Upheaval in the bacterial nucleoid. An active chromosome segregation mechanism

Recent advances have completely overturned the classical view of chromosome segregation in bacteria. Far from being a passive process involving gradual separation of the chromosomes, an active, possibly mitotic-like machinery is now known to exist. Soon after the initiation of DNA replication, the newly replicated copies of the oriC region, behaving rather like eukaryotic centromeres, move rapidly apart towards opposite poles of the cell. They then determine the positions that will be taken up by the newly formed sister nucleoids when DNA replication has been completed. Thus, the gradual expansion of the diffuse nucleoid camouflages an underlying active mechanism. Several genes involved in chromosome segregation in bacteria have now been defined; their possible functions are discussed.     

Recombination and chromosome segregation

The duplication of DNA and faithful segregation of newly replicated chromosomes at cell division is frequently dependent on recombinational processes. The rebuilding of broken or stalled replication forks is universally dependent on homologous recombination proteins. In bacteria with circular chromosomes, crossing over by homologous recombination can generate dimeric chromosomes, which cannot be segregated to daughter cells unless they are converted to monomers before cell division by the conserved Xer site-specific recombination system. Dimer resolution also requires FtsK, a division septum-located protein, which coordinates chromosome segregation with cell division, and uses the energy of ATP hydrolysis to activate the dimer resolution reaction. FtsK can also translocate DNA, facilitate synapsis of sister chromosomes and minimize entanglement and catenation of newly replicated sister chromosomes. The visualization of the replication/recombination-associated proteins, RecQ and RarA, and specific genes within living Escherichia coli cells, reveals further aspects of the processes that link replication with recombination, chromosome segregation and cell division, and provides new insight into how these may be coordinated.     

The segregation of the Escherichia coli origin and terminus of replication

Escherichia coli chromosome replication forks are tethered to the cell centre. Two opposing models describe how the chromosomes segregate. In the extrusion-capture model, newly replicated DNA is fed bi-directionally from the forks toward the cell poles, forming new chromosomes in each cell half. Starting with the origins, chromosomal regions segregate away from their sisters progressively as they are replicated. The termini segregate last. In the sister chromosome cohesion model, replication produces sister chromosomes that are paired along much of their length. The origins and most other chromosomal regions remain paired until late in the replication cycle, and all segregate together. We use a combination of microscopy and flow cytometry to determine the relationship of origin and terminus segregation to the cell cycle. Origin segregation frequently follows closely after initiation, in strong support of the extrusion-capture model. The spatial disposition of the origin and terminus sequences also fits this model. Terminus segregation occurs extremely late in the cell cycle as the daughter cells separate. As the septum begins to invaginate, the termini of the completed sister chromosomes are transiently held apart at the cell centre, on opposite sides of the cell. This may facilitate the resolution of topological linkages between the chromosomes.     

SMC is recruited to oriC by ParB and promotes chromosome segregation in Streptococcus pneumoniae

Segregation of replicated chromosomes is an essential process in all organisms. How bacteria, such as the oval-shaped human pathogen Streptococcus pneumoniae, efficiently segregate their chromosomes is poorly understood. Here we show that the pneumococcal homologue of the DNA-binding protein ParB recruits S. pneumoniae condensin (SMC) to centromere-like DNA sequences (parS) that are located near the origin of replication, in a similar fashion as was shown for the rod-shaped model bacterium Bacillus subtilis. In contrast to B. subtilis, smc is not essential in S. pneumoniae, and Δsmc cells do not show an increased sensitivity to gyrase inhibitors or high temperatures. However, deletion of smc and/or parB results in a mild chromosome segregation defect. Our results show that S. pneumoniae contains a functional chromosome segregation machine that promotes efficient chromosome segregation by recruitment of SMC via ParB. Intriguingly, the data indicate that other, as of yet unknown mechanisms, are at play to ensure proper chromosome segregation in this organism.     

The two chromosomes of Vibrio cholerae are initiated at different time points in the cell cycle

The bacterium Vibrio cholerae, the cause of the diarrhoeal disease cholera, has its genome divided between two chromosomes, a feature uncommon for bacteria. The two chromosomes are of different sizes and different initiator molecules control their replication independently. Using novel methods for analysing flow cytometry data and marker frequency analysis, we show that the small chromosome II is replicated late in the C period of the cell cycle, where most of chromosome I has been replicated. Owing to the delay in initiation of chromosome II, the two chromosomes terminate replication at approximately the same time and the average number of replication origins per cell is higher for chromosome I than for chromosome II. Analysis of cell-cycle parameters shows that chromosome replication and segregation is exceptionally fast in V. cholerae. The divided genome and delayed replication of chromosome II may reduce the metabolic burden and complexity of chromosome replication by postponing DNA synthesis to the last part of the cell cycle and reducing the need for overlapping replication cycles during rapid proliferation.     

Cytological and functional aspects of telomere maintenance

The fact that eukaryotic chromosomes are linear poses a special problem for their maintenance: the natural ends of chromosomes must be distinguished from ends generated by chromosomal breakage and somehow, the chromosome ends must also be fully replicated to maintain their integrity. Telomeres, the complex structures at the ends of chromosomes are thought to be instrumental for both of these functions. However, recent insights in telomere biology suggest that these terminal structures do much more than just fulfill these two basic functions. Cytological data demonstrate that telomeres may play leading roles in chromatin organization and nuclear architecture during mitosis and meiosis. Moreover, non-functional telomeres may lead to genetic instability, a common prelude to cancer. Here, we review the basic functions of telomeres during chromosome replication and discuss the cytological aspects of telomere function during mitosis and meiosis.     

Mapping the driving forces of chromosome structure and segregation in Escherichia coli

The mechanism responsible for the accurate partitioning of newly replicated Escherichia coli chromosomes into daughter cells remains a mystery. In this article, we use automated cell cycle imaging to quantitatively analyse the cell cycle dynamics of the origin of replication (oriC) in hundreds of cells. We exploit the natural stochastic fluctuations of the chromosome structure to map both the spatial and temporal dependence of the motional bias segregating the chromosomes. The observed map is most consistent with force generation by an active mechanism, but one that generates much smaller forces than canonical molecular motors, including those driving eukaryotic chromosome segregation.     

Excess SeqA leads to replication arrest and a cell division defect in Vibrio cholerae

Although most bacteria contain a single circular chromosome, some have complex genomes, and all Vibrio species studied so far contain both a large and a small chromosome. In recent years, the divided genome of Vibrio cholerae has proven to be an interesting model system with both parallels to and novel features compared with the genome of Escherichia coli. While factors influencing the replication and segregation of both chromosomes have begun to be elucidated, much remains to be learned about the maintenance of this genome and of complex bacterial genomes generally. An important aspect of replicating any genome is the correct timing of initiation, without which organisms risk aneuploidy. During DNA replication in E. coli, newly replicated origins cannot immediately reinitiate because they undergo sequestration by the SeqA protein, which binds hemimethylated origin DNA. This DNA is already methylated by Dam on the template strand and later becomes fully methylated; aberrant amounts of Dam or the deletion of seqA leads to asynchronous replication. In our study, hemimethylated DNA was detected at both origins of V. cholerae, suggesting that these origins are also subject to sequestration. The overproduction of SeqA led to a loss of viability, the condensation of DNA, and a filamentous morphology. Cells with abnormal DNA content arose in the population, and replication was inhibited as determined by a reduced ratio of origin to terminus DNA in SeqA-overexpressing cells. Thus, excessive SeqA negatively affects replication in V. cholerae and prevents correct progression to downstream cell cycle events such as segregation and cell division.     

The bacterial nucleoid: a highly organized and dynamic structure

Recent advances in bacterial cell biology have revealed unanticipated structural and functional complexity, reminiscent of eukaryotic cells. Particular progress has been made in understanding the structure, replication, and segregation of the bacterial chromosome. It emerged that multiple mechanisms cooperate to establish a dynamic assembly of supercoiled domains, which are stacked in consecutive order to adopt a defined higher-level organization. The position of genetic loci on the chromosome is thereby linearly correlated with their position in the cell. SMC complexes and histone-like proteins continuously remodel the nucleoid to reconcile chromatin compaction with DNA replication and gene regulation. Moreover, active transport processes ensure the efficient segregation of sister chromosomes and the faithful restoration of nucleoid organization while DNA replication and condensation are in progress.     

Chromosome dynamics in multichromosome bacteria

On the basis of limited information, bacteria were once assumed to have no more than one chromosome. In the era of genomics, it has become clear that some, like eukaryotes, have more than one chromosome. Multichromosome bacteria provide opportunities to investigate how split genomes emerged, whether the individual chromosomes communicate to coordinate their replication and segregation, and what selective advantages split genomes might provide. Our current knowledge of these topics comes mostly from studies in Vibrio cholerae, which has two chromosomes, chr1 and chr2. Chr1 carries out most of the house-keeping functions and is considered the main chromosome, whereas chr2 appears to have originated from a plasmid and has acquired genes of mostly unknown origin and function. Nevertheless, unlike plasmids, chr2 replicates once and only once per cell cycle, like a bona fide chromosome. The two chromosomes replicate and segregate using separate programs, unlike eukaryotic chromosomes. They terminate replication synchronously, suggesting that there might be communication between them. Replication of the chromosomes is affected by segregation genes but in a chromosome specific fashion, a new development in the field of DNA replication control. The split genome allows genome duplication to complete in less time and with fewer replication forks, which could be beneficial for genome maintenance during rapid growth, which is the norm for V. cholerae in broth cultures and in the human host. In the latter, the expression of chr2 genes increases preferentially. Studies of chromosome maintenance in multichromosomal bacteria, although in their infancy, are already broadening our view of chromosome biology. This article is part of a Special Issue entitled: Chromatin in time and space.     

Spatial and Temporal Organization of Chromosome Duplication and Segregation in the Cyanobacterium Synechococcus elongatus PCC 7942

The spatial and temporal control of chromosome duplication and segregation is crucial for proper cell division. While this process is well studied in eukaryotic and some prokaryotic organisms, relatively little is known about it in prokaryotic polyploids such as Synechococcus elongatus PCC 7942, which is known to possess one to eight copies of its single chromosome. Using a fluorescent repressor-operator system, S. elongatus chromosomes and chromosome replication forks were tagged and visualized. We found that chromosomal duplication is asynchronous and that the total number of chromosomes is correlated with cell length. Thus, replication is independent of cell cycle and coupled to cell growth. Replication events occur in a spatially random fashion. However, once assembled, replisomes move in a constrained manner. On the other hand, we found that segregation displays a striking spatial organization in some cells. Chromosomes transiently align along the major axis of the cell and timing of alignment was correlated to cell division. This mechanism likely contributes to the non-random segregation of chromosome copies to daughter cells.     

Extended sister-chromosome catenation leads to massive reorganization of the E. coli genome

In bacteria, chromosome segregation occurs progressively from the origin to terminus within minutes of replication of each locus. Between replication and segregation, sister loci are held in an apparent cohesive state by topological links. The decatenation activity of topoisomerase IV (Topo IV) is required for segregation of replicated loci, yet little is known about the structuring of the chromosome maintained in a cohesive state. In this work, we investigated chromosome folding in cells with altered decatenation activities. Within minutes after Topo IV inactivation, massive chromosome reorganization occurs, associated with increased in contacts between nearby loci, likely trans-contacts between sister chromatids, and in long-range contacts between the terminus and distant loci. We deciphered the respective roles of Topo III, MatP and MukB when TopoIV activity becomes limiting. Topo III reduces short-range inter-sister contacts suggesting its activity near replication forks. MatP, the terminus macrodomain organizing system, and MukB, the Escherichia coli SMC, promote long-range contacts with the terminus. We propose that the large-scale conformational changes observed under these conditions reveal defective decatenation attempts involving the terminus area. Our results support a model of spatial and temporal partitioning of the tasks required for sister chromosome segregation.     

Mitotic chromosome size scaling in Xenopus

As rapid divisions without growth generate progressively smaller cells within an embryo, mitotic chromosomes must also decrease in size to permit their proper segregation, but this scaling phenomenon is poorly understood. We demonstrated previously that nuclear and spindle size scale between egg extracts of the related frog species Xenopus tropicalis and Xenopus laevis, but show here that dimensions of isolated mitotic sperm chromosomes do not differ. This is consistent with the hypothesis that chromosome scaling does not occur in early embryonic development when cell and spindles sizes are large and anaphase B segregates chromosomes long distances. To recapitulate chromosome scaling during development, we combined nuclei isolated from different stage Xenopus laevis embryos with metaphase-arrested egg extracts. Mitotic chromosomes derived from nuclei of cleaving embryos through the blastula stage were similar in size to replicated sperm chromosomes, but decreased in area approximately 50% by the neurula stage, reproducing the trend in size changes observed in fixed embryos. Allowing G2 nuclei to swell in interphase prior to mitotic condensation did not increase mitotic chromosome size, but progression through a full cell cycle in egg extract did, suggesting that epigenetic mechanisms determining chromosome size can be altered during DNA replication. Comparison of different sized mitotic chromosomes assembled in vitro provides a tractable system to elucidate underlying molecular mechanisms.     

New genes with old modus operandi. The connection between supercoiling and partitioning of DNA in Escherichia coli

The process of partitioning bacterial sister chromosomes into daughter cells seems to be distinct from chromatid segregation during eukaryotic mitosis. In Escherichia coli, partitioning starts soon after initiation of replication, when the two newly replicated oriCs move from the cell centre to quarter positions within the cell. As replication proceeds, domains of the compact, supercoiled chromosome are locally decondensed ahead of the replication fork. The nascent daughter chromosomes are recondensed and moved apart through the concerted activities of topoisomerases and the SeqA (sequestration) and MukB (chromosome condensation) proteins, all of which modulate nucleoid superhelicity. Thus, genes involved in chromosome topology, once set aside as ‘red herrings’ in the search for ‘true’ partition functions, are again recognized as being important for chromosome partitioning in E. coli.     

Nse1, Nse2, and a novel subunit of the Smc5-Smc6 complex, Nse3, play a crucial role in meiosis

The structural maintenance of chromosomes (SMC) family of proteins play key roles in the organization, packaging, and repair of chromosomes. Cohesin (Smc1+3) holds replicated sister chromatids together until mitosis, condensin (Smc2+4) acts in chromosome condensation, and Smc5+6 performs currently enigmatic roles in DNA repair and chromatin structure. The SMC heterodimers must associate with non-SMC subunits to perform their functions. Using both biochemical and genetic methods, we have isolated a novel subunit of the Smc5+6 complex, Nse3. Nse3 is an essential nuclear protein that is required for normal mitotic chromosome segregation and cellular resistance to a number of genotoxic agents. Epistasis with Rhp51 (Rad51) suggests that like Smc5+6, Nse3 functions in the homologous recombination based repair of DNA damage. We previously identified two non-SMC subunits of Smc5+6 called Nse1 and Nse2. Analysis of nse1-1, nse2-1, and nse3-1 mutants demonstrates that they are crucial for meiosis. The Nse1 mutant displays meiotic DNA segregation and homologous recombination defects. Spore viability is reduced by nse2-1 and nse3-1, without affecting interhomolog recombination. Finally, genetic interactions shared by the nse mutants suggest that the Smc5+6 complex is important for replication fork stability.     

Chromosome segregation in<Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis, a Gram-positive bacterium commonly found in soil, is an excellent model organism for the study of basic cell processes, such as cell division and cell differentiation, called sporulation. In B. subtilis the essential genetic information is carried on a single circular chromosome, the correct segregation of which is crucial for both vegetative growth and sporulation. The proper completion of life cycle requires each daughter cell to obtain identical genetic information. The consequences of inaccurate chromosome segregation can lead to formation of anucleate cells, cells with two chromosomes, or cells with incomplete chromosomes. Although bacteria miss the classical eukaryotic mitotic apparatus, the chromosome segregation is undeniably an active process tightly connected to other cell processes as DNA replication and compaction. To fully understand the chromosome segregation, it is necessary to study this process in a wider context and to examine the role of different proteins at various cell life cycle stages. The life cycle of B. subtilis is characteristic by its specific cell differentiation process where, two slightly different segregation mechanisms exist, specialized in vegetative growth and in sporulation.     

Studies of mammalian chromosome replication

Sister chromatids of metaphase chromosomes can be differentially stained if the cells have replicated their DNA semiconservatively for two cell cycles in a medium containing 5-bromodeoxyuridine (BrdU). When prematurely condensed chromosomes (PCC) are induced in cells during the second S phase after BrdU is added to the medium, the replicated chromosome segments show sister chromatid differential (SCD) staining. Employing this PCC-SCD system on synchronous and asynchronous Chinese hamster ovary (CHO) cells, we have demonstrated that the replication patterns of the CHO cells can be categorized into G₁/S, early, early-mid, mid-late, and late S phase patterns according to the amount of replicated chromosomes. During the first 4 h of the S phase, the replication patterns show SCD staining in chains of small chromosome segments. The amount of replicated chromosomes increase during the mid-late and late S categories (last 4 h). Significantly, small SCD segments are also present during these late intervals of the S phase. Measurements of these replicated segments indicate the presence of characteristic chromosome fragment sizes between 0.2 to 1.2 m in all S phase cells except those at G₁/S which contain no SCD fragments. These small segments are operationally defined as chromosome replicating units or chromosomal replicons. They are interpreted to be composed of clusters of molecular DNA replicons. The larger SCD segments in the late S cells may arise by the joining of adjacent chromosomal replicons. Further application of this PCC-SCD method to study the chromosome replication process of two other rodents, Peromyscus eremicus and Microtus agrestis, with peculiar chromosomal locations of heterochromatin has demonstrated an ordered sequence of chromosome replication. The euchromatin and heterochromatin of the two species undergo two separate sequences of decondensation, replication, and condensation during the early-mid and mid-late intervals respectively of the S phase. Similar-sized chromosomal replicons are present in both types of chromatin. These data suggest that mammalian chromosomes are replicated in groups of replicating units, or chromosomal replicons, along their lengths. The organization and structure of these chromosomal replicons with respect to those of the interphase nucleus and metaphase chromosomes are discussed.     

Evidence for Two Different Regulatory Mechanisms Linking Replication and Segregation of Vibrio cholerae Chromosome II

Understanding the mechanisms that coordinate replication initiation with subsequent segregation of chromosomes is an important biological problem. Here we report two replication-control mechanisms mediated by a chromosome segregation protein, ParB2, encoded by chromosome II of the model multichromosome bacterium, Vibrio cholerae. We find by the ChIP-chip assay that ParB2, a centromere binding protein, spreads beyond the centromere and covers a replication inhibitory site (a 39-mer). Unexpectedly, without nucleation at the centromere, ParB2 could also bind directly to a related 39-mer. The 39-mers are the strongest inhibitors of chromosome II replication and they mediate inhibition by binding the replication initiator protein. ParB2 thus appears to promote replication by out-competing initiator binding to the 39-mers using two mechanisms: spreading into one and direct binding to the other. We suggest that both these are novel mechanisms to coordinate replication initiation with segregation of chromosomes.     

SMC and the bactofilin/PadC scaffold have distinct yet redundant functions in chromosome segregation and organization in Myxococcus xanthus

In bacteria, ParABS systems and structural maintenance of chromosome (SMC) condensin-like complexes are important for chromosome segregation and organization. The rod-shaped Myxococcus xanthus cells have a unique chromosome arrangement in which a scaffold composed of the BacNOP bactofilins and PadC positions the essential ParB∙parS segregation complexes and the DNA segregation ATPase ParA in the subpolar regions. We identify the Smc and ScpAB subunits of the SMC complex in M. xanthus and demonstrate that SMC is conditionally essential, with Δsmc or ΔscpAB mutants being temperature sensitive. Inactivation of SMC caused defects in chromosome segregation and organization. Lack of the BacNOP/PadC scaffold also caused chromosome segregation defects but this scaffold is not essential for viability. Inactivation of SMC was synthetic lethal with lack of the BacNOP/PadC scaffold. Lack of SMC interfered with formation of the BacNOP/PadC scaffold while lack of this scaffold did not interfere with chromosome association by SMC. Altogether, our data support that three systems function together to enable chromosome segregation in M. xanthus. ParABS constitutes the basic and essential machinery. SMC and the BacNOP/PadC scaffold have different yet redundant roles in chromosome segregation with SMC supporting individualization of daughter chromosomes and BacNOP/PadC making the ParABS system operate more robustly.