Multiple essential roles for EzrA in cell division of Staphylococcus aureus

In Bacillus subtilis, EzrA is involved in preventing aberrant formation of FtsZ rings and has also been implicated in the localization cycle of Pbp1. We have identified the orthologue of EzrA in Staphylococcus aureus to be essential for growth and cell division in this organism. Phenotypic analyses following titration of EzrA levels in S. aureus have shown that the protein is required for peptidoglycan synthesis as well as for assembly of the divisome at the midcell and cytokinesis. Protein interaction studies revealed that EzrA forms a complex with both the cytoplasmic components of the division machinery and those with periplasmic domains, suggesting that EzrA may be a scaffold molecule permitting the assembly of the division complex and forming an interface between the cytoplasmic cytoskeletal element FtsZ and the peptidoglycan biosynthetic apparatus active in the periplasm.     

EzrA prevents aberrant cell division by modulating assembly of the cytoskeletal protein FtsZ

In response to a cell cycle signal, the cytoskeletal protein FtsZ assembles into a ring structure that establishes the location of the division site and serves as a framework for assembly of the division machinery. A battery of factors control FtsZ assembly to ensure that the ring forms in the correct position and at the precise time. EzrA, a negative regulator of FtsZ ring formation, is important for ensuring that the ring forms only once per cell cycle and that cytokinesis is restricted to mid-cell. EzrA is distributed throughout the plasma membrane and localizes to the ring in an FtsZ-dependent manner, suggesting that it interacts directly with FtsZ to modulate assembly. We have performed a series of experiments examining the interaction between EzrA and FtsZ. As little as twofold overexpression of EzrA blocks FtsZ ring formation in a sensitized genetic background, consistent with its predicted function. A purified EzrA fusion protein interacts directly with FtsZ to block assembly in vitro. Although EzrA is able to inhibit FtsZ assembly, it is unable to disassemble preformed polymers. These data support a model in which EzrA interacts directly with FtsZ at the plasma membrane to prevent polymerization and aberrant FtsZ ring formation.     

A membrane protein, EzrA, regulates assembly dynamics of FtsZ by interacting with the C-terminal tail of FtsZ

FtsZ polymerizes to form a dynamic ring structure called the Z-ring at the midcell of bacteria. EzrA, a membrane protein, has been shown to prevent the formation of aberrant Z-rings in the low GC Gram-positive bacteria by inhibiting FtsZ assembly. In this study, we show that Bacillus subtilis (B. subtilis) EzrA inhibited the assembly and bundling of B. subtilis FtsZ. It increased the critical concentration of FtsZ assembly and depolymerized the preformed FtsZ polymers in vitro. We obtained evidence suggesting that B. subtilis EzrA forms complex with B. subtilis FtsZ in vitro. EzrA was found to bind to FtsZ at a single site with a dissociation constant of 4.3 +/- 0.6 microM. EzrA-FtsZ interaction has a significant electrostatic contribution as apparent from the effect of salt on their binding interactions. To elucidate the site of interaction between EzrA and FtsZ, we deleted 16 amino acid residues from the extreme C-terminal tail of B. subtilis FtsZ, which are conserved in FtsZ orthologues. EzrA did not inhibit the assembly of C-terminal truncated B. subtilis FtsZ. It also did not bind to the C-terminal truncated FtsZ detectably, suggesting that EzrA interacts with FtsZ through its conserved C-terminal tail residues. Further, a 17-residue synthetic peptide (365-382) of the C-terminal tail of FtsZ (CTP17) was used to probe the interaction of EzrA with the C-terminal tail of FtsZ. CTP17 bound to EzrA, inhibited the binding of EzrA to FtsZ, and surmounted the inhibitory effects of EzrA on the assembly of FtsZ in vitro. The data together showed that EzrA binds to the C-terminal tail of FtsZ. FtsA, a positive regulator of FtsZ assembly, is also known to interact with the C-terminal tail of FtsZ. The results indicated an interesting possibility that the assembly dynamics of FtsZ in the Z-ring is regulated by the competition between positive and negative regulators sharing the same binding site on FtsZ.     

The division inhibitor EzrA contains a seven-residue patch required for maintaining the dynamic nature of the medial FtsZ ring

The essential cytoskeletal protein FtsZ assembles into a ring-like structure at the nascent division site and serves as a scaffold for the assembly of the prokaryotic division machinery. We previously characterized EzrA as an inhibitor of FtsZ assembly in Bacillus subtilis. EzrA interacts directly with FtsZ to prevent aberrant FtsZ assembly and cytokinesis at cell poles. EzrA also concentrates at the cytokinetic ring in an FtsZ-dependent manner, although its precise role at this position is not known. Here, we identified a conserved patch of amino acids in the EzrA C terminus that is essential for localization to the FtsZ ring. Mutations in this patch (designated the “QNR patch”) abolish EzrA localization to midcell but do not significantly affect EzrA's ability to inhibit FtsZ assembly at cell poles. ezrA QNR patch mutant cells exhibit stabilized FtsZ assembly at midcell and are significantly longer than wild-type cells, despite lacking extra FtsZ rings. These results indicate that EzrA has two distinct activities in vivo: (i) preventing aberrant FtsZ ring formation at cell poles through inhibition of de novo FtsZ assembly and (ii) maintaining proper FtsZ assembly dynamics within the medial FtsZ ring, thereby rendering it sensitive to the factors responsible for coordinating cell growth and cell division.     

Functional Domain Analysis of the Cell Division Inhibitor EzrA

The precise spatial and temporal control of bacterial cell division is achieved through the balanced actions of factors that inhibit assembly of the tubulin-like protein FtsZ at aberrant subcellular locations or promote its assembly at the future sites of division. In Bacillus subtilis, the membrane anchored cell division protein EzrA, interacts directly with FtsZ to prevent aberrant FtsZ assembly at cell poles and contributes to the inherently dynamic nature of the cytokinetic ring. Recent work suggests EzrA also serves as a scaffolding protein to coordinate lateral growth with cell wall biosynthesis through interactions with a host of proteins, a finding consistent with EzrA''s four extensive coiled-coil domains. In a previous study we identified a conserved patch of residues near EzrA''s C-terminus (the QNR motif) that are critical for maintenance of a dynamic cytokinetic ring, but dispensable for EzrA-mediated inhibition of FtsZ assembly at cell poles. In an extension of this work, here we report that EzrA''s two C-terminal coiled-coils function in concert with the QNR motif to mediate interactions with FtsZ and maintain the dynamic nature of the cytokinetic ring. In contrast, EzrA''s two N-terminal coiled-coils are dispensable for interaction between EzrA and FtsZ in vitro and in vivo, but required for EzrA mediated inhibition of FtsZ assembly at cell poles. Finally, chimeric analysis indicates that EzrA''s transmembrane anchor plays a generic role: concentrating EzrA at the plasma membrane where presumably it can most effectively modulate FtsZ assembly.     

Bacillus subtilis SepF Binds to the C-Terminus of FtsZ

Bacterial cell division is mediated by a multi-protein machine known as the "divisome", which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z-ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull-down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.     

Assembly dynamics of FtsZ rings in Bacillus subtilis and Escherichia coli and effects of FtsZ-regulating proteins

FtsZ is the major cytoskeletal component of the bacterial cell division machinery. It forms a ring-shaped structure (the Z ring) that constricts as the bacterium divides. Previous in vivo experiments with green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching have shown that the Escherichia coli Z ring is extremely dynamic, continually remodeling itself with a half time of 30 s, similar to microtubules in the mitotic spindle. In the present work, under different experimental conditions, we have found that the half time for fluorescence recovery of E. coli Z rings is even shorter (approximately 9 s). As before, the turnover appears to be coupled to GTP hydrolysis, since the mutant FtsZ84 protein, with reduced GTPase in vitro, showed an approximately 3-fold longer half time. We have also extended the studies to Bacillus subtilis and found that this species exhibits equally rapid dynamics of the Z ring (half time, approximately 8 s). Interestingly, null mutations of the FtsZ-regulating proteins ZapA, EzrA, and MinCD had only modest effects on the assembly dynamics. This suggests that these proteins do not directly regulate FtsZ subunit exchange in and out of polymers. In B. subtilis, only 30 to 35% of the FtsZ protein was in the Z ring, from which we conclude that a Z ring only 2 or 3 protofilaments thick can function for cell division.     

Mechanism of regulation of prokaryotic tubulin-like GTPase FtsZ by membrane protein EzrA

At initiation of cell division, FtsZ, a tubulin-like GTPase, assembles into a so-called Z-ring structure at the site of division. The formation of Z ring is negatively regulated by EzrA, which ensures only one ring at the midcell per cell cycle. The mechanism leading to the negative regulation of Z-ring formation by EzrA has been analyzed. Our data reveal that the interaction between EzrA and FtsZ not only reduces the GTP-binding ability of FtsZ but also accelerates the rate of GTP hydrolysis, both of which are unfavorable for the polymerization of FtsZ. Moreover, the acceleration in rate of GTP hydrolysis by EzrA is attributed to stabilization of the transition state for GTP hydrolysis and reduction in the affinity of GDP for FtsZ. Clearly, EzrA is able to modify the GTP hydrolysis cycle of FtsZ. On the basis of these results, a model for how EzrA acts to negatively regulate Z-ring formation is proposed.     

Cytological and biochemical characterization of the FtsA cell division protein of Bacillus subtilis

The actin-like protein FtsA is present in many eubacteria, and genetic experiments have shown that it plays an important, sometimes essential, role in cell division. Here, we show that Bacillus subtilis FtsA is targeted to division sites in both vegetative and sporulating cells. As in other organisms FtsA is probably recruited immediately after FtsZ. In sporulating cells of B. subtilis FtsZ is recruited to potential division sites at both poles of the cell, but asymmetric division occurs at only one pole. We have now found that FtsA is recruited to only one cell pole, suggesting that it may play an important role in the generation of asymmetry in this system. FtsA is present in much higher quantities in B. subtilis than in Escherichia coli, with approximately one molecule of FtsA for five of FtsZ. This means that there is sufficient FtsA to form a complete circumferential ring at the division site. Therefore, FtsA may have a direct structural role in cell division. We have purified FtsA and shown that it behaves as a dimer and that it has both ATP-binding and ATP-hydrolysis activities. This suggests that ATP hydrolysis by FtsA is required, together with GTP hydrolysis by FtsZ, for cell division in B. subtilis (and possibly in most eubacteria).     

FtsZ polymer-bundling by the Escherichia coli ZapA orthologue, YgfE, involves a conformational change in bound GTP

Cell division is a fundamental process for both eukaryotic and prokaryotic cells. In bacteria, cell division is driven by a dynamic, ring-shaped, cytoskeletal element (the Z-ring) made up of polymers of the tubulin-like protein FtsZ. It is thought that lateral associations between FtsZ polymers are important for function of the Z-ring in vivo, and that these interactions are regulated by accessory cell division proteins such as ZipA, EzrA and ZapA. We demonstrate that the putative Escherichia coli ZapA orthologue, YgfE, exists in a dimer/tetramer equilibrium in solution, binds to FtsZ polymers, strongly promotes FtsZ polymer bundling and is a potent inhibitor of the FtsZ GTPase activity. We use linear dichroism, a technique that allows structure analysis of molecules within linear polymers, to reveal a specific conformational change in GTP bound to FtsZ polymers, upon bundling by YgfE. We show that the consequences of FtsZ polymer bundling by YgfE and divalent cations are very similar in terms of GTPase activity, bundle morphology and GTP orientation and therefore propose that this conformational change in bound GTP reveals a general mechanism of FtsZ bundling.     

A new FtsZ-interacting protein, YlmF, complements the activity of FtsA during progression of cell division in Bacillus subtilis

The assembly of ring-like structures, composed of FtsZ proteins (i.e. the Z ring), is the earliest and most essential process in bacterial cytokinesis. It has been shown that this process is directly regulated by the FtsZ-binding proteins, FtsA, ZapA, and EzrA, in Bacillus subtilis. In this study, protein complexes that are involved in Z-ring formation were chemically cross-linked in vivo, purified by affinity chromatography, and analysed by mass spectrometry. Analysis of the results identified YlmF as a new component of the FtsZ complex. Yeast two-hybrid analysis and fluorescence microscopy of YFP-YlmF in B. subtilis cells indicated YlmF localizes to the division site in an FtsZ-dependent manner. A single disruption of YlmF resulted in a slight elongation of cells; however, simultaneous inactivation of both YlmF and FtsA showed synthetic lethality caused by complete blockage of cell division due to the defect in Z-ring formation. In contrast, the ftsA-null mutant phenotype, caused by inefficient Z-ring formation, could be complemented by overexpression of YlmF. These results suggest that YlmF has an overlapping function with FtsA in stimulating the formation of Z rings in B. subtilis.     

Control of the cell elongation-division cycle by shuttling of PBP1 protein in Bacillus subtilis

The characteristic shape of bacterial cells is mainly determined by the cell wall, the synthesis of which is orchestrated by penicillin-binding proteins (PBPs). Rod-shaped bacteria have two distinct modes of cell wall synthesis, involved in cell elongation and cell division, which are believed to employ different sets of PBPs. A long-held question has been how these different modes of growth are co-ordinated in space and time. We have now identified the cell division protein, EzrA, and a newly discovered protein, GpsB, as key players in the elongation-division cycle of Bacillus subtilis. Mutations in these genes have a synthetic phenotype with defects in both cell division and cell elongation. They also have an unusual bulging phenotype apparently due to a failure in properly completing cell pole maturation. We show that these phenotypes are tightly associated with disturbed localization of the major transglycosylase/transpeptidase of the cell, PBP1. EzrA and GpsB have partially differentiated roles in the localization cycle of PBP1, with EzrA mainly promoting the recruitment of PBP1 to division sites, and GpsB facilitating its removal from the cell pole, after the completion of pole maturation.     

Cytokinesis in bacteria.

Work on two diverse rod-shaped bacteria, Escherichia coli and Bacillus subtilis, has defined a set of about 10 conserved proteins that are important for cell division in a wide range of eubacteria. These proteins are directed to the division site by the combination of two negative regulatory systems. Nucleoid occlusion is a poorly understood mechanism whereby the nucleoid prevents division in the cylindrical part of the cell, until chromosome segregation has occurred near midcell. The Min proteins prevent division in the nucleoid-free spaces near the cell poles in a manner that is beginning to be understood in cytological and biochemical terms. The hierarchy whereby the essential division proteins assemble at the midcell division site has been worked out for both E. coli and B. subtilis. They can be divided into essentially three classes depending on their position in the hierarchy and, to a certain extent, their subcellular localization. FtsZ is a cytosolic tubulin-like protein that polymerizes into an oligomeric structure that forms the initial ring at midcell. FtsA is another cytosolic protein that is related to actin, but its precise function is unclear. The cytoplasmic proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E. coli and possibly EzrA of B. subtilis, which have a single membrane span but a cytoplasmic C-terminal domain. The remaining proteins are either integral membrane proteins or transmembrane proteins with their major domains outside the cell. The functions of most of these proteins are unclear with the exception of at least one penicillin-binding protein, which catalyzes a key step in cell wall synthesis in the division septum.     

Early targeting of Min proteins to the cell poles in germinated spores of Bacillus subtilis: evidence for division apparatus-independent recruitment of Min proteins to the division site

The earliest event in bacterial cell division is the assembly of a tubulin-like protein, FtsZ, at mid-cell to form a ring. In rod-shaped bacteria, the Min system plays an important role in division site placement by inhibiting FtsZ ring formation specifically at the polar regions of the cell. The Min system comprises MinD and MinC, which form an inhibitor complex and, in Bacillus subtilis, DivIVA, which ensures that division is inhibited only in the polar regions. All three proteins localize to the division site at mid-cell and to cell poles. Their recruitment to the division site is dependent on localization of both 'early' and 'late' division proteins. We have examined the temporal and spatial localization of DivIVA relative to that of FtsZ during the first and second cell division after germination and outgrowth of B. subtilis spores. We show that, although the FtsZ ring assembles at mid-cell about halfway through the cell cycle, DivIVA assembles at this site immediately before cell division and persists there during Z-ring constriction and completion of division. We also show that both DivIVA and MinD localize to the cell poles immediately upon spore germination, well before a Z ring forms at mid-cell. Furthermore, these proteins were found to be present in mature, dormant spores. These results suggest that targeting of Min proteins to division sites does not depend directly on the assembly of the division apparatus, as suggested previously, and that potential polar division sites are blocked at the earliest possible stage in the cell cycle in germinated spores as a mechanism to ensure that equal-sized daughter cells are produced upon cell division.     

A new assembly pathway for the cytokinetic Z ring from a dynamic helical structure in vegetatively growing cells of Bacillus subtilis

The earliest event in bacterial cell division is the formation of a Z ring, composed of the tubulin-like FtsZ protein, at the division site at midcell. This ring then recruits several other division proteins and together they drive the formation of a division septum between two replicated chromosomes. Here we show that, in addition to forming a cytokinetic ring, FtsZ localizes in a helical-like pattern in vegetatively growing cells of Bacillus subtilis. FtsZ moves rapidly within this helix-like structure. Examination of FtsZ localization in individual live cells undergoing a single cell cycle suggests a new assembly mechanism for Z ring formation that involves a cell cycle-mediated multistep remodelling of FtsZ polymers. Our observations suggest that initially FtsZ localizes in a helical pattern, with movement of FtsZ within this structure occurring along the entire length of the cell. Next, movement of FtsZ in a helical-like pattern is restricted to a central region of the cell. Finally the FtsZ ring forms precisely at midcell. We further show that another division protein, FtsA, shown to interact with FtsZ prior to Z ring formation in B. subtilis, also localizes to similar helical patterns in vegetatively growing cells.     

SepF, a novel FtsZ-interacting protein required for a late step in cell division

Cell division in nearly all bacteria is initiated by polymerization of the conserved tubulin-like protein FtsZ into a ring-like structure at midcell. This Z-ring functions as a scaffold for a group of conserved proteins that execute the synthesis of the division septum (the divisome). Here we describe the identification of a new cell division protein in Bacillus subtilis. This protein is conserved in Gram positive bacteria, and because it has a role in septum development, we termed it SepF. sepF mutants are viable but have a cell division defect, in which septa are formed slowly and with a severely abnormal morphology. Yeast two-hybrid analysis showed that SepF can interact with itself and with FtsZ. Accordingly, fluorescence microscopy showed that SepF accumulates at the site of cell division, and this localization depends on the presence of FtsZ. Combination of mutations in sepF and ezrA, encoding another Z-ring interacting protein, had a synthetic lethal division effect. We conclude that SepF is a new member of the Gram positive divisome, required for proper execution of septum synthesis.     

A metabolic sensor governing cell size in bacteria

Nutrient availability is one of the strongest determinants of cell size. When grown in rich media, single-celled organisms such as yeast and bacteria can be up to twice the size of their slow-growing counterparts. The ability to modulate size in a nutrient-dependent manner requires cells to: (1) detect when they have reached the appropriate mass for a given growth rate and (2) transmit this information to the division apparatus. We report the identification of a metabolic sensor that couples nutritional availability to division in Bacillus subtilis. A key component of this sensor is an effector, UgtP, which localizes to the division site in a nutrient-dependent manner and inhibits assembly of the tubulin-like cell division protein FtsZ. This sensor serves to maintain a constant ratio of FtsZ rings to cell length regardless of growth rate and ensures that cells reach the appropriate mass and complete chromosome segregation prior to cytokinesis.