The requirement for pneumococcal MreC and MreD is relieved by inactivation of the gene encoding PBP1a

MreC and MreD, along with the actin homologue MreB, are required to maintain the shape of rod-shaped bacteria. The depletion of MreCD in rod-shaped bacteria leads to the formation of spherical cells and the accumulation of suppressor mutations. Ovococcus bacteria, such as Streptococcus pneumoniae, lack MreB homologues, and the functions of the S. pneumoniae MreCD (MreCD(Spn)) proteins are unknown. mreCD are located upstream from the pcsB cell division gene in most Streptococcus species, but we found that mreCD and pcsB are transcribed independently. Similarly to rod-shaped bacteria, we show that mreCD are essential in the virulent serotype 2 D39 strain of S. pneumoniae, and the depletion of MreCD results in cell rounding and lysis. In contrast, laboratory strain R6 contains suppressors that allow the growth of ΔmreCD mutants, and bypass suppressors accumulate in D39 ΔmreCD mutants. One class of suppressors eliminates the function of class A penicillin binding protein 1a (PBP1a). Unencapsulated Δpbp1a D39 mutants have smaller diameters than their pbp1a(+) parent or Δpbp2a and Δpbp1b mutants, which lack other class A PBPs and do not show the suppression of ΔmreCD mutations. Suppressed ΔmreCD Δpbp1a double mutants form aberrantly shaped cells, some with misplaced peptidoglycan (PG) biosynthesis compared to that of single Δpbp1a mutants. Quantitative Western blotting showed that MreC(Spn) is abundant (≈8,500 dimers per cell), and immunofluorescent microscopy (IFM) located MreCD(Spn) to the equators and septa of dividing cells, similarly to the PBPs and PG pentapeptides indicative of PG synthesis. These combined results are consistent with a model in which MreCD(Spn) direct peripheral PG synthesis and control PBP1a localization or activity.     

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.     

Differential localization of Mre proteins with PBP2 in Rhodobacter sphaeroides

In Rhodobacter sphaeroides, MreB, MreC, MreD, PBP2, and RodA are encoded at the same locus. The localizations of PBP2, MreB, and MreC, which have all been implicated in the synthesis of the peptidoglycan layer, were investigated under different growth conditions to gain insight into the relationships between these proteins. Immunofluorescence microscopy showed that PBP2 localized to specific sites at the midcell of elongating cells under both aerobic and photoheterotrophic conditions. Visualizing PBP2 at different stages of the cell cycle showed that in elongating cells, PBP2 was found predominately at the midcell, with asymmetric foci and bands across the cell. PBP2 remained at midcell until the start of septation, after which it moved to midcell of the daughter cells. Deconvolution and three-dimensional reconstructions suggested that PBP2 forms a partial ring at the midcell of newly divided cells and elongated cells, while in septating cells, partial PBP2 rings were present at one-quarter and three-quarter positions. Due to the diffraction limits of light microscopy, these partial rings could represent unresolved helices. Colocalization studies showed that MreC always colocalized with PBP2, while MreB colocalized with PBP2 only during elongation; during septation, MreB remained at the septation site, whereas PBP2 relocalized to the one-quarter and three-quarter positions. These results suggest that PBP2 and MreC are involved in peptidoglycan synthesis during elongation and that this occurs at specific sites close to midcell in R. sphaeroides.     

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.     

MreB, the cell shape-determining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus

The bacterial actin homologue, MreB, is required for the maintenance of a rod-shaped cell and has been shown to form spirals that traverse along the longitudinal axis of Bacillus subtilis and Escherichia coli cells. The depletion of MreB in Caulobacter crescentus resulted in lemon-shaped cells that possessed defects in the integrity of the cell wall. MreB localization appeared as bands or spirals that encircled the cell along its entire length and switched to a mid-cell location at a time that coincided with the initiation of cell division. The formation of smaller MreB spirals or bands at the mid-cell was dependent on the presence on the cytokinetic protein, FtsZ. Penicillin-binding protein 2 (PBP2) also formed band-like structures perpendicular to the cell periphery that resembled, and depended upon, MreB localization. PBP2 co-immunoprecipitated with several other penicillin-binding proteins, suggesting that these proteins are in association in Caulobacter cells. We hypothesize that MreB filaments function as a cytoskeleton that serves as an organizer or tracking device for the PBP2-peptidoglycan biosynthesis complex.     

Growth of Escherichia coli: significance of peptidoglycan degradation during elongation and septation

We have found a striking difference between the modes of action of amdinocillin (mecillinam) and compound A22, both of which inhibit cell elongation. This was made possible by employment of a new method using an Escherichia coli peptidoglycan (PG)-recycling mutant, lacking ampD, to analyze PG degradation during cell elongation and septation. Using this method, we have found that A22, which is known to prevent MreB function, strongly inhibited PG synthesis during elongation. In contrast, treatment of elongating cells with amdinocillin, which inhibits penicillin-binding protein 2 (PBP2), allowed PG glycan synthesis to proceed at a nearly normal rate with concomitant rapid degradation of the new glycan strands. By treating cells with A22 to inhibit sidewall synthesis, the method could also be applied to study septum synthesis. To our surprise, over 30% of newly synthesized septal PG was degraded during septation. Thus, excess PG sufficient to form at least one additional pole was being synthesized and rapidly degraded during septation. We propose that during cell division, rapid removal of the excess PG serves to separate the new poles of the daughter cells. We have also employed this new method to demonstrate that PBP2 and RodA are required for the synthesis of glycan strands during elongation and that the periplasmic amidases that aid in cell separation are minor players, cleaving only one-sixth of the PG that is turned over by the lytic transglycosylases.     

PBP Deletion Mutants of Escherichia coli Exhibit Irregular Distribution of MreB at the Deformed Zones

MreB is a cytoskeletal protein, which is responsible for maintaining proper cellular morphology and is essential for cell survival. Likewise, penicillin-binding protein 5 (PBP5) helps in maintaining cell shape, though non-essential for survival. The contradicting feature of these two proteins paves the way for this study, wherein we attempt to draw a relation on the nature of distribution of MreB in PBP deletion mutants. The study revealed that the uniform MreB helices/patches were destabilized/disturbed at the zone of deformities of the PBP mutants, whereas the helical patterns were retained at the regions maintaining a rod shape. We interpret that MreB remains functional irrespective of its distribution being misguided by the aberrant shapes of PBP mutants.     

Several distinct localization patterns for penicillin-binding proteins in Bacillus subtilis

Bacterial cell shape is determined by a rigid external cell wall. In most non-coccoid bacteria, this shape is also determined by an internal cytoskeleton formed by the actin homologues MreB and/or Mbl. To gain further insights into the topological control of cell wall synthesis in bacteria, we have constructed green fluorescent protein (GFP) fusions to all 11 penicillin-binding proteins (PBPs) expressed during vegetative growth of Bacillus subtilis. The localization of these fusions was studied in a wild-type background as well as in strains deficient in FtsZ, MreB or Mbl. PBP3 and PBP4a localized specifically to the lateral wall, in distinct foci, whereas PBP1 and PBP2b localized specifically to the septum. All other PBPs localized to both the septum and the lateral cell wall, sometimes with irregular distribution along the lateral wall or a preference for the septum. This suggests that cell wall synthesis is not dispersed but occurs at specific places along the lateral cell wall. The results implicate PBP3, PBP5 and PBP4a, and possibly PBP4, in lateral wall growth. Localization of PBPs to the septum was found to be dependent on FtsZ, but the GFP-PBP fluorescence patterns were not detectably altered in the absence of MreB or Mbl.     

Localization of MreB in Rhodobacter sphaeroides under conditions causing changes in cell shape and membrane structure

MreB is thought to be a bacterial actin homolog that defines the morphology of rod-shaped bacteria. Rhodobacter sphaeroides changes shape, from a rod to coccobacillus, and undergoes extensive cytoplasmic membrane invagination when it switches from aerobic to photoheterotrophic growth. The role of MreB in defining R. sphaeroides shape was therefore investigated. Attempts at deleting or insertionally inactivating mreB were unsuccessful under all growth conditions. Immunofluorescence microscopy showed MreB localized to mid-cell in elongating cells under both aerobic and photoheterotrophic conditions. Three-dimensional reconstruction showed that MreB formed a ring at mid-cell. MreB remained at mid-cell as septation began but localized to new sites in the daughter cells before the completion of septation. MreB localized to putative septation sites in cephalexin-treated filamentous cells. Genomic single-copy mreB was replaced with gfp-mreB, and green fluorescent protein (GFP)-MreB localized in the same pattern, as seen with immunofluorescence microscopy. Some of the cells expressing GFP-MreB were abnormal, principally displaying an increase in cell width, suggesting that the fusion was not fully functional in all cells. GFP-MreB localized to swellings at mid-cell in cells treated with the penicillin-binding protein 2 inhibitor amdinocillin. These data suggest that MreB is essential in R. sphaeroides, performing a role at mid-cell in elongating cells, and in early septation, putatively in the cytoplasmic control of the peptidoglycan synthetic complexes.     

Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria

How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (>80 nm) helical filaments exist near or along either surface of the inner membrane. We also use correlated cryo-fluorescent light microscopy (cryo-fLM) and electron cryo-tomography (ECT) to identify cytoplasmic bundles of MreB, showing that MreB filaments are detectable by ECT. In light of these results, the structure and function of MreB must be reconsidered: instead of acting as a large, rigid scaffold that localizes cell-wall synthetic machinery, moving MreB complexes may apply tension to growing peptidoglycan strands to ensure their orderly, linear insertion.     

Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell

Cell shape in most eubacteria is maintained by a tough external peptidoglycan cell wall. Recently, cell shape determining proteins of the MreB family were shown to form helical, actin-like cables in the cell. We used a fluorescent derivative of the antibiotic vancomycin as a probe for nascent peptidoglycan synthesis in unfixed cells of various Gram-positive bacteria. In the rod-shaped bacterium B. subtilis, synthesis of the cylindrical part of the cell wall occurs in a helical pattern governed by an MreB homolog, Mbl. However, a few rod-shaped bacteria have no MreB system. Here, a rod-like shape can be achieved by a completely different mechanism based on use of polar growth zones derived from the division machinery. These results provide insights into the diverse molecular strategies used by bacteria to control their cellular morphology, as well as suggesting ways in which these strategies may impact on growth rates and cell envelope structure.     

Control of cell septation by lateral wall extension in a pH-conditional morphology mutant of Klebsiella pneumoniae.

The pH-conditional morphology mutant of Klebsiella pneumoniae strain MirM7 grows as cocci at pH 7 and as rods at pH 5.8. The mutant has a high-level mecillinam resistance (50% lethal dose greater than 200 micrograms/ml) in both forms. When broth cultures of the rod-shaped mutant were grown with 0.7 microgram of mecillinam per ml, cells assumed a round shape and continued to divided at a higher rate than the untreated control. A MirM7 rod-shaped revertant (MirA12), when treated with the same antibiotic concentration, changed to coccal shape and stopped dividing. The penicillin-binding proteins (PBPs) of strains MirA12 and MirM7 were analyzed. K. pneumoniae had six major PBPs quite similar to those of Escherichia coli. No differences were seen in the PBPs of MirM7 cocci and rods and MirA12 cells. In particular, PBP2 was found to be present and similar in MirM7 rods and cocci and MirA12 cells. We suggest that that in gram-negative rods, a control mechanism exists which prevents further septation in the absence of lateral cell wall elongation. The unique behavior of MirM7 is due to the fact that the control mechanism is not active in this strain. This model allows us to explain the preservation of shape in bacterial rods under various conditions of growth and the mechanism of bacterial killing by mecillinam.     

Cell shape determination in Escherichia coli

The rigid cell wall peptidoglycan (murein) is a single giant macromolecule whose shape determines the shape of the bacterial cell. Insight into morphogenetic mechanism(s) responsible for determining the shape of the murein sacculus itself has begun to emerge only in recent years. The discovery that MfreB and Mbl are cytoskeletal actin homologues that form helical structures extending from pole to pole in rod-shaped cells has opened an exciting new field of microbial cell biology. MreB (in Gram-negative rods) and Mbl (in Gram-positive species) are essential for murein synthesis along the lateral wall and hence, the rod shape of the cell. Known members of the morphogenetic system include MreB (or Mbl), MreC, MreD and PBP2, but Rod A and murein biosynthetic enzymes involved in peptidoglycan precursor synthesis and assembly are likely to be recruited to the same multimolecular apparatus. However, the actual role of MreB in assembly of the morphogenetic complex is still not clear and little is known about regulatory mechanisms controlling the switch from lateral murein elongation to septa1 murein synthesis at the time of cell division.     

MreB Drives De Novo Rod Morphogenesis in Caulobacter crescentus via Remodeling of the Cell Wall

MreB, the bacterial actin-like cytoskeleton, is required for the rod morphology of many bacterial species. Disruption of MreB function results in loss of rod morphology and cell rounding. Here, we show that the widely used MreB inhibitor A22 causes MreB-independent growth inhibition that varies with the drug concentration, culture medium conditions, and bacterial species tested. MP265, an A22 structural analog, is less toxic than A22 for growth yet equally efficient for disrupting the MreB cytoskeleton. The action of A22 and MP265 is enhanced by basic pH of the culture medium. Using this knowledge and the rapid reversibility of drug action, we examined the restoration of rod shape in lemon-shaped Caulobacter crescentus cells pretreated with MP265 or A22 under nontoxic conditions. We found that reversible restoration of MreB function after drug removal causes extensive morphological changes including a remarkable cell thinning accompanied with elongation, cell branching, and shedding of outer membrane vesicles. We also thoroughly characterized the composition of C. crescentus peptidoglycan by high-performance liquid chromatography and mass spectrometry and showed that MreB disruption and recovery of rod shape following restoration of MreB function are accompanied by considerable changes in composition. Our results provide insight into MreB function in peptidoglycan remodeling and rod shape morphogenesis and suggest that MreB promotes the transglycosylase activity of penicillin-binding proteins.Most bacteria have characteristic cell morphologies maintained during growth (67). The peptidoglycan (PG) component of the cell wall represents in most cases the physical support of various bacterial shapes. PG is a mesh-like polymeric macromolecule which opposes the osmotic pressure of the bacterial cytoplasm and prevents lysis in hypotonic growth environments (29). Isolated PG cell walls (sacculi) retain the shapes of the cells from which they originate while PG disruption causes the formation of osmotically labile spheroplasts, underscoring PG''s essential role in cell shape determination and cellular integrity maintenance. PG is composed of long glycan chains that are oriented roughly along the short axis of rod-shaped Gram-negative bacteria and that are connected by short peptide cross-links (21, 60). Bacterial growth and division necessitate the expansion and division of the PG cell wall, which requires the insertion of new PG material in the preexisting, covalently linked mesh (29). New PG synthesis requires two enzymatic reactions performed by penicillin-binding proteins (PBPs). Glycan chain synthesis is achieved by transglycosylation activity while cross-linkage of glycan chains to the existing mesh is achieved by transpeptidation activity (47). Class A PBPs, called bifunctional or bimodular PBPs (e.g., PBP1a and 1b of Escherichia coli), possess both transpeptidase and transglycosylase domains while class B PBPs, such as PBP2 and PBP3 of E. coli, can perform only transpeptidase reactions (47). Controlled degradation of the PG by cell wall hydrolases is necessary for incorporation of new PG material during growth. Tight coordination between PG synthesis and degradation is required to maintain the integrity of the mesh at all times (29).The bacterial cytoskeleton also plays a central role in cell shape determination and maintenance (7). MreB is a bacterial actin homolog that forms dynamic helical structures underneath the cytoplasmic membrane in most rod-shaped bacteria (8, 34, 37, 56). In some species, the spatial distribution of MreB varies during the cell cycle, changing from a helical/patchy localization pattern throughout the cell to a ring-like distribution near midcell (20, 22, 50, 58). MreB is required for rod shape maintenance as deletion of the MreB-encoding gene or depletion of MreB causes loss of rod shape and cell rounding (20, 22, 34, 63). Other proteins, including MreC, MreD, RodA, PBP2, and RodZ, function along with MreB to maintain rod shape as loss of their function also results in cell rounding (2, 5, 33, 48, 62). Among these rod-morphogenic proteins, only PBP2 has a known enzymatic function, being involved in PG synthesis as an elongation-specific transpeptidase; the others are membrane-spanning or integral membrane proteins (2, 5, 15, 48). The overall involvement of these morphogenetic proteins in rod shape maintenance has led to a model in which they are part of the elongase complex, a PG synthesizing machine that elongates the PG side wall (2, 5, 15, 48, 59). The elongase complex would include PG lytic enzymes and at least one bifunctional PBP required for glycan strand synthesis (15, 59). In Bacillus subtilis, MreB homologs were found to associate with the bifunctional PBP1 (36) and to regulate the localization of the PG hydrolase LytE (9). However, it is still unclear how MreB functions in the context of the proposed elongase complex to determine and maintain rod shape.It has been previously shown that repletion of MreB in lemon-shaped, MreB-depleted Caulobacter crescentus cells leads to the formation of cell filaments that present branches and ectopic stalks (64). To examine how MreB can drive de novo rod shape morphogenesis, we followed a similar strategy except that we used drug treatment to interfere with MreB function. The small molecule 3,4-dichlorobenzyl carbamimidothioate, also known as A22, has been shown to rapidly disrupt MreB localization in vivo and to induce growth-dependent rounding in several Gram-negative bacteria (23, 32, 41, 45, 52). Furthermore, genetic and biochemical experiments have shown that MreB is the direct molecular target of A22 and that A22 binds to MreB''s ATP-binding pocket, inducing a state with low affinity for polymerization (3, 23). As removal of A22 is followed within minutes by recovery of the normal MreB localization pattern (23), this drug represents a convenient tool for rapid and reversible inhibition of MreB function. However, A22 was found to inhibit the growth of an mreB deletion mutant of E. coli, suggesting that it can have MreB-independent toxic effects (35). In this study, we show that the toxicity of A22 varies with the drug concentration, culture medium conditions, and Gram-negative species tested. We identify a similarly potent but less toxic structural analog, MP265 (4-chlorobenzyl carbamimidothioate), as well as nontoxic concentrations and conditions for both A22 and MP265 that induce loss of rod cell morphology in C. crescentus. We also show that recovery of rod shape after drug removal is accompanied by intensive remodeling of PG morphology and composition.     

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.     

FtsZ directs a second mode of peptidoglycan synthesis in Escherichia coli

Certain penicillin binding protein mutants of Escherichia coli grow with spirillum-like morphologies when the FtsZ protein is inhibited, suggesting that FtsZ might govern aspects of cell wall growth other than those strictly associated with septation. While investigating the mechanism of spiral cell formation, we discovered conditions for visualizing this second function of FtsZ. Normally, inhibiting the cytoskeleton protein MreB forces E. coli cells to grow as smoothly enlarging spheres from which the poles disappear, yielding coccoid or lemon-shaped forms. However, when FtsZ and MreB were inhibited simultaneously in a strain lacking PBP 5 and PBP 7, the resulting cells ballooned outward but retained conspicuous rod-shaped extensions at sites representing the original poles. This visual phenotype was paralleled by the biochemistry of sacculus growth. Muropeptides are usually inserted homogeneously into the lateral cell walls, but when FtsZ polymerization was inhibited, the incorporation of new material occurred mainly in the central regions of cells and was significantly lower in those portions of side walls abutting a pole. Thus, reduced precursor incorporation into side walls near the poles explained why these regions retained their rod-like morphology while the rest of the cell grew spherically. Also, inhibiting FtsZ increased the amount of pentapeptides in sacculi by about one-third. Finally, the MreB protein directed the helical or diagonal incorporation of new peptidoglycan into the wall, but the location of that incorporation depended on whether FtsZ was active. In sum, the results indicate that in addition to nucleating cell septation in E. coli, FtsZ can direct the insertion of new peptidoglycan into portions of the lateral wall.     

Bacterial cell wall synthesis: new insights from localization studies.

In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall that consists mainly of the cross-linked polymer peptidoglycan (PG). The importance of PG for the maintenance of bacterial cell shape is underscored by the fact that, for various bacteria, several mutations affecting PG synthesis are associated with cell shape defects. In recent years, the application of fluorescence microscopy to the field of PG synthesis has led to an enormous increase in data on the relationship between cell wall synthesis and bacterial cell shape. First, a novel staining method enabled the visualization of PG precursor incorporation in live cells. Second, penicillin-binding proteins (PBPs), which mediate the final stages of PG synthesis, have been localized in various model organisms by means of immunofluorescence microscopy or green fluorescent protein fusions. In this review, we integrate the knowledge on the last stages of PG synthesis obtained in previous studies with the new data available on localization of PG synthesis and PBPs, in both rod-shaped and coccoid cells. We discuss a model in which, at least for a subset of PBPs, the presence of substrate is a major factor in determining PBP localization.     

Bacterial Cell Wall Synthesis: New Insights from Localization Studies

In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall that consists mainly of the cross-linked polymer peptidoglycan (PG). The importance of PG for the maintenance of bacterial cell shape is underscored by the fact that, for various bacteria, several mutations affecting PG synthesis are associated with cell shape defects. In recent years, the application of fluorescence microscopy to the field of PG synthesis has led to an enormous increase in data on the relationship between cell wall synthesis and bacterial cell shape. First, a novel staining method enabled the visualization of PG precursor incorporation in live cells. Second, penicillin-binding proteins (PBPs), which mediate the final stages of PG synthesis, have been localized in various model organisms by means of immunofluorescence microscopy or green fluorescent protein fusions. In this review, we integrate the knowledge on the last stages of PG synthesis obtained in previous studies with the new data available on localization of PG synthesis and PBPs, in both rod-shaped and coccoid cells. We discuss a model in which, at least for a subset of PBPs, the presence of substrate is a major factor in determining PBP localization.     

A Highlights from MBoC Selection: Motion of variable-length MreB filaments at the bacterial cell membrane influences cell morphology

The maintenance of rod-cell shape in many bacteria depends on actin-like MreB proteins and several membrane proteins that interact with MreB. Using superresolution microscopy, we show that at 50-nm resolution, Bacillus subtilis MreB forms filamentous structures of length up to 3.4 μm underneath the cell membrane, which run at angles diverging up to 40° relative to the cell circumference. MreB from Escherichia coli forms at least 1.4-μm-long filaments. MreB filaments move along various tracks with a maximal speed of 85 nm/s, and the loss of ATPase activity leads to the formation of extended and static filaments. Suboptimal growth conditions lead to formation of patch-like structures rather than extended filaments. Coexpression of wild-type MreB with MreB mutated in the subunit interface leads to formation of shorter MreB filaments and a strong effect on cell shape, revealing a link between filament length and cell morphology. Thus MreB has an extended-filament architecture with the potential to position membrane proteins over long distances, whose localization in turn may affect the shape of the cell wall.     

MreB: pilot or passenger of cell wall synthesis?

The discovery that the bacterial cell shape determinant MreB is related to actin spurred new insights into bacterial morphogenesis and development. The trafficking and mechanical roles of the eukaryotic cytoskeleton were hypothesized to have a functional ancestor in MreB based on evidence implicating MreB as an organizer of cell wall synthesis. Genetic, biochemical and cytological studies implicate MreB as a coordinator of a large multi-protein peptidoglycan (PG) synthesizing holoenzyme. Recent advances in microscopy and new biochemical evidence, however, suggest that MreB may function differently than previously envisioned. This review summarizes our evolving knowledge of MreB and attempts to refine the generalized model of the proteins organizing PG synthesis in bacteria. This is generally thought to be conserved among eubacteria and the majority of the discussion will focus on studies from a few well-studied model organisms.