may be secondary to the disturbance in cell width. These results suggest that the major role of MreB in B. subtilis lies in the control of cell diameter.     

The Bacterial Actin-Like Cytoskeleton

Recent advances have shown conclusively that bacterial cells possess distant but true homologues of actin (MreB, ParM, and the recently uncovered MamK protein). Despite weak amino acid sequence similarity, MreB and ParM exhibit high structural homology to actin. Just like F-actin in eukaryotes, MreB and ParM assemble into highly dynamic filamentous structures in vivo and in vitro. MreB-like proteins are essential for cell viability and have been implicated in major cellular processes, including cell morphogenesis, chromosome segregation, and cell polarity. ParM (a plasmid-encoded actin homologue) is responsible for driving plasmid-DNA partitioning. The dynamic prokaryotic actin-like cytoskeleton is thought to serve as a central organizer for the targeting and accurate positioning of proteins and nucleoprotein complexes, thereby (and by analogy to the eukaryotic cytoskeleton) spatially and temporally controlling macromolecular trafficking in bacterial cells. In this paper, the general properties and known functions of the actin orthologues in bacteria are reviewed.     

Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system

Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is modified. In agreement with ATP-dependent filament formation, the depletion of ATP in the cells lead to rapid dissociation of MreB filaments. Extended induction of MreB resulted in the formation of membrane protrusions, showing that like actin, MreB can exert force against the cell membrane. Mbl also formed membrane associated filaments, while MreBH formed filaments within the cytosol. When co-expressed, MreB, Mbl and MreBH built up mixed filaments underneath the cell membrane. Membrane protein RodZ localized to endosomes in S2 cells, but localized to the cell membrane when co-expressed with Mbl, showing that bacterial MreB/Mbl structures can recruit a protein to the cell membrane. Thus, MreB paralogs form a self-organizing and dynamic filamentous scaffold underneath the membrane that is able to recruit other proteins to the cell surface.     

The Helical MreB Cytoskeleton in Escherichia coli MC1000/pLE7 Is an Artifact of the N-Terminal Yellow Fluorescent Protein Tag

Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarity, and motility. Here we use electron cryotomography to show that MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not. In contrast, mCherry fused to an internal loop (MreB-RFP^SW) does not induce helices. The helices are therefore an artifact of the placement of the fluorescent protein tag. YFP-MreB helices were also clearly distinguishable from the punctate, “patchy” localization patterns of MreB-RFP^SW, even by standard light microscopy. The many interpretations in the literature of such punctate patterns as helices should therefore be reconsidered.     

The small protein MbiA interacts with MreB and modulates cell shape in Caulobacter crescentus

In Caulobacter crescentus, the actin homologue MreB is critical for cell shape maintenance. Despite the central importance of MreB for cell morphology and viability, very little is known about MreB-interacting factors. Here, we use an overexpression approach to identify a novel MreB interactor, MbiA. MbiA interacts with MreB in both biochemical and genetic assays, colocalizes with MreB throughout the cell cycle, and relies on MreB for its localization. MbiA overexpression mimics the loss of MreB function, severely perturbing cell morphology, inhibiting growth and inducing cell lysis. Additionally, mbiA deletion shows a synthetic growth phenotype with a hypomorphic allele of the MreB interactor RodZ, suggesting that these two MreB-interacting proteins either have partially redundant functions or participate in the same functional complex. Our work thus establishes MbiA as a novel cell shape regulator that appears to function through regulating MreB, and opens avenues for discovery of more MreB-regulating factors by showing that overexpression screens are a valuable tool for uncovering potentially redundant cell shape effectors.     

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.     

Exclusion of assembled MreB by anionic phospholipids at cell poles confers cell polarity for bidirectional growth

Cell polarity determines the direction of cell growth in bacteria. MreB actin spatially regulates peptidoglycan synthesis to enable cells to elongate bidirectionally. MreB densely localizes in the cylindrical part of the rod cell and not in polar regions in Escherichia coli. When treated with A22, which inhibits MreB polymerization, rod‐shaped cells became round and MreB was diffusely distributed throughout the cytoplasmic membrane. A22 removal resulted in restoration of the rod shape. Initially, diffuse MreB started to re‐assemble, and MreB‐free zones were subsequently observed in the cytoplasmic membrane. These MreB‐free zones finally became cell poles, allowing the cells to elongate bidirectionally. When MreB was artificially located at the cell poles, an additional pole was created, indicating that artificial localization of MreB at the cell pole induced local peptidoglycan synthesis. It was found that the anionic phospholipids (aPLs), phosphatidylglycerol and cardiolipin, which were enriched in cell poles preferentially interact with monomeric MreB compared with assembled MreB in vitro. MreB tended to localize to cell poles in cells lacking both aPLs, resulting in production of Y‐shaped cells. Their findings indicated that aPLs exclude assembled MreB from cell poles to establish cell polarity, thereby allowing cells to elongate in a particular direction.     

Bacterial mitotic machineries

Here, we review recent progress that yields fundamental new insight into the molecular mechanisms behind plasmid and chromosome segregation in prokaryotic cells. In particular, we describe how prokaryotic actin homologs form mitotic machineries that segregate DNA before cell division. Thus, the ParM protein of plasmid R1 forms F actin-like filaments that separate and move plasmid DNA from mid-cell to the cell poles. Evidence from three different laboratories indicate that the morphogenetic MreB protein may be involved in segregation of the bacterial chromosome.     

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.