Bacterial chromosomes are organised as two replichores of opposite polarity that coincide with the replication arms from the ori to the ter region. Here, we investigated the effects of asymmetry in replichore organisation in Escherichia coli. We show that large chromosome inversions from the terminal junction of the replichores disturb the ongoing post-replicative events, resulting in inhibition of both cell division and cell elongation. This is accompanied by alterations of the segregation pattern of loci located at the inversion endpoints, particularly of the new replichore junction. None of these defects is suppressed by restoration of termination of replication opposite oriC, indicating that they are more likely due to the asymmetry of replichore polarity than to asymmetric replication. Strikingly, DNA translocation by FtsK, which processes the terminal junction of the replichores during cell division, becomes essential in inversion-carrying strains. Inactivation of the FtsK translocation activity leads to aberrant cell morphology, strongly suggesting that it controls membrane synthesis at the division septum. Our results reveal that FtsK mediates a reciprocal control between processing of the replichore polarity junction and cell division.
In most bacteria, chromosomes consist of a single replication unit. Replication initiates at a single ori and terminates in the diametrically opposite zone, ter, defining two replication arms. These are also called replichores to account for base composition and sequence motif polarity biases from ori to ter. This organisation as symmetrical replichores is conserved in bacteria. Here, we have investigated the effect of replichore asymmetry in E. coli. We show that it blocks the cell cycle at a step posterior to chromosome replication. This phenotype is not corrected by restoration of the termination of replication opposite ori, strongly suggesting that it is due to the asymmetry of replichore polarity. FtsK, a DNA-translocase associated with the division septum that processes the terminal junction of replichore polarity, is essential for growth and for the controlled blockage of cell growth in cells with asymmetric replichores. These results reveal a reciprocal control between the processing of the terminal junction of the replichores by FtsK and the progression of the cell cycle.
Chromosome organizations of related bacterial genera are well conserved despite a very long divergence period. We have assessed the forces limiting bacterial genome plasticity in Escherichia coli by measuring the respective effect of altering different parameters, including DNA replication, compositional skew of replichores, coordination of gene expression with DNA replication, replication-associated gene dosage, and chromosome organization into macrodomains. Chromosomes were rearranged by large inversions. Changes in the compositional skew of replichores, in the coordination of gene expression with DNA replication or in the replication-associated gene dosage have only a moderate effect on cell physiology because large rearrangements inverting the orientation of several hundred genes inside a replichore are only slightly detrimental. By contrast, changing the balance between the two replication arms has a more drastic effect, and the recombinational rescue of replication forks is required for cell viability when one of the chromosome arms is less than half than the other one. Macrodomain organization also appears to be a major factor restricting chromosome plasticity, and two types of inverted configurations severely affect the cell cycle. First, the disruption of the Ter macrodomain with replication forks merging far from the normal replichore junction provoked chromosome segregation defects. The second major problematic configurations resulted from inversions between Ori and Right macrodomains, which perturb nucleoid distribution and early steps of cytokinesis. Consequences for the control of the bacterial cell cycle and for the evolution of bacterial chromosome configuration are discussed.
Genomic analyses have revealed that bacterial genomes are dynamic entities that evolve through various processes including intrachromosome genetic rearrangements, gene duplication, and gene loss or acquisition by gene transfer. Nevertheless, comparison of bacterial chromosomes from related genera revealed a conservation of genetic organization. Most bacterial genomes are circular molecules, and DNA replication proceeds bidirectionally from a single origin to an opposite region where replication forks meet. The replication process imprints the bacterial chromosome because initiation and termination at defined loci result in strand biases due to the mutational differences occurring during leading and lagging strands synthesis. We analyze the strength of different parameters that may limit genome plasticity. We show that the preferential positioning of essential genes on the leading strand, the proximity of genes involved in transcription and translation to the origin of replication on the leading strand, and the presence of biased motifs along the replichores operate only as long-term positive selection determinants. By contrast, selection operates to maintain replication arms of similar lengths. Finally, we demonstrate that spatial structuring of the chromosome impedes strongly genome plasticity. Genetic evidence supports the presence of two steps in the cell cycle controlled by the spatial organization of the chromosome.
The mechanism of Escherichia coli chromosome segregation remains elusive. We present results on the simultaneous tracking of segregation of multiple loci in the ori region of the chromosome in cells growing under conditions in which a single round of replication is initiated and completed in the same generation. Loci segregated as expected for progressive replication-segregation from oriC, with markers placed symmetrically on either side of oriC segregating to opposite cell halves at the same time, showing that sister locus cohesion in the origin region is local rather than extensive. We were unable to observe any influence on segregation of the proposed centromeric site, migS, or indeed any other potential cis-acting element on either replication arm (replichore) in the AB1157 genetic background. Site-specific inhibition of replication close to oriC on one replichore did not prevent segregation of loci on the other replichore. Inhibition of RNA synthesis and inhibition of the dynamic polymerization of the actin homolog MreB did not affect ori and bulk chromosome segregation.
Cells closely coordinate cell division with chromosome replication and segregation; however, the mechanisms responsible for this coordination still remain largely unknown. Here, we analyzed the spatial arrangement and temporal dynamics of the 9.1 Mb circular chromosome in the rod-shaped cells of Myxococcus xanthus. For chromosome segregation, M. xanthus uses a parABS system, which is essential, and lack of ParB results in chromosome segregation defects as well as cell divisions over nucleoids and the formation of anucleate cells. From the determination of the dynamic subcellular location of six genetic loci, we conclude that in newborn cells ori, as monitored following the ParB/parS complex, and ter regions are localized in the subpolar regions of the old and new cell pole, respectively and each separated from the nearest pole by approximately 1 µm. The bulk of the chromosome is arranged between the two subpolar regions, thus leaving the two large subpolar regions devoid of DNA. Upon replication, one ori region remains in the original subpolar region while the second copy segregates unidirectionally to the opposite subpolar region followed by the rest of the chromosome. In parallel, the ter region of the mother chromosome relocates, most likely passively, to midcell, where it is replicated. Consequently, after completion of replication and segregation, the two chromosomes show an ori-ter-ter-ori arrangement with mirror symmetry about a transverse axis at midcell. Upon completion of segregation of the ParB/parS complex, ParA localizes in large patches in the DNA-free subpolar regions. Using an Ssb-YFP fusion as a proxy for replisome localization, we observed that the two replisomes track independently of each other from a subpolar region towards ter. We conclude that M. xanthus chromosome arrangement and dynamics combine features from previously described systems with new features leading to a novel spatiotemporal arrangement pattern.
Work on several model organisms has revealed that bacterial chromosomes are spatially highly arranged throughout the cell cycle in a dynamic yet reproducible manner. These analyses have also demonstrated significant differences between chromosome arrangements and dynamics in different bacterial species. Here, we show that the Myxococcus xanthus genome is arranged about a longitudinal axis with ori in a subpolar region and ter in the opposite subpolar region. Upon replication, one ori remains at the original subpolar region while the second copy in a directed and parABS-dependent manner segregates to the opposite subpolar region followed by the rest of the chromosome. In parallel, ter relocates from a subpolar region to midcell. Replication involves replisomes that track independently of each other from the ori-containing subpolar region towards ter. Moreover, we find that the parABS system is essential in M. xanthus and ParB depletion not only results in chromosome segregation defects but also in cell division defects with cell divisions occurring over nucleoids. In M. xanthus the dynamics of chromosome replication and segregation combine features from previously described systems leading to a novel spatiotemporal arrangement pattern.
The circular Escherichia coli chromosome is organized by bidirectional replication into two equal left and right arms (replichores). Each arm occupies a separate cell half, with the origin of replication (oriC) at mid-cell. E. coli MukBEF belongs to the ubiquitous family of SMC protein complexes that play key roles in chromosome organization and processing. In mukBEF mutants, viability is restricted to low temperature with production of anucleate cells, reflecting chromosome segregation defects. We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole. Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells. Furthermore, we show that MukB and the oriC region generally colocalize throughout the cell cycle, even when oriC localization is aberrant. We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region.
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.
Cell division in Escherichia coli begins with the assembly of FtsZ proteins into a ring-like structure, the Z-ring. Remarkably, the Z-ring localizes with very high precision at midcell. Currently, two molecular systems, nucleoid occlusion and the Min system, are known to localize the Z-ring. Here, we explore whether there are additional divisome localization systems in E. coli. Using quantitative fluorescence imaging, we show that slow growing cells lacking both known positioning systems continue to divide accurately at midcell. We find that the terminus region of the chromosome moves first to mid-cell where it functions as a positional landmark for the subsequent localization of the Z-ring. Furthermore, we provide evidence that this divisome positioning system involves MatP, ZapB, and ZapA proteins. Our work shows that E. coli can divide without the canonical mechanisms for localizing its cytokinetic ring. 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.
Analogously to chromosome cohesion in eukaryotes, newly replicated DNA in E. coli is held together by inter-sister linkages before partitioning into daughter nucleoids. In both cases, initial joining is apparently mediated by DNA catenation, in which replication-induced positive supercoils diffuse behind the fork, causing newly replicated duplexes to twist around each other. Type-II topoisomerase-catalyzed sister separation is delayed by the well-characterized cohesin complex in eukaryotes, but cohesion control in E. coli is not currently understood. We report that the abundant fork tracking protein SeqA is a strong positive regulator of cohesion, and is responsible for markedly prolonged cohesion observed at “snap” loci. Epistasis analysis suggests that SeqA stabilizes cohesion by antagonizing Topo IV-mediated sister resolution, and possibly also by a direct bridging mechanism. We show that variable cohesion observed along the E. coli chromosome is caused by differential SeqA binding, with oriC and snap loci binding disproportionally more SeqA. We propose that SeqA binding results in loose inter-duplex junctions that are resistant to Topo IV cleavage. Lastly, reducing cohesion by genetic manipulation of Topo IV or SeqA resulted in dramatically slowed sister locus separation and poor nucleoid partitioning, indicating that cohesion has a prominent role in chromosome segregation.
Sister chromosome cohesion in eukaryotes maintains genome stability by mediating chromosome segregation and homologous recombination-dependent DNA repair. Here we have investigated the mechanism of cohesion regulation in E. coli by measuring cohesion timing in a broad set of candidate mutant strains. Using a sensitive DNA replication and segregation assay, we show that cohesion is controlled by the conserved DNA decatenation enzyme Topo IV and the abundant DNA binding protein SeqA. Results suggest that cohesion occurs in E. coli by twisting of replicated duplexes around each other behind the replication fork, and immediate resolution of cohered regions is blocked by SeqA. SeqA binds to a sliding 300–400 kb window of hemimethylated DNA behind the fork, and regions binding more SeqA experience longer cohesion periods. An analogous decatenation inhibition function is carried out by the cohesin complex in eukaryotes, indicating that cells mediate pairing and separation of replicated DNA by a conserved mechanism. In both cases, mismanaged cohesion results in failed or inefficient chromosome segregation.
The organization of the Escherichia coli chromosome into a ring composed of four macrodomains and two less-structured regions influences the segregation of sister chromatids and the mobility of chromosomal DNA. The structuring of the terminus region (Ter) into a macrodomain relies on the interaction of the protein MatP with a 13-bp target called matS repeated 23 times in the 800-kb-long domain. Here, by using a new method that allows the transposition of any chromosomal segment at a defined position on the genetic map, we reveal a site-specific system that restricts to the Ter region a constraining process that reduces DNA mobility and delays loci segregation. Remarkably, the constraining process is regulated during the cell cycle and occurs only when the Ter MD is associated with the division machinery at mid-cell. The change of DNA properties does not rely on the presence of a trans-acting mechanism but rather involves a cis-effect acting at a long distance from the Ter region. Two specific 12-bp sequences located in the flanking Left and Right macrodomains and a newly identified protein designated YfbV conserved with MatP through evolution are required to impede the spreading of the constraining process to the rest of the chromosome. Our results unravel a site-specific system required to restrict to the Ter region the consequences of anchoring the Ter MD to the division machinery.
The large size of genomes compared to cell dimensions imposes an extensive compaction of chromosomes compatible with various processes of DNA metabolism, such as gene expression or segregation of the genetic information. Most bacterial genomes are circular molecules, and DNA replication proceeds bidirectionally from a single origin to an opposite region where replication forks meet. In the bacteria Escherichia coli, the long-range organization of the chromosome relies on the presence of mechanisms that structure large regions called macrodomains. The macrodomain containing the terminus of replication is structured by a specific organization system involving the binding of the protein MatP to 23 matS sites scattered over the 800-kb-long Ter region. In this report, we describe a site-specific insulation system that restricts to the Ter region the consequences of the mechanism structuring the Ter macrodomain. We identified two 12-bp sequences flanking the Ter macrodomain and one protein that are required to isolate the Ter region from the other parts of the chromosome.
The Escherichia coli SeqA protein forms complexes with new, hemimethylated DNA behind replication forks and is important for successful replication during rapid growth. Here, E. coli cells with two simultaneously replicating chromosomes (multifork DNA replication) and YFP tagged SeqA protein was studied. Fluorescence microscopy showed that in the beginning of the cell cycle cells contained a single focus at midcell. The focus was found to remain relatively immobile at midcell for a period of time equivalent to the duration of origin sequestration. Then, two abrupt relocalization events occurred within 2–6 minutes and resulted in SeqA foci localized at each of the cell’s quarter positions. Imaging of cells containing an additional fluorescent tag in the origin region showed that SeqA colocalizes with the origin region during sequestration. This indicates that the newly replicated DNA of first one chromosome, and then the other, is moved from midcell to the quarter positions. At the same time, origins are released from sequestration. Our results illustrate that newly replicated sister DNA is segregated pairwise to the new locations. This mode of segregation is in principle different from that of slowly growing bacteria where the newly replicated sister DNA is partitioned to separate cell halves and the decatenation of sisters a prerequisite for, and possibly a mechanistic part of, segregation.
A prevalent view of DNA replication has been that it is carried out in fixed “replication factories.” By tracking the progression of sister replication forks with respect to genetic loci in live Escherichia coli, we show that at initiation replisomes assemble at replication origins irrespective of where the origins are positioned within the cell. Sister replisomes separate and move to opposite cell halves shortly after initiation, migrating outwards as replication proceeds and both returning to midcell as replication termination approaches. DNA polymerase is maintained at stalled replication forks, and over short intervals of time replisomes are more dynamic than genetic loci. The data are inconsistent with models in which replisomes associated with sister forks act within a fixed replication factory. We conclude that independent replication forks follow the path of the compacted chromosomal DNA, with no structure other than DNA anchoring the replisome to any particular cellular region.
DNA; CELLCYCLE; MICROBIO
The Escherichia coli
structural maintenance of chromosome (SMC) complex, MukBEF, and topoisomerase IV (TopoIV) interact in vitro through a direct contact between the MukB dimerization hinge and the C-terminal domain of ParC, the catalytic subunit of TopoIV. The interaction stimulates catalysis by TopoIV in vitro. Using live-cell quantitative imaging, we show that MukBEF directs TopoIV to ori, with fluorescent fusions of ParC and ParE both forming cellular foci that colocalize with those formed by MukBEF throughout the cell cycle and in cells unable to initiate DNA replication. Removal of MukBEF leads to loss of fluorescent ParC/ParE foci. In the absence of functional TopoIV, MukBEF forms multiple foci that are distributed uniformly throughout the nucleoid, whereas multiple catenated oris cluster at midcell. Once functional TopoIV is restored, the decatenated oris segregate to positions that are largely coincident with the MukBEF foci, thereby providing support for a mechanism by which MukBEF acts in chromosome segregation by positioning newly replicated and decatenated oris. Additional evidence for such a mechanism comes from the observation that in TopoIV-positive (TopoIV+) cells, newly replicated oris segregate rapidly to the positions of MukBEF foci. Taken together, the data implicate MukBEF as a key component of the DNA segregation process by acting in concert with TopoIV to promote decatenation and positioning of newly replicated oris.
Mechanistic understanding of how newly replicated bacterial chromosomes are segregated prior to cell division is incomplete. In this work, we provide in vivo experimental support for the view that topoisomerase IV (TopoIV), which decatenates newly replicated sister duplexes as a prelude to successful segregation, is directed to the replication origin region of the Escherichia coli chromosome by the SMC (structural maintenance of chromosome) complex, MukBEF. We provide in vivo data that support the demonstration in vitro that the MukB interaction with TopoIV stimulates catalysis by TopoIV. Finally, we show that MukBEF directs the normal positioning of sister origins after their replication and during their segregation. Overall, the data support models in which the coordinate and sequential action of TopoIV and MukBEF plays an important role during bacterial chromosome segregation.
The terminus region of the Escherichia coli chromosome is the scene of frequent homologous recombination. This can be demonstrated by formation of deletions between directly repeated sequences which flank a genetic marker whose loss can be easily detected. We report here that terminal recombination events are restricted to a relatively large terminal recombination zone (TRZ). On one side of the TRZ, the transition from the region with a high excision rate to the normal (low) excision rates of the rest of the chromosome occurs along a DNA stretch of less than 1 min. No specific border of this domain has been defined. To identify factors inducing terminal recombination, we examined its relation to two other phenomena affecting the same region, site-specific recombination at the dif locus and site-specific replication pausing. Both the location and the efficiency of terminal recombination remained unchanged after inactivation of the dif-specific recombination system. Similarly, inactivation of site-specific replication pausing or displacement of the replication fork trap so that termination occurs about 200 kb away from the normal region had no clear effect on this phenomenon. Therefore, terminal recombination is not a direct consequence of either dif-specific recombination or replication termination. Furthermore, deletions encompassing the wild-type TRZ do not eliminate hyperrecombination. Terminal recombination therefore cannot be attributed to the activity of some unique sequence of the region. A possible explanation of terminal hyperrecombination involves nucleoid organization and its remodeling after replication: we propose that post replicative reconstruction of the nucleoid organization results in a displacement of the catenation links between sister chromosomes to the last chromosomal domain to be rebuilt. Unrelated to replication termination, this process would facilitate interactions between the catenated molecules and would make the domain highly susceptible to recombination between sister chromosomes.
The replication terminus region (Ter) of the unique chromosome of most bacteria locates at mid-cell at the time of cell division. In several species, this localization participates in the necessary coordination between chromosome segregation and cell division, notably for the selection of the division site, the licensing of the division machinery assembly and the correct alignment of chromosome dimer resolution sites. The genome of Vibrio cholerae, the agent of the deadly human disease cholera, is divided into two chromosomes, chrI and chrII. Previous fluorescent microscopy observations suggested that although the Ter regions of chrI and chrII replicate at the same time, chrII sister termini separated before cell division whereas chrI sister termini were maintained together at mid-cell, which raised questions on the management of the two chromosomes during cell division. Here, we simultaneously visualized the location of the dimer resolution locus of each of the two chromosomes. Our results confirm the late and early separation of chrI and chrII Ter sisters, respectively. They further suggest that the MatP/matS macrodomain organization system specifically delays chrI Ter sister separation. However, TerI loci remain in the vicinity of the cell centre in the absence of MatP and a genetic assay specifically designed to monitor the relative frequency of sister chromatid contacts during constriction suggest that they keep colliding together until the very end of cell division. In contrast, we found that even though it is not able to impede the separation of chrII Ter sisters before septation, the MatP/matS macrodomain organization system restricts their movement within the cell and permits their frequent interaction during septum constriction.
The genome of Vibrio cholerae is divided into two circular chromosomes, chrI and chrII. ChrII is derived from a horizontally acquired mega-plasmid, which raised questions on the necessary coordination of the processes that ensure its segregation with the cell division cycle. Here, we show that the MatP/matS macrodomain organization system impedes the separation of sister copies of the terminus region of chrI before the initiation of septum constriction. In its absence, however, chrI sister termini remain sufficiently close to mid-cell to be processed by the FtsK cell division translocase. In contrast, we show that MatP cannot impede the separation of chrII sister termini before constriction. However, it restricts their movements within the cell, which allows for their processing by FtsK at the time of cell division. These results suggest that multiple redundant factors, including MatP in the enterobacteriaceae and the Vibrios, ensure that sister copies of the terminus region of bacterial chromosomes remain sufficiently close to mid-cell to be processed by FtsK.
The effects of quinolone antibiotics on nucleoid segregation in growing Escherichia coli were examined by using fleroxacin (Ro 23-6240, AM 833) as a prototype compound. At levels that were close to its MIC and induced growth arrest and filamentation, fleroxacin caused large nucleoids to appear in midcell, suggesting inhibition of nucleoid segregation. With increasing fleroxacin concentrations, nucleoids became progressively smaller, suggesting inhibition of DNA replication. Removal of fleroxacin restored normal cell and nucleoid morphology in filaments with large nucleoids but not in filaments with small nucleoids. The results are consistent with inhibition of chromosome decatenation at low quinolone concentrations (bacteriostatic effect) and DNA supercoiling at high concentrations (bactericidal effect).
We followed the position of the replication complex in the pathogenic bacterium Helicobacter pylori using antibodies raised against the single-stranded DNA binding protein (HpSSB) and the replicative helicase (HpDnaB). The position of the replication origin, oriC, was also localized in growing cells by fluorescence in situ hybridization (FISH) with fluorescence-labeled DNA sequences adjacent to the origin. The replisome assembled at oriC near one of the cell poles, and the two forks moved together toward the cell center as replication progressed in the growing cell. Termination and resolution of the forks occurred near midcell, on one side of the septal membrane. The duplicated copies of oriC did not separate until late in elongation, when the daughter chromosomes segregated into bilobed nucleoids, suggesting sister chromatid cohesion at or near the oriC region. Components of the replication machinery, viz., HpDnaB and HpDnaG (DNA primase), were found associated with the cell membrane. A model for the assembly and location of the H. pylori replication machinery during chromosomal duplication is presented.
In Escherichia coli, assembly of the FtsZ ring (Z ring) at the cell division site is negatively regulated by the nucleoid in a phenomenon called nucleoid occlusion (NO). Previous studies have indicated that chromosome packing plays a role in NO, as mukB mutants grown in rich medium often exhibit FtsZ rings on top of diffuse, unsegregated nucleoids. To address the potential role of overall nucleoid structure on NO, we investigated the effects of disrupting chromosome structure on Z-ring positioning. We found that NO was mostly normal in cells with inactivated DNA gyrase or in mukB-null mutants lacking topA, although some suppression of NO was evident in the latter case. Previous reports suggesting that transcription, translation, and membrane insertion of proteins (“transertion”) influence nucleoid structure prompted us to investigate whether disruption of these activities had effects on NO. Blocking transcription caused nucleoids to become diffuse, and FtsZ relocalized to multiple bands on top of these nucleoids, biased towards midcell. This suggested that these diffuse nucleoids were defective in NO. Blocking translation with chloramphenicol caused characteristic nucleoid compaction, but FtsZ rarely assembled on top of these centrally positioned nucleoids. This suggested that NO remained active upon translation inhibition. Blocking protein secretion by thermoinduction of a secA(Ts) strain caused a chromosome segregation defect similar to that in parC mutants, and NO was active. Although indirect effects are certainly possible with these experiments, the above data suggest that optimum NO activity may require specific organization and structure of the nucleoid.
Chromosome and replisome dynamics were examined in synchronized E. coli cells undergoing a eukaryotic-like cell cycle. Sister chromosomes remain tightly colocalized for much of S phase and then separate, in a single coordinate transition. Origin and terminus regions behave differently, as functionally independent domains. During separation, sister loci move far apart and the nucleoid becomes bilobed. Origins and terminus regions also move. We infer that sisters are initially linked and that loss of cohesion triggers global chromosome reorganization. This reorganization creates the 2-fold symmetric, ter-in/ori-out conformation which, for E. coli, comprises sister segregation. Analogies with eukaryotic prometaphase suggest that this could be a primordial segregation mechanism to which microtubule-based processes were later added. We see no long-lived replication “factory”; replication initiation timing does not covary with cell mass, and we identify changes in nucleoid position and state that are tightly linked to cell division. We propose that cell division licenses the next round of replication initiation via these changes.
We know that stochastic feedback impairs children’s associative stimulus–response (S–R) learning (Crone et al., 2004a; Eppinger et al., 2009), but the impact of stochastic feedback on sequence learning that involves deductive reasoning has not been not tested so far. In the current study, 8- to 11-year-old children (N = 171) learned a sequence of four left and right button presses, LLRR, RRLL, LRLR, RLRL, LRRL, and RLLR, which needed to be deduced from feedback because no directional cues were given. One group of children experienced consistent feedback only (deterministic feedback, 100% correct). In this condition, green feedback on the screen indicated that the children had been right when they were right, and red feedback indicated that the children had been wrong when they were wrong. Another group of children experienced inconsistent feedback (stochastic feedback, 85% correct, 15% false), where in some trials, green feedback on the screen could signal that children were right when in fact they were wrong, and red feedback could indicate that they were wrong when in fact they had been right. Independently of age, children’s sequence learning in the stochastic condition was initially much lower than in the deterministic condition, but increased gradually and improved with practice. Responses toward positive vs. negative feedback varied with age. Children were increasingly able to understand that they could have been wrong when feedback indicated they were right (self-reflection), but they remained unable to understand that they could have been right when feedback indicated they were wrong (self-assertion).
sequence learning; learning under uncertainty; stochastic feedback; positive and negative feedback
Condensin—an SMC-kleisin complex—is essential for efficient segregation of sister chromatids in eukaryotes [1–4]. In Escherichia coli and Bacillus subtilis, deletion of condensin subunits results in severe growth phenotypes and the accumulation of cells lacking nucleoids [5, 6]. In many other bacteria and under slow growth conditions, however, the reported phenotypes are much milder or virtually absent [7–10]. This raises the question of what role prokaryotic condensin might play during chromosome segregation under various growth conditions. In B. subtilis and Streptococcus pneumoniae, condensin complexes are enriched on the circular chromosome near the single origin of replication by ParB proteins bound to parS sequences [11, 12]. Using conditional alleles of condensin in B. subtilis, we demonstrate that depletion of its activity results in an immediate and severe defect in the partitioning of replication origins. Multiple copies of the chromosome remain unsegregated at or near the origin of replication. Surprisingly, the growth and chromosome segregation defects in rich medium are suppressed by a reduction of replication fork velocity but not by partial inhibition of translation or transcription. Prokaryotic condensin likely prevents the formation of sister DNA interconnections at the replication fork or promotes their resolution behind the fork.
•Smc-ScpAB inactivation causes a severe chromosome segregation defect in B. subtilis•Replication origins remain interconnected in the absence of prokaryotic condensin•Defects in chromosome segregation are highly dependent on growth conditions•Reduction of replication fork velocity rescues segregation of replication origins
Gruber et al. show that conditional inactivation of prokaryotic condensin in B. subtilis results in immediate and severe defects in chromosome segregation under conditions promoting fast growth. The separation of replication origins is blocked in the absence of Smc-ScpAB but can be rescued by artificial reduction of replication fork speed.
The bacterial SeqA protein binds to hemi-methylated GATC sequences that arise in newly synthesized DNA upon passage of the replication machinery. In Escherichia coli K-12, the single replication origin oriC is a well-characterized target for SeqA, which binds to multiple hemi-methylated GATC sequences immediately after replication has initiated. This sequesters oriC, thereby preventing reinitiation of replication. However, the genome-wide DNA binding properties of SeqA are unknown, and hence, here, we describe a study of the binding of SeqA across the entire Escherichia coli K-12 chromosome, using chromatin immunoprecipitation in combination with DNA microarrays. Our data show that SeqA binding correlates with the frequency and spacing of GATC sequences across the entire genome. Less SeqA is found in highly transcribed regions, as well as in the ter macrodomain. Using synchronized cultures, we show that SeqA distribution differs with the cell cycle. SeqA remains bound to some targets after replication has ceased, and these targets locate to genes encoding factors involved in nucleotide metabolism, chromosome replication, and methyl transfer.
DNA replication in bacteria is a highly regulated process. In many bacteria, a protein called SeqA plays a key role by binding to newly replicated DNA. Thus, at the origin of DNA replication, SeqA binding blocks premature reinitiation of replication rounds. Although most investigators have focused on the role of SeqA at replication origins, it has long been suspected that SeqA has a more pervasive role. In this study, we describe how we have been able to identify scores of targets, across the entire Escherichia coli chromosome, to which SeqA binds. Using synchronously growing cells, we show that the distribution of SeqA between these targets alters as replication of the chromosome progresses. This suggests that sequential changes in SeqA distribution orchestrate a program of gene expression that ensures coordinated DNA replication and cell division.
Genome structure variation has profound impacts on phenotype in organisms ranging from microbes to humans, yet little is known about how natural selection acts on genome arrangement. Pathogenic bacteria such as Yersinia pestis, which causes bubonic and pneumonic plague, often exhibit a high degree of genomic rearrangement. The recent availability of several Yersinia genomes offers an unprecedented opportunity to study the evolution of genome structure and arrangement. We introduce a set of statistical methods to study patterns of rearrangement in circular chromosomes and apply them to the Yersinia. We constructed a multiple alignment of eight Yersinia genomes using Mauve software to identify 78 conserved segments that are internally free from genome rearrangement. Based on the alignment, we applied Bayesian statistical methods to infer the phylogenetic inversion history of Yersinia. The sampling of genome arrangement reconstructions contains seven parsimonious tree topologies, each having different histories of 79 inversions. Topologies with a greater number of inversions also exist, but were sampled less frequently. The inversion phylogenies agree with results suggested by SNP patterns. We then analyzed reconstructed inversion histories to identify patterns of rearrangement. We confirm an over-representation of “symmetric inversions”—inversions with endpoints that are equally distant from the origin of chromosomal replication. Ancestral genome arrangements demonstrate moderate preference for replichore balance in Yersinia. We found that all inversions are shorter than expected under a neutral model, whereas inversions acting within a single replichore are much shorter than expected. We also found evidence for a canonical configuration of the origin and terminus of replication. Finally, breakpoint reuse analysis reveals that inversions with endpoints proximal to the origin of DNA replication are nearly three times more frequent. Our findings represent the first characterization of genome arrangement evolution in a bacterial population evolving outside laboratory conditions. Insight into the process of genomic rearrangement may further the understanding of pathogen population dynamics and selection on the architecture of circular bacterial chromosomes.
Whole-genome sequencing has revealed that organisms exhibit extreme variability in chromosome structure. One common type of chromosome structure variation is genome arrangement variation: changes in the ordering of genes on the chromosome. Not only do we find differences in genome arrangement across species, but in some organisms, members of the same species have radically different genome arrangements. We studied the evolution of genome arrangement in pathogenic bacteria from the genus Yersinia. The Yersinia exhibit substantial variation in genome arrangement both within and across species. We reconstructed the history of genome rearrangement by inversion in a group of eight Yersinia, and we statistically quantified the forces shaping their genome arrangement evolution. In particular, we discovered an excess of rearrangement activity near the origin of chromosomal replication and found evidence for a preferred configuration for the relative orientations of the origin and terminus of replication. We also found real inversions to be significantly shorter than expected. Finally, we discovered that no single reconstruction of inversion history is parsimonious with respect to the total number of inversion mutations, but on average, reconstructed genome arrangements favor “balanced” genomes—where the replication origin is positioned opposite the terminus on the circular chromosome.
The bacterial stringent response, triggered by nutritional deprivation, causes an accumulation of the signaling nucleotides pppGpp and ppGpp. We characterize the replication arrest that occurs during the stringent response in Escherichia coli. Wild type cells undergo a RelA-dependent arrest after treatment with serine hydroxamate to contain an integer number of chromosomes and a replication origin-to-terminus ratio of 1. The growth rate prior to starvation determines the number of chromosomes upon arrest. Nucleoids of these cells are decondensed; in the absence of the ability to synthesize ppGpp, nucleoids become highly condensed, similar to that seen after treatment with the translational inhibitor chloramphenicol. After induction of the stringent response, while regions corresponding to the origins of replication segregate, the termini remain colocalized in wild-type cells. In contrast, cells arrested by rifampicin and cephalexin do not show colocalized termini, suggesting that the stringent response arrests chromosome segregation at a specific point. Release from starvation causes rapid nucleoid reorganization, chromosome segregation, and resumption of replication. Arrest of replication and inhibition of colony formation by ppGpp accumulation is relieved in seqA and dam mutants, although other aspects of the stringent response appear to be intact. We propose that DNA methylation and SeqA binding to non-origin loci is necessary to enforce a full stringent arrest, affecting both initiation of replication and chromosome segregation. This is the first indication that bacterial chromosome segregation, whose mechanism is not understood, is a step that may be regulated in response to environmental conditions.
Management of cell growth and division in response to environmental conditions is important for all cells. In bacteria, nutritional downturns are signaled by accumulation of the nucleotide ppGpp. Amino acid starvation causes a programmed change in transcription, known as the “stringent response”; ppGpp also causes an arrest of cell cycle in bacteria, whose mechanism has not been thoroughly investigated. Here, we show that E. coli cells, when the stringent response is in effect, complete chromosomal replication but do not initiate new rounds and arrest with an integer number of chromosomes. The number of chromosomes corresponds to the growth rate prior to arrest. In polyploid arrested cells, the chromosomal regions at which replication initiates are segregated, whereas the termini regions remain colocalized. The E. coli chromosome remains decondensed and unsegregated during arrest and rapidly resumes replication and segregation, concomitant with chromosome condensation, upon release. The protein SeqA, a DNA binding protein and negative regulator of replication, is necessary for enforcing this arrest.
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.
How organisms regulate biological processes so that they occur at the correct place within the cell is a fundamental question in research. Spatial regulation of cell division is vital to ensure equal partitioning of DNA into newborn cells. Correct positioning of the division site at the cell centre in rod-shaped bacteria is generally believed to occur via the combined action of two factors: (i) nucleoid (chromosome) occlusion and (ii) a set of proteins known collectively as the Min system. The earliest stage in bacterial cell division is the assembly of a ring, called the Z ring, at the division site. Nucleoid occlusion and Min work by preventing Z ring assembly at all sites along the cell other than the cell centre. Here we make the surprising discovery that, in the absence of both these factors, Z rings are positioned correctly at the division site, but there is a delay in this process and it is less efficient. We propose that a separate mechanism identifies the division site at midcell in rod-shaped bacteria, and nucleoid occlusion and Min ensure that the Z ring forms there and only there, at the right time and every time.
Asymmetrical segregation of differentiated sister chromatids is thought to be important for cellular differentiation in higher eukaryotes. Similarly, in fission yeast, cellular differentiation involves the asymmetrical segregation of a chromosomal imprint. This imprint has been shown to consist of two ribonucleotides that are incorporated into the DNA during lagging-strand synthesis in response to a replication pause, but the underlying mechanism remains unknown. Here we present key novel discoveries important for unravelling this process. Our data show that cis-acting sequences within the mat1 cassette mediate pausing of replication forks at the proximity of the imprinting site, and the results suggest that this pause dictates specific priming at the position of imprinting in a sequence-independent manner. Also, we identify a novel type of cis-acting spacer region important for the imprinting process that affects where subsequent primers are put down after the replication fork is released from the pause. Thus, our data suggest that the imprint is formed by ligation of a not-fully-processed Okazaki fragment to the subsequent fragment. The presented work addresses how differentiated sister chromatids are established during DNA replication through the involvement of replication barriers.
Differentiated sister chromatids, coupled with non-random segregation, have been proposed to control cell fate during differentiation in multicellular organisms as well as fission yeast. However, while nothing is known about how the differentiated sister chromatids are established in higher eukaryotes, the nature of the epigenetic mark that is required for the asymmetrical switching pattern of Schizosaccharomyces pombe is known. We have previously shown that two ribonucleotides are introduced in a strand- and site-specific manner during DNA replication at the mat1 locus in only one of the two sister chromatids synthesized. However, the molecular mechanism by which the imprint is introduced is elusive. We know that imprinting involves a site-specific pause of the replication fork, but how the replication fork is paused and how this leads to imprinting were unknown. Here we present key novel discoveries important for unravelling this mechanism. Our data suggest that site-specific replication pausing leads to lagging-strand priming at the site of imprinting and that this RNA primer is converted into the imprint by ligation of a not-fully-processed Okazaki fragment to the subsequent fragment. Potentially, this could be a more general mechanism for formation of epigenetic marks.
Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells.
A deletion of the sbcC gene rendered Bacillus subtilis cells sensitive to DNA damage caused by Mitomycin C (MMC) or by gamma irradiation. The deletion of the sbcC gene in a recN mutant background increased the sensitivity of the single recN mutant strain. SbcC was also non-epistatic with AddAB (analog of Escherichia coli RecBCD), but epistatic with RecA. A deletion of the ykoV gene encoding the B. subtilis Ku protein in a sbcC mutant strain did not resulted in an increase in sensitivity towards MMC and gamma irradiation, but exacerbated the phenotype of a recN or a recA mutant strain. In exponentially growing cells, SbcC-GFP was present throughout the cells, or as a central focus in rare cases. Upon induction of DNA damage, SbcC formed 1, rarely 2, foci on the nucleoids. Different to RecN protein, which forms repair centers at any location on the nucleoids, SbcC foci mostly co-localized with the DNA polymerase complex. In contrast to this, AddA-GFP or AddB-GFP did not form detectable foci upon addition of MMC.
Our experiments show that SbcC plays an important role in the repair of DNA inter-strand cross-links (induced by MMC), most likely through HR, and suggest that NHEJ via Ku serves as a backup DNA repair system. The cell biological experiments show that SbcC functions in close proximity to the replication machinery, suggesting that SbcC may act on stalled or collapsed replication forks. Our results show that different patterns of localization exist for DNA repair proteins, and that the B. subtilis SMC proteins RecN and SbcC play distinct roles in the repair of DNA damage.