Coordinating DNA replication with cell division: lessons from outgrowing spores

Progress in solving the long-standing puzzle of how a cell coordinates chromosome replication with cell division is significantly aided by the use of synchronous cell populations. Currently three systems are employed for obtaining such populations: the Escherichia coli 'baby machine', the developmentally-controlled cell cycle of Caulobacter crescentus, and Bacillus subtilis germinated and outgrowing spores. This review examines our current understanding of the relationship between replication and division and how the use of B. subtilis outgrowing spores and, more recently its combination with immunofluorescence microscopy, has contributed significantly to this important area of biology. About 20 years ago, and also more recently, this system was used to show convincingly that termination of DNA replication is not essential for a central septum to form, raising the possibility that the early stages of division occur well before termination. It has also been demonstrated that there is no major synthesis of the division initiation proteins, FtsZ and DivIB, linked to initiation, progression or completion of the first round of chromosome replication accompanying spore outgrowth. This has led to the suggestion that the primary link between chromosome replication and cell division at midcell is not likely to occur through a control over the levels of these proteins. Very recent work has employed a combination of the use of B. subtilis outgrowing spores with immunofluorescence microscopy to investigate the relationship between midcell Z ring assembly and the round of chromosome replication linked to it. The results of this work suggest a role for initiation and progression into the round of replication in blocking midcell Z ring formation until the round is complete or almost complete, thereby ensuring that cell division occurs between two equally-partitioned chromosomes.     

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.     

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.     

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.     

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.     

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.     

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.     

Cell division in Bacillus subtilis: FtsZ and FtsA association is Z-ring independent, and FtsA is required for efficient midcell Z-Ring assembly

The earliest stage in cell division in bacteria is the assembly of a Z ring at the division site at midcell. Other division proteins are also recruited to this site to orchestrate the septation process. FtsA is a cytosolic division protein that interacts directly with FtsZ. Its function remains unknown. It is generally believed that FtsA localization to the division site occurs immediately after Z-ring formation or concomitantly with it and that FtsA is responsible for recruiting the later-assembling membrane-bound division proteins to the division site. Here, we report the development of an in vivo chemical cross-linking assay to examine the association between FtsZ and FtsA in Bacillus subtilis cells. We subsequently use this assay in a synchronous cell cycle to show that these two proteins can interact prior to Z-ring formation. We further show that in a B. subtilis strain containing an ftsA deletion, FtsZ localized at regular intervals along the filament but the majority of Z rings were abnormal. FtsA in this organism is therefore critical for the efficient formation of functional Z rings. This is the first report of abnormal Z-ring formation resulting from the loss of a single septation protein. These results suggest that in this organism, and perhaps others, FtsA ensures recruitment of the membrane-bound division proteins by ensuring correct formation of the Z ring.     

RefZ Facilitates the Switch from Medial to Polar Division during Spore Formation in Bacillus subtilis

During sporulation, Bacillus subtilis redeploys the division protein FtsZ from midcell to the cell poles, ultimately generating an asymmetric septum. Here, we describe a sporulation-induced protein, RefZ, that facilitates the switch from a medial to a polar FtsZ ring placement. The artificial expression of RefZ during vegetative growth converts FtsZ rings into FtsZ spirals, arcs, and foci, leading to filamentation and lysis. Mutations in FtsZ specifically suppress RefZ-dependent division inhibition, suggesting that RefZ may target FtsZ. During sporulation, cells lacking RefZ are delayed in polar FtsZ ring formation, spending more time in the medial and transition stages of FtsZ ring assembly. A RefZ-green fluorescent protein (GFP) fusion localizes in weak polar foci at the onset of sporulation and as a brighter midcell focus at the time of polar division. RefZ has a TetR DNA binding motif, and point mutations in the putative recognition helix disrupt focus formation and abrogate cell division inhibition. Finally, chromatin immunoprecipitation assays identified sites of RefZ enrichment in the origin region and near the terminus. Collectively, these data support a model in which RefZ helps promote the switch from medial to polar division and is guided by the organization of the chromosome. Models in which RefZ acts as an activator of FtsZ ring assembly near the cell poles or as an inhibitor of the transient medial ring at midcell are discussed.     

Mycobacterium tuberculosis cells growing in macrophages are filamentous and deficient in FtsZ rings

FtsZ, a bacterial homolog of tubulin, forms a structural element called the FtsZ ring (Z ring) at the predivisional midcell site and sets up a scaffold for the assembly of other cell division proteins. The genetic aspects of FtsZ-catalyzed cell division and its assembly dynamics in Mycobacterium tuberculosis are unknown. Here, with an M. tuberculosis strain containing FtsZ(TB) tagged with green fluorescent protein as the sole source of FtsZ, we examined FtsZ structures under various growth conditions. We found that midcell Z rings are present in approximately 11% of actively growing cells, suggesting that the low frequency of Z rings is reflective of their slow growth rate. Next, we showed that SRI-3072, a reported FtsZ(TB) inhibitor, disrupted Z-ring assembly and inhibited cell division and growth of M. tuberculosis. We also showed that M. tuberculosis cells grown in macrophages are filamentous and that only a small fraction had midcell Z rings. The majority of filamentous cells contained nonring, spiral-like FtsZ structures along their entire length. The levels of FtsZ in bacteria grown in macrophages or in broth were comparable, suggesting that Z-ring formation at midcell sites was compromised during intracellular growth. Our results suggest that the intraphagosomal milieu alters the expression of M. tuberculosis genes affecting Z-ring formation and thereby cell division.     

A moving DNA replication factory in Caulobacter crescentus

The in vivo intracellular location of components of the Caulobacter replication apparatus was visualized during the cell cycle. Replisome assembly occurs at the chromosomal origin located at the stalked cell pole, coincident with the initiation of DNA replication. The replisome gradually moves to midcell as DNA replication proceeds and disassembles upon completion of DNA replication. Although the newly replicated origin regions of the chromosome are rapidly moved to opposite cell poles by an active process, the replisome appears to be an untethered replication factory that is passively displaced towards the center of the cell by the newly replicated DNA. These results are consistent with a model in which unreplicated DNA is pulled into the replication factory and newly replicated DNA is bidirectionally extruded from the complex, perhaps contributing to chromosome segregation.     

The Min system is not required for precise placement of the midcell Z ring in Bacillus subtilis

In bacteria, the Min system plays a role in positioning the midcell division site by inhibiting the formation of the earliest precursor of cell division, the Z ring, at the cell poles. However, whether the Min system also contributes to establishing the precise placement of the midcell Z ring is unresolved. We show that the Z ring is positioned at midcell with a high degree of precision in Bacillus subtilis, and this is completely maintained in the absence of the Min system. Min is therefore not required for correct midcell Z ring placement in B. subtilis. Our results strongly support the idea that the primary role of the Min system is to block Z ring formation at the cell poles and that a separate mechanism must exist to ensure cell division occurs precisely at midcell.     

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.     

Diverse paths to midcell: assembly of the bacterial cell division machinery

At the heart of bacterial cell division is a dynamic ring-like structure of polymers of the tubulin homologue FtsZ. This ring forms a scaffold for assembly of at least ten additional proteins at midcell, the majority of which are likely to be involved in remodeling the peptidoglycan cell wall at the division site. Together with FtsZ, these proteins are thought to form a cell division complex, or divisome. In Escherichia coli, the components of the divisome are recruited to midcell according to a strikingly linear hierarchy that predicts a step-wise assembly pathway. However, recent studies have revealed unexpected complexity in the assembly steps, indicating that the apparent linearity does not necessarily reflect a temporal order. The signals used to recruit cell division proteins to midcell are diverse and include regulated self-assembly, protein-protein interactions, and the recognition of specific septal peptidoglycan substrates. There is also evidence for a complex web of interactions among these proteins and at least one distinct subcomplex of cell division proteins has been defined, which is conserved among E. coli, Bacillus subtilis and Streptococcus pneumoniae.     

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.     

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.     

Spatial and temporal organization of the Bacillus subtilis replication cycle

DNA replication occurs at discrete sites in the cell. To gain insight into the spatial and temporal organization of the Bacillus subtilis replication cycle, we simultaneously visualized replication origins and the replication machinery (replisomes) inside live cells. We found that the origin of replication is positioned near midcell prior to replication. After initiation, the replisome colocalizes with the origin, confirming that replication initiates near midcell. The replisome remains near midcell after duplicated origins separate. Artificially mispositioning the origin region leads to mislocalization of the replisome indicating that the location of the origin at the time of initiation establishes the position of the replisome. Time-lapse microscopy revealed that a single replisome focus reversibly splits into two closely spaced foci every few seconds in many cells, including cells that recently initiated replication. Thus, sister replication forks are likely not intimately associated with each other throughout the replication cycle. Fork dynamics persisted when replication elongation was halted, and is thus independent of the relative movement of DNA through the replisome. Our results provide new insights into how the replisome is positioned in the cell and refine our current understanding of the spatial and temporal events of the B. subtilis replication cycle.     

MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter

Correct positioning of the division plane is a prerequisite for the generation of daughter cells with a normal chromosome complement. Here, we present a mechanism that coordinates assembly and placement of the FtsZ cytokinetic ring with bipolar localization of the newly duplicated chromosomal origins in Caulobacter. After replication of the polarly located origin region, one copy moves rapidly to the opposite end of the cell in an MreB-dependent manner. A previously uncharacterized essential protein, MipZ, forms a complex with the partitioning protein ParB near the origin of replication and localizes with the duplicated origin regions to the cell poles. MipZ directly interferes with FtsZ polymerization, thereby restricting FtsZ ring formation to midcell, the region of lowest MipZ concentration. The cellular localization of MipZ thus serves the dual function of positioning the FtsZ ring and delaying formation of the cell division apparatus until chromosome segregation has initiated.     

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.