Roles for replichores and macrodomains in segregation of the Escherichia coli chromosome.
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ABSTRACT:
SUBMITTER: Lesterlin C
PROVIDER: S-EPMC1369093 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The mechanisms underlying chromosome segregation in prokaryotes remain a subject of debate and no unifying view has yet emerged. Given that the initial disentanglement of duplicated chromosomes could be achieved by purely entropic forces, even the requirement of an active prokaryotic segregation machinery has been questioned. Using computer simulations, we show that entropic forces alone are not sufficient to achieve and maintain full separation of chromosomes. This is, however, possible by assuming repeated binding of chromosomes along a gradient of membrane-associated tethering sites toward the poles. We propose that, in Escherichia coli, such a gradient of membrane tethering sites may be provided by the oscillatory Min system, otherwise known for its role in selecting the cell division site. Consistent with this hypothesis, we demonstrate that MinD binds to DNA and tethers it to the membrane in an ATP-dependent manner. Taken together, our combined theoretical and experimental results suggest the existence of a novel mechanism of chromosome segregation based on the Min system, further highlighting the importance of active segregation of chromosomes in prokaryotic cell biology.
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Project description:In slow-growing Escherichia coli cells the chromosome is organized with its left (L) and right (R) arms lying separated in opposite halves of the nucleoid and with the origin (O) in-between, giving the pattern L-O-R. During replication one of the arms has to pass the other to obtain the same organization in the daughter cells: L-O-R L-O-R. To determine the movement of arms during segregation six strains were constructed carrying three colored loci: the left and right arms were labeled with red and cyan fluorescent-proteins, respectively, on loci symmetrically positioned at different distances from the central origin, which was labeled with green-fluorescent protein. In non-replicating cells with the predominant spot pattern L-O-R, initiation of replication first resulted in a L-O-O-R pattern, soon changing to O-L-R-O. After replication of the arms the predominant spot patterns were, L-O-R L-O-R, O-R-L R-O-L or O-L-R L-O-R indicating that one or both arms passed an origin and the other arm. To study the driving force for these movements cell growth was inhibited with rifampicin allowing run-off DNA synthesis. Similar spot patterns were obtained in growing and non-growing cells, indicating that the movement of arms is not a growth-sustained process, but may result from DNA synthesis itself. The distances between loci on different arms (LR-distances) and between duplicated loci (LL- or RR-distances) as a function of their distance from the origin, indicate that in slow-growing cells DNA is organized according to the so-called sausage model and not according to the doughnut model.
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Project description: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.
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Project description:Structural Maintenance of Chromosomes (SMC) complexes act ubiquitously to compact DNA linearly, thereby facilitating chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE proteins that interact with kleisins. The ATPase activity of the Escherichia coli SMC protein, MukB, which is essential for its in vivo function, requires its interaction with the dimeric kleisin, MukF that in turn interacts with the KITE protein, MukE. Here we demonstrate that, in addition, MukB interacts specifically with Acyl Carrier Protein (AcpP) that has essential functions in fatty acid synthesis. We characterize the AcpP interaction at the joint of the MukB coiled-coil and show that the interaction is necessary for MukB ATPase and for MukBEF function in vivo.
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Project description:Escherichia coli possesses two type 1A topoisomerases, Topo I (topA) and Topo III (topB). Topo I relaxes excess negative supercoiling, and topA mutants can grow only in the presence of compensatory mechanisms, such as gyrase mutations. topB mutants grow as well as wild-type cells. In vitro, Topo III, but not Topo I, can efficiently decatenate DNA during replication. However, in vivo, a chromosome segregation defect is seen only when both type 1A topoisomerases are absent. Here we present experimental evidence for an interplay between gyrase and type 1A topoisomerases in chromosome segregation. We found that both the growth defect and the Par(-) phenotypes of a gyrB(Ts) mutant at nonpermissive temperatures were significantly corrected by deleting topA, but only when topB was present. Overproducing Topo IV, the major cellular decatenase, could not substitute for topB. We also show that overproducing Topo III at a very high level could suppress the Par(-) phenotype. We previously found that the growth and chromosome segregation defects of a triple topA rnhA gyrB(Ts) mutant in which gyrase supercoiling activity was strongly inhibited could be corrected by overproducing Topo III (V. Usongo, F. Nolent, P. Sanscartier, C. Tanguay, S. Broccoli, I. Baaklini, K. Drlica, and M. Drolet, Mol. Microbiol. 69:968-981, 2008). We show here that this overproduction could be bypassed by substituting the gyrB(Ts) allele for a gyrB(+) one or by growing cells in a minimal medium, conditions that reduced both topA- and rnhA-dependent unregulated replication. Altogether, our data point to a role for Topo III in chromosome segregation when gyrase is inefficient and suggest that Topo I plays an indirect role via supercoiling regulation.
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Project description:Characterized bacteria, unlike eukaryotes and some archaea, initiate replication bidirectionally from a single replication origin contained within a circular or linear chromosome. We constructed Escherichia coli cells with two WT origins separated by 1 Mb in their 4.64-Mb chromosome. Productive bidirectional replication initiated synchronously at both spatially separate origins. Newly replicated DNA from both origins was segregated sequentially as replication progressed, with two temporally and spatially separate replication termination events. Replication initiation occurred at a cell volume identical to that of cells with a single WT origin, showing that initiation control is independent of cellular and chromosomal oriC concentration. Cells containing just the ectopic origin initiated bidirectional replication at the expected cell mass and at the normal cellular location of that region. In all strains, spatial separation of sister loci adjacent to active origins occurred shortly after their replication, independently of whether replication initiated at the normal origin, the ectopic origin, or both origins.
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Project description:Homologous recombination between the circular chromosomes of bacteria can generate chromosome dimers. They are resolved by a recombination event at a specific site in the replication terminus of chromosomes, dif, by dedicated tyrosine recombinases. The reaction is under the control of a cell division protein, FtsK, which assembles into active DNA pumps at mid-cell during septum formation. Previous studies suggested that activation of Xer recombination at dif was restricted to chromosome dimers in Escherichia coli but not in Vibrio cholerae, suggesting that FtsK mainly acted on chromosome dimers in E. coli but frequently processed monomeric chromosomes in V. cholerae. However, recent microscopic studies suggested that E. coli FtsK served to release the MatP-mediated cohesion and/or cell division apparatus-interaction of sister copies of the dif region independently of chromosome dimer formation. Here, we show that these apparently paradoxical observations are not linked to any difference in the dimer resolution machineries of E. coli and V. cholerae but to differences in the timing of segregation of their chromosomes. V. cholerae harbours two circular chromosomes, chr1 and chr2. We found that whatever the growth conditions, sister copies of the V. cholerae chr1 dif region remain together at mid-cell until the onset of constriction, which permits their processing by FtsK and the activation of dif-recombination. Likewise, sister copies of the dif region of the E. coli chromosome only separate after the onset of constriction in slow growth conditions. However, under fast growth conditions the dif sites separate before constriction, which restricts XerCD-dif activity to resolving chromosome dimers.
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Project description:The Escherichia coli chromosome is organized into four macrodomains (Ori, Ter, Right and Left) and two non-structured regions. This organization influences the segregation of sister chromatids, the mobility of chromosomal DNA, and the cellular localization of the chromosome. The organization of the Ter and Ori macrodomains relies on two specific systems, MatP/matS for the Ter domain and MaoP/maoS for the Ori domain, respectively. Here by constructing strains with chromosome rearrangements to reshuffle the distribution of chromosomal segments, we reveal that the difference between the non-structured regions and the Right and Left lateral macrodomains relies on their position on the chromosome. A change in the genetic location of oriC generated either by an inversion within the Ori macrodomain or by the insertion of a second oriC modifies the position of Right and Left macrodomains, as the chromosome region the closest to oriC are always non-structured while the regions further away behave as macrodomain regardless of their DNA sequence. Using fluorescent microscopy we estimated that loci belonging to a non-structured region are significantly closer to the Ori MD than loci belonging to a lateral MD. Altogether, our results suggest that the origin of replication plays a prominent role in chromosome organization in E. coli, as it determines structuring and localization of macrodomains in growing cell.
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Project description:The type II topoisomerase TopoIV, which has an essential role in Escherichia coli chromosome decatenation, interacts with MukBEF, an SMC (structural maintenance of chromosomes) complex that acts in chromosome segregation. We have characterized the intracellular dynamics of individual TopoIV molecules and the consequences of their interaction with MukBEF clusters by using photoactivated-localization microscopy. We show that ~15 TopoIV molecules per cell are associated with MukBEF clusters that are preferentially localized to the replication origin region (ori), close to the long axis of the cell. A replication-dependent increase in the fraction of immobile molecules, together with a proposed catalytic cycle of ~1.8 s, is consistent with the majority of active TopoIV molecules catalyzing decatenation, with a minority maintaining steady-state DNA supercoiling. Finally, we show that the MukB-ParC interaction is crucial for timely decatenation and segregation of newly replicated ori DNA.
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