Nucleoid Occlusion Research Articles

Influence of the nucleoid and the early stages of DNA replication on positioning the division site in Bacillus subtilis

Although division site positioning in rod-shaped bacteria is generally believed to occur through the combined effect of nucleoid occlusion and the Min system, several lines of evidence suggest the existence of additional mechanisms. Studies using outgrown spores of Bacillus subtilis have shown that inhibiting the early stages of DNA replication, leading up to assembly of the replisome at oriC, influences Z ring positioning. Here we examine whether Z ring formation at midcell under various conditions of DNA replication inhibition is solely the result of relief of nucleoid occlusion. We show that midcell Z rings form preferentially over unreplicated nucleoids that have a bilobed morphology (lowering DNA concentration at midcell), whereas acentral Z rings form beside a single-lobed nucleoid. Remarkably however, when the DnaB replication initiation protein is inactivated midcell Z rings never form over bilobed nucleoids. Relieving nucleoid occlusion by deleting noc increased midcell Z ring frequency for all situations of DNA replication inhibition, however not to the same extent, with the DnaB-inactivated strain having the lowest frequency of midcell Z rings. We propose an additional mechanism for Z ring positioning in which the division site becomes increasingly potentiated for Z ring formation as initiation of replication is progressively completed.

Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing

The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re-localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re-targeted to alternative sites such as the inner membrane in the presence of site-specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage-free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations.

Fully efficient chromosome dimer resolution in Escherichia coli cells lacking the integral membrane domain of FtsK

In bacteria, septum formation frequently initiates before the last steps of chromosome segregation. This is notably the case when chromosome dimers are formed by homologous recombination. Chromosome segregation then requires the activity of a double-stranded DNA transporter anchored at the septum by an integral membrane domain, FtsK. It was proposed that the transmembrane segments of proteins of the FtsK family form pores across lipid bilayers for the transport of DNA. Here, we show that truncated Escherichia coli FtsK proteins lacking all of the FtsK transmembrane segments allow for the efficient resolution of chromosome dimers if they are connected to a septal targeting peptide through a sufficiently long linker. These results indicate that FtsK does not need to transport DNA through a pore formed by its integral membrane domain. We propose therefore that FtsK transports DNA before membrane fusion, at a time when there is still an opening in the constricted septum.

Noc protein binds to specific DNA sequences to coordinate cell division with chromosome segregation

Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis, the nucleoid occlusion protein Noc protects the chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP-on-Chip and bioinformatics, we have identified a consensus Noc-binding DNA sequence (NBS), and have shown that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus. Purified Noc bound specifically to an NBS in vitro. NBSs inserted near the replication terminus bound Noc-YFP and caused a delay in cell division. An autonomous plasmid carrying an NBS array recruited Noc-YFP and conferred a severe Noc-dependent inhibition of cell division. This shows that Noc is a potent inhibitor of division, but that its activity is strictly localized by the interaction with NBS sites in vivo. We propose that Noc serves not only as a spatial regulator of cell division to protect the nucleoid, but also as a timing device with an important role in the coordination of chromosome segregation and cell division.

Bacillus subtilis MinC destabilizes FtsZ-rings at new cell poles and contributes to the timing of cell division

Division site selection in rod-shaped bacteria depends on nucleoid occlusion, which prevents division over the chromosome and MinCD, which prevent division at the poles. MinD is thought to localize MinC to the cell poles where it prevents FtsZ assembly. Time-lapse microscopy demonstrates that in Bacillus subtilis transient polar FtsZ rings assemble adjacent to recently completed septa and that in minCD strains these persist and are used for division, producing a minicell. This suggests that MinC acts when division proteins are released from newly completed septa to prevent their immediate reassembly at new cell poles. The minCD mutant appears to uncouple FtsZ ring assembly from cell division and thus shows a variable interdivisional time and a rapid loss of cell cycle synchrony. Functional MinC-GFP expressed from the chromosome minCD locus is dynamic. It is recruited to active division sites before septal biogenesis, rotates around the septum, and moves away from completed septa. Thus high concentrations of MinC are found primarily at the septum and, more transiently, at the new cell pole. DivIVA and MinD recruit MinC to division sites, rather than mediating the stable polar localization previously thought to restrict MinC activity to the pole. Together, our results suggest that B. subtilis MinC does not inhibit FtsZ assembly at the cell poles, but rather prevents polar FtsZ rings adjacent to new cell poles from supporting cell division.

A novel component of the division‐site selection system of Bacillus subtilis and a new mode of action for the division inhibitor MinCD

Cell division in bacteria is governed by a complex cytokinetic machinery in which the key player is a tubulin homologue, FtsZ. Most rod-shaped bacteria divide precisely at mid-cell between segregated sister chromosomes. Selection of the correct site for cell division is thought to be determined by two negative regulatory systems: the nucleoid occlusion system, which prevents division in the vicinity of the chromosomes, and the Min system, which prevents inappropriate division at the cell poles. In Bacillus subtilis recruitment of the division inhibitor MinCD to cell poles depends on DivIVA, and these proteins were thought to be sufficient for Min function. We have now identified a novel component of the division-site selection system, MinJ, which bridges DivIVA and MinD. minJ mutants are impaired in division because MinCD activity is no longer restricted to cell poles. Although MinCD was thought to act specifically on FtsZ assembly, analysis of minJ and divIVA mutants showed that their block in division occurs downstream of FtsZ. The results support a model in which the main function of the Min system lies in allowing only a single round of division per cell cycle, and that MinCD acts at multiple levels to prevent inappropriate division.

Bacterial Division: Another Way to Box in the Ring

Proper placement of the cell division site in some rod-shaped bacteria requires two different negative regulatory systems, nucleoid occlusion and the Min proteins. Caulobacter crescentus lacks these systems, but recent work has uncovered a novel regulator that achieves the same goals.

Bacillus subtilis EzrA and FtsL synergistically regulate FtsZ ring dynamics during cell division

Previous work has shown that the Bacillus subtilis EzrA protein directly inhibits FtsZ ring assembly, which is required for normal cell division, and that loss of EzrA results in hyperstabilization of the FtsZ polymer in vivo. Here, it was found that in ezrA-disrupted cells, artificial expression of YneA, which suppresses cell division during the SOS response, and disruption of noc (yyaA), which acts as an effector of nucleoid occlusion, resulted in accumulation of multiple non-constricting FtsZ rings, inhibition of cell division, and synthetic lethality. Overexpression of the essential cell division protein FtsL suppressed the effect of ezrA disruption. FtsL overexpression recovered the delayed FtsZ ring constriction seen in ezrA-disrupted wild-type cells. Conversely, the absence of EzrA caused lethality in cells producing a lower amount of FtsL than wild-type cells. It has previously been reported that FtsL is recruited to the division site during the later stages of cell division, although its exact role is currently unknown. The results of this study suggest that FtsL and EzrA synergistically regulate the FtsZ ring constriction in B. subtilis. Interestingly, FtsL overexpression also suppressed the cell division inhibition due to YneA expression or Noc inactivation in ezrA-disrupted cells.

In vivo monitoring of PHA granule formation using GFP-labeled PHA synthases

For the first time a functional protein was fused to a PHA synthase resulting in PHA granule formation and display of the respective function at the PHA granule surface. The GFP reporter protein was N-terminally fused to the class I PHA synthase of Cupriavidus necator (PhaC) and the class II PHA synthase of Pseudomonas aeruginosa PAO1 (PhaC1), respectively, while maintaining PHA synthase activity and PHA granule formation. Fluorescence microscopy studies of GFP–PHA synthase attached to emerging PHA granules indicated that emerging PHA granules locate to cell poles and to midcell representing the future cell poles. A rapid oscillating movement of GFP–PHA synthase foci from pole to pole was observed. In cell division impaired Escherichia coli, PHA granules were localized between nucleoids at regular spacing suggesting that nucleoid occlusion occurred. Accordingly, anucleate regions of the E. coli mukB mutant showed no regular spacing, but PHA granules with twofold increased diameter were formed. First evidence was provided that the cell division and the localization of GFP–PHA synthase foci are in vivo co-located.

SlmA, a Nucleoid-Associated, FtsZ Binding Protein Required for Blocking Septal Ring Assembly over Chromosomes in E. coli

Cell division in Escherichia coli begins with assembly of the tubulin-like FtsZ protein into a ring structure just underneath the cell membrane. Spatial control over Z ring assembly is achieved by two partially redundant negative regulatory systems, the Min system and nucleoid occlusion (NO), which cooperate to position the division site at midcell. In contrast to the well-studied Min system, almost nothing is known about how Z ring assembly is blocked in the vicinity of nucleoids to effect NO. Reasoning that Min function might become essential in cells impaired for NO, we screened for mutations synthetically lethal with a defective Min system (slm mutants). By using this approach, we identified SlmA (Ttk) as the first NO factor in E. coli. Our combined genetic, cytological, and biochemical results suggest that SlmA is a DNA-associated division inhibitor that is directly involved in preventing Z ring assembly on portions of the membrane surrounding the nucleoid.

Effects of perturbing nucleoid structure on nucleoid occlusion-mediated toporegulation of FtsZ ring assembly.

In Escherichia coli, assembly of the FtsZ ring (Z ring) at the cell division site is negatively regulated by the nucleoid in a phenomenon called nucleoid occlusion (NO). Previous studies have indicated that chromosome packing plays a role in NO, as mukB mutants grown in rich medium often exhibit FtsZ rings on top of diffuse, unsegregated nucleoids. To address the potential role of overall nucleoid structure on NO, we investigated the effects of disrupting chromosome structure on Z-ring positioning. We found that NO was mostly normal in cells with inactivated DNA gyrase or in mukB-null mutants lacking topA, although some suppression of NO was evident in the latter case. Previous reports suggesting that transcription, translation, and membrane insertion of proteins ("transertion") influence nucleoid structure prompted us to investigate whether disruption of these activities had effects on NO. Blocking transcription caused nucleoids to become diffuse, and FtsZ relocalized to multiple bands on top of these nucleoids, biased towards midcell. This suggested that these diffuse nucleoids were defective in NO. Blocking translation with chloramphenicol caused characteristic nucleoid compaction, but FtsZ rarely assembled on top of these centrally positioned nucleoids. This suggested that NO remained active upon translation inhibition. Blocking protein secretion by thermoinduction of a secA(Ts) strain caused a chromosome segregation defect similar to that in parC mutants, and NO was active. Although indirect effects are certainly possible with these experiments, the above data suggest that optimum NO activity may require specific organization and structure of the nucleoid.

Coordination of Cell Division and Chromosome Segregation by a Nucleoid Occlusion Protein in Bacillus subtilis

A range of genetical and physiological experiments have established that diverse bacterial cells possess a function called nucleoid occlusion, which acts to prevent cell division in the vicinity of the nucleoid. We have identified a specific effector of nucleoid occlusion in Bacillus subtilis, Noc (YyaA), as an inhibitor of division that is also a nonspecific DNA binding protein. Under various conditions in which the cell cycle is perturbed, Noc prevents the division machinery from assembling in the vicinity of the nucleoid. Unexpectedly, cells lacking both Noc and the Min system (which prevents division close to the cell poles) are blocked for division, apparently because they establish multiple nonproductive accumulations of division proteins. The results help to explain how B. subtilis specifies the division site under a range of conditions and how it avoids catastrophic breakage of the chromosome by division through the nucleoid.

A hypothesis to explain division site selection in Escherichia coli by combining nucleoid occlusion and Min

The positioning of the site of cell division in Escherichia coli results, it is generally believed, from the operation of nucleoid occlusion in combination with the Min system. Nucleoid occlusion prevents division over the nucleoids and directs it by default to the mid-cell region between segregating nucleoids or to polar regions while the Min system prevents division in polar regions. Unresolved questions include how these systems interact to control the earliest known event in division, the assembly at the membrane of the tubulin-like protein, FtsZ, and, more importantly, what exactly constitutes a division site. Evidence exists that (1) the coupled transcription, translation and insertion of proteins into membrane (transertion), can structure the cytoplasmic membrane into phospholipid domains, (2) the MinD protein can convert vesicles into tubes and (3) a variety of membranous structures can be observed at mid-cell. These data support a model in which transertion from the segregating daughter chromosomes leads to the formation of a distinct proteolipid domain between them at mid-cell; the composition of this domain allows phospholipid tubes to extend like fingers into the cytoplasm; these tubes then become the substrate for the dynamic assembly and disassembly of FtsZ which converts them into the invaginating fold responsible for division; the Min system inhibits division at unwanted sites and times by removing these tubes especially at the cell poles.

Evidence for polar positional information independent of cell division and nucleoid occlusion.

We present evidence that, in Escherichia coli, polar positional information is present at midcell independent of known cell division factors. In filamented cells, IcsA, which is normally polar, localizes at or near potential cell division sites. Because the cell pole is derived from the septum, the sites to which IcsA localizes in filaments correspond to future poles. IcsA localization to these sites is independent of FtsZ, MinCDE, septation, and nucleoid occlusion, indicating that positional information for the future pole is independent of cell division and chromosome positioning. Upon IcsA localization to these sites, septation is inhibited, suggesting that IcsA recognition of this polar positional information may influence cell division.

Localizing cell division in spherical Escherichia coli by nucleoid occlusion

The spatial relationship between FtsZ localization and nucleoid segregation was followed in Escherichia coli thyA cells, made spheroidal by brief exposure to mecillinam and after manipulating chromosome replication time using changes (‘steps’) in thymine concentration [Zaritsky et al., Microbiology 145 (1999) 1015–1022]. In such cells, fluorescent FtsZ-GFP arcs did not overlap the DAPI-stained nucleoids. It is concluded that FtsZ rings are deposited between segregating nucleoids, consistent with the nucleoid occlusion model [Woldringh et al., J. Bacteriol. 176 (1994) 6030–6038].

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.

Division in bacteria is determined by hyperstructure dynamics and membrane domains

The migration of the “tubulin-like” GTPase, FtsZ, from the cytoplasm to a site between the future daughter chromosomes is the earliest known stage of cell division in bacteria [1]. Polymerization of FtsZ from this site forms a ring of FtsZ around the cell [2] and recruits at least nine different proteins to a division complex, the so-called “divisome” or “septalsome” [3, 4]; these proteins include the “actin-like” molecules FtsA [5], FtsI, FtsN, FtsQ, FtsK, FtsL and ZipA [6]. The nature of the events that lie upstream of FtsZ migration and polymerization remain elusive and cannot simply be attributed to the action of yet another protein without risking an infinite regression. One approach to the problem of division is that of “nucleoid occlusion” in which the nucleoid would inhibit formation of a division site on the surrounding membrane (by an unknown mechanism) such that subsequent separation of the segregating nucleoids would allow this site to form between them [7, 8]; this would be consistent with the observed repression of peptidoglycan synthesis around the nucleoids [9]. There are other possibilities. It has been argued that certain genes, their products and associated ions and lipids interact to structure membranes and cytoplasm into hyperstructures [10] (see Fig. 1). Hyperstructures constitute a level intermediate between macromolecules and cells and correspond to the modules recently proposed to occupy a similar level in eukaryotic cells [11]. In searching for events upstream of FtsZ activation and the operation of the divisome, here we propose a hypothesis in which FtsZ is released by certain hyperstructures to be attracted by membrane domains produced by other hyperstructures. Division in bacteria is determined by hyperstructure dynamics and membrane domains

Bacterial cell division: regulating Z-ring formation.

The earliest stage of cell division in bacteria is the formation of a Z ring, composed of a polymer of the FtsZ protein, at the division site. Z rings appear to be synthesized in a bi-directional manner from a nucleation site (NS) located on the inside of the cytoplasmic membrane. It is the utilization of a NS specifically at the site of septum formation that determines where and when division will occur. However, a Z ring can be made to form at positions other than at the division site. How does a cell regulate utilization of a NS at the correct location and at the right time? In rod-shaped bacteria such as Escherichia coli and Bacillus subtilis, two factors involved in this regulation are the Min system and nucleoid occlusion. It is suggested that in B. subtilis, the main role of the Min proteins is to inhibit division at the nucleoid-free cell poles. In E. coli it is currently not clear whether the Min system can direct a Z ring to the division site at mid-cell or whether its main role is to ensure that division inhibition occurs away from mid-cell, a role analogous to that in B. subtilis. While the nucleoid negatively influences Z-ring formation in its vicinity in these rod-shaped organisms, the exact relationship between nucleoid occlusion and the ability to form a mid-cell Z ring is unresolved. Recent evidence suggests that in B. subtilis and Caulobacter crescentus, utilization of the NS at the division site is intimately linked to the progress of a round of chromosome replication and this may form the basis of achieving co-ordination between chromosome replication and cell division.

Mid-cell Z ring assembly in the absence of entry into the elongation phase of the round of replication in bacteria: co-ordinating chromosome replication with cell division.

We have shown previously that, when spores of a thymine-requiring strain of Bacillus subtilis were grown out in the absence of thymine, mid-cell Z rings formed over the nucleoid and much earlier than might be expected with respect to progression into the round of replication. It is now shown that such conditions allow no replication of oriC. Rather than replication, partial degradation of the oriC region occurs, suggesting that the status of this region is connected with the 'premature' mid-cell Z ring assembly. A correlation was observed between entry into the replication elongation phase and a block to mid-cell Z rings. The conformation of the nucleoid under various conditions of DNA replication inhibition or limitation suggests that relief of nucleoid occlusion is not primarily responsible for mid-cell Z ring formation in the absence of thymine. We propose the existence of a specific structure at mid-cell that defines the Z ring nucleation site (NS). It is suggested that this NS is normally masked by the replisome upon initiation of replication or soon after entry into the elongation phase, and subsequently unmasked relatively late in the round. During spore outgrowth in the absence of thymine, this checkpoint control over mid-cell Z ring assembly breaks down prematurely.

Magnesium-induced Linear Self-association of the FtsZ Bacterial Cell Division Protein Monomer: THE PRIMARY STEPS FOR FtsZ ASSEMBLY

The bacterial cell division protein FtsZ from Escherichia coli has been purified with a new calcium precipitation method. The protein contains one GDP and one Mg(2+) bound, it shows GTPase activity, and requires GTP and Mg(2+) to polymerize into long thin filaments at pH 6.5. FtsZ, with moderate ionic strength and low Mg(2+) concentrations, at pH 7.5, is a compact and globular monomer. Mg(2+) induces FtsZ self-association into oligomers, which has been studied by sedimentation equilibrium over a wide range of Mg(2+) and FtsZ concentrations. The oligomer formation mechanism is best described as an indefinite self-association, with binding of an additional Mg(2+) for each FtsZ monomer added to the growing oligomer, and a slight gradual decrease of the affinity of addition of a protomer with increasing oligomer size. The sedimentation velocity of FtsZ oligomer populations is compatible with a linear single-stranded arrangement of FtsZ monomers and a spacing of 4 nm. It is proposed that these FtsZ oligomers and the polymers formed under assembly conditions share a similar axial interaction between monomers (like in the case of tubulin, the eukaryotic homolog of FtsZ). Similar mechanisms may apply to FtsZ assembly in vivo, but additional factors, such as macromolecular crowding, nucleoid occlusion, or specific interactions with other cellular components active in septation have to be invoked to explain FtsZ assembly into a division ring.