The ParB homologs,Spo0J and Noc,together prevent premature midcell Z ring assembly when the early stages of replication are blocked in Bacillus subtilis

Precise cell division in coordination with DNA replication and segregation is of utmost importance for all organisms. The earliest stage of cell division is the assembly of a division protein FtsZ into a ring, known as the Z ring, at midcell. What still eludes us, however, is how bacteria precisely position the Z ring at midcell. Work in B. subtilis over the last two decades has identified a link between the early stages of DNA replication and cell division. A recent model proposed that the progression of the early stages of DNA replication leads to an increased ability for the Z ring to form at midcell. This model arose through studies examining Z ring position in mutants blocked at different steps of the early stages of DNA replication. Here, we show that this model is unlikely to be correct and the mutants previously studied generate nucleoids with different capacity for blocking midcell Z ring assembly. Importantly, our data suggest that two proteins of the widespread ParB family, Noc and Spo0J are required to prevent Z ring assembly over the bacterial nucleoid and help fine tune the assembly of the Z ring at midcell during the cell cycle.     

The Min system and nucleoid occlusion are not required for identifying the division site in Bacillus subtilis but ensure its efficient utilization

Precise temporal and spatial control of cell division is essential for progeny survival. The current general view is that precise positioning of the division site at midcell in rod-shaped bacteria is a result of the combined action of the Min system and nucleoid (chromosome) occlusion. Both systems prevent assembly of the cytokinetic Z ring at inappropriate places in the cell, restricting Z rings to the correct site at midcell. Here we show that in the bacterium Bacillus subtilis Z rings are positioned precisely at midcell in the complete absence of both these systems, revealing the existence of a mechanism independent of Min and nucleoid occlusion that identifies midcell in this organism. We further show that Z ring assembly at midcell is delayed in the absence of Min and Noc proteins, while at the same time FtsZ accumulates at other potential division sites. This suggests that a major role for Min and Noc is to ensure efficient utilization of the midcell division site by preventing Z ring assembly at potential division sites, including the cell poles. Our data lead us to propose a model in which spatial regulation of division in B. subtilis involves identification of the division site at midcell that requires Min and nucleoid occlusion to ensure efficient Z ring assembly there and only there, at the right time in the cell cycle.     

ZapA and ZapB form an FtsZ‐independent structure at midcell

Cell division in Escherichia coli begins with the polymerization of FtsZ into a ring‐like structure, the Z‐ring, at midcell. All other division proteins are thought to require the Z‐ring for recruitment to the future division site. Here, it is reported that the Z‐ring associated proteins ZapA and ZapB form FtsZ‐independent structures at midcell. Upon Z‐ring disruption by the FtsZ polymerization antagonist SulA, ZapA remained at midcell as a cloud‐like accumulation. Using ZapA(N60Y), a variant defective for interaction with FtsZ, it was established that these ZapA structures form without a connection to the Z‐ring. Furthermore, midcell accumulations of GFP‐ZapA(N60Y) often preceded Z‐rings at midcell and required ZapB to assemble, suggesting that ZapB polymers form the foundation of these structures. In the absence of MatP, a DNA‐binding protein that links ZapB to the chromosomal terminus region, cloud‐like ZapA structures still formed but failed to track with the chromosome terminus and did not consistently precede FtsZ at midcell. Taken together, the results suggest that FtsZ‐independent structures of ZapA–ZapB provide additional positional cues for Z‐ring formation and may help coordinate its assembly with chromosome replication and segregation.     

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.     

PomZ,a ParA‐like protein,regulates Z‐ring formation and cell division in Myxococcus xanthus

Accurate positioning of the division site is essential to generate appropriately sized daughter cells with the correct chromosome number. In bacteria, division generally depends on assembly of the tubulin homologue FtsZ into the Z‐ring at the division site. Here, we show that lack of the ParA‐like protein PomZ in Myxococcus xanthus resulted in division defects with the formation of chromosome‐free minicells and filamentous cells. Lack of PomZ also caused reduced formation of Z‐rings and incorrect positioning of the few Z‐rings formed. PomZ localization is cell cycle regulated, and PomZ accumulates at the division site at midcell after chromosome segregation but prior to FtsZ as well as in the absence of FtsZ. FtsZ displayed cooperative GTP hydrolysis in vitro but did not form detectable filaments in vitro. PomZ interacted with FtsZ in M. xanthus cell extracts. These data show that PomZ is important for Z‐ring formation and is a spatial regulator of Z‐ring formation and cell division. The cell cycle‐dependent localization of PomZ at midcell provides a mechanism for coupling cell cycle progression and Z‐ring formation. Moreover, the data suggest that PomZ is part of a system that recruits FtsZ to midcell, thereby, restricting Z‐ring formation to this position.     

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.     

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.     

FtsZ rings in mukB mutants with or without the Min system

The site of cell division in Escherichia coli is defined by formation of the Z ring between the two segregated daughter nucleoids. Positioning of the Z ring, composed of the highly conserved and tubulin-like FtsZ protein, appears to be negatively regulated by both the nucleoid and the oscillating MinCD inhibitor proteins. MukB protein is probably involved in nucleoid condensation, and in the absence of MukB, the negative effect of the nucleoid on Z rings appears to be partially suppressed. In this study, we examined the localization of Z rings in cells lacking both the Min system and MukB. In the Deltamin DeltamukB double null mutant, essentially all nucleoid-free zones, either at the cell poles or at non-polar sites between nucleoids, contained Z rings. However, a significant proportion of Z rings also formed on top of nucleoids. Interestingly, Z ring clusters often formed at gaps between nucleoids, and some of the rings within the clusters were clearly positioned on top of nucleoids. These results provide further evidence that the negative topological effect of nucleoids in cells lacking MukB is partially but not totally suppressed, and that the absence of the Min system allows more promiscuous Z ring formation.     

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.     

Influence of the nucleoid on placement of FtsZ and MinE rings in Escherichia coli

We previously presented evidence that replicating but unsegregated nucleoids, along with the Min system, act as topological inhibitors to restrict assembly of the FtsZ ring (Z ring) to discrete sites in the cell. To test if nonreplicating nucleoids have similar exclusion effects, we examined Z rings in dnaA (temperature sensitive) mutants. Z rings were excluded from centrally localized nucleoids and were often observed at nucleoid edges. Cells with nonreplicating nucleoids formed filaments, some of which contained large nucleoid-free areas in which Z rings were positioned at regular intervals. Because MinE may protect FtsZ from the action of the MinC inhibitor in these nucleoid-free zones, we examined the localization of a MinE-green fluorescent protein (GFP) fusion with respect to Z rings and nucleoids. Like Z rings, MinE-GFP appeared to localize independently of nucleoid position, forming rings at regular intervals in nucleoid-free regions. Unlike FtsZ, however, MinE-GFP often localized on top of nucleoids, replicating or not, suggesting that MinE is relatively insensitive to the nucleoid inhibition effect. These data suggest that both replicating and nonreplicating nucleoids are capable of topologically excluding Z rings but not MinE.     

Mathematical model for positioning the FtsZ contractile ring in Escherichia coli

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

Subcellular localization of Dna-initiation proteins of Bacillus subtilis: evidence that chromosome replication begins at either edge of the nucleoids

We examined the intracellular distribution of Bacillus subtilis Dna-initiation proteins by immunofluorescence microscopy to visualize the initiation complex of replication in vivo. DnaA was distributed throughout the cytoplasm, but both DnaB and DnaI were always detected as foci during the cell-division cycle. Interaction of DnaI with the DnaC helicase by the yeast two-hybrid assay suggests that DnaI acts as a helicase loader. The number of DnaB and DnaI foci within the cell exceeded that of oriC. Although the foci were not always co-localized with oriC, they seemed to be localized near the outer or inner edges of the nucleoids at initiation of replication. When the replication cycle was synchronized in cells using a temperature-sensitive dnaA mutant, duplication of the oriC region was observed predominantly near an edge of the nucleoid. Before initiation occurred, each one of the DnaB and DnaI foci was frequently observed near there. Furthermore, DnaX-GFP (DnaX is a component of DNA polymerase III) foci were detected near either of the edges of the nucleoids at the onset of replication. These results suggest that the replisome is recruited into oriC near either edge of the nucleoids to initiate chromosome replication in B. subtilis.     

Overview of controls in the Escherichia coli cell cycle

The harmonious growth and cell-to-cell uniformity of steady-state bacterial populations indicate the existence of a well-regulated cell cycle, responding to a set of internal signals. In Escherichia coli, the key events of this cycle are the initiation of DNA replication, nucleoid segregation and the initiation of cell division. The replication initiator is the DnaA protein. In nucleoid segregation, the MukB protein, required for proper partitioning, may be a member of the myosin-kinesin superfamily of mechanoenzymes. In cell division, the FtsZ protein has a tubulin motif, is a GTPase and polymerizes in a ring around midcell during septation; the FtsA protein has an actin-like structure. The nature of the internal signals triggering these events is not known but candidates include cell mass, the superhelical density of the chromosome and the concentration of two regulatory nucleotides, cyclic AMP and ppGpp. The involvement of cytoskeletal-like proteins in key cycle events encourages the notion of a fundamental biological unity in cell cycle regulation in all organisms.     

Evidence for Divisome Localization Mechanisms Independent of the Min System and SlmA in Escherichia coli

Cell division in Escherichia coli starts with assembly of FtsZ protofilaments into a ring-like structure, the Z-ring. Positioning of the Z-ring at midcell is thought to be coordinated by two regulatory systems, nucleoid occlusion and the Min system. In E. coli, nucleoid occlusion is mediated by the SlmA proteins. Here, we address the question of whether there are additional positioning systems that are capable of localizing the E. coli divisome with respect to the cell center. Using quantitative fluorescence imaging we show that slow growing cells lacking functional Min and SlmA nucleoid occlusion systems continue to divide preferentially at midcell. We find that the initial Z-ring assembly occurs over the center of the nucleoid instead of nucleoid-free regions under these conditions. We determine that Z-ring formation begins shortly after the arrival of the Ter macrodomain at the nucleoid center. Removal of either the MatP, ZapB, or ZapA proteins significantly affects the accuracy and precision of Z-ring positioning relative to the nucleoid center in these cells in accordance with the idea that these proteins link the Ter macrodomain and the Z-ring. Interestingly, even in the absence of Min, SlmA, and the putative Ter macrodomain – Z-ring link, there remains a weak midcell positioning bias for the Z-ring. Our work demonstrates that additional Z-ring localization systems are present in E. coli than are known currently. In particular, we identify that the Ter macrodomain acts as a landmark for the Z-ring in the presence of MatP, ZapB and ZapA proteins.     

A conserved coiled‐coil protein pair focuses the cytokinetic Z‐ring in Caulobacter crescentus

The cytoskeletal GTPase FtsZ assembles at midcell, recruits the division machinery and directs envelope invagination for bacterial cytokinesis. ZapA, a conserved FtsZ‐binding protein, promotes Z‐ring stability and efficient division through a mechanism that is not fully understood. Here, we investigated the function of ZapA in Caulobacter crescentus. We found that ZapA is encoded in an operon with a small coiled‐coil protein we named ZauP. ZapA and ZauP co‐localized at the division site and were each required for efficient division. ZapA interacted directly with both FtsZ and ZauP. Neither ZapA nor ZauP influenced FtsZ dynamics or bundling, in vitro, however. Z‐rings were diffuse in cells lacking zapA or zauP and, conversely, FtsZ was enriched at midcell in cells overproducing ZapA and ZauP. Additionally, FtsZ persisted at the poles longer when ZapA and ZauP were overproduced, and frequently colocalized with MipZ, a negative regulator of FtsZ polymerization. We propose that ZapA and ZauP promote efficient cytokinesis by stabilizing the midcell Z‐ring through a bundling‐independent mechanism. The zauPzapA operon is present in diverse Gram‐negative bacteria, indicating a common mechanism for Z‐ring assembly.     

Co-ordinating DNA replication with cell division in bacteria: a link between the early stages of a round of replication and mid-cell Z ring assembly

Spores of a thymine-requiring strain of Bacillus subtilis 168, which is also temperature sensitive for the initiation of chromosome replication, were germinated and allowed to grow out at the permissive temperature in a minimal medium containing no added thymine. Under these conditions, there was no or very limited progression into the elongation phase of the first round of replication. In a significant proportion of the outgrown cells, a Z ring formed precisely at mid-cell and over the centrally positioned nucleoid, leading eventually to the formation of a mature division septum. When initiation of the first round of replication was blocked through a temperature shift and with thymine present, the Z ring was positioned acentrally. The central Z ring that formed in the absence of thymine was blocked by the presence of a DNA polymerase III inhibitor. It is concluded that the very early stages of a round of replication (initiation plus possibly limited progression into the elongation phase) play a key role in the precise positioning of the Z ring at mid-cell and between replicating daughter chromosomes.     

The midcell replication factory in Bacillus subtilis is highly mobile: implications for coordinating chromosome replication with other cell cycle events

During vegetative growth, rod-shaped bacterial cells such as Escherichia coli and Bacillus subtilis divide precisely at midcell. It is the Z ring that defines the position of the division site. We previously demonstrated that the early stages of chromosome replication are linked to midcell Z ring assembly in B. subtilis and proposed a direct role for the centrally located replication factory in masking and subsequently unmasking the midcell site for Z ring assembly. We now show that the replication factory is significantly more scattered about the cell centre than the Z ring in both vegetative cells and outgrown spores of B. subtilis. This finding is inconsistent with the midcell replication factory acting as a direct physical block to Z ring assembly. Time-lapse experiments demonstrated that the lower precision of replication factory positioning results from its high mobility around the cell centre. Various aspects of this mobility are presented and the results are discussed in the light of current views on the determinants of positional information required for accurate chromosome segregation and cell division.     

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.