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.     

Bacillus subtilis MreB paralogues have different filament architectures and lead to shape remodelling of a heterologous cell system

Like many bacteria, Bacillus subtilis cells contain three actin-like MreB proteins. We show that the three paralogues, MreB, Mbl and MreBH, have different filament architectures in a heterologous cell system, and form straight filaments, helices or ring structures, different from the regular helical arrangement in B. subtilis cells. However, when coexpressed, they colocalize into a single filamentous helical structure, showing that the paralogues influence each other's filament architecture. Ring-like MreBH structures can be converted into MreB-like helical filaments by a single point mutation affecting subunit contacts, showing that MreB paralogues feature flexible filament arrangements. Time-lapse and FRAP experiments show that filaments can extend as well as shrink at both ends, and also show internal rearrangement, suggesting that filaments consist of overlapping bundles of shorter filaments that continuously turn over. Upon induction in Escherichia coli cells, B. subtilis MreB (BsMreB) filaments push the cells into strikingly altered cell morphology, showing that MreB filaments can change cell shape. E. coli cells with a weakened cell wall were ruptured upon induction of BsMreB filaments, suggesting that the bacterial actin orthologue may exert force against the cell membrane and envelope, and thus possibly plays an additional mechanical role in bacteria.     

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.     

Regulation of cell wall morphogenesis in Bacillus subtilis by recruitment of PBP1 to the MreB helix

The bacterial actin homologue MreB plays a key role in cell morphogenesis. In Bacillus subtilis MreB is essential under normal growth conditions and mreB mutants are defective in the control of cell diameter. However, the precise role of MreB is still unclear. Analysis of the lethal phenotypic consequences of mreB disruption revealed an unusual bulging phenotype that precedes cell death. A similar phenotype was seen in wild-type cells at very low Mg²⁺ concentrations. We found that inactivation of the major bi-functional penicillin-binding protein (PBP) PBP1 of B. subtilis restored the viability of an mreB null mutant as well as preventing bulging in both mutant and wild-type backgrounds. Bulging was associated with delocalization of PBP1. We show that the normal pattern of localization of PBP1 is dependent on MreB and that the proteins can physically interact using in vivo pull-down and bacterial two-hybrid approaches. Interactions between MreB and several other PBPs were also detected. Our results suggest that MreB filaments associate directly with the peptidoglycan biosynthetic machinery in B. subtilis as part of the mechanism that brings about controlled cell elongation.     

Dynamic movement of actin-like proteins within bacterial cells

Actin proteins are present in pro- and eukaryotes, and have been shown to perform motor-like functions in eukaryotic cells in a variety of processes. Bacterial actin homologues are essential for cell viability and have been implicated in the formation of rod cell shape, as well as in segregation of plasmids and whole chromosomes. We have generated functional green fluorescent protein fusions of all three Bacillus subtilis actin-like proteins (MreB, Mbl and MreBH), and show that all three proteins form helical filaments underneath the cell membrane, the pattern of which is distinct for each protein. Time-lapse microscopy showed that the filaments are highly dynamic structures. A number of separate filaments of MreB and Mbl continuously move through the cell along helical tracks underneath the cell membrane. The speed of extension of the growing end of filaments is within the range of known actin polymerization (0.1 microm/s), generating a potential poleward or centreward pushing velocity at 0.24 microm/min for MreB or Mbl, respectively. During nutritional downshift and a block in topoisomerase IV activity, the filaments rapidly disintegrated, showing that movement occurs only in growing cells. Contrary to Mbl and MreBH filaments, MreB filaments were generally absent in cells lacking DNA, providing a further distinction between the three orthologues.     

Bacillus subtilis possesses a second determinant with extensive sequence similarity to the Escherichia coli mreB morphogene.

A gene with substantial sequence similarity to the mreB morphogene of Bacillus subtilis has been identified at 302 degrees on the chromosomal map by A. Decatur, B. Kunkel, and R. Losick (Harvard University; personal communication). Our characterization has revealed that the protein product of this determinant (termed mbl for mreB-like) is 55 and 53% identical in sequence to the MreB proteins of B. subtilis and Escherichia coli, respectively. The protein is 86% identical to a protein identified as MreB from Bacillus cereus, suggesting that the B. cereus protein is actually Mbl. Insertional inactivation of mbl indicated that this gene is not essential for cell viability or sporulation. Cells bearing mutant mbl alleles display a decreased growth rate and an altered cellular morphology. The cells appear bloated and are frequently twisted. Intergenic suppressor mutations which restore the growth rate to an approximately normal level arise within the mutant population. A second site mutation, designated som-1, was mapped to the hisA-mbl region of the chromosome by transduction.     

The YvcK protein is required for morphogenesis via localization of PBP1 under gluconeogenic growth conditions in Bacillus subtilis

The YvcK protein was previously shown to be dispensable when B. subtilis cells are grown on glycolytic carbon sources but essential for growth and normal shape on gluconeogenic carbon sources. Here, we report that YvcK is localized as a helical-like pattern in the cell. This localization seems independent of the actin-like protein, MreB. A YvcK overproduction restores a normal morphology in an mreB mutant strain when bacteria are grown on PAB medium. Reciprocally, an additional copy of mreB restores a normal growth and morphology in a yvcK mutant strain when bacteria are grown on a gluconeogenic carbon source like gluconate. Furthermore, as already observed for the mreB mutant, the deletion of the gene encoding the penicillin-binding protein PBP1 restores growth and normal shape of a yvcK mutant on gluconeogenic carbon sources. The PBP1 is delocalized in an mreB mutant grown in the absence of magnesium and in a yvcK mutant grown on gluconate medium. Interestingly, its proper localization can be rescued by YvcK overproduction. Therefore, in gluconeogenic growth conditions, YvcK is required for the correct localization of PBP1 and hence for displaying a normal rod shape.     

Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis

In the absence of an overt cytoskeleton, the external cell wall of bacteria has traditionally been assumed to be the primary determinant of cell shape. In the Gram-positive bacterium Bacillus subtilis, two related genes, mreB and mbl, were shown to be required for different aspects of cell morphogenesis. Subcellular localization of the MreB and Mbl proteins revealed that each forms a distinct kind of filamentous helical structure lying close to the cell surface. The distribution of the proteins in different species of bacteria, and the similarity of their sequence to eukaryotic actins, suggest that the MreB-like proteins have a cytoskeletal, actin-like role in bacterial cell morphogenesis.     

Actin homolog MreBH governs cell morphogenesis by localization of the cell wall hydrolase LytE

MreB proteins are bacterial actin homologs involved in cell morphogenesis and various other cellular processes. However, the effector proteins used by MreBs remain largely unknown. Bacillus subtilis has three MreB isoforms. Mbl and possibly MreB have previously been shown to be implicated in cell wall synthesis. We have now found that the third isoform, MreBH, colocalizes with the two other MreB isoforms in B. subtilis and also has an important role in cell morphogenesis. MreBH can physically interact with a cell wall hydrolase, LytE, and is required for its helical pattern of extracellular localization. Moreover, lytE and mreBH mutants exhibit similar cell-wall-related defects. We propose that controlled elongation of rod-shaped B. subtilis depends on the coordination of cell wall synthesis and hydrolysis in helical tracts defined by MreB proteins. Our data also suggest that physical interactions with intracellular actin bundles can influence the later localization pattern of extracellular effectors.     

蜜蜂螺原体细胞骨架相关基因mreB的克隆表达及在螺原体二种形态时的表达差异

【目的】在大肠杆菌中克隆表达蜜蜂螺原体细胞骨架相关基因mreB1?5,并预测所编码蛋白的理化性质,分析这些基因在螺原体螺旋状和非螺旋状时的表达水平,为进一步分析该基因的功能奠定基础。【方法】通过PCR扩增,从Spiroplasma melliferum CH-1基因组中获得mreB1?5基因,构建的重组表达载体pETmreB1?5分别在大肠杆菌BL21中诱导表达,利用镍亲和树脂纯化重组蛋白,通过在线工具预测MreB蛋白质的理化性质和功能域。利用Real-Time PCR比较螺原体CH-1在两种不同形态时mreB1?5基因的表达量。【结果】成功克隆到5个mreB基因,并在大肠杆菌BL21中高效表达。MreB蛋白分子量分别为36、23、23、37和25 kD,可能均为疏水性的蛋白,属于MreB/Mbl蛋白质家族。荧光定量PCR结果显示,螺原体在非螺旋状时mreB1?5基因的表达水平均远低于在螺旋状时基因的表达水平。【结论】本文第一次克隆表达了螺原体细胞骨架相关基因mreB1?5,初步表明这些基因在螺原体形态方面可能具有重要作用,为后续研究螺原体mreB基因在其运动和形态方面的功能提供了重要信息。     

<Emphasis Type="Italic">Bacillus subtilis</Emphasis> actin-like protein MreB influences the positioning of the replication machinery and requires membrane proteins MreC/D and other actin-like proteins for proper localization

Background  

Bacterial actin-like proteins have been shown to perform essential functions in several aspects of cellular physiology. They affect cell growth, cell shape, chromosome segregation and polar localization of proteins, and localize as helical filaments underneath the cell membrane. Bacillus subtilis MreB and Mbl have been shown to perform dynamic motor like movements within cells, extending along helical tracks in a time scale of few seconds.     

Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis

MreB proteins are bacterial actin homologues thought to have a role in cell shape determination by positioning the cell wall synthetic machinery. Many bacteria, particularly Gram-positives, have more than one MreB isoform. Bacillus subtilis has three, MreB, Mbl and MreBH, which colocalize in a single helical structure. We now show that the helical pattern of peptidoglycan (PG) synthesis in the cylindrical part of the rod-shaped cell is governed by the redundant action of the three MreB isoforms. Single mutants for any one of mreB isoforms can still incorporate PG in a helical pattern and generate a rod shape. However, after depletion of MreB in an mbl mutant (or depletion of all three isoforms) lateral wall PG synthesis was impaired and the cells became spherical and lytic. Overexpression of any one of the MreB isoforms overcame the lethality as well as the defects in lateral PG synthesis and cell shape. Furthermore, MreB and Mbl can associate with the peptidoglycan biosynthetic machinery independently. However, no single MreB isoform was able to support normal growth under various stress conditions, suggesting that the multiple isoforms are used to allow cells to maintain proper growth and morphogenesis under changing and sometimes adverse conditions.     

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.     

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.     

Presence of multiple sites containing polar material in spherical Escherichia coli cells that lack MreB

In rod-shaped bacteria, certain proteins are specifically localized to the cell poles. The nature of the positional information that leads to the proper localization of these proteins is unclear. In a screen for factors required for the localization of the Shigella sp. actin assembly protein IcsA to the bacterial pole, a mutant carrying a transposon insertion in mreB displayed altered targeting of IcsA. The phenotype of cells containing a transposon insertion in mreB was indistinguishable from that of cells containing a nonpolar mutation in mreB or that of wild-type cells treated with the MreB inhibitor A22. In cells lacking MreB, a green fluorescent protein (GFP) fusion to a cytoplasmic derivative of IcsA localized to multiple sites. Secreted full-length native IcsA was present in multiple faint patches on the surfaces of these cells in a pattern similar to that seen for the cytoplasmic IcsA-GFP fusion. EpsM, the polar Vibrio cholerae inner membrane protein, also localized to multiple sites in mreB cells and colocalized with IcsA, indicating that localization to multiple sites is not unique to IcsA. Our results are consistent with the requirement, either direct or indirect, for MreB in the restriction of certain polar material to defined sites within the cell and, in the absence of MreB, with the formation of ectopic sites containing polar material.     

The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex

MreB proteins of Escherichia coli, Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface. The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis. In B. subtilis and C. crescentus, the mreB gene is essential. However, in E. coli, mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD-encoded components were essential. We found that cells depleted of mreBCD became spherical, enlarged and finally lysed. Depletion of each mre gene separately conferred similar gross changes in cell morphology and viability. Thus, the three proteins encoded by mreBCD are all essential and function in the same morphogenetic pathway. Interestingly, the presence of a multicopy plasmid carrying the ftsQAZ genes suppressed the lethality of deletions in the mre operon. Using GFP and cell fractionation methods, we showed that the MreC and MreD proteins were associated with the cell membrane. Using a bacterial two-hybrid system, we found that MreC interacted with both MreB and MreD. In contrast, MreB and MreD did not interact in this assay. Thus, we conclude that the E. coli MreBCD form an essential membrane-bound complex. Curiously, MreB did not form cables in cell depleted for MreC, MreD or RodA, indicating a mutual interdependency between MreB filament morphology and cell shape. Based on these and other observations we propose a model in which the membrane-associated MreBCD complex directs longitudinal cell wall synthesis in a process essential to maintain cell morphology.     

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.     

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.     

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.