Membrane reconstitution of FtsZ–ZipA complex inside giant spherical vesicles made of E. coli lipids: Large membrane dilation and analysis of membrane plasticity

During the division process of Escherichia coli, the globular protein FtsZ is early recruited at the constriction site. The Z-ring, based on FtsZ filaments associated to the inner cell membrane, has been postulated to exert constriction forces. Membrane anchoring is mediated by ZipA, an essential transmembrane protein able to specifically bind FtsZ. In this work, an artificial complex of FtsZ–ZipA has been reconstituted at the inner side of spherical giant unilamellar vesicles made of E. coli lipids. Under these conditions, FtsZ polymerization, triggered when a caged GTP analogue is UV-irradiated, was followed by up to 40% vesicle inflation. The homogeneous membrane dilation was accompanied by the visualization of discrete FtsZ assemblies at the membrane. Complementary rheological data revealed enhanced elasticity under lateral dilation. This explains why vesicles can undergo large dilations in the regime of mechanical stability. A mechanical role for FtsZ polymers as promoters of membrane softening and plasticization is hypothesized.     

Crystal Structure and Site-directed Mutational Analysis Reveals Key Residues Involved in Escherichia coli ZapA Function

FtsZ is an essential cell division protein in Escherichia coli, and its localization, filamentation, and bundling at the mid-cell are required for Z-ring stability. Once assembled, the Z-ring recruits a series of proteins that comprise the bacterial divisome. Zaps (FtsZ-associated proteins) stabilize the Z-ring by increasing lateral interactions between individual filaments, bundling FtsZ to provide a scaffold for divisome assembly. The x-ray crystallographic structure of E. coli ZapA was determined, identifying key structural differences from the existing ZapA structure from Pseudomonas aeruginosa, including a charged α-helix on the globular domains of the ZapA tetramer. Key helix residues in E. coli ZapA were modified using site-directed mutagenesis. These ZapA variants significantly decreased FtsZ bundling in protein sedimentation assays when compared with WT ZapA proteins. Electron micrographs of ZapA-bundled FtsZ filaments showed the modified ZapA variants altered the number of FtsZ filaments per bundle. These in vitro results were corroborated in vivo by expressing the ZapA variants in an E. coli ΔzapA strain. In vivo, ZapA variants that altered FtsZ bundling showed an elongated phenotype, indicating improper cell division. Our findings highlight the importance of key ZapA residues that influence the extent of FtsZ bundling and that ultimately affect Z-ring formation in dividing cells.     

Localization and Function of Early Cell Division Proteins in Filamentous Escherichia coli Cells Lacking Phosphatidylethanolamine

Escherichia coli cells that contain the pss-93 null mutation are completely deficient in the major membrane phospholipid phosphatidylethanolamine (PE). Such cells are defective in cell division. To gain insight into how a phospholipid defect could block cytokinesis, we used fluorescence techniques on whole cells to investigate which step of the cell division cycle was affected. Several proteins essential for early steps in cytokinesis, such as FtsZ, ZipA, and FtsA, were able to localize as bands to potential division sites in pss-93 filaments, indicating that the generation and localization of potential division sites was not grossly affected by the absence of PE. However, there was no evidence of constriction at most of these potential division sites. FtsZ and green fluorescent protein (GFP) fusions to FtsZ and ZipA often formed spiral structures in these mutant filaments. This is the first report of spirals formed by wild-type FtsZ expressed at normal levels and by ZipA-GFP. The results suggest that the lack of PE may affect the correct interaction of FtsZ with membrane nucleation sites and alter FtsZ ring structure so as to prevent or delay its constriction.     

Assembly and architecture of Escherichia coli divisome proteins FtsA and FtsZ

During Escherichia coli cell division, an intracellular complex of cell division proteins known as the Z-ring assembles at midcell during early division and serves as the site of constriction. While the predominant protein in the Z-ring is the widely conserved tubulin homolog FtsZ, the actin homolog FtsA tethers the Z-ring scaffold to the cytoplasmic membrane by binding to FtsZ. While FtsZ is known to function as a dynamic, polymerized GTPase, the assembly state of its partner, FtsA, and the role of ATP are still unclear. We report that a substitution mutation in the FtsA ATP-binding site impairs ATP hydrolysis, phospholipid vesicle remodeling in vitro, and Z-ring assembly in vivo. We demonstrate by transmission electron microscopy and Förster Resonance Energy Transfer that a truncated FtsA variant, FtsA(ΔMTS) lacking a C-terminal membrane targeting sequence, self assembles into ATP-dependent filaments. These filaments coassemble with FtsZ polymers but are destabilized by unassembled FtsZ. These findings suggest a model wherein ATP binding drives FtsA polymerization and membrane remodeling at the lipid surface, and FtsA polymerization is coregulated with FtsZ polymerization. We conclude that the coordinated assembly of FtsZ and FtsA polymers may serve as a key checkpoint in division that triggers cell wall synthesis and division progression.     

FtsZ polymers bound to lipid bilayers through ZipA form dynamic two dimensional networks

Bacteria divide by forming a contractile ring around their midcell region. FtsZ, a cytoskeletal soluble protein structurally related to tubulin, is the main component of this division machinery. It forms filaments that bundle at the inner side of the cytoplasmic membrane. These FtsZ bundles do not attach to bare lipid surfaces. In Escherichia coli they remain near the membrane surface by attaching to the membrane protein ZipA and FtsA. In order to study the structure and dynamics of the ZipA-FtsZ bundles formed on a lipid surface, we have oriented a soluble form of ZipA (sZipA), with its transmembrane domain substituted by a histidine tag, on supported lipid membranes. Atomic force microscopy has been used to visualize the polymers formed on top of this biomimetic surface. In the presence of GTP, when sZipA is present, FtsZ polymers restructure forming higher order structures. The lipid composition of the underlying membrane affects the aggregation kinetics and the shape of the structures formed. On the negatively charged E. coli lipid membranes, filaments condense from initially disperse material to form a network that is more dynamic and flexible than the one formed on phosphatidyl choline bilayers. These FtsZ-ZipA filament bundles are interconnected, retain their capacity to dynamically restructure, to fragment, to anneal and to condense laterally.     

Mathematical model for positioning the FtsZ contractile ring in Escherichia coli

During bacterial cytokinesis, a proteinaceous contractile ring assembles in the cell middle. The Z ring tethers to the membrane and contracts, when triggered, to form two identical daughter cells. One mechanism for positioning the ring involves the MinC, MinD and MinE proteins, which oscillate between cell poles to inhibit ring assembly. Averaged over time, the concentration of the inhibitor MinC is lowest at midcell, restricting ring assembly to this region. A second positioning mechanism, called Nucleoid Occlusion, acts through protein SlmA to inhibit ring polymerization in the location of the nucleoid. Here, a mathematical model was developed to explore the interactions between Min oscillations, nucleoid occlusion, Z ring assembly and positioning. One-dimensional advection-reaction-diffusion equations were built to simulate the spatio-temporal concentrations of Min proteins and their effect on various forms of FtsZ. The resulting partial differential equations were numerically solved using a finite volume method. The reduced chemical model assumed that the ring is composed of overlapping FtsZ filaments and that MinC disrupts lateral interactions between filaments. SlmA was presumed to break long FtsZ filaments into shorter units. A term was developed to account for the movement of FtsZ subunits in membrane-bound filaments as they touch and align with other filaments. This alignment was critical in forming sharp stable rings. Simulations qualitatively reproduced experimental results showing the incorrect positioning of rings when Min proteins were not expressed, and the formation of multiple rings when FtsZ was overexpressed.     

Negative-stain electron microscopy of inside-out FtsZ rings reconstituted on artificial membrane tubules show ribbons of protofilaments

FtsZ, the primary cytoskeletal element of the Z ring, which constricts to divide bacteria, assembles into short, one-stranded filaments in vitro. These must be further assembled to make the Z ring in bacteria. Conventional electron microscopy (EM) has failed to image the Z ring or resolve its substructure. Here we describe a procedure that enabled us to image reconstructed, inside-out FtsZ rings by negative-stain EM, revealing the arrangement of filaments. We took advantage of a unique lipid that spontaneously forms 500 nm diameter tubules in solution. We optimized conditions for Z-ring assembly with fluorescence light microscopy and then prepared specimens for negative-stain EM. Reconstituted FtsZ rings, encircling the tubules, were clearly resolved. The rings appeared as ribbons of filaments packed side by side with virtually no space between neighboring filaments. The rings were separated by variable expanses of empty tubule as seen by light microscopy or EM. The width varied considerably from one ring to another, but each ring maintained a constant width around its circumference. The inside-out FtsZ rings moved back and forth along the tubules and exchanged subunits with solution, similarly to Z rings reconstituted outside or inside tubular liposomes. FtsZ from Escherichia coli and Mycobacterium tuberculosis assembled rings of similar structure, suggesting a universal structure across bacterial species. Previous models for the Z ring in bacteria have favored a structure of widely scattered filaments that are not in contact. The ribbon structure that we discovered here for reconstituted inside-out FtsZ rings provides what to our knowledge is new evidence that the Z ring in bacteria may involve lateral association of protofilaments.     

Macromolecular interactions of the bacterial division FtsZ protein: from quantitative biochemistry and crowding to reconstructing minimal divisomes in the test tube

The division of Escherichia coli is an essential process strictly regulated in time and space. It requires the association of FtsZ with other proteins to assemble a dynamic ring during septation, forming part of the functionally active division machinery, the divisome. FtsZ reversibly interacts with FtsA and ZipA at the cytoplasmic membrane to form a proto-ring, the first molecular assembly of the divisome, which is ultimately joined by the rest of the division-specific proteins. In this review we summarize the quantitative approaches used to study the activity, interactions, and assembly properties of FtsZ under well-defined solution conditions, with the aim of furthering our understanding of how the behavior of FtsZ is controlled by nucleotides and physiological ligands. The modulation of the association and assembly properties of FtsZ by excluded-volume effects, reproducing in part the natural crowded environment in which this protein has evolved to function, will be described. The subsequent studies on the reactivity of FtsZ in membrane-like systems using biochemical, biophysical, and imaging technologies are reported. Finally, we discuss the experimental challenges to be met to achieve construction of the minimum protein set needed to initiate bacterial division, without cells, in a cell-like compartment. This integrated approach, combining quantitative and synthetic strategies, will help to support (or dismiss) conclusions already derived from cellular and molecular analysis and to complete our understanding on how bacterial division works.     

The Cell Division Protein FtsZ from Streptococcus pneumoniae Exhibits a GTPase Activity Delay

The cell division protein FtsZ assembles in vitro by a mechanism of cooperative association dependent on GTP, monovalent cations, and Mg²⁺. We have analyzed the GTPase activity and assembly dynamics of Streptococcus pneumoniae FtsZ (SpnFtsZ). SpnFtsZ assembled in an apparently cooperative process, with a higher critical concentration than values reported for other FtsZ proteins. It sedimented in the presence of GTP as a high molecular mass polymer with a well defined size and tended to form double-stranded filaments in electron microscope preparations. GTPase activity depended on K⁺ and Mg²⁺ and was inhibited by Na⁺. GTP hydrolysis exhibited a delay that included a lag phase followed by a GTP hydrolysis activation step, until reaction reached the GTPase rate. The lag phase was not found in polymer assembly, suggesting a transition from an initial non-GTP-hydrolyzing polymer that switches to a GTP-hydrolyzing polymer, supporting models that explain FtsZ polymer cooperativity.     

The Kil Peptide of Bacteriophage λ Blocks Escherichia coli Cytokinesis via ZipA-Dependent Inhibition of FtsZ Assembly

Assembly of the essential, tubulin-like FtsZ protein into a ring-shaped structure at the nascent division site determines the timing and position of cytokinesis in most bacteria and serves as a scaffold for recruitment of the cell division machinery. Here we report that expression of bacteriophage λ kil, either from a resident phage or from a plasmid, induces filamentation of Escherichia coli cells by rapid inhibition of FtsZ ring formation. Mutant alleles of ftsZ resistant to the Kil protein map to the FtsZ polymer subunit interface, stabilize FtsZ ring assembly, and confer increased resistance to endogenous FtsZ inhibitors, consistent with Kil inhibiting FtsZ assembly. Cells with the normally essential cell division gene zipA deleted (in a modified background) display normal FtsZ rings after kil expression, suggesting that ZipA is required for Kil-mediated inhibition of FtsZ rings in vivo. In support of this model, point mutations in the C-terminal FtsZ-interaction domain of ZipA abrogate Kil activity without discernibly altering FtsZ-ZipA interactions. An affinity-tagged-Kil derivative interacts with both FtsZ and ZipA, and inhibits sedimentation of FtsZ filament bundles in vitro. Together, these data inspire a model in which Kil interacts with FtsZ and ZipA in the cell to prevent FtsZ assembly into a coherent, division-competent ring structure. Phage growth assays show that kil⁺ phage lyse ∼30% later than kil mutant phage, suggesting that Kil delays lysis, perhaps via its interaction with FtsZ and ZipA.     

Localization of division protein FtsZ in Mycoplasma hominis

The localization of FtsZ protein in M. hominis cells was studied by immunoelectron microscopy with polyclonal antibodies to this protein. Cell polymorphism typical for mycoplasmas was seen on electron microscopic pictures. Among the diversity of cell shapes, we distinguished dumbbell-shaped dividing cells and cells connected with each other by membrane tubules (former constrictions). The label was predominantly observed in the constriction area of dividing M. hominis cells and on thin membrane tubules. A septum and the gold labeling of this structure have not been described before in mycoplasma cells. For the first time, in some rounded and oval cells, colloidal gold particles labeled the entire plasma membrane, probably marking a submembranous contractile ring (Z ring). These observations confirm the implication of FtsZ protein in M. hominis cytokinesis. In some cells, the spiral-like distribution of gold particles was observed. Most likely, FtsZ protofilaments in M. hominis cells form spiral structures similar to Z spirals in Bacillus subtilis and Escherichia coli. Their presence in mycoplasma cells may be considered to be an important argument in favor of Z ring assembly through the reorganization of Z spirals. FtsZ as a bacterial cytoskeleton protein binding with membrane directly or through intermediates may be engaged in maintenance of M. hominis cell shape.     

Influence of FtsZ proteins from some mycoplasma species on the division process in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

FtsZ is a highly conserved protein that performs one of the key roles in cell division of most prokaryotes. At the present time it is believed that the main role of FtsZ in the cells of the microorganisms studied (Escherichia coli, Bacillus subtilis, etc.) is to control the process of cell wall synthesis at the site of future septum. In this regard, the presence of FtsZ in the cells of mycoplasmas – bacteria that lack the cell wall, looks intriguing. In the present work we investigated the influence of FtsZ proteins of three mycoplasmas – Mycoplasma hominis, Mycoplasma gallisepticum and Acholeplasma laidlawii – on the cytokinesis of E. coli. FtsZ proteins were able to interact with the E. coli division machinery, including inhibiting the cytokinesis process, while demonstrating various patterns of distribution in the cell. The data obtained support the hypothesis that FtsZ plays a significant role in the division of mycoplasma cells.     

OmpA: Gating and dynamics via molecular dynamics simulations

Outer membrane proteins (OMPs) of Gram-negative bacteria have a variety of functions including passive transport, active transport, catalysis, pathogenesis and signal transduction. Whilst the structures of ∼ 25 OMPs are currently known, there is relatively little known about their dynamics in different environments. The outer membrane protein, OmpA from Escherichia coli has been studied extensively in different environments both experimentally and computationally, and thus provides an ideal test case for the study of the dynamics and environmental interactions of outer membrane proteins. We review molecular dynamics simulations of OmpA and its homologues in a variety of different environments and discuss possible mechanisms of pore gating. The transmembrane domain of E. coli OmpA shows subtle differences in dynamics and interactions between a detergent micelle and a lipid bilayer environment. Simulations of the crystallographic unit cell reveal a micelle-like network of detergent molecules interacting with the protein monomers. Simulation and modelling studies emphasise the role of an electrostatic-switch mechanism in the pore-gating mechanism. Simulation studies have been extended to comparative models of OmpA homologues from Pseudomonas aeruginosa (OprF) and Pasteurella multocida (PmOmpA), the latter model including the periplasmic C-terminal domain.     

FtsZ filament structures in different nucleotide states reveal the mechanism of assembly dynamics

Treadmilling protein filaments perform essential cellular functions by growing from one end while shrinking from the other, driven by nucleotide hydrolysis. Bacterial cell division relies on the primitive tubulin homolog FtsZ, a target for antibiotic discovery that assembles into single treadmilling filaments that hydrolyse GTP at an active site formed upon subunit association. We determined high-resolution filament structures of FtsZ from the pathogen Staphylococcus aureus in complex with different nucleotide analogs and cations, including mimetics of the ground and transition states of catalysis. Together with mutational and biochemical analyses, our structures reveal interactions made by the GTP γ-phosphate and Mg²⁺ at the subunit interface, a K⁺ ion stabilizing loop T7 for co-catalysis, new roles of key residues at the active site and a nearby crosstalk area, and rearrangements of a dynamic water shell bridging adjacent subunits upon GTP hydrolysis. We propose a mechanistic model that integrates nucleotide hydrolysis signaling with assembly-associated conformational changes and filament treadmilling. Equivalent assembly mechanisms may apply to more complex tubulin and actin cytomotive filaments that share analogous features with FtsZ.

Bacterial cell division critically relies on the tubulin homolog FtsZ, which assembles into filaments that treadmill, fuelled by GTP hydrolysis. This structural and biochemical study of FtsZ from Staphylocuccus aureus reveals the mechanism of GTP hydrolysis and its connection with filament dynamics.     

Energetics and geometry of FtsZ polymers: nucleated self-assembly of single protofilaments

Essential cell division protein FtsZ is an assembling GTPase which directs the cytokinetic ring formation in dividing bacterial cells. FtsZ shares the structural fold of eukaryotic tubulin and assembles forming tubulin-like protofilaments, but does not form microtubules. Two puzzling problems in FtsZ assembly are the nature of protofilament association and a possible mechanism for nucleated self-assembly of single-stranded protofilaments above a critical FtsZ concentration. We assembled two-dimensional arrays of FtsZ on carbon supports, studied linear polymers of FtsZ with cryo-electron microscopy of vitrified unsupported solutions, and formulated possible polymerization models. Nucleated self-assembly of FtsZ from Escherichia coli with GTP and magnesium produces flexible filaments 4-6 nm-wide, only compatible with a single protofilament. This agrees with previous scanning transmission electron microscopy results and is supported by recent cryo-electron tomography studies of two bacterial cells. Observations of double-stranded FtsZ filaments in negative stain may come from protofilament accretion on the carbon support. Preferential protofilament cyclization does not apply to FtsZ assembly. The apparently cooperative polymerization of a single protofilament with identical intermonomer contacts is explained by the switching of one inactive monomer into the active structure preceding association of the next, creating a dimer nucleus. FtsZ behaves as a cooperative linear assembly machine.     

The antimicrobial peptide Temporin L impairs E. coli cell division by interacting with FtsZ and the divisome complex

BackgroundThe comprehension of the mechanism of action of antimicrobial peptides is fundamental for the design of new antibiotics. Studies performed looking at the interaction of peptides with bacterial cells offer a faithful picture of what really happens in nature.MethodsIn this work we focused on the interaction of the peptide Temporin L with E. coli cells, using a variety of biochemical and biophysical techniques that include: functional proteomics, docking, optical microscopy, TEM, DLS, SANS, fluorescence.ResultsWe identified bacterial proteins specifically interacting with the peptides that belong to the divisome machinery; our data suggest that the GTPase FtsZ is the specific peptide target. Docking experiments supported the FtsZ-TL interaction; binding and enzymatic assays using recombinant FtsZ confirmed this hypothesis and revealed a competitive inhibition mechanism. Optical microscopy and TEM measurements demonstrated that, upon incubation with the peptide, bacterial cells are unable to divide forming long necklace-like cell filaments. Dynamic light scattering studies and Small Angle Neutron Scattering experiments performed on treated and untreated bacterial cells, indicated a change at the nanoscale level of the bacterial membrane.ConclusionsThe peptide temporin L acts by a non-membrane-lytic mechanism of action, inhibiting the divisome machinery.General significanceIdentification of targets of antimicrobial peptides is pivotal to the tailored design of new antimicrobials.     

A Multi-layered Protein Network Stabilizes the Escherichia coli FtsZ-ring and Modulates Constriction Dynamics

The prokaryotic tubulin homolog, FtsZ, forms a ring-like structure (FtsZ-ring) at midcell. The FtsZ-ring establishes the division plane and enables the assembly of the macromolecular division machinery (divisome). Although many molecular components of the divisome have been identified and their interactions extensively characterized, the spatial organization of these proteins within the divisome is unclear. Consequently, the physical mechanisms that drive divisome assembly, maintenance, and constriction remain elusive. Here we applied single-molecule based superresolution imaging, combined with genetic and biophysical investigations, to reveal the spatial organization of cellular structures formed by four important divisome proteins in E. coli: FtsZ, ZapA, ZapB and MatP. We show that these interacting proteins are arranged into a multi-layered protein network extending from the cell membrane to the chromosome, each with unique structural and dynamic properties. Further, we find that this protein network stabilizes the FtsZ-ring, and unexpectedly, slows down cell constriction, suggesting a new, unrecognized role for this network in bacterial cell division. Our results provide new insight into the structure and function of the divisome, and highlight the importance of coordinated cell constriction and chromosome segregation.     

Organization of FtsZ Filaments in the Bacterial Division Ring Measured from Polarized Fluorescence Microscopy

Cytokinesis in bacteria is accomplished by a ring-shaped cell-division complex (the Z-ring). The primary component of the Z-ring is FtsZ, a filamentous tubulin homolog that serves as a scaffold for the recruitment of other cell-division-related proteins. FtsZ forms filaments and bundles. In the cell, it has been suggested that FtsZ filaments form the arcs of the ring and are aligned in the cell-circumferential direction. Using polarized fluorescence microscopy in live Escherichia coli cells, we measure the structural organization of FtsZ filaments in the Z-ring. The data suggest a disordered organization: a substantial portion of FtsZ filaments are aligned in the cell-axis direction. FtsZ organization in the Z-ring also appears to depend on the bacterial species. Taken together, the unique arrangement of FtsZ suggests novel unexplored mechanisms in bacterial cell division.     

Investigating intracellular dynamics of FtsZ cytoskeleton with photoactivation single-molecule tracking

Using photoactivatable fluorescent protein as an intracellular protein label for single-molecule tracking offers several advantages over the traditional methods. Here we demonstrate the technique of photoactivation single-molecule tracking by investigating the mobility dynamics of intracellular FtsZ protein molecules in live Escherichia coli cells. FtsZ is a prokaryotic cytoskeleton protein (a homolog of tubulin) and plays important roles in cytokinesis. We demonstrate two heterogeneous subpopulations of FtsZ molecules with distinct diffusional dynamics. The FtsZ molecules forming the Z-rings near the center of the cell were mostly stationary, consistent with the assumption that they are within polymeric filamentous structures. The rest of the FtsZ molecules, on the other hand, undergo Brownian motion spanning the whole cell length. Surprisingly, the diffusion of FtsZ is spatially restricted to helical-shaped regions, implying an energy barrier for free diffusion. Consistently, the measured mean-square displacements of FtsZ showed anomalous diffusion characteristics. These results demonstrated the feasibility and advantages of photoactivation single-molecule tracking, and suggested new levels of complexity in the prokaryotic membrane organization.     

Organization of FtsZ Filaments in the Bacterial Division Ring Measured from Polarized Fluorescence Microscopy

Cytokinesis in bacteria is accomplished by a ring-shaped cell-division complex (the Z-ring). The primary component of the Z-ring is FtsZ, a filamentous tubulin homolog that serves as a scaffold for the recruitment of other cell-division-related proteins. FtsZ forms filaments and bundles. In the cell, it has been suggested that FtsZ filaments form the arcs of the ring and are aligned in the cell-circumferential direction. Using polarized fluorescence microscopy in live Escherichia coli cells, we measure the structural organization of FtsZ filaments in the Z-ring. The data suggest a disordered organization: a substantial portion of FtsZ filaments are aligned in the cell-axis direction. FtsZ organization in the Z-ring also appears to depend on the bacterial species. Taken together, the unique arrangement of FtsZ suggests novel unexplored mechanisms in bacterial cell division.