The Bacterial Cell Cycle: DNA Replication,Nucleoid Segregation,and Cell Division

Data on the bacterial cell cycle published in the last 10–15 years are considered, with a special stress on studies of nucleoid segregation between dividing cells. The degree of similarity between the eukaryotic mitotic apparatus and the apparatus performing nucleoid separation is discussed.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 437–451.Original Russian Text Copyright © 2005 by Prozorov.     

Genomewide phenotypic analysis of growth,cell morphogenesis,and cell cycle events in Escherichia coli

Cell size, cell growth, and cell cycle events are necessarily intertwined to achieve robust bacterial replication. Yet, a comprehensive and integrated view of these fundamental processes is lacking. Here, we describe an image‐based quantitative screen of the single‐gene knockout collection of Escherichia coli and identify many new genes involved in cell morphogenesis, population growth, nucleoid (bulk chromosome) dynamics, and cell division. Functional analyses, together with high‐dimensional classification, unveil new associations of morphological and cell cycle phenotypes with specific functions and pathways. Additionally, correlation analysis across ~4,000 genetic perturbations shows that growth rate is surprisingly not predictive of cell size. Growth rate was also uncorrelated with the relative timings of nucleoid separation and cell constriction. Rather, our analysis identifies scaling relationships between cell size and nucleoid size and between nucleoid size and the relative timings of nucleoid separation and cell division. These connections suggest that the nucleoid links cell morphogenesis to the cell cycle.     

Spiral architecture of the nucleoid in Bdellovibrio bacteriovorus

We present a cryo-electron tomographic analysis of the three-dimensional architecture of a strain of the Gram-negative bacterium Bdellovibrio bacteriovorus in which endogenous MreB2 was replaced with monomeric teal fluorescent protein (mTFP)-labeled MreB2. In contrast to wild-type Bdellovibrio cells that predominantly displayed a compact nucleoid region, cells expressing mTFP-labeled MreB2 displayed a twisted spiral organization of the nucleoid. The more open structure of the MreB2-mTFP nucleoids enabled clear in situ visualization of ribosomes decorating the periphery of the nucleoid. Ribosomes also bordered the edges of more compact nucleoids from both wild-type cells and mutant cells. Surprisingly, MreB2-mTFP localized to the interface between the spiral nucleoid and the cytoplasm, suggesting an intimate connection between nucleoid architecture and MreB arrangement. Further, in contrast to wild-type cells, where a single tight chemoreceptor cluster localizes close to the single polar flagellum, MreB2-mTFP cells often displayed extended chemoreceptor arrays present at one or both poles and displayed multiple or inaccurately positioned flagella. Our findings provide direct structural evidence for spiral organization of the bacterial nucleoid and suggest a possible role for MreB in regulation of nucleoid architecture and localization of the chemotaxis apparatus.     

Partitioning of P1 plasmids by gradual distribution of the ATPase ParA

Recently, it has been reported that prokaryotes also have a mitotic-like apparatus in which polymerized fibres govern the bipolar movement of chromosomes and plasmids. Here, we show evidence that a non-mitotic-like apparatus that does not form polymerized filaments carries out plasmid partitioning. P1 ParA, which is a DNA-binding ATPase protein, was found to be distributed through the whole nucleoid and formed a dense spot at the centre of the nucleoid. The fluorescent intensity of the ParA spot blinked, and then the spot gradually migrated from the midcell to a cell quarter position. Such distribution was not observed in anucleate cells, suggesting that the nucleoid could be a matrix for gradual distribution of ParA. Plasmid DNA constantly colocalized at the spot of ParA and migrated according to spot migration and separation. Thus, the gradient distribution of ParA determines the destination of partitioning plasmids and may direct plasmids to the cell quarters.     

Does a structural bridge exist between the DNA and the specialized cytoplasmic organelles during the early part of their development? A mechanism for the positioning of flagella and possibly other cytoplasmic organelles

Cell differentiation involves the development of a new cytoplasm containing a set of specialized organelles such as cilia and flagella which are placed in the cell with a predetermined orientation. Arguments are put forward to show that the orientation of the flagellar apparatus could be brought about by a macromolecular structural bridge between the nucleoid and the assembling flagellar apparatus, the orientation being determined by the spatial geometry inherent in the folding of the DNA. An analysis of differentiation in unicelled eukaryotes suggests that the same basic mechanism of a structural bridge could also apply to the orientation of their cilia and flagella and perhaps may have a more general application in the positioning of cytoplasmic organelles.     

Nucleoid-independent identification of cell division sites in Escherichia coli

The mechanism used by Escherichia coli to determine the correct site for cell division is unknown. In this report, we have attempted to distinguish between a model in which septal position is determined by the position of the nucleoids and a model in which septal position is predetermined by a mechanism that does not involve nucleoid position. To do this, filaments with extended nucleoid-free regions adjacent to the cell poles were produced by simultaneous inactivation of cell division and DNA replication. The positions of septa that formed within the nucleoid-free zones after division was allowed to resume were then analyzed. The results showed that septa were formed at a uniform distance from cell poles when division was restored, with no relation to the distance from the nearest nucleoid. In some cells, septa were formed directly over nucleoids. These results are inconsistent with models that invoke nucleoid positioning as the mechanism for determining the site of division site formation.     

MinCD-independent inhibition of cell division by a protein that fuses MalE to the topological specificity factor MinE.

We report that MinE, the topological specificity factor of cell division in Escherichia coli, inhibits septation when fused to the C terminus of the maltose-binding protein MalE. This contrasts with overexpression of MinE alone, which affects growth but has no effect on division. Inhibition by MalE-MinE was minCD independent and depended on MinE segments involved in dimerization and prevention of MinCD division inhibition. The SOS and the heat shock responses were not involved, suggesting that the inhibition comes from a direct interaction of MalE-MinE with the septation apparatus. MalE-MinE lethality was suppressed by overexpression of ftsZ, as well as by overexpression of ftsN, a suppressor of temperature-sensitive mutations in genes ftsQ, ftsA, and ftsI. We also report that high-level synthesis of MalE disturbs nucleoid partitioning.     

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.     

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.     

Nucleoid partitioning in Escherichia coli during steady-state growth and upon recovery from chloramphenicol treatment

To distinguish between a gradual or an abrupt movement of the Escherichia coli nucleoid during partitioning we determined the distances between nucleoid borders and cell poles. Measurements were performed on fixed but hydrated cells and on living cells growing in steady state. The distance between nucleoid outer border and cell pole remained constant in cells with either one or two nucleoids. Thus the nucleoid outer borders moved gradually during the partition process. To study partitioning during recovery from protein-synthesis inhibition cells were treated with chloramphenicol. After growth resumption, cells and nucleoids first elongated before partitioning occurred. Again, no indication of a rapid displacement of the nucleoid to one-quarter and three-quarter positions in the cell was observed.     

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.     

Variations in chloroplast nucleoid number during the cell cycle in the algaScenedesmus quadricauda grown under different light conditions

Summary Synchronous cultures of the green algaScenedesmus quadricauda were grown at different mean irradiances (ranging from 15 Wm^–2 to 130Wm^–2). At each irradiance, the algae were exposed to illumination regimes which differed in light duration and dark intervals (222 to 240 hours). The cells from these cultures were sampled during their cycles, stained with DAPI and the number of nuclei and chloroplast nucleoids estimated.The nucleoids divided semisynchronously in steps which represented doublings in their number. For each doubling a constant amount of light energy (defined as the product of irradiance and light duration) had to be converted by the cells to become committed to this division. The times to the start of the nucleoid divisions were therefore inversely proportional to the irradiances applied and the final number of nucleoids was proportional to the light duration.Temporal relationships between nuclear and nucleoid divisions were also light dependent. Shortage of light energy caused delay in nucleoid division. The cell division rate was higher than the rate of nucleoid division and consequently, the cells tended to decrease their nucleoid number with decreasing irradiance. With increasing irradiance the start of nucleoid division was gradually shifted toward the beginning of the cell cycle. The rate of nucleoid division exceeded the rate of nuclear and cellular division, thus with increasing irradiance cells with increasing numbers of nucleoids were formed.Abbreviations DAPI 46-diamidino-2-phenylindole - pt-DNA chloroplast DNA     

Large amounts of apicoplast nucleoid DNA and its segregation inToxoplasma gondii

Summary Apicoplasts (apicomplexan plastids) are nonphotosynthetic, secondary endosymbiotic plastids that are found in most apicomplexans. Although these organelles are essential for parasite survival, their functions, activities, and structures are not well understood. We examined the apicoplast nucleoid ofToxoplasma gondii from a morphological aspect by high-resolution epifluorescence microscopy and electron microscopy. We found unexpectedly large amounts of DNA in the nucleoid and the presence of several division-related structures. Initially, we identified the organellar nucleoids by staining with the DNA-specific dye 4,6-diamidino-2-phenylindole. A single nucleoid was observed per apicoplast, and the fluorescent spot representing the nucleoid was bright and spherical in contrast to the weak and filamentous spot representing the mitochondrial nucleoid. We also measured the DNA content of each apicoplast nucleoid by a video-intensified microscope photon-counting system and determined that the genomic copy number was at least 25, a figure over four times greater than that reported previously. Moreover, several groups of apicoplasts had significantly higher genomic copy numbers. The DNA molecules were accurately divided into two daughter apicoplasts just before nuclear division. In addition, we examined nucleoid segregation and the division apparatus using electron microscopy. However, we failed to observe nucleoid structures, suggesting that the apicoplasts are predominantly composed of nucleoid material. In addition, we observed cap structures at the termini of dividing apicoplasts, a possible plastid-dividing ring, and a microbody-like granule around the constriction. These structures may be involved in apicoplast division.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system - PD ring plastid-dividing ring     

The cell cycle of the planctomycete Gemmata obscuriglobus with respect to cell compartmentalization

Background  

Gemmata obscuriglobus is a distinctive member of the divergent phylum Planctomycetes, all known members of which are peptidoglycan-less bacteria with a shared compartmentalized cell structure and divide by a budding process. G. obscuriglobus in addition shares the unique feature that its nucleoid DNA is surrounded by an envelope consisting of two membranes forming an analogous structure to the membrane-bounded nucleoid of eukaryotes and therefore G. obscuriglobus forms a special model for cell biology. Draft genome data for G. obscuriglobus as well as complete genome sequences available so far for other planctomycetes indicate that the key bacterial cell division protein FtsZ is not present in these planctomycetes, so the cell division process in planctomycetes is of special comparative interest. The membrane-bounded nature of the nucleoid in G. obscuriglobus also suggests that special mechanisms for the distribution of this nuclear body to the bud and for distribution of chromosomal DNA might exist during division. It was therefore of interest to examine the cell division cycle in G. obscuriglobus and the process of nucleoid distribution and nuclear body formation during division in this planctomycete bacterium via light and electron microscopy.     

A mechanism for ParB-dependent waves of ParA,a protein related to DNA segregation during cell division in prokaryotes

Prokaryotic plasmids encode partitioning (par) loci involved in segregation of DNA to daughter cells at cell division. A functional fusion protein consisting of Walker-type ParA ATPase and green fluorescent protein (Gfp) oscillates back and forth within nucleoid regions with a wave period of about 20 minutes. A model is discussed which is based on cooperative non-specific binding of ParA to the nucleoid, and local ParB initiated generation of ParA oligomer degradation products, which act autocatalytically on the degradation reaction. The model yields self-initiated spontaneous pattern formation, based on Turing's mechanism, and these patterns are destroyed by the degradation products, only to initiate a new pattern at the opposite nucleoid region. A recurrent wave thus emerges. This may be a particular example of a more general class of pattern forming mechanisms, based on protein oligomerization upon a template (membranes, DNA a.o.) with resulting enhanced NTPase function in the oligomer state, which may bring the oligomer into an unstable internal state. An effector initializes destabilization of the oligomer to yield degradation products, which act as seeds for further degradation in an autocatalytic process. We discuss this mechanism in relation to recent models for MinDE oscillations in E.coli and to microtubule degradation in mitosis. The study points to an ancestral role for the presented pattern types in generating bipolarity in prokaryotes and eukaryotes.     

Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus

Progression through the Caulobacter crescentus cell cycle is coupled to a cellular differentiation program. The swarmer cell is replicationally quiescent, and DNA replication initiates at the swarmer-to-stalked cell transition. There is a very short delay between initiation of DNA replication and movement of one of the newly replicated origins to the opposite pole of the cell, indicating the absence of cohesion between the newly replicated origin-proximal parts of the Caulobacter chromosome. The terminus region of the chromosome becomes located at the invaginating septum in predivisional cells, and the completely replicated terminus regions stay associated with each other after chromosome replication is completed, disassociating very late in the cell cycle shortly before the final cell division event. Invagination of the cytoplasmic membrane occurs earlier than separation of the replicated terminus regions and formation of separate nucleoids, which results in trapping of a chromosome on either side of the cell division septum, indicating that there is not a nucleoid exclusion phenotype.     

Partitioning, Movement, and Positioning of Nucleoids in Mycoplasma capricolum

The nucleoids in Mycoplasma capricolum cells were visualized by phase-combined fluorescence microscopy of DAPI (4', 6-diamidino-2-phenylindole)-stained cells. Most growing cells in a rich medium had one or two nucleoids in a cell, and no anucleate cells were found. The nucleoids were positioned in the center in mononucleoid cells and at one-quarter and three-quarters of the cell length in binucleoid cells. These formations may have the purpose of ensuring delivery of replicated DNA to daughter cells. Internucleoid distances in binucleoid cells correlated with the cell lengths, and the relationship of DNA content to cell length showed that cell length depended on DNA content in binucleoid cells but not in mononucleoid cells. These observations suggest that cell elongation takes place in combination with nucleoid movement. Lipid synthesis was inhibited by transfer of cells to a medium lacking supplementation for lipid synthesis. The transferred cells immediately stopped dividing and elongated while regular spaces were maintained between the nucleoids for 1 h. After 1 h, the cells changed their shapes from rod-like to round, but the proportion of multinucleoid cells increased. Inhibition of protein synthesis by chloramphenicol induced nucleoid condensation and abnormal positioning, although partitioning was not inhibited. These results suggest that nucleoid partitioning does not require lipid or protein synthesis, while regular positioning requires both. When DNA replication was inhibited, the cells formed branches, and the nucleoids were positioned at the branching points. A model for the reproduction process of M. capricolum, including nucleoid migration and cell division, is discussed.     

The mitochondrial genome. The nucleoid

Mitochondrial DNA (mtDNA) in cells is organized in nucleoids containing DNA and various proteins. This review discusses questions of organization and structural dynamics of nucleoids as well as their protein components. The structures of mt-nucleoid from different organisms are compared. The currently accepted model of nucleoid organization is described and questions needing answers for better understanding of the fine mechanisms of the mitochondrial genetic apparatus functioning are discussed.     

DNA replication in mammalian cells after the action of factors of a physical, chemical or biological nature. V. The structural reorganization of replicating DNA in HeLa cells after incubation with 5-fluorodeoxyuridine

A study was made of sedimentation properties of the nucleoid (chromatin) of HeLa cells with radio- and thermostable mode of DNA synthesis induced by 5-fluorodeoxyuridine (FUdR). After the incubation of HeLa cells with FUdR (10(-6) M, 6 h or 24 h) the rate of nucleoid sedimentation was shown to rise by 40 and 25%, respectively. Maximum relaxation of the nucleoid was observed under 5 mg/ml ethidium bromide concentration in sucrose gradients. After the incubation with FUdR the nucleoid relaxes to a lesser extent, and after irradiation its response to ethidium bromide in various concentrations was similar to that of intact nucleoid, and by this property the "FUdR nucleoid" differs essentially from the irradiated "normal nucleoid". A model of chromatin structure of cells exposed to FUdR is proposed, based on the transformation of large domains in small ones, for the explanation of radioresistant DNA synthesis.     

ATP-regulated interactions between P1 ParA, ParB and non-specific DNA that are stabilized by the plasmid partition site, parS

Localization of the P1 plasmid requires two proteins, ParA and ParB, which act on the plasmid partition site, parS. ParB is a site-specific DNA-binding protein and ParA is a Walker-type ATPase with non-specific DNA-binding activity. In vivo ParA binds the bacterial nucleoid and forms dynamic patterns that are governed by the ParB-parS partition complex on the plasmid. How these interactions drive plasmid movement and localization is not well understood. Here we have identified a large protein-DNA complex in vitro that requires ParA, ParB and ATP, and have characterized its assembly by sucrose gradient sedimentation and light scattering assays. ATP binding and hydrolysis mediated the assembly and disassembly of this complex, while ADP antagonized complex formation. The complex was not dependent on, but was stabilized by, parS. The properties indicate that ParA and ParB are binding and bridging multiple DNA molecules to create a large meshwork of protein-DNA molecules that involves both specific and non-specific DNA. We propose that this complex represents a dynamic adaptor complex between the plasmid and nucleoid, and further, that this interaction drives the redistribution of partition proteins and the plasmid over the nucleoid during partition.