Long-range chromosome organization in E. coli: a site-specific system isolates the Ter macrodomain

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.     

Differential Management of the Replication Terminus Regions of the Two Vibrio cholerae Chromosomes during Cell Division

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 role of MatP,ZapA and ZapB in chromosomal organization and dynamics in Escherichia coli

Despite extensive research over several decades, a comprehensive view of how the Escherichia coli chromosome is organized within the nucleoid, and how two daughter chromosomes segregate has yet to emerge. Here we investigate the role of the MatP, ZapA and ZapB proteins in organizing the replication terminus (Ter) region and in the chromosomal segregation process. Quantitative image analysis of the fluorescently labeled Ter region shows that the replication terminus attaches to the divisome in a single segment along the perimeter of the cell in a MatP, ZapA and ZapB-dependent manner. The attachment does not significantly affect the bulk chromosome segregation in slow growth conditions. With or without the attachment, two chromosomal masses separate from each other at a speed comparable to the cell growth. The separation starts even before the replication terminus region positions itself at the center of the nucleoid. Modeling of the segregation based on conformational entropy correctly predicts the positioning of the replication terminus region within the nucleoid. However, the model produces a distinctly different chromosomal density distribution than the experiment, indicating that the conformational entropy plays a limited role in segregating the chromosomes in the late stages of replication.     

Pirt, a phosphoinositide-binding protein, functions as a regulatory subunit of TRPV1

The organization of the Escherichia coli chromosome into insulated macrodomains influences the segregation of sister chromatids and the mobility of chromosomal DNA. Here, we report that organization of the Terminus region (Ter) into a macrodomain relies on the presence of a 13 bp motif called matS repeated 23 times in the 800-kb-long domain. matS sites are the main targets in the E. coli chromosome of a newly identified protein designated MatP. MatP accumulates in the cell as a discrete focus that colocalizes with the Ter macrodomain. The effects of MatP inactivation reveal its role as main organizer of the Ter macrodomain: in the absence of MatP, DNA is less compacted, the mobility of markers is increased, and segregation of Ter macrodomain occurs early in the cell cycle. Our results indicate that a specific organizational system is required in the Terminus region for bacterial chromosome management during the cell cycle.     

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.     

Spatial and temporal organization of replicating Escherichia coli chromosomes

The positions of DNA regions close to the chromosome replication origin and terminus in growing cells of Escherichia coli have been visualized simultaneously, using new widely applicable reagents. Furthermore, the positions of these regions with respect to a replication factory-associated protein have been analysed. Time-lapse analysis has allowed the fate of origins, termini and the FtsZ ring to be followed in a lineage-specific manner during the formation of microcolonies. These experiments reveal new aspects of the E. coli cell cycle and demonstrate that the replication terminus region is frequently located asymmetrically, on the new pole side of mid-cell. This asymmetry could provide a mechanism by which the chromosome segregation protein FtsK, located at the division septum, can act directionally to ensure that the septal region is free of DNA before the completion of cell division.     

Interpretation of organizational role of proteins on E. coli nucleoid via Hi-C integrated model

Several proteins in Escherichia coli work together to maintain the complex organization of its chromosome. However, the individual roles of these so-called nucleoid-associated proteins (NAPs) in chromosome architectures are not well characterized. Here, we quantitatively dissect the organizational roles of Heat Unstable (HU), a ubiquitous protein in E. coli and MatP, an NAP specifically binding to the Ter macrodomain of the chromosome. Toward this end, we employ a polymer physics-based computer model of wild-type chromosome and their HU- and MatP-devoid counterparts by incorporating their respective experimentally derived Hi-C contact matrix, cell dimensions, and replication status of the chromosome commensurate with corresponding growth conditions. Specifically, our model for the HU-devoid chromosome corroborates well with the microscopy observation of compaction of chromosome at short genomic range but diminished long-range interactions, justifying precedent hypothesis of segregation defect upon HU removal. Control simulations point out that the change in cell dimension and chromosome content in the process of HU removal holds the key to the observed differences in chromosome architecture between wild-type and HU-devoid cells. On the other hand, simulation of MatP-devoid chromosome led to locally enhanced contacts between Ter and its flanking macrodomains, consistent with previous recombination assay experiments and MatP’s role in insulation of the Ter macrodomain from the rest of the chromosome. However, the simulation indicated no change in matS sites’ localization. Rather, a set of designed control simulations showed that insulation of Ter is not caused by bridging of distant matS sites, also lending credence to a recent mobility experiment on various loci of the E. coli chromosome. Together, the investigations highlight the ability of an integrative model of the bacterial genome in elucidating the role of NAPs and in reconciling multiple experimental observations.     

MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves

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.     

Chromosomal macrodomains and associated proteins: implications for DNA organization and replication in gram negative bacteria

The Escherichia coli chromosome is organized into four macrodomains, the function and organisation of which are poorly understood. In this review we focus on the MatP, SeqA, and SlmA proteins that have recently been identified as the first examples of factors with macrodomain-specific DNA-binding properties. In particular, we review the evidence that these factors contribute towards the control of chromosome replication and segregation by specifically targeting subregions of the genome and contributing towards their unique properties. Genome sequence analysis of multiple related bacteria, including pathogenic species, reveals that macrodomain-specific distribution of SeqA, SlmA, and MatP is conserved, suggesting common principles of chromosome organisation in these organisms. This discovery of proteins with macrodomain-specific binding properties hints that there are other proteins with similar specificity yet to be unveiled. We discuss the roles of the proteins identified to date as well as strategies that may be employed to discover new factors.     

Organization of the Escherichia coli chromosome into macrodomains and its possible functional implications

Recent advances in fluorescent microscopy have revealed the non-random organization of chromosomes in bacterial cells. In Escherichia coli, segments included in two large regions show similar localization patterns allowing the definition of two macrodomains centered, respectively, on the centromere-like site migS (the Ori domain) and the replication terminus (the Ter domain). A genetic system measuring long distance DNA interactions confirmed the macrodomain organization of the chromosome and revealed the existence of two additional macrodomains flanking the Ter domain. Altogether, the E. coli chromosome appears to have an open ring-like conformation. Recent developments regarding various stages of chromosome biology such as replication, sister chromatid cohesion, segregation, and chromosome choreography, have considerably improved our understanding of the coordination of chromosome dynamics with the cell cycle. The possible involvement of macrodomains in these various processes is discussed.     

Sister chromatid interactions in bacteria revealed by a site-specific recombination assay

The process of Sister Chromosome Cohesion (SCC), which holds together sister chromatids upon replication, is essential for chromosome segregation and DNA repair in eukaryotic cells. Although cohesion at the molecular level has never been described in E. coli, previous studies have reported that sister sequences remain co-localized for a period after their replication. Here, we have developed a new genetic recombination assay that probes the ability of newly replicated chromosome loci to interact physically. We show that Sister Chromatid Interaction (SCI) occurs exclusively within a limited time frame after replication. Importantly, we could differentiate sister cohesion and co-localization since factors such as MatP and MukB that reduced the co-localization of markers had no effect on molecular cohesion. The frequency of sister chromatid interactions were modulated by the activity of Topo-IV, revealing that DNA topology modulates cohesion at the molecular scale in bacteria.     

A Replication-Inhibited Unsegregated Nucleoid at Mid-Cell Blocks Z-Ring Formation and Cell Division Independently of SOS and the SlmA Nucleoid Occlusion Protein in Escherichia coli

Chromosome replication and cell division of Escherichia coli are coordinated with growth such that wild-type cells divide once and only once after each replication cycle. To investigate the nature of this coordination, the effects of inhibiting replication on Z-ring formation and cell division were tested in both synchronized and exponentially growing cells with only one replicating chromosome. When replication elongation was blocked by hydroxyurea or nalidixic acid, arrested cells contained one partially replicated, compact nucleoid located mid-cell. Cell division was strongly inhibited at or before the level of Z-ring formation. DNA cross-linking by mitomycin C delayed segregation, and the accumulation of about two chromosome equivalents at mid-cell also blocked Z-ring formation and cell division. Z-ring inhibition occurred independently of SOS, SlmA-mediated nucleoid occlusion, and MinCDE proteins and did not result from a decreased FtsZ protein concentration. We propose that the presence of a compact, incompletely replicated nucleoid or unsegregated chromosome masses at the normal mid-cell division site inhibits Z-ring formation and that the SOS system, SlmA, and MinC are not required for this inhibition.     

Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome

Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle.     

SpbR overproduction reveals the importance of proteolytic degradation for cell pole development and chromosome segregation in Caulobacter crescentus

In most rod‐shaped bacteria, DNA replication is quickly followed by chromosome segregation, when one of the newly duplicated centromeres moves across the cell to the opposite (or ‘new’) pole. Two proteins in Caulobacter crescentus, PopZ and TipN, provide directional cues at the new pole that guide the translocating chromosome to its destination. We show that centromere translocation can be inhibited by an evolutionarily conserved pole‐localized protein that we have named SpbR. When overproduced, SpbR exhibits aberrant accumulation at the old pole, where it physically interacts with PopZ. This prevents the relocation of PopZ to the new pole, thereby eliminating a positional cue for centromere translocation. Consistent with this, the centromere translocation phenotype of SpbR overproducing cells is strongly enhanced in a ?tipN mutant background. We find that pole‐localized SpbR is normally cleared by ClpXP‐mediated proteolysis before the time of chromosome segregation, indicating that SpbR turnover is part of the cell cycle‐dependent program of polar development. This work demonstrates the importance of proteolysis as a housekeeping activity that removes outgoing factors from the developing cell pole, and provides an example of a substrate that can inhibit polar functions if it is insufficiently cleared.     

A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control

We have examined a replication terminus (psiL1) located on the left arm of the chromosome of Bacillus subtilis and within the yxcC gene and at or near the left replication checkpoint that is activated under stringent conditions. The psiL1 sequence appears to bind to two dimers of the replication terminator protein (RTP) rather weakly and seems to possess overlapping core and auxiliary sites that have some sequence similarities with normal Ter sites. Surprisingly, the asymmetrical, isolated psiL1 site arrested replication forks in vivo in both orientations and independent of stringent control. In vitro, the sequence arrested DnaB helicase in both orientations, albeit more weakly than the normal Ter1 terminus. The key points of mechanistic interest that emerge from the present work are: (i) strong binding of a Ter (psiL1) sequence to RTP did not appear to be essential for fork arrest and (ii) polarity of fork arrest could not be correlated in this case with just symmetrical protein-DNA interaction at the core and auxiliary sites of psiL1. On the basis of the result it would appear that the weak RTP-L1Ter interaction cannot by itself account for fork arrest, thus suggesting a role for DnaB-RTP interaction.     

ZapA and ZapB form an FtsZ‐independent structure at midcell

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

Spatial arrangement and macrodomain organization of bacterial chromosomes

Recent developments in fluorescence microscopy have shown that bacterial chromosomes have a defined spatial arrangement that preserves the linear order of genes on the genetic map. These approaches also revealed that large portions of the chromosome in Escherichia coli or Bacillus subtilis are concentrated in the same cellular space, suggesting an organization as large regions defined as macrodomains. In E. coli, two macrodomains of 1 Mb containing the replication origin (Ori) and the replication terminus (Ter) have been shown to relocalize at specific steps of the cell cycle. A genetic analysis of the collision probability between distant DNA sites in E. coli has confirmed the presence of macrodomains by revealing the existence of large regions that do not collide with each other. Two macrodomains defined by the genetic approach coincide with the Ori and Ter macrodomains, and two new macrodomains flanking the Ter macrodomain have been identified. Altogether, these results indicate that the E. coli chromosome has a ring organization with four structured and two less-structured regions. Implications for chromosome dynamics during the cell cycle and future prospects for the characterization and understanding of macrodomain organization are discussed.     

FtsK controls metastable recombination provoked by an extra Ter site in the Escherichia coli chromosome terminus

The FtsK protein is required for septum formation in Escherichia coli and as a DNA translocase for chromosome processing while the septum closes. Its domain of action on the chromosome overlaps the replication terminus region, which lies between replication pause sites TerA and TerC. An extra Ter site, PsrA*, has been inserted at a position common to the FtsK and terminus domains. It is well tolerated, although it compels replication forks travelling clockwise from oriC to stall and await arrival of counter-clockwise forks. Elevated recombination has been detected at the stalled fork. Analysis of PsrA*-induced homologous recombination by an excision test revealed unique features. (i) rates of excision near PsrA* may fluctuate widely from clone to clone, a phenomenon we term whimsicality, (ii) excision rates are nevertheless conserved for many generations, a phenomenon we term memorization; their metastability at the clone level is explainable by frequent shifting between three cellular states--high, medium and low probability of excision, (iii) PsrA*-induced excision is RecBC-independent and is strongly counteracted by FtsK, which in addition is involved in its whimsicality and (iv) whimsicality disappears as the distance from the pause site increases. Action of FtsK at a replication fork was unexpected because the factor was thought to act on the chromosome only at septation, i.e. after replication is completed. Idiosyncrasy of PsrA*-induced recombination is discussed with respect to possible intermingling of replication, repair and post-replication steps of bacterial chromosome processing during the cell cycle.     

Replication arrests during a single round of replication of the Escherichia coli chromosome in the absence of DnaC activity

We used a flow cytometric assay to determine the frequency of replication fork arrests during a round of chromosome replication in Escherichia coli. After synchronized initiation from oriC in a dnaC(Ts) strain, non-permissive conditions were imposed, such that active DnaC was not available during elongation. Under these conditions, about 18% of the cells failed to complete chromosome replication. The sites of replication arrests were random and occurred on either arm of the bidirectionally replicating chromosome, as stalled forks accumulated at the terminus from both directions. The forks at the terminal Ter sites disappeared in the absence of Tus protein, as the active forks could then pass through the terminus to reach the arrest site, and the unfinished rounds of replication would be completed without DnaC. In a dnaC2(Ts)rep double mutant, almost all cells failed to complete chromosome replication in the absence of DnaC activity. As inactivation of Rep helicase (the rep gene product) has been shown to cause frequent replication arrests inducing double-strand breaks (DSBs) in a replicating chromosome, DnaC activity appears to be essential for replication restart from DSBs during elongation.     

Bacterial DNA segregation: its motors and positional control

A model for DNA segregation in bacteria is proposed which involves not merely growth of the cell membrane and wall, as previously assumed, but also the active movement of one of the two chromosome sister origins by a DNA helicase enzyme and of the chromosome termini and the bulk of the chromosomes by supercoiling tension exerted by DNA gyrase. This provides a unified mechanism for DNA chromosome movement in prosthecate budding bacteria as well as for bacteria that undergo binary fission. The positional control of DNA segregation and the plane of cell division depend, I suggest, on four things: (1) the attachment of the daughter chromosome termini to the cell wall in a position adjacent to the new cell poles at about the time of septation, (2) the displacement of the parental chromosome terminus from this attachment site by the mobile origin, which attaches itself instead to the wall at that point, (3) the movement of the chromosome terminus to a new location in between the daughter origins by the tension of supercoiling, and (4) the determination of the location of the future septum at the position occupied by the chromosome terminus at the time of septal initiation; septum-initiation proteins are postulated to achieve this by binding directly or indirectly to the chromosome terminus. This mechanism automatically ensures ordered DNA segregation in rapidly growing bacteria with more than two sister origins of replication.