Condensation of FtsZ filaments can drive bacterial cell division

Forces are important in biological systems for accomplishing key cell functions, such as motility, organelle transport, and cell division. Currently, known force generation mechanisms typically involve motor proteins. In bacterial cells, no known motor proteins are involved in cell division. Instead, a division ring (Z-ring) consists of mostly FtsZ, FtsA, and ZipA is used to exerting a contractile force. The mechanism of force generation in bacterial cell division is unknown. Using computational modeling, we show that Z-ring formation results from the colocalization of FtsZ and FtsA mediated by the favorable alignment of FtsZ polymers. The model predicts that the Z-ring undergoes a condensation transition from a low-density state to a high-density state and generates a sufficient contractile force to achieve division. FtsZ GTP hydrolysis facilitates monomer turnover during the condensation transition, but does not directly generate forces. In vivo fluorescence measurements show that FtsZ density increases during division, in accord with model results. The mechanism is akin to van der Waals picture of gas-liquid condensation, and shows that organisms can exploit microphase transitions to generate mechanical forces.     

Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ, an ancestral tubulin

Little is known about the division of eukaryotic cell organelles and up to now neither in animals nor in plants has a gene product been shown to mediate this process. A cDNA encoding a homolog of the bacterial cell division protein FtsZ, an ancestral tubulin, was isolated from the eukaryote Physcomitrella patens and used to disrupt efficiently the genomic locus in this terrestrial seedless plant. Seven out of 51 transgenics obtained were knockout plants generated by homologous recombination; they were specifically impeded in plastid division with no detectable effect on mitochondrial division or plant morphology. Implications on the theory of endosymbiosis and on the use of reverse genetics in plants are discussed.     

Evolutionary linkage between eukaryotic cytokinesis and chloroplast division by dynamin proteins

Chloroplasts have evolved from a cyanobacterial endosymbiont and been retained for more than 1 billion years by coordinated chloroplast division in multiplying eukaryotic cells. Chloroplast division is performed by ring structures at the division site, encompassing both the inside and the outside of the two envelopes. A part of the division machinery is derived from the cyanobacterial cytokinetic activity based on the FtsZ protein. In contrast, other parts of the division machinery involve proteins specific to eukaryotes, including a member of the dynamin family. Each member of the dynamin family is involved in the division or fusion of a distinct eukaryotic membrane system. To gain insight into the kind of ancestral dynamin protein and eukaryotic membrane activity that evolved to regulate chloroplast division, we investigated the functions of the dynamin proteins that are most closely related to chloroplast division proteins. These proteins in the amoeba Dictyostelium discoideum and Arabidopsis thaliana localize at the sites of cell division, where they are involved in cytokinesis. Our results suggest that the dynamin for chloroplast division is derived from that involved in eukaryotic cytokinesis. Therefore, the chloroplast division machinery is a mixture of bacterial and eukaryotic cytokinesis components, with the latter a key factor in the synchronization of endosymbiotic cell division with host cell division, thus helping to establish the permanent endosymbiotic relationship.     

Z-ring force and cell shape during division in rod-like bacteria

The life cycle of bacterial cells consists of repeated elongation, septum formation, and division. Before septum formation, a division ring called the Z-ring, which is made of a filamentous tubulin analog, FtsZ, is seen at the mid cell. Together with several other proteins, FtsZ is essential for cell division. Visualization of strains with GFP-labeled FtsZ shows that the Z-ring contracts before septum formation and pinches the cell into two equal halves. Thus, the Z-ring has been postulated to act as a force generator, although the magnitude of the contraction force is unknown. In this article, we develop a mathematical model to describe the process of growth and Z-ring contraction in rod-like bacteria. The elasticity and growth of the cell wall is incorporated in the model to predict the contraction speed, the cell shape, and the contraction force. With reasonable parameters, the model shows that a small force from the Z-ring (8 pN in Escherichia coli) is sufficient to accomplish division.     

Force generation by a dynamic Z-ring in Escherichia coli cell division

FtsZ, a bacterial homologue of tubulin, plays a central role in bacterial cell division. It is the first of many proteins recruited to the division site to form the Z-ring, a dynamic structure that recycles on the time scale of seconds and is required for division to proceed. FtsZ has been recently shown to form rings inside tubular liposomes and to constrict the liposome membrane without the presence of other proteins, particularly molecular motors that appear to be absent from the bacterial proteome. Here, we propose a mathematical model for the dynamic turnover of the Z-ring and for its ability to generate a constriction force. Force generation is assumed to derive from GTP hydrolysis, which is known to induce curvature in FtsZ filaments. We find that this transition to a curved state is capable of generating a sufficient force to drive cell-wall invagination in vivo and can also explain the constriction seen in the in vitro liposome experiments. Our observations resolve the question of how FtsZ might accomplish cell division despite the highly dynamic nature of the Z-ring and the lack of molecular motors.     

Presence of a mitochondrial-type 70-kDa heat shock protein in Trichomonas vaginalis suggests a very early mitochondrial endosymbiosis in eukaryotes

Molecular phylogenetic analyses, based mainly on ribosomal RNA, show that three amitochondriate protist lineages, diplomonads, microsporidia, and trichomonads, emerge consistently at the base of the eukaryotic tree before groups having mitochondria. This suggests that these groups could have diverged before the mitochondrial endosymbiosis. Nevertheless, since all these organisms live in anaerobic environments, the absence of mitochondria might be due to secondary loss, as demonstrated for the later emerging eukaryote Entamoeba histolytica. We have now isolated from Trichomonas vaginalis a gene encoding a chaperone protein (HSP70) that in other lineages is addressed to the mitochondrial compartment. The phylogenetic reconstruction unambiguously located this HSP70 within a large set of mitochondrial sequences, itself a sister-group of α-purple bacteria. In addition, the T. vaginalis protein exhibits the GDAWV sequence signature, so far exclusively found in mitochondrial HSP70 and in proteobacterial dnaK. Thus mitochondrial endosymbiosis could have occurred earlier than previously assumed. The trichomonad double membrane-bounded organelles, the hydrogenosomes, could have evolved from mitochondria.     

SlmA forms a higher-order structure on DNA that inhibits cytokinetic Z-ring formation over the nucleoid

The spatial and temporal control of Filamenting temperature sensitive mutant Z (FtsZ) Z-ring formation is crucial for proper cell division in bacteria. In Escherichia coli, the synthetic lethal with a defective Min system (SlmA) protein helps mediate nucleoid occlusion, which prevents chromosome fragmentation by binding FtsZ and inhibiting Z-ring formation over the nucleoid. However, to perform its function, SlmA must be bound to the nucleoid. To deduce the basis for this chromosomal requirement, we performed biochemical, cellular, and structural studies. Strikingly, structures show that SlmA dramatically distorts DNA, allowing it to bind as an orientated dimer-of-dimers. Biochemical data indicate that SlmA dimer-of-dimers can spread along the DNA. Combined structural and biochemical data suggest that this DNA-activated SlmA oligomerization would prevent FtsZ protofilament propagation and bundling. Bioinformatic analyses localize SlmA DNA sites near membrane-tethered chromosomal regions, and cellular studies show that SlmA inhibits FtsZ reservoirs from forming membrane-tethered Z rings. Thus, our combined data indicate that SlmA DNA helps block Z-ring formation over chromosomal DNA by forming higher-order protein-nucleic acid complexes that disable FtsZ filaments from coalescing into proper structures needed for Z-ring creation.Accurate cell division demands a tight synchronization of chromosome replication, segregation, and septum formation. In bacteria, cell division is mediated by the tubulin-like protein, FtsZ (1–5). FtsZ self-assembles into linear protofilaments (pfs) in a GTP-dependent manner by the interaction of the plus end of one subunit with the minus end of another subunit. These pfs combine to form a septal ring-like structure called the Z ring, which drives cell division (1). The precise organization of FtsZ filaments in the Z ring has not been resolved. However, recent cryo-electron microscopy (EM) tomography and superresolution imaging data suggest that lateral contacts between FtsZ molecules in the Z ring are loosely arranged (6–9). Notably, the intracellular levels of FtsZ remain unchanged during the cell cycle and exceed the critical concentration required for Z-ring formation (3). Hence, Z-ring assembly is affected by a diverse repertoire of FtsZ binding proteins that ensure that the ring forms at the correct time and place during cell division (2, 4, 10). Two key regulatory systems, the Min and nucleoid occlusion (NO) systems, ensure that Z rings do not form at cell poles and over chromosomal DNA (11–13). In Escherichia coli, the Min system creates a gradient of FtsZ polymer inhibition at the cell poles (14). In contrast to the pole proximal effect of the Min system, NO prevents Z rings from forming over chromosomal DNA (15).Although the effects of NO are well known, only in recent years have effectors of this process been identified in both gram-negative and gram-positive model organisms. Surprisingly, the gram-positive model organism, Bacillus subtilis, uses a ParB-like protein called Noc, whereas in E. coli the TetR-family member, SlmA, has been shown to be involved in NO (13, 16–18). Recent studies showed that SlmA binds directly to FtsZ and that this interaction antagonizes Z-ring formation when SlmA is bound to specific DNA sites (16, 17). These DNA sequences, called SlmA binding sites (SBSs), are evenly distributed on the chromosome, with the notable exception of the Ter chromosomal region, which is the last to partition. Similar to SlmA, the unrelated Noc protein also binds specific DNA sites that are distributed in all chromosomal regions but the Ter containing domain (18, 19). Thus, NO factors coordinate DNA segregation and cell division.How SlmA prevents Z-ring formation by FtsZ is unclear. However, a key finding was that this inhibition necessitates that SlmA be bound to its specific DNA sites (16, 17). In fact, SlmA alone does not affect FtsZ filament formation, making SlmA the only known FtsZ assembly/disassembly factor that requires DNA for its function. To determine the molecular basis for the specific chromosomal DNA requirement for SlmA’s NO function, we performed structural, bioinformatic, cellular, and biochemical studies. Strikingly, these data show that SlmA interacts with specific SBS sites to form a higher-order structure that can spread on the DNA. Combined with biochemical and bioinformatics analyses, these findings suggest a molecular model for how SlmA participates in NO in gram-negative bacteria.     

FtsZ filament capping by MciZ,a developmental regulator of bacterial division

Cytoskeletal structures are dynamically remodeled with the aid of regulatory proteins. FtsZ (filamentation temperature-sensitive Z) is the bacterial homolog of tubulin that polymerizes into rings localized to cell-division sites, and the constriction of these rings drives cytokinesis. Here we investigate the mechanism by which the Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitor of FtsZ), blocks assembly of FtsZ. The X-ray crystal structure reveals that MciZ binds to the C-terminal polymerization interface of FtsZ, the equivalent of the minus end of tubulin. Using in vivo and in vitro assays and microscopy, we show that MciZ, at substoichiometric levels to FtsZ, causes shortening of protofilaments and blocks the assembly of higher-order FtsZ structures. The findings demonstrate an unanticipated capping-based regulatory mechanism for FtsZ.The discovery that bacteria have actin-, tubulin-, and intermediate filament-like proteins demonstrated that the cytoskeleton is an ancient invention, predating the divergence between prokaryotes and eukaryotes (1). The GTPase FtsZ (filamentation temperature-sensitive Z) was the first prokaryotic protein to be recognized as a cytoskeletal element (2, 3). FtsZ is a tubulin-like protein, which is widely conserved in bacteria and the main component of the bacterial cytokinesis machine, or “divisome.” FtsZ self-assembles into single-stranded protofilaments and these associate further inside cells to form a superstructure known as the Z ring (4, 5). FtsZ alone can generate a constriction force to initiate division (6). The Z ring also provides a scaffold onto which several other components of the divisome—mostly cell wall synthesizing enzymes—are recruited and oriented so as to build the division septum, a cross-wall separating a progenitor cell into two isogenic daughter cells (7).FtsZ and tubulin share several essential properties: their assembly is cooperative, stimulated by GTP, and leads to GTP hydrolysis; they form dynamic polymers whose turnover is dependent on nucleotide hydrolysis (8); they use essentially the same bond for polymer formation (9); and recent evidence indicates that they undergo similar allosteric transitions upon polymerization (10, 11). Not surprisingly, however, the functional specialization of these proteins led to some significant differences between them, the most prominent being that FtsZ exists as single protofilaments, whereas tubulin always adopts a multifilament tubular structure. This difference in their higher-order structure implies that the reactions that lead to cooperativity and subunit turnover are likely different. It has also represented a significant technical challenge for the study of FtsZ. Because FtsZ filaments are smaller than the resolution of optical microscopy, so far it has been impossible to determine essential properties associated with its dynamic behavior.Similarly to actin filaments and microtubules, the assembly of FtsZ protofilaments into a Z ring is regulated by a number of proteins that bind directly to FtsZ and modulate its polymerization (4). Among these proteins, negative modulators have attracted the most attention because of their crucial role in determining when and where a Z ring should form. The strikingly precise positioning of the division site in rod-shaped bacteria, such as Escherichia coli and Bacillus subtilis, is because of the combined action of Min and nucleoid occlusion systems, two negative modulators that work together to ensure that the Z ring will be formed only at midcell. The Min system, whose core component is the polarly localized MinCD complex, prevents the Z ring from forming at the cell poles, reducing the chances of minicell formation (12, 13). The nucleoid occlusion system, in turn, is based on DNA binding proteins (Noc in B. subtilis and SlmA in E. coli) that recognize specific sequence signatures and inhibit Z ring formation around the bacterial chromosomes (14–16). The combination of Min and nucleoid occlusion inhibition prevent division from happening at the cell poles and over the chromosomes, leaving only the central region of the cell free for Z-ring formation. Min/nucleoid occlusion also provide a means to regulate the cell-cycle timing of Z-ring formation because the creation of an inhibitor-free zone at midcell depends on proper DNA replication and segregation. Another well-studied FtsZ modulator is the checkpoint protein SulA (suppressor of Lon A), which makes bacterial cytokinesis responsive to environmental stresses. SulA is expressed in response to DNA damage as part of the SOS system in E. coli and halts Z-ring formation and cell division until DNA damage is repaired by the cell (17, 18). More recently, other negative modulators of FtsZ have been reported whose function is to control cytokinesis in response to the nutritional [UgtP (19), KidO (20), and OpgH (21)] and developmental [MciZ (mother cell inhibitor of FtsZ) (22) and Maf (23)] state of the cell.Eukaryotic cytoskeletal modulators use a variety of strategies to achieve their effect, including nucleation, monomer sequestration, filament capping, and severing (24, 25). The conservation of the structure and principles that govern cytoskeletal filament formation suggest that the general mechanisms operating in eukaryotes should also be present in prokaryotes. However, little is known about the molecular details of how modulators affect FtsZ assembly. SulA is one of the few inhibitors whose mechanism has been studied in detail. The crystal structure of SulA in complex with FtsZ showed that it binds to the C-terminal polymerization interface of FtsZ (26). In addition, in vitro experiments demonstrated that SulA inhibits FtsZ polymerization by a simple sequestration mechanism (27, 28).Here we focused on MciZ (mother cell inhibitor of FtsZ), a poorly understood 40-aa peptide discovered in a yeast two-hybrid screen for FtsZ binding partners (22). MciZ is an intriguing example of a developmentally regulated division inhibitor. It is expressed during sporulation in B. subtilis and blocks Z-ring formation after cells commit to the terminally differentiated spore fate. Although it has been shown that MciZ directly inhibits FtsZ polymerization in vitro (22, 29), the nature of its interaction with FtsZ and its inhibition mechanism are still unknown. We determined the crystal structure of MciZ in complex with FtsZ from B. subtilis and, by using fluorescence and electron microscopy as well as biochemical experiments, showed that MciZ binds to the C-terminal subdomain of FtsZ and acts as a capper of the minus end of FtsZ filaments. Minus-end capping is an unusual way to inhibit filament formation and indicates that annealing plays an important role in FtsZ filament dynamics.     

Posttranslational regulation impacts the fate of duplicated genes

Gene and genome duplications create novel genetic material on which evolution can work and have therefore been recognized as a major source of innovation for many eukaryotic lineages. Following duplication, the most likely fate is gene loss; however, a considerable fraction of duplicated genes survive. Not all genes have the same probability of survival, but it is not fully understood what evolutionary forces determine the pattern of gene retention. Here, we use genome sequence data as well as large-scale phosphoproteomics data from the baker’s yeast Saccharomyces cerevisiae, which underwent a whole-genome duplication ∼100 mya, and show that the number of phosphorylation sites on the proteins they encode is a major determinant of gene retention. Protein phosphorylation motifs are short amino acid sequences that are usually embedded within unstructured and rapidly evolving protein regions. Reciprocal loss of those ancestral sites and the gain of new ones are major drivers in the retention of the two surviving duplicates and in their acquisition of distinct functions. This way, small changes in the sequences of unstructured regions in proteins can contribute to the rapid rewiring and adaptation of regulatory networks.     

A mitochondrial-like chaperonin 60 gene in Giardia lamblia: Evidence that diplomonads once harbored an endosymbiont related to the progenitor of mitochondria

Diplomonads, parabasalids, as represented by trichomonads, and microsporidia are three protist lineages lacking mitochondria that branch earlier than all other eukaryotes in small subunit rRNA and elongation factor phylogenies. The absence of mitochondria and plastids in these organisms suggested that they diverged before the origin of these organelles. However, recent discoveries of mitochondrial-like heat shock protein 70 and/or chaperonin 60 (cpn60) genes in trichomonads and microsporidia imply that the ancestors of these two groups once harbored mitochondria or their endosymbiotic progenitors. In this report, we describe a mitochondrial-like cpn60 homolog from the diplomonad parasite Giardia lamblia. Northern and Western blots reveal that the expression of cpn60 is independent of cellular stress and, except during excystation, occurs throughout the G. lamblia life cycle. Phylogenetic analyses position the G. lamblia cpn60 in a clade that includes mitochondrial and hydrogenosomal cpn60 proteins. The most parsimonious interpretation of these data is that the cpn60 gene was transferred from the endosymbiotic ancestors of mitochondria to the nucleus early in eukaryotic evolution, before the divergence of the diplomonads and trichomonads from other extant eukaryotic lineages. A more complicated explanation requires that these genes originated from distinct α-proteobacterial endosymbioses that formed transiently within these protist lineages.     

Ancient dynamin segments capture early stages of host–mitochondrial integration

Eukaryotic cells use dynamins—mechano-chemical GTPases—to drive the division of endosymbiotic organelles. Here we probe early steps of mitochondrial and chloroplast endosymbiosis by tracing the evolution of dynamins. We develop a parsimony-based phylogenetic method for protein sequence reconstruction, with deep time resolution. Using this, we demonstrate that dynamins diversify through the punctuated transformation of sequence segments on the scale of secondary-structural elements. We find examples of segments that have remained essentially unchanged from the 1.8-billion-y-old last eukaryotic common ancestor to the present day. Stitching these together, we reconstruct three ancestral dynamins: The first is nearly identical to the ubiquitous mitochondrial division dynamins of extant eukaryotes, the second is partially preserved in the myxovirus-resistance-like dynamins of metazoans, and the third gives rise to the cytokinetic dynamins of amoebozoans and plants and to chloroplast division dynamins. The reconstructed sequences, combined with evolutionary models and published functional data, suggest that the ancestral mitochondrial division dynamin also mediated vesicle scission. This bifunctional protein duplicated into specialized mitochondrial and vesicle variants at least three independent times—in alveolates, green algae, and the ancestor of fungi and metazoans—accompanied by the loss of the ancient prokaryotic mitochondrial division protein FtsZ. Remarkably, many extant species that retain FtsZ also retain the predicted ancestral bifunctional dynamin. The mitochondrial division apparatus of such organisms, including amoebozoans, red algae, and stramenopiles, seems preserved in a near-primordial form.Eukaryotes arose through the acquisition of mitochondria by an archaeal host cell about 2 billion y ago (1, 2), a watershed moment in the evolution of the modern compartmentalized cell plan (3). A second transformative endosymbiotic event, the acquisition of a cyanobacterium by a eukaryotic host to form chloroplasts, gave rise to the photosynthetic eukaryotic lineages (4). As the endosymbionts became integrated with their hosts, their growth and division became regulated by host–cellular machinery (5). Proteins of the dynamin superfamily were central to this process: Mitochondria and chloroplasts originally divided using a constricting ring of the prokaryotic cytoskeletal protein FtsZ, but dynamins have been recruited to these roles in all extant eukaryotes (6, 7). By reconstructing the evolutionary history of dynamins, we can probe the process of endosymbiont integration.The dynamin superfamily is diverse (8, 9), and different dynamin variants remodel membranes at different cellular locations (Table S1 and primary references therein). A major class of dynamins is essential for mitochondrial and peroxisomal division. Another large group drives the scission of clathrin-coated vesicles in organisms such as fungi and alveolates. A related group, the so-called “classical” dynamins that drive clathrin-coated vesicle scission in metazoans and land plants, contains a membrane-targeted pleckstrin homology (PH) domain. Members of the phragmoplastin class of dynamins participate in cell plate formation in land plants. The myxovirus-resistance-like dynamins are implicated in antiviral activity in vertebrates. A truncated dynamin variant is involved in cytokinesis in amoebozoans and plants, as well as in chloroplast fission in photosynthetic lineages; another truncated variant drives mitochondrial inner membrane fusion in fungi and metazoans. Finally, mitofusins and the related bacterial dynamin-like proteins (BDLPs) are potentially ancient members of the dynamin superfamily (10); these are excluded from our study because they are highly diverged at the sequence level.Here we present the most comprehensive analysis of dynamin evolution yet reported, including thousands of functionally diverse dynamins from hundreds of broadly sampled eukaryotic species. We reconstruct the series of events that led from the primordial dynamins of the 1.8-billion-y-old last eukaryotic common ancestor (LECA) (11) to the great variety of present-day dynamins. The outcome is a nuanced picture of protein diversification, mirroring key events in the evolution of eukaryotes themselves and shedding light on the earliest stages of endosymbiont integration.     

A unique cell division machinery in the Archaea

In contrast to the cell division machineries of bacteria, euryarchaea, and eukaryotes, no division components have been identified in the second main archaeal phylum, Crenarchaeota. Here, we demonstrate that a three-gene operon, cdv, in the crenarchaeon Sulfolobus acidocaldarius, forms part of a unique cell division machinery. The operon is induced at the onset of genome segregation and division, and the Cdv proteins then polymerize between segregating nucleoids and persist throughout cell division, forming a successively smaller structure during constriction. The cdv operon is dramatically down-regulated after UV irradiation, indicating division inhibition in response to DNA damage, reminiscent of eukaryotic checkpoint systems. The cdv genes exhibit a complementary phylogenetic range relative to FtsZ-based archaeal division systems such that, in most archaeal lineages, either one or the other system is present. Two of the Cdv proteins, CdvB and CdvC, display homology to components of the eukaryotic ESCRT-III sorting complex involved in budding of luminal vesicles and HIV-1 virion release, suggesting mechanistic similarities and a common evolutionary origin.     

Kinetochore asymmetry defines a single yeast lineage

Asymmetric cell division is of fundamental importance in biology as it allows for the establishment of separate cell lineages during the development of multicellular organisms. Although microbial systems, including the yeast Saccharomyces cerevisiae, are excellent models of asymmetric cell division, this phenotype occurs in all cell divisions; consequently, models of lineage-specific segregation patterns in these systems do not exist. Here, we report the first example of lineage-specific asymmetric division in yeast. We used fluorescent tags to show that components of the yeast kinetochore, the protein complex that anchors chromosomes to the mitotic spindle, divide asymmetrically in a single postmeiotic lineage. This phenotype is not seen in vegetatively dividing haploid or diploid cells. This kinetochore asymmetry suggests a mechanism for the selective segregation of sister centromeres to daughter cells to establish different cell lineages or fates. These results provide a mechanistic link between lineage-defining asymmetry of metazoa with unicellular eukaryotes.     

Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein

In the current model for bacterial cell division, FtsZ protein forms a ring that marks the division plane, creating a cytoskeletal framework for the subsequent action of other proteins such as FtsA. This putative protein complex ultimately generates the division septum. Herein we report that FtsZ and FtsA proteins tagged with green fluorescent protein (GFP) colocalize to division-site ring-like structures in living bacterial cells in a visible space between the segregated nucleoids. Cells with higher levels of FtsZ–GFP or with FtsA–GFP plus excess wild-type FtsZ were inhibited for cell division and often exhibited bright fluorescent spiral tubules that spanned the length of the filamentous cells. This suggests that FtsZ may switch from a septation-competent localized ring to an unlocalized spiral under some conditions and that FtsA can bind to FtsZ in both conformations. FtsZ–GFP also formed nonproductive but localized aggregates at a higher concentration that could represent FtsZ nucleation sites. The general domain structure of FtsZ–GFP resembles that of tubulin, since the C terminus of FtsZ is not required for polymerization but may regulate polymerization state. The N-terminal portion of Rhizobium FtsZ polymerized in Escherichia coli and appeared to copolymerize with E. coli FtsZ, suggesting a degree of interspecies functional conservation. Analysis of several deletions of FtsA–GFP suggests that multiple segments of FtsA are important for its localization to the FtsZ ring.     

Rapid assembly dynamics of the Escherichia coli FtsZ-ring demonstrated by fluorescence recovery after photobleaching

FtsZ, the major cytoskeletal component of the bacterial cell-division machine, assembles into a ring (the Z-ring) that contracts at septation. FtsZ is a bacterial homolog of tubulin, with similar tertiary structure, GTP hydrolysis, and in vitro assembly. We used green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching to show that the E. coli Z-ring is extremely dynamic, continually remodeling itself with a half-time of 30 s. ZipA, a membrane protein involved in cell division that colocalizes with FtsZ, was equally dynamic. The Z-ring of the mutant ftsZ84, which has 1/10 the guanosine triphosphatase activity of wild-type FtsZ in vitro, showed a 9-fold slower turnover in vivo. This finding implies that assembly dynamics are determined primarily by GTP hydrolysis. Despite the greatly reduced assembly dynamics, the ftsZ84 cells divide with a normal cell-cycle time.     

Structural and genetic analyses reveal the protein SepF as a new membrane anchor for the Z ring

A key step in bacterial cell division is the polymerization of the tubulin homolog FtsZ at midcell. FtsZ polymers are anchored to the cell membrane by FtsA and are required for the assembly of all other cell division proteins. In Gram-positive and cyanobacteria, FtsZ filaments are aligned by the protein SepF, which in vitro polymerizes into large rings that bundle FtsZ filaments. Here we describe the crystal structure of the only globular domain of SepF, located within the C-terminal region. Two-hybrid data revealed that this domain comprises the FtsZ binding site, and EM analyses showed that it is sufficient for ring formation, which is explained by the filaments in the crystals of SepF. Site-directed mutagenesis, gel filtration, and analytical ultracentrifugation indicated that dimers form the basic units of SepF filaments. High-resolution structured illumination microscopy suggested that SepF is membrane associated, and it turned out that purified SepF not only binds to lipid membranes, but also recruits FtsZ. Further genetic and biochemical analyses showed that an amphipathic helix at the N terminus functions as the membrane-binding domain, making SepF a unique membrane anchor for the FtsZ ring. This clarifies why Bacillus subtilis grows without FtsA or the putative membrane anchor EzrA and why bacteria lacking FtsA contain SepF homologs. Both FtsA and SepF use an amphipathic helix for membrane binding. These helices prefer positively curved membranes due to relaxed lipid density; therefore this type of membrane anchor may assist in keeping the Z ring positioned at the strongly curved leading edge of the developing septum.One of the first steps in bacterial cell division is the polymerization of the conserved protein FtsZ at midcell. FtsZ shares structural homology with eukaryotic tubulin and uses GTP to polymerize into filaments close to the cell membrane. These filaments then assemble into a ring-like structure, the Z ring, which recruits other proteins needed for the division septum (1). Several cell division proteins support the formation of a stable Z ring, such as ZapA that forms cross-links between FtsZ filaments (2, 3). In Gram-positive bacteria and cyanobacteria, the protein SepF also stimulates bundling of FtsZ polymers (4–6). Electron microscopic (EM) studies have shown that SepF assembles into large and regular protein rings with diameters of about 50 nm. In vitro, these rings are able to bundle FtsZ protofilaments into long tubular structures (4). Furthermore, SepF is essential for the synthesis of regular and smooth division septa (7–9).The Z ring is associated with the cell membrane and the best-characterized membrane anchor is the conserved protein FtsA. This protein binds to FtsZ directly and contains a C-terminal amphipathic helix that binds to lipid bilayers in a membrane-potential-dependent manner (10–12). In Escherichia coli, FtsA is essential, but in Bacillus subtilis, ftsA can be deleted, although this affects Z-ring formation and cells become elongated. Because B. subtilis can grow without FtsA, there must be another protein that links the Z ring to the cell membrane. The essential E. coli cell division protein ZipA binds to FtsZ and contains an N-terminal transmembrane domain (13). The necessity for ZipA can be bypassed by a gain-of-function mutation in FtsA (14). Gram-positive bacteria contain EzrA, which shows a similar topology to that of ZipA, with an N-terminal transmembrane helix and a large C-terminal domain that binds to the FtsZ C terminus (15). It therefore seemed likely that EzrA functions as an alternative membrane anchor for the Z ring in B. subtilis when FtsA is absent.Comparison of the SepF amino acid sequence against protein databases did not reveal conserved motifs that provide clues to potential molecular mechanisms. To gain insight into the residues that are important for FtsZ interaction, we used a yeast two-hybrid screen. This revealed that the conserved C-terminal part of SepF comprises the FtsZ binding site. We were able to obtain diffracting crystals of this domain that revealed a tight dimer structure. Site-directed mutagenesis indicated that the protein polymerizes as units of dimers. Although the FtsZ interaction domain constitutes only 60% of the full-length protein, it appeared to be also sufficient for the formation of the large protein rings. This raised the question of what the function is of the N-terminal domain of SepF. Interestingly, high-resolution fluorescent-light microscopy suggested that SepF associates with the cell membrane. Subsequent analyses indicated that the membrane-binding domain resides in the N-terminal domain of SepF. Biochemical experiments using purified SepF showed that the protein binds specifically to lipid membranes. These data suggested that SepF might function as a membrane anchor for the Z ring. Interestingly, we were able to delete both ftsA and the putative FtsZ-membrane anchor ezrA in B. subtilis and this did not affect viability. However, it turned SepF into an essential protein. Indeed, using purified SepF we were able to show that the protein recruits FtsZ to artificial lipid membranes. We conclude that SepF is a unique membrane anchor for FtsZ and the Z ring.     

Genome-wide screen of Saccharomyces cerevisiae null allele strains identifies genes involved in selenomethionine resistance

Selenomethionine (SeMet) is a potentially toxic amino acid, and yet it is a valuable tool in the preparation of labeled proteins for multiwavelength anomalous dispersion or single-wavelength anomalous dispersion phasing in X-ray crystallography. The mechanism by which high levels of SeMet exhibits its toxic effects in eukaryotic cells is not fully understood. Attempts to use Saccharomyces cerevisiae for the preparation of fully substituted SeMet proteins for X-ray crystallography have been limited. A screen of the viable S. cerevisiae haploid null allele strain collection for resistance to SeMet was performed. Deletion of the CYS3 gene encoding cystathionine gamma-lyase resulted in the highest resistance to SeMet. In addition, deletion of SSN2 resulted in both increased resistance to SeMet as well as reduced levels of Cys3p. A methionine auxotrophic strain lacking CYS3 was able to grow in media with SeMet as the only source of Met, achieving essentially 100% occupancy in total proteins. The CYS3 deletion strain provides advantages for an easy and cost-effective method to prepare SeMet-substituted protein in yeast and perhaps other eukaryotic systems.     

Nucleoid occlusion factor SlmA is a DNA-activated FtsZ polymerization antagonist

The tubulin-like FtsZ protein initiates assembly of the bacterial cytokinetic machinery by polymerizing into a ring structure, the Z ring, at the prospective site of division. To block Z-ring formation over the nucleoid and help coordinate cell division with chromosome segregation, Escherichia coli employs the nucleoid-associated division inhibitor, SlmA. Here, we investigate the mechanism by which SlmA regulates FtsZ assembly. We show that SlmA disassembles FtsZ polymers in vitro. In addition, using chromatin immunoprecipitation (ChIP), we identified 24 SlmA-binding sequences (SBSs) on the chromosome. Remarkably, SlmA binding to SBSs dramatically enhanced its ability to interfere with FtsZ polymerization, and ChIP studies indicate that SlmA regulates FtsZ assembly at these sites in vivo. Because of the dynamic and highly organized nature of the chromosome, coupling SlmA activation to specific DNA binding provides a mechanism for the precise spatiotemporal control of its anti-FtsZ activity within the cell.     

Localization of eukaryote-specific ribosomal proteins in a 5.5-Å cryo-EM map of the 80S eukaryotic ribosome

Protein synthesis in all living organisms occurs on ribonucleoprotein particles, called ribosomes. Despite the universality of this process, eukaryotic ribosomes are significantly larger in size than their bacterial counterparts due in part to the presence of 80 r proteins rather than 54 in bacteria. Using cryoelectron microscopy reconstructions of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome, we have localized and modeled 74/80 (92.5%) of the ribosomal proteins, encompassing 12 archaeal/eukaryote-specific small subunit proteins as well as the complete complement of the ribosomal proteins of the eukaryotic large subunit. Near-complete atomic models of the 80S ribosome provide insights into the structure, function, and evolution of the eukaryotic translational apparatus.