Overproduction of FtsZ induces minicell formation in E. coli

The ftsZ gene in E. coli K-12 is an essential cell division gene. We report that a two to sevenfold increase in the level of the FtsZ protein resulted in induction of the minicell phenotype. An increase in the level of FtsZ beyond this range resulted in an inhibition of all cell division. Unlike the classical minicell mutant, the formation of minicells induced by increased levels of FtsZ did not occur at the expense of normal divisions, indicating that increasing FtsZ resulted in additional division events per cell cycle. In addition, increased FtsZ caused cell division to be initiated earlier in the cell cycle. These results are consistent with the level or activity of FtsZ controlling the frequency of cell division in E. coli.     

FtsZ regulates frequency of cell division in Escherichia coli.

Cell division is regulated so that it occurs only once per cell cycle. In Escherichia coli, a rod-shaped bacterium, division normally takes place at the center of the long axis of the cell; however, in the minicell mutant, division can also take place at the cell pole. Such divisions take place at the expense of normal divisions, resulting in an overall increase in nucleated cell length. We report here that increasing the level of FtsZ can completely suppress the cell length of the minicell mutant by increasing the frequency at which cell division events take place. This result suggests that the level of FtsZ controls the frequency of cell division in E. coli.     

Deletion of the min operon results in increased thermosensitivity of an ftsZ84 mutant and abnormal FtsZ ring assembly, placement, and disassembly

To investigate the interaction between FtsZ and the Min system during cell division of Escherichia coli, we examined the effects of combining a well-known thermosensitive mutation of ftsZ, ftsZ84, with DeltaminCDE, a deletion of the entire min locus. Because the Min system is thought to down-regulate Z-ring assembly, the prediction was that removing minCDE might at least partially suppress the thermosensitivity of ftsZ84, which can form colonies below 42 degrees C but not at or above 42 degrees C. Contrary to expectations, the double mutant was significantly more thermosensitive than the ftsZ84 single mutant. When shifted to the new lower nonpermissive temperature, the double mutant formed long filaments mostly devoid of Z rings, suggesting a likely cause of the increased thermosensitivity. Interestingly, even at 22 degrees C, many Z rings were missing in the double mutant, and the rings that were present were predominantly at the cell poles. Of these, a large number were present only at one pole. These cells exhibited a higher than expected incidence of polar divisions, with a bias toward the newest pole. Moreover, some cells exhibited dramatically elongated septa that stained for FtsZ, suggesting that the double mutant is defective in Z-ring disassembly, and providing a possible mechanism for the polar bias. Thermoresistant suppressors of the double mutant arose that had modestly increased levels of FtsZ84. These cells also exhibited elongated septa and, in addition, produced a high frequency of branched cells. A thermoresistant suppressor of the ftsZ84 single mutant also synthesized more FtsZ84 and produced branched cells. The evidence from this study indicates that removing the Min system exposes and exacerbates the inherent defects of the FtsZ84 protein, resulting in clear septation phenotypes even at low growth temperatures. Increasing levels of FtsZ84 can suppress some, but not all, of these phenotypes.     

Extreme C terminus of bacterial cytoskeletal protein FtsZ plays fundamental role in assembly independent of modulatory proteins

Bacterial cell division typically requires assembly of the cytoskeletal protein FtsZ into a ring (Z-ring) at the nascent division site that serves as a foundation for assembly of the division apparatus. High resolution imaging suggests that the Z-ring consists of short, single-stranded polymers held together by lateral interactions. Several proteins implicated in stabilizing the Z-ring enhance lateral interactions between FtsZ polymers in vitro. Here we report that residues at the C terminus of Bacillus subtilis FtsZ (C-terminal variable region (CTV)) are both necessary and sufficient for stimulating lateral interactions in vitro in the absence of modulatory proteins. Swapping the 6-residue CTV from B. subtilis FtsZ with the 4-residue CTV from Escherichia coli FtsZ completely abolished lateral interactions between chimeric B. subtilis FtsZ polymers. The E. coli FtsZ chimera readily formed higher order structures normally seen only in the presence of molecular crowding agents. CTV-mediated lateral interactions are important for the integrity of the Z-ring because B. subtilis cells expressing the B. subtilis FtsZ chimera had a low frequency of FtsZ ring formation and a high degree of filamentation relative to wild-type cells. Site-directed mutagenesis of the B. subtilis CTV suggests that electrostatic forces are an important determinant of lateral interaction potential.     

Visualizing multiple constrictions in spheroidal Escherichia coli cells

An Escherichia coli cell grows by elongation and divides in a perpendicular plane. Alternating planes of successive divisions in three dimensions can only be ascertained when multiple constrictions exist simultaneously in large, spheroidal cells (with extended constriction process), if the division signals are enhanced. Large, spheroidal cells are obtained by a brief mecillinam treatment, and more frequent divisions are achieved by manipulating the rate of chromosome replication without affecting cell mass growth rate. Such a procedure has recently been performed by thymine-limitation of E. coli K12 strain CR34 (Zaritsky et al., Microbiology 145 (1999), 1052-1022). Enhancing the replication rate in cells with multi-forked replicating chromosomes (by addition of deoxyguanosine) shortens the intervals between successive terminations and thus triggers divisions more frequently. Monoclonal antibodies against FtsZ were used to visualize the rings of secondary constrictions, but apparent shortage of FtsZ to complete rings over wide cells allowed assembly of arcs only. The arcs observed were not parallel nor perpendicular; the tilted constriction planes are consistent with our 3-D 'nucleoid segregation'model for division under conditions which relieve the cylindrical constraint for nucleoid segregation by the bacillari peptidoglycan sacculus (Woldringh et al. , J. Bacteriol. 176 (1994) 6030-6038). The shortage in FtsZ may explain the longer time required to complete the division process in wide cells with long circumferences, observed during thymine step-up. Overexpression of fusion protein FtsZ-GFP on a multi-copy plasmid should circumvent the shortage.     

Assembly of the FtsZ ring at the central division site in the absence of the chromosome

The FtsZ ring assembles between segregated daughter chromosomes in prokaryotic cells and is essential for cell division. To understand better how the FtsZ ring is influenced by chromosome positioning and structure in Escherichia coli , we investigated its localization in parC and mukB mutants that are defective for chromosome segregation. Cells of both mutants at non-permissive temperatures were either filamentous with unsegregated nucleoids or short and anucleate. In parC filaments, FtsZ rings tended to localize only to either side of the central unsegregated nucleoid and rarely to the cell midpoint; however, medial rings reappeared soon after switching back to the permissive temperature. Filamentous mukB cells were usually longer and lacked many potential rings. At temperatures permissive for mukB viability, medial FtsZ rings assembled despite the presence of apparently unsegregated nucleoids. However, a significant proportion of these FtsZ rings were mislocalized or structurally abnormal. The most surprising result of this study was revealed upon further examination of FtsZ ring positioning in anucleate cells generated by the parC and mukB mutants: many of these cells, despite having no chromosome, possessed FtsZ rings at their midpoints. This discovery strongly suggests that the chromosome itself is not required for the proper positioning and development of the medial division site.     

Novel coiled-coil cell division factor ZapB stimulates Z ring assembly and cell division

Formation of the Z ring is the first known event in bacterial cell division. However, it is not yet known how the assembly and contraction of the Z ring are regulated. Here, we identify a novel cell division factor ZapB in Escherichia coli that simultaneously stimulates Z ring assembly and cell division. Deletion of zapB resulted in delayed cell division and the formation of ectopic Z rings and spirals, whereas overexpression of ZapB resulted in nucleoid condensation and aberrant cell divisions. Localization of ZapB to the divisome depended on FtsZ but not FtsA, ZipA or FtsI, and ZapB interacted with FtsZ in a bacterial two-hybrid analysis. The simultaneous inactivation of FtsA and ZipA prevented Z ring assembly and ZapB localization. Time lapse microscopy showed that ZapB–GFP is present at mid-cell in a pattern very similar to that of FtsZ. Cells carrying a zapB deletion and the ftsZ84 ^ts allele exhibited a synthetic sick phenotype and aberrant cell divisions. The crystal structure showed that ZapB exists as a dimer that is 100% coiled-coil. In vitro , ZapB self-assembled into long filaments and bundles. These results raise the possibility that ZapB stimulates Z ring formation directly via its capacity to self-assemble into larger structures.     

The developmental regulator PatD modulates assembly of the cell-division protein FtsZ in the cyanobacterium Anabaena sp. PCC 7120

FtsZ is a tubulin-like GTPase that polymerizes to initiate the process of cell division in bacteria. Heterocysts are terminally differentiated cells of filamentous cyanobacteria that have lost the capacity for cell division and in which the ftsZ gene is downregulated. However, mechanisms of FtsZ regulation during heterocyst differentiation have been scarcely investigated. The patD gene is NtcA dependent and involved in the optimization of heterocyst frequency in Anabaena sp. PCC 7120. Here, we report that the inactivation of patD caused the formation of multiple FtsZ-rings in vegetative cells, cell enlargement, and the retention of peptidoglycan synthesis activity in heterocysts, whereas its ectopic expression resulted in aberrant FtsZ polymerization and cell division. PatD interacted with FtsZ, increased FtsZ precipitation in sedimentation assays, and promoted the formation of thick straight FtsZ bundles that differ from the toroidal aggregates formed by FtsZ alone. These results suggest that in the differentiating heterocysts, PatD interferes with the assembly of FtsZ. We propose that in Anabaena FtsZ is a bifunctional protein involved in both vegetative cell division and regulation of heterocyst differentiation. In the differentiating cells PatD-FtsZ interactions appear to set an FtsZ activity that is insufficient for cell division but optimal to foster differentiation.     

Cell division control in Escherichia coli K-12: some properties of the ftsZ84 mutation and suppression of this mutation by the product of a newly identified gene.

The Fts proteins play an important role in the control of cell division in Escherichia coli. These proteins, which possibly form a functional complex, are encoded by genes that form an operon. In this study, we examined the properties of the temperature-sensitive mutation ftsZ84 harbored by low- or high-copy-number plasmids. Cells of strain AB1157, which had the ftsZ84 mutation, did not form colonies on salt-free L agar at 30 degrees C. When a low-copy-number plasmid containing the ftsZ84 mutation was present in these mutant cells, colony formation was restored on this medium at 30 degrees C, suggesting that FtsZ84 is probably less active than the wild-type protein and is therefore limiting in its capacity to trigger cell divisions. On the other hand, when the ftsZ84 mutation was harbored by the high-copy-number plasmid pBR325, colony formation was prevented on salt-free L agar plates whether the recipients were ftsZ84 mutant or parental cells, suggesting that, at high levels, FtsZ84 acts as a division inhibitor. The fact that colony formation was also prevented at 42 degrees C indicates that the FtsZ84 protein is not inactivated at the nonpermissive temperature. The possibility that FtsZ84 is a more efficient division inhibitor than the wild-type FtsZ is discussed. Evidence is also presented showing that a gene adjacent to mutT codes for a product that, under certain conditions, suppresses the ftsZ84 mutation.     

The min locus, which confers topological specificity to cell division, is not involved in its coupling with nucleoid separation.

In Escherichia coli, nucleoid separation and cell constriction remain tightly linked when division is retarded by altering the level of synthesis of the protein FtsZ. In this study, we have examined the role of the min locus, which is responsible for the inactivation of polar division sites, in the partition-septation coupling mechanism. We conclude that the coupling persists in a delta min strain and that its timing relative to replication remains dependent on the level of FtsZ synthesis. We suggest that the retarded nucleoid segregation observed in min mutants is the result of this coupling in cells with a perturbed pattern of nonpolar divisions.     

FtsZ ring formation without subsequent cell division after replication runout in Escherichia coli

In this report, we have investigated cell division after inhibition of initiation of chromosome replication in Escherichia coli. In a culture grown to the stationary phase, cells containing more than one chromosome were able to divide some time after restart of growth, under conditions not allowing initiation of chromosome replication. This shows that there is no requirement for cell division to take place within a certain time after initiation of chromosome replication. Continued growth without initiation of replication resulted in filamented cells that generally did not have any constrictions. Interestingly, FtsZ rings were formed in a majority of these cells as they reached a certain cell length. These rings appeared and were maintained for some time at the cell quarter positions on both sides of the centrally localized nucleoid. These results confirm previous findings that cell division sites are formed independently of chromosome replication and indicate that FtsZ ring assembly is dependent on cell size rather than on the capacity of the cell to divide. Disruption of the mukB gene caused a significant increase in the region occupied by DNA after the replication runout, consistent with a role of MukB in chromosome condensation. The aberrant nucleoid structure was accompanied by a shift in FtsZ ring positioning, indicating an effect of the nucleoid on the positioning of the FtsZ ring. A narrow cell length interval was found, under and over which primarily central and non-central FtsZ rings, respectively, were observed. This finding correlates well with the previously observed oscillatory movement of MinC and MinD in short and long cells.     

Bacterial SOS Checkpoint Protein SulA Inhibits Polymerization of Purified FtsZ Cell Division Protein

Cell division of Escherichia coli is inhibited when the SulA protein is induced in response to DNA damage as part of the SOS checkpoint control system. The SulA protein interacts with the tubulin-like FtsZ division protein. We investigated the effects of purified SulA upon FtsZ. SulA protein inhibits the polymerization and the GTPase activity of FtsZ, while point mutant SulA proteins show little effect on either of these FtsZ activities. SulA did not inhibit the polymerization of purified FtsZ2 mutant protein, which was originally isolated as insensitive to SulA. These studies define polymerization assays for FtsZ which respond to an authentic cellular regulator. The observations presented here support the notion that polymerization of FtsZ is central to its cellular role and that direct, reversible inhibition of FtsZ polymerization by SulA may account for division inhibition.     

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.     

Evidence of conformational switch in Streptococcus pneumoniae FtsZ during polymerization

FtsZ, the master coordinator of bacterial cell division, assembles into filaments in the presence of nucleotide. FtsZ from Streptococcus pneumoniae bears two tryptophan residues (W294 and W378) in its amino acid sequence. The tryptophan fluorescence of FtsZ increases during the assembly of FtsZ. We hypothesized that this increase in the fluorescence intensity was due to the change in the environment of one or both tryptophan residues. To examine this, we constructed two mutants (W294F and W378F) of FtsZ by individually replacing tryptophan with phenylalanine. The mutants displayed similar secondary structures, GTPase activity, and polymerization ability as the wild type FtsZ. During the polymerization, only one tryptophan (W294) showed an increase in its fluorescence intensity. Using time‐correlated single‐photon counting, the fluorescence lifetime of W294 was found to be significantly higher than W378, indicating that W294 was more buried in the structure than W378. The lifetime of W294 further increased during polymer formation, while that of W378 remained unchanged. Fluorescence quenching experiment suggested that the solvent exposure of W294 reduced during the polymerization of FtsZ. W294 is located near the T‐7 loop of the protein, a region important for the monomer‐monomer interaction during the formation of a protofilament. The results indicated that the region around W294 of S. pneumoniae FtsZ undergoes a conformational switch during polymerization as seen for FtsZ from other bacteria.     

Analysis of the interaction of FtsZ with itself, GTP, and FtsA.

The interaction of FtsZ with itself, GTP, and FtsA was examined by analyzing the sensitivity of FtsZ to proteolysis and by using the yeast two-hybrid system. The N-terminal conserved domain consisting of 320 amino acids bound GTP, and a central region of FtsZ, encompassing slightly more than half of the protein, was cross-linked to GTP. Site-directed mutagenesis revealed that none of six highly conserved aspartic acid and asparagine residues were required for GTP binding. These results indicate that the specificity determinants for GTP binding are different than those for the GTPase superfamily. The N-terminal conserved domain of FtsZ contained a site for self-interaction that is conserved between FtsZ proteins from distantly related bacterial species. FtsZ320, which was truncated at the end of the conserved domain, was a potent inhibitor of division although it expressed normal GTPase activity and could polymerize. FtsZ was also found to interact directly with FtsA, and this interaction could also be observed between these proteins from distantly related bacterial species.     

Ruthenium red-induced bundling of bacterial cell division protein, FtsZ

The assembly of FtsZ plays a major role in bacterial cell division, and it is thought that the assembly dynamics of FtsZ is a finely regulated process. Here, we show that ruthenium red is able to modulate FtsZ assembly in vitro. In contrast to the inhibitory effects of ruthenium red on microtubule polymerization, we found that a substoichiometric concentration of ruthenium red strongly increased the light-scattering signal of FtsZ assembly. Further, sedimentable polymer mass was increased by 1.5- and 2-fold in the presence of 2 and 10 microm ruthenium red, respectively. In addition, ruthenium red strongly reduced the GTPase activity and prevented dilution-induced disassembly of FtsZ polymers. Electron microscopic analysis showed that 4-10 microm of ruthenium red produced thick bundles of FtsZ polymers. The significant increase in the light-scattering signal and pelletable polymer mass in the presence of ruthenium red seemed to be due to the bundling of FtsZ protofilaments into larger polymers rather than the actual increase in the level of polymeric FtsZ. Furthermore, ruthenium red was found to copolymerize with FtsZ, and the copolymerization of substoichiometric amounts of ruthenium red with FtsZ polymers promoted cooperative assembly of FtsZ that produced large bundles. Calcium inhibited the binding of ruthenium red to FtsZ. However, a concentration of calcium 1000-fold higher than that of ruthenium red was required to produce similar effects on FtsZ assembly. Ruthenium red strongly modulated FtsZ polymerization, suggesting the presence of an important regulatory site on FtsZ and suggesting that a natural ligand, which mimics the action of ruthenium red, may regulate the assembly of FtsZ in bacteria.     

Berberine targets assembly of Escherichia coli cell division protein FtsZ

The ever increasing problem of antibiotic resistance necessitates a search for new drug molecules that would target novel proteins in the prokaryotic system. FtsZ is one such target protein involved in the bacterial cell division machinery. In this study, we have shown that berberine, a natural plant alkaloid, targets Escherichia coli FtsZ, inhibits the assembly kinetics of the Z-ring, and perturbs cytokinesis. It also destabilizes FtsZ protofilaments and inhibits the FtsZ GTPase activity. Saturation transfer difference NMR spectroscopy of the FtsZ-berberine complex revealed that the dimethoxy groups, isoquinoline nucleus, and benzodioxolo ring of berberine are intimately involved in the interaction with FtsZ. Berberine perturbs the Z-ring morphology by disturbing its typical midcell localization and reduces the frequency of Z-rings per unit cell length to half. Berberine binds FtsZ with high affinity ( K D approximately 0.023 microM) and displaces bis-ANS, suggesting that it may bind FtsZ in a hydrophobic pocket. Isothermal titration calorimetry suggests that the FtsZ-berberine interaction occurs spontaneously and is enthalpy/entropy-driven. In silico molecular modeling suggests that the rearrangement of the side chains of the hydrophobic residues in the GTP binding pocket may facilitate the binding of the berberine to FtsZ and lead to inhibition of the association between FtsZ monomers. Together, these results clearly indicate the inhibitory role of berberine on the assembly function of FtsZ, establishing it as a novel FtsZ inhibitor that halts the first stage in bacterial cell division.     

SulA inhibits assembly of FtsZ by a simple sequestration mechanism

We have investigated the inhibition by SulA of the assembly of Escherichia coli FtsZ. Using quantitative GTPase and fluorescence assays, we found that SulA inhibition resulted in an increase in the apparent critical concentration for FtsZ assembly. The increase in apparent critical concentration was always less than the total amount of SulA added, suggesting that the association of SulA and FtsZ was of modest affinity. Isothermal titration calorimetry gave a value of 0.78 μM for the dissociation constant of the FtsZ-SulA complex, similar in magnitude to the 0.72 μM critical concentration of FtsZ protofilament assembly at steady state. We modeled the reaction as an equilibrium competition between (a) FtsZ subunits assembling onto protofilaments or (b) binding SulA. When FtsZ was assembled in GMPCPP or in EDTA, the inhibition by SulA was reduced. The reduced inhibition could be explained by a 3- and 10-fold weaker binding of SulA to FtsZ. The mutant D212G, which has no GTPase activity and therefore minimal subunit cycling, was shown here to assemble one-stranded protofilaments, and the assembly was blocked by SulA. We also assayed the SulA and FtsZ proteins from Pseudomonas. The SulA inhibition was stronger than with the E. coli proteins, and the model indicated a 5-fold higher affinity of Pseudomonas SulA for FtsZ.     

FtsZ Dynamics during the Division Cycle of Live Escherichia coli Cells

The dynamics and assembly of bacterial cell division protein FtsZ were monitored in individual, growing and dividing Escherichia coli cells in real time by microculture of a merodiploid strain expressing green fluorescent protein (GFP)-tagged FtsZ. Cells expressing FtsZ-GFP at levels less than or equivalent to that of wild-type FtsZ were able to grow and divide over multiple generations, with their FtsZ rings visualized by fluorescence. During the late stages of cytokinesis, which constituted the last one-fourth of the cell cycle, the lumen of the FtsZ ring disappeared as the whole structure condensed. At this time, loops of FtsZ-GFP polymers emanated outward from the condensing ring structure and other unstable fluorescent structures elsewhere in the cell were also observed. Assembly of FtsZ rings at new division sites occurred within 1 min, from what appeared to be single points. Interestingly, this nucleation often took place in the predivisional cell at the same time the central FtsZ ring was in its final contraction phase. This demonstrates directly that, at least when FtsZ-GFP is being expressed, new division sites have the capacity to become fully functional for FtsZ targeting and assembly before cell division of the mother cell is completed. The results suggest that the timing of FtsZ assembly may be normally controlled in part by cellular FtsZ concentration. The use of wide-field optical sectioning microscopy to obtain sharp fluorescence images of FtsZ structures is also discussed.