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.     

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.     

Direct interaction of FtsZ and MreB is required for septum synthesis and cell division in Escherichia coli

How bacteria coordinate cell growth with division is not well understood. Bacterial cell elongation is controlled by actin–MreB while cell division is governed by tubulin–FtsZ. A ring‐like structure containing FtsZ (the Z ring) at mid‐cell attracts other cell division proteins to form the divisome, an essential protein assembly required for septum synthesis and cell separation. The Z ring exists at mid‐cell during a major part of the cell cycle without contracting. Here, we show that MreB and FtsZ of Escherichia coli interact directly and that this interaction is required for Z ring contraction. We further show that the MreB–FtsZ interaction is required for transfer of cell‐wall biosynthetic enzymes from the lateral to the mature divisome, allowing cells to synthesise the septum. Our observations show that bacterial cell division is coupled to cell elongation via a direct and essential interaction between FtsZ and MreB.     

Escherichia coli low-molecular-weight penicillin-binding proteins help orient septal FtsZ, and their absence leads to asymmetric cell division and branching

Escherichia coli cells lacking low-molecular-weight penicillin-binding proteins (LMW PBPs) exhibit morphological alterations that also appear when the septal protein FtsZ is mislocalized, suggesting that peptidoglycan modification and division may work together to produce cell shape. We found that in strains lacking PBP5 and other LMW PBPs, higher FtsZ concentrations increased the frequency of branched cells and incorrectly oriented Z rings by 10- to 15-fold. Invagination of these rings produced improperly oriented septa, which in turn gave rise to asymmetric cell poles that eventually elongated into branches. Branches always originated from the remnants of abnormal septation events, cementing the relationship between aberrant cell division and branch formation. In the absence of PBP5, PBP6 and DacD localized to nascent septa, suggesting that these PBPs can partially substitute for the loss of PBP5. We propose that branching begins when mislocalized FtsZ triggers the insertion of inert peptidoglycan at unusual positions during cell division. Only later, after normal cell wall elongation separates the patches, do branches become visible. Thus, a relationship between the LMW PBPs and cytoplasmic FtsZ ultimately affects cell division and overall shape.     

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.     

Characterization of HMW-PBPs from the rod-shaped actinomycete Corynebacterium glutamicum: peptidoglycan synthesis in cells lacking actin-like cytoskeletal structures

Analysis of the complete genome sequence of Corynebacterium glutamicum indicated that, in addition to ftsI, there are eight proteins with sequence motifs that are strongly conserved in penicillin binding proteins (PBPs): four genes that code for high-molecular-weight (HMW)-PBPs (PBP1a, PBP1b, PBP2a and PBP2b), two genes encoding low-molecular-weight PBPs (PBP4 and PBP4b) and two probable beta-lactamases (PBP5 and PBP6). Here, the function of the four HMW-PBPs in C. glutamicum was investigated using a combination of genetic knockouts, enhanced green fluorescent protein 2 (EGFP2) fusions and penicillin staining of membrane preparations. The four HMW-PBPs were expressed in a growing culture of C. glutamicum, but none of four pbp genes was individually essential for the growth of the bacterium, and only the simultaneous disruption of both pbp1b and pbp2b was lethal. The fused EGFP2-PBP proteins were functional in vivo, which allowed correct determination of their cellular localization. EGFP2 fusions to PBP1a, PBP1b and PBP2b localized at the poles and at the septum, whereas EGFP2-PBP2a was predominantly found at the septum. Cefsulodin treatment specifically delocalized PBP1a and PBP1b (class A HMW-PBPs), whereas mecillinam caused the specific delocalization of PBP2b and PBP2a (class B HMW-PBPs). The results provide new insight into the mechanisms involved in the synthesis of the cell wall in this bacterial species, which lacks a known actin-like cytoskeletal structure.     

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.     

A new slant to the Z ring and bacterial cell branch formation

Rod-shaped bacteria such as Escherichia coli accurately maintain their shape from generation to generation. The cytoskeletal proteins MreB and FtsZ, which respectively guide parallel growth of the sidewall and perpendicular growth of the division septum, are important to maintain a straight sidewall and uniformly rounded cell poles. FtsZ normally assembles into a ring at the cell midpoint, called the Z ring, which is oriented perpendicular to the cell's axis and is thus in perfect position to guide growth of a perpendicular septum. In this issue of Molecular Microbiology, Potluri et al. show that low molecular weight penicillin binding proteins, particularly PBP5, have a role in maintaining the perpendicular geometry of the Z ring and subsequent septum in E. coli. When these factors are absent or perturbed, division septa are readily deformed, which results in abnormal cell poles that often bifurcate over time to generate branches. The data suggest that cellular branching in E. coli is specifically induced by aberrant septation events caused by mis-oriented Z rings and not by deformation of a growing cell pole or emergence of new tips from the sidewall, which are likely mechanisms of branching in other bacterial families.     

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.     

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.     

A new morphogenesis pathway in bacteria: unbalanced activity of cell wall synthesis machineries leads to coccus-to-rod transition and filamentation in ovococci

Bacteria display a variety of shapes, which have biological relevance. In most eubacteria, cell shape is maintained by the tough peptidoglycan (PG) layer of the cell wall, the sacculus. The organization of PG synthesis machineries, orchestrated by different cytoskeletal elements, determines the specific shapes of sacculi. In rod-shaped bacteria, the actin-like (MreB) and the tubuline-like (FtsZ) cytoskeletons control synthesis of the sidewall (elongation) and the crosswall (septation) respectively. Much less is known concerning cell morphogenesis in cocci, which lack MreB proteins. While spherical cocci exclusively display septal growth, ovococci additionally display peripheral growth, which is responsible of the slight longitudinal expansion that generates their ovoid shape. Here, we report that the ovococcus Lactococcus lactis has the ability to become rod-shaped. L. lactis IL1403 wild-type cells form long aseptate filaments during both biofilm and planktonic growth in a synthetic medium. Nascent PG insertion and the division protein FtsK localize in multiple peripheral rings regularly spaced along the filaments. We show that filamentation results from septation inhibition, and that penicillin-binding proteins PBP2x and PBP2b play a direct role in this process. We propose a model for filament formation in L. lactis, and discuss the possible biological role of such morphological differentiation.     

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.     

The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes

MreB, the bacterial actin homologue, is thought to function in spatially co-ordinating cell morphogenesis in conjunction with MreC, a protein that wraps around the outside of the cell within the periplasmic space. In Caulobacter crescentus, MreC physically associates with penicillin-binding proteins (PBPs) which catalyse the insertion of intracellularly synthesized precursors into the peptidoglycan cell wall. Here we show that MreC is required for the spatial organization of components of the peptidoglycan-synthesizing holoenzyme in the periplasm and MreB directs the localization of a peptidoglycan precursor synthesis protein in the cytosol. Additionally, fluorescent vancomycin (Van-FL) labelling revealed that the bacterial cytoskeletal proteins MreB and FtsZ, as well as MreC and RodA, were required for peptidoglycan synthetic activity. MreB and FtsZ were found to be required for morphogenesis of the polar stalk. FtsZ was required for a cell cycle-regulated burst of peptidoglycan synthesis early in the cell cycle resulting in the synthesis of cross-band structures, whereas MreB was required for lengthening of the stalk. Thus, the bacterial cytoskeleton and cell shape-determining proteins such as MreC, function in concert to orchestrate the localization of cell wall synthetic complexes resulting in spatially co-ordinated and efficient peptidoglycan synthetic activity.     

Dimeric structure of the cell shape protein MreC and its functional implications

The bacterial actin homologue MreB forms helical filaments in the cytoplasm of rod-shaped bacteria where it helps maintain the shape of the cell. MreB is co-transcribed with mreC that encodes a bitopic membrane protein with a major periplasmic domain. Like MreB, MreC is localized in a helical pattern and might be involved in the spatial organization of the peptidoglycan synthesis machinery. Here, we present the structure of the major, periplasmic part of MreC from Listeria monocytogenes at 2.5 A resolution. MreC forms a dimer through an intimate contact along an N-terminal alpha-helix that connects the transmembrane region with two C-terminal beta-domains. The translational relationship between the molecules enables, in principle, filament formation. One of the beta-domains shows structural similarity to the chymotrypsin family of proteins and possesses a highly conserved Thr Ser dipeptide. Unexpectedly, mutagenesis studies show that the dipeptide is dispensable for maintaining cell shape and viability in both Escherichia coil and Bacillus subtilis. Bacterial two-hybrid experiments reveal that MreC Interacts with high-molecular-weight penicillin-binding proteins (PBPs), rather than with low-molecular-weight endo- and carboxypeptidases, indicating that MreC might act as a scaffold to which the murein synthases are recruited in order to spatially organize the synthesis of new cell wall material. Deletion analyses indicate which domains of B. subtilis MreC are required for interaction with MreD as well as with the PBPs.     

Discrete and overlapping functions of peptidoglycan synthases in growth,cell division and virulence of Listeria monocytogenes

Upon ingestion of contaminated food, Listeria monocytogenes can cause serious infections in humans that are normally treated with β‐lactam antibiotics. These target Listeria's five high molecular weight penicillin‐binding proteins (HMW PBPs), which are required for peptidoglycan biosynthesis. The two bi‐functional class A HMW PBPs PBP A1 and PBP A2 have transglycosylase and transpeptidase domains catalyzing glycan chain polymerization and peptide cross‐linking, respectively, whereas the three class B HMW PBPs B1, B2 and B3 are monofunctional transpeptidases. The precise roles of these PBPs in the cell cycle are unknown. Here we show that green fluorescent protein (GFP)‐PBP fusions localized either at the septum, the lateral wall or both, suggesting distinct and overlapping functions. Genetic data confirmed this view: PBP A1 and PBP A2 could not be inactivated simultaneously, and a conditional double mutant strain is largely inducer dependent. PBP B1 is required for rod‐shape and PBP B2 for cross‐wall biosynthesis and viability, whereas PBP B3 is dispensable for growth and cell division. PBP B1 depletion dramatically increased β‐lactam susceptibilities and stimulated spontaneous autolysis but had no effect on peptidoglycan cross‐linkage. Our in vitro virulence assays indicated that the complete set of all HMW PBPs is required for maximal virulence.     

Bacterial shape

In free-living eubacteria an external shell of peptidoglycan opposes internal hydrostatic pressure and prevents membrane rupture and death. At the same time, this wall imposes on each cell a shape. Because shape is both stable and heritable, as is the ability of many organisms to execute defined morphological transformations, cells must actively choose from among a large repertoire of available shapes. How they do so has been debated for decades, but recently experiment has begun to catch up with theory. Two discoveries are particularly informative. First, specific protein assemblies, nucleated by FtsZ, MreB or Mbl, appear to act as internal scaffolds that influence cell shape, perhaps by correctly localizing synthetic enzymes. Second, defects in cell shape are correlated with the presence of inappropriately placed, metabolically inert patches of peptidoglycan. When combined with what we know about mutants affecting cellular morphology, these observations suggest that bacteria may fabricate specific shapes by directing the synthesis of two kinds of cell wall: a long-lived, rigid framework that defines overall topology, and a metabolically plastic peptidoglycan whose shape is directed by internal scaffolds.     

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.     

Deciphering morphological determinants of the helix-shaped Leptospira

Leptospira spp. are thin, highly motile, slow-growing spirochetes that can be distinguished from other bacteria on the basis of their unique helical shape. Defining the mechanisms by which these bacteria generate and maintain this atypical morphology should greatly enhance our understanding of the fundamental physiology of these pathogens. In this study, we showed that peptidoglycan sacculi from Leptospira spp. retain the helical shape of intact cells. Interestingly, the distribution of muropeptides was different from that in the Escherichia coli model, indicating that specific enzymes might be active on the peptidoglycan macromolecule. We could alter the shape of Leptospira biflexa with the broad-spectrum β-lactam antibiotic penicillin G and with amdinocillin and aztreonam, which are β-lactams that preferentially target penicillin-binding protein 2 (PBP2) and PBP3, respectively, in some species. Although genetic manipulations of Leptospira spp. are scarce, we were able to obtain mutants with alterations in genes encoding PBPs, including PBP3. Loss of this protein resulted in cell elongation. We also generated an L. biflexa strain that conditionally expresses MreB. Loss of the MreB function was correlated with morphological abnormalities such as a localized increased diameter and heterogeneous length. A prolonged depletion of MreB resulted in cell lysis, suggesting that this protein is essential. These findings indicate that important aspects of leptospiral cell morphology are determined by the cytoskeleton and the murein layer, thus providing a starting point for a better understanding of the morphogenesis in these atypical bacteria.