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.     

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.     

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.     

Proteins encoded by the mre gene cluster in Streptomyces coelicolor A3(2) cooperate in spore wall synthesis

It is still an open question how an intracellular cytoskeleton directs the synthesis of the peptidoglycan exoskeleton. In contrast to MreB of rod-shaped bacteria, which is essential for lateral cell wall synthesis, MreB of Streptomyces coelicolor has a role in sporulation. To study the function of the S. coelicolor mre gene cluster consisting of mreB, mreC, mreD, pbp2 and sfr, we generated non-polar replacement mutants. The individual mutants were viable and growth of substrate mycelium was not affected. However, all mutants produced enlarged spores, which frequently germinated prematurely and were sensitive to heat, high osmolarity and cell wall damaging agents. Protein-protein interaction assays by bacterial two-hybrid analyses indicated that the S. coelicolor Mre proteins form a spore wall synthesizing complex, which closely resembles the lateral wall synthesizing complex of rod-shaped bacteria. Screening of a genomic library identified several novel putative components of this complex. One of them (sco2097) was deleted. The Δsco2097 mutant formed sensitive spores with an aberrant morphology, demonstrating that SCO2097 is a new player in cell morphogenesis of Streptomyces. Our results suggest that all Mre proteins cooperate with the newly identified proteins in the synthesis of the thickened spore wall required to resist detrimental environmental conditions.     

Bactofilins,a ubiquitous class of cytoskeletal proteins mediating polar localization of a cell wall synthase in Caulobacter crescentus

The cytoskeleton has a key function in the temporal and spatial organization of both prokaryotic and eukaryotic cells. Here, we report the identification of a new class of polymer-forming proteins, termed bactofilins, that are widely conserved among bacteria. In Caulobacter crescentus, two bactofilin paralogues cooperate to form a sheet-like structure lining the cytoplasmic membrane in proximity of the stalked cell pole. These assemblies mediate polar localization of a peptidoglycan synthase involved in stalk morphogenesis, thus complementing the function of the actin-like cytoskeleton and the cell division machinery in the regulation of cell wall biogenesis. In other bacteria, bactofilins can establish rod-shaped filaments or associate with the cell division apparatus, indicating considerable structural and functional flexibility. Bactofilins polymerize spontaneously in the absence of additional cofactors in vitro, forming stable ribbon- or rod-like filament bundles. Our results suggest that these structures have evolved as an alternative to intermediate filaments, serving as versatile molecular scaffolds in a variety of cellular pathways.     

Changes of lipid domains in Bacillus subtilis cells with disrupted cell wall peptidoglycan

The cell wall is responsible for cell integrity and the maintenance of cell shape in bacteria. The Gram-positive bacterial cell wall consists of a thick peptidoglycan layer located on the outside of the cytoplasmic membrane. Bacterial cell membranes, like eukaryotic cell membranes, are known to contain domains of specific lipid and protein composition. Recently, using the membrane-binding fluorescent dye FM4-64, helix-like lipid structures extending along the long axis of the cell and consisting of negatively charged phospholipids were detected in the rod-shaped bacterium Bacillus subtilis. It was also shown that the cardiolipin-specific dye, nonyl acridine orange (NAO), is preferentially distributed at the cell poles and in the septal regions in both Escherichia coli and B. subtilis. These results suggest that phosphatidylglycerol is the principal component of the observed spiral domains in B. subtilis. Here, using the fluorescent dyes FM4-64 and NAO, we examined whether these lipid domains are linked to the presence of cell wall peptidoglycan. We show that in protoplasted cells, devoid of the peptidoglycan layer, helix-like lipid structures are not preserved. Specific lipid domains are also missing in cells depleted of MurG, an enzyme involved in peptidoglycan synthesis, indicating a link between lipid domain formation and peptidoglycan synthesis.     

The different shapes of cocci

The shape of bacteria is determined by their cell wall and can be very diverse. Even among genera with the suffix 'cocci', which are the focus of this review, different shapes exist. While staphylococci or Neisseria cells, for example, are truly round-shaped, streptococci, lactococci or enterococci have an ovoid shape. Interestingly, there seems to be a correlation between the shape of an organism and its set of penicillin-binding proteins--the enzymes that assemble the peptidoglycan, the main constituent of the cell wall. While only one peptidoglycan biosynthesis machinery seems to exist in staphylococci, two of these machineries are proposed to function in ovoid-shaped bacteria, reinforcing the intrinsic differences regarding the morphogenesis of different classes of cocci. The present review aims to integrate older ultra-structural data with recent localization studies, in order to clarify the relation between the mechanisms of cell wall synthesis and the determination of cell shape in various cocci.     

Taking shape: control of bacterial cell wall biosynthesis

The characteristic shape of a bacterial cell is a function of the three dimensional architectures of the cell envelope and is determined by the balance between lateral wall extension and synthesis of peptidoglycan at the division septum. The three dimensional patterns of cell wall synthesis in the bacterium Bacillus subtilis is influenced by actin-like proteins that form helical coils in the cell and by the MreCD membrane proteins that link the cytoskeletal elements with the penicillin-binding proteins that carry out peptidoglycan synthesis. Recent genetic studies have provided important clues as to how these proteins are arranged in the cell and how they function to regulate cell shape.     

Role of penicillin-binding proteins in bacterial cell morphogenesis

The penicillin-binding proteins (PBPs) polymerize and modify peptidoglycan, the stress-bearing component of the bacterial cell wall. As part of this process, the PBPs help to create the morphology of the peptidoglycan exoskeleton together with cytoskeleton proteins that regulate septum formation and cell shape. Genetic and microscopic studies reveal clear morphological responsibilities for class A and class B PBPs and suggest that the mechanism of shape determination involves differential protein localization and interactions with specific cell components. In addition, the low molecular weight PBPs, by varying the substrates on which other PBPs act, alter peptidoglycan synthesis or turnover, with profound effects on morphology.     

Essential role of DivIVA in polar growth and morphogenesis in Streptomyces coelicolor A3(2)

Streptomycetes grow by cell wall extension at hyphal tips. The molecular basis for such polar growth in prokaryotes is largely unknown. It is reported here that DivIVASC, the Streptomyces coelicolor homologue of the Bacillus subtilis protein DivIVA, is essential and directly involved in hyphal tip growth and morphogenesis. A DivIVASC-EGFP hybrid was distinctively localized to hyphal tips and lateral branches. Reduction of divIVASC expression to about 10% of the normal level produced a phenotype strikingly similar to that of many tip growth mutants in fungi, including irregular curly hyphae and apical branching. Overexpression of the gene dramatically perturbed determination of cell shape at the growing tips. Furthermore, staining of nascent peptidoglycan with a fluorescent vancomycin conjugate revealed that induction of overexpression in normal hyphae disturbed tip growth, and gave rise to several new sites of cell wall assembly, effectively causing hyperbranching. The results show that DivIVASC is a novel bacterial morphogene, and it is localized at or very close to the apical sites of peptidoglycan assembly in Streptomyces hyphae.     

Interaction of Penicillin‐Binding Protein 2x and Ser/Thr protein kinase StkP,two key players in Streptococcus pneumoniae R6 morphogenesis

Bacterial cell growth and division require the co‐ordinated action of peptidoglycan biosynthetic enzymes and cell morphogenesis proteins. However, the regulatory mechanisms that allow generating proper bacterial shape and thus preserving cell integrity remain largely uncharacterized, especially in ovococci. Recently, the conserved eukaryotic‐like Ser/Thr protein kinase of Streptococcus pneumoniae (StkP) was demonstrated to play a major role in cell shape and division. Here, we investigate the molecular mechanisms underlying the regulatory function(s) of StkP and show that it involves one of the essential actors of septal peptidoglycan synthesis, Penicillin‐Binding Protein 2x (PBP2x). We demonstrate that StkP and PBP2x interact directly and are present in the same membrane‐associated complex in S. pneumoniae. We further show that they both display a late‐division localization pattern at the division site and that the positioning of PBP2x depends on the presence of the extracellular PASTA domains of StkP. We demonstrate that StkP and PBP2x interaction is mediated by their extracellular regions and that the complex formation is inhibited in vitro in the presence of cell wall fragments. These data suggest that the role of StkP in cell division is modulated by an interaction with PBP2x.     

沉默FNBP1表达诱导7703细胞形态重塑

FNBP1在真核细胞中广泛表达,能通过诱导细胞膜管状化、内陷或变形及后续膜曲率依赖性肌动蛋白聚合过程,参与细胞内吞和运动;还可参与端粒维持及FasL与溶酶体结合过程的调控。研究发现,在肝癌细胞7703中,利用RNA干扰（RNA interference technology,RNAi）技术使内源FNBPI基因表达沉默以后,7703细胞形态发生重塑,由上皮样转变为树状分枝的纤维状,表明FNBP1在细胞形态维持过程中亦具有不可或缺的潜在作用;通过对已知FNBP1蛋白相互作用情况的分析推断,FNBP1可能是通过对肌动蛋白骨架动态组装过程的分子调控参与7703细胞形态的控制。     

Bacterial cell curvature through mechanical control of cell growth

The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament‐like protein, is required for the curved shape of Caulobacter crescentus and localizes to the inner cell curvature. Here, we show that crescentin forms a single filamentous structure that collapses into a helix when detached from the cell membrane, suggesting that it is normally maintained in a stretched configuration. Crescentin causes an elongation rate gradient around the circumference of the sidewall, creating a longitudinal cell length differential and hence curvature. Such curvature can be produced by physical force alone when cells are grown in circular microchambers. Production of crescentin in Escherichia coli is sufficient to generate cell curvature. Our data argue for a model in which physical strain borne by the crescentin structure anisotropically alters the kinetics of cell wall insertion to produce curved growth. Our study suggests that bacteria may use the cytoskeleton for mechanical control of growth to alter morphology.     

Bridging cell wall biosynthesis and bacterial morphogenesis

The bacterial cell wall is a complex three-dimensional structure that protects the cell from environmental stress and ensures its shape. The biosynthesis of its main component, the peptidoglycan, involves the coordination of activities of proteins present in the cytoplasm, the membrane, and the periplasm, some of which also interact with the bacterial cytoskeleton. The sheer complexity of the cell wall elongation process, which is the main focus of this review, has created a significant challenge for the study of the macromolecular interactions that regulate peptidoglycan biosynthesis. The availability of new structural and biochemical data on a number of components of peptidoglycan assembly machineries, including a complex between MreB and RodZ as well as structures of penicillin-binding proteins (PBPs) from a number of pathogenic species, now provide novel insight into the underpinnings of an intricate molecular machinery.     

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.     

Differentiated roles for MreB‐actin isologues and autolytic enzymes in Bacillus subtilis morphogenesis

Cell morphogenesis in most bacteria is governed by spatiotemporal growth regulation of the peptidoglycan cell wall layer. Much is known about peptidoglycan synthesis but regulation of its turnover by hydrolytic enzymes is much less well understood. Bacillus subtilis has a multitude of such enzymes. Two of the best characterized are CwlO and LytE: cells lacking both enzymes have a lethal block in cell elongation. Here we show that activity of CwlO is regulated by an ABC transporter, FtsEX, which is required for cell elongation, unlike cell division as in Escherichia coli. Actin‐like MreB proteins are thought to play a key role in orchestrating cell wall morphogenesis. B. subtilis has three MreB isologues with partially differentiated functions. We now show that the three MreB isologues have differential roles in regulation of the CwlO and LytE systems and that autolysins control different aspects of cell morphogenesis. The results add major autolytic activities to the growing list of functions controlled by MreB isologues in bacteria and provide new insights into the different specialized functions of essential cell wall autolysins.     

Physics of Bacterial Morphogenesis

Summary: Bacterial cells utilize three-dimensional (3D) protein assemblies to perform important cellular functions such as growth, division, chemoreception, and motility. These assemblies are composed of mechanoproteins that can mechanically deform and exert force. Sometimes, small-nucleotide hydrolysis is coupled to mechanical deformations. In this review, we describe the general principle for an understanding of the coupling of mechanics with chemistry in mechanochemical systems. We apply this principle to understand bacterial cell shape and morphogenesis and how mechanical forces can influence peptidoglycan cell wall growth. We review a model that can potentially reconcile the growth dynamics of the cell wall with the role of cytoskeletal proteins such as MreB and crescentin. We also review the application of mechanochemical principles to understand the assembly and constriction of the FtsZ ring. A number of potential mechanisms are proposed, and important questions are discussed.     

Shape determination in Bacillus subtilis

The discovery of cytoskeletal elements in prokaryotes has dramatically changed the way we think about bacterial cell morphogenesis. The rod shape of Bacillus subtilis is maintained by the two major polymers (peptidoglycan and teichoic acids) of its thick cell wall and determined by the way these are inserted during growth. The current view is that the dynamic tubulin-like (FtsZ) and actin-like (MreB) cytoskeletons orchestrate, both in time and space, the assembly of macromolecular machineries that effect cell wall synthesis and hydrolysis during cell division and cell elongation, respectively.     

Studying cytoskeletal dynamics in living cells using green fluorescent protein

Microfilaments, intermediate filaments, and microtubules are three major cytoskeletal systems providing cells with stability to maintain proper shape. Although the word “cytoskeleton” implicates rigidity, it is quite dynamic exhibiting constant changes within cells. In addition to providing cell stability, it participates in a variety of essential and dynamic cellular processes including cell migration, cell division, intracellular transport, vesicular trafficking, and organelle morphogenesis. During the past eight years since the green fluorescent protein (GFP) was first used as a marker for the exogenous gene expression, it has been an especially booming era for live cell observations of intracellular movement of many proteins. Because of the dynamic behavior of the cytoskeleton in the cell, GFP has naturally been a vital part of the studies of the cytoskeleton and its associated proteins. In this article, we will describe the advantage of using GFP and how it has been used to study cytoskeletal proteins.     

Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria

Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super-resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms.