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1.
Matthew W. Bailey Paola Bisicchia Boyd T. Warren David J. Sherratt Jaan M?nnik 《PLoS genetics》2014,10(8)
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. 相似文献
2.
The Ori region of bacterial genomes is segregated early in the replication cycle of bacterial chromosomes. Consequently, Ori region positioning plays a pivotal role in chromosome dynamics. The Ori region of the E. coli chromosome is organized as a macrodomain with specific properties concerning DNA mobility, segregation of loci and long distance DNA interactions. Here, by using strains with chromosome rearrangements and DNA mobility as a read-out, we have identified the MaoP/maoS system responsible for constraining DNA mobility in the Ori region and limiting long distance DNA interactions with other regions of the chromosome. MaoP belongs to a group of proteins conserved in the Enterobacteria that coevolved with Dam methylase including SeqA, MukBEF and MatP that are all involved in the control of chromosome conformation and segregation. Analysis of DNA rings excised from the chromosome demonstrated that the single maoS site is required in cis on the chromosome to exert its effect while MaoP can act both in cis and in trans. The position of markers in the Ori region was affected by inactivating maoP. However, the MaoP/maoS system was not sufficient for positioning the Ori region at the ¼–¾ regions of the cell. We also demonstrate that the replication and the resulting expansion of bulk DNA are localized centrally in the cell. Implications of these results for chromosome positioning and segregation in E. coli are discussed. 相似文献
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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. 相似文献
5.
SeqA proteins of Escherichia coli bound to the hemimethylated GATC sequences (hemi-sites) interact with each other and eventually form an aggregate. SeqA foci, which are suggested to be formed by aggregation, play important roles in the regulation of chromosome replication and segregation. We found that aggregation of SeqA proteins was preceded by cooperative interactions between these proteins bound to hemi-sites. Positively charged amino acids in the hinge region, which connects the N-terminal and C-terminal domain of SeqA, were critical for SeqA aggregation on hemimethylated DNA. Although the substitution of positively charged amino acids with negatively charged or neutral amino acids maintained the binding and cooperative interaction of mutant proteins, these proteins were defective in aggregation and foci formation in vitro and in vivo, respectively. Our results suggest that in vivo SeqA foci were formed by aggregation following cooperative interactions between SeqA proteins bound to hemi-sites. 相似文献
6.
The SeqA protein regulates chromosome initiation and is involved in segregation in Escherichia coli. One SeqA protein binds to two hemi-methylated GATC sequences to form a stable SeqA-DNA complex. We found that binding induced DNA bending, which was pronounced when the two sequences were on the same face of the DNA. Two SeqA molecules bound cooperatively to each pair of hemi-methylated sites when the spacing between the sites was < or = 30 bp. This cooperative binding was able to stabilize the binding of a wild type to a single hemi-methylated site, or mutant form of SeqA protein to hemi-methylated sites, although such binding did not occur without cooperative interaction. Two cooperatively bound SeqA molecules interacted with another SeqA bound up to 185 bp away from the two bound SeqA proteins, and this was followed by aggregation of free SeqA proteins onto the bound proteins. These results suggest that the stepwise interaction of SeqA proteins with hemi-methylated GATC sites enhances their interaction and leads to the formation of SeqA aggregates. Cooperative interaction followed by aggregation may be the driving force for formation of the SeqA foci that appear to be located behind replication forks. 相似文献
7.
Assembly of the Z-ring over unsegregated nucleoids is prevented by a process called nucleoid occlusion (NO), which in Escherichia coli is partially mediated by SlmA. SlmA is a Z ring antagonist that is spatially regulated and activated by binding to specific DNA sequences (SlmA binding sites, SBSs) more abundant in the origin proximal region of the chromosome. However, the mechanism by which SBS bound SlmA (activated form) antagonizes Z ring assembly is controversial. Here, we report the isolation and characterization of two FtsZ mutants, FtsZ-K190V and FtsZ-D86N that confer resistance to activated SlmA. In trying to understand the basis of resistance of these mutants, we confirmed that activated SlmA antagonizes FtsZ polymerization and determined these mutants were resistant, even though they still bind SlmA. Investigation of SlmA binding to FtsZ revealed activated SlmA binds to the conserved C-terminal tail of FtsZ and that the ability of activated SlmA to antagonize FtsZ assembly required the presence of the tail. Together, these results lead to a model in which SlmA binding to an SBS is activated to bind the tail of FtsZ resulting in further interaction with FtsZ leading to depolymerization of FtsZ polymers. This model is strikingly similar to the model for the inhibitory mechanism of the spatial inhibitor MinCD. 相似文献
8.
Jackson Buss Carla Coltharp Gleb Shtengel Xinxing Yang Harald Hess Jie Xiao 《PLoS genetics》2015,11(4)
The prokaryotic tubulin homolog, FtsZ, forms a ring-like structure (FtsZ-ring) at midcell. The FtsZ-ring establishes the division plane and enables the assembly of the macromolecular division machinery (divisome). Although many molecular components of the divisome have been identified and their interactions extensively characterized, the spatial organization of these proteins within the divisome is unclear. Consequently, the physical mechanisms that drive divisome assembly, maintenance, and constriction remain elusive. Here we applied single-molecule based superresolution imaging, combined with genetic and biophysical investigations, to reveal the spatial organization of cellular structures formed by four important divisome proteins in E. coli: FtsZ, ZapA, ZapB and MatP. We show that these interacting proteins are arranged into a multi-layered protein network extending from the cell membrane to the chromosome, each with unique structural and dynamic properties. Further, we find that this protein network stabilizes the FtsZ-ring, and unexpectedly, slows down cell constriction, suggesting a new, unrecognized role for this network in bacterial cell division. Our results provide new insight into the structure and function of the divisome, and highlight the importance of coordinated cell constriction and chromosome segregation. 相似文献
9.
Segregation in Escherichia coli, the process of separating the replicated chromosomes into daughter progeny cells, seems to start long before the duplication of the genome reaches completion. Soon after initiation in mid-cell region, the daughter oriCs rapidly move apart to fixed positions inside the cell (quarter length positions from each pole) and are anchored there by yet unknown mechanism(s). As replication proceeds, the rest of the chromosome is sequentially unwound and then refolded. At termination, the two sister chromosomes are unlinked by decatenation and separated by supercoiling and/or condensation. Muk and Seq proteins are involved in different stages of this replication-cum-partition process and thus can be categorized as important partition proteins along with topoisomerases. E. coli strains, lacking mukB or seqA functions, are defective in segregation and cell division. The nucleoids in these mutant strains exhibit altered condensation and superhelicity as can be demonstrated by sedimentation analysis and by fluorescence microscopy. As the supercoiling of an extrachromosomal element (a plasmid DNA) was also influenced by the mukB and seqA mutations we concluded that the MukB and SeqA proteins are possibly involved in maintaining the general supercoiling activity in the cell. The segregation of E. coli chromosome might therefore be predominantly driven by factors that operate by affecting the superhelicity and condensation of the nucleoid (MukB, SeqA, topoisomerases and additional unknown proteins). A picture thus emerges in which replication and partition are no longer compartmentalized into separable stages with clear gaps (S and M phases in eukaryotes) but are parallel processes that proceed concomitantly through a cell cycle continuum. 相似文献
10.
H Lee S Kang S H Bae B S Choi D S Hwang 《The Journal of biological chemistry》2001,276(37):34600-34606
The binding of SeqA protein to hemimethylated GATC sequences is important in the negative modulation of chromosomal initiation at oriC, and in the formation of SeqA foci necessary for Escherichia coli chromosome segregation. Using gel-filtration chromotography and glycerol gradient sedimentation, we demonstrate that SeqA exists as a homotetramer. SeqA tetramers are able to aggregate or multimerize in a reversible, concentration-dependent manner. Using a bacterial two-hybrid system, we demonstrate that the N-terminal region of SeqA, especifically the 9th amino acid residue, glutamic acid, is required for functional SeqA-SeqA interaction. Although the SeqA(E9K) mutant protein, containing lysine rather than glutamic acid at the 9th amino acid residue, exists as a tetramer, the mutant protein binds to hemimethylated DNA with altered binding patterns as compared with wild-type SeqA. Aggregates of SeqA(E9K) are defective in hemimethylated DNA binding. Here we demonstrate that proper interaction between SeqA tetramers is required for both hemimethylated DNA binding and formation of active aggregates. SeqA tetramers and aggregates might be involved in the formation of SeqA foci required for the segregation of chromosomal DNA as well as the regulation of chromosomal initiation. 相似文献
11.
Espéli O Borne R Dupaigne P Thiel A Gigant E Mercier R Boccard F 《The EMBO journal》2012,31(14):3198-3211
Initiation of chromosome segregation in bacteria is achieved by proteins acting near the origin of replication. Here, we report that the precise choreography of the terminus region of the Escherichia coli chromosome is also tightly controlled. The segregation of the terminus (Ter) macrodomain (MD) involves the structuring factor MatP. We characterized that migration of the Ter MD from the new pole to mid-cell and its subsequent persistent localization at mid-cell relies on several processes. First, the replication of the Ter DNA is concomitant with its recruitment from the new pole to mid-cell in a sequential order correlated with the position on the genetic map. Second, using a strain carrying a linear chromosome with the Ter MD split in two parts, we show that replisomes are repositioned at mid-cell when replication of the Ter occurs. Third, we demonstrate that anchoring the Ter MD at mid-cell depends on the specific interaction of MatP with the division apparatus-associated protein ZapB. Our results reveal how segregation of the Ter MD is integrated in the cell-cycle control. 相似文献
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Tonthat NK Arold ST Pickering BF Van Dyke MW Liang S Lu Y Beuria TK Margolin W Schumacher MA 《The EMBO journal》2011,30(1):154-164
In Escherichia coli, cytokinesis is orchestrated by FtsZ, which forms a Z-ring to drive septation. Spatial and temporal control of Z-ring formation is achieved by the Min and nucleoid occlusion (NO) systems. Unlike the well-studied Min system, less is known about the anti-DNA guillotining NO process. Here, we describe studies addressing the molecular mechanism of SlmA (synthetic lethal with a defective Min system)-mediated NO. SlmA contains a TetR-like DNA-binding fold, and chromatin immunoprecipitation analyses show that SlmA-binding sites are dispersed on the chromosome except the Ter region, which segregates immediately before septation. SlmA binds DNA and FtsZ simultaneously, and the SlmA-FtsZ structure reveals that two FtsZ molecules sandwich a SlmA dimer. In this complex, FtsZ can still bind GTP and form protofilaments, but the separated protofilaments are forced into an anti-parallel arrangement. This suggests that SlmA may alter FtsZ polymer assembly. Indeed, electron microscopy data, showing that SlmA-DNA disrupts the formation of normal FtsZ polymers and induces distinct spiral structures, supports this. Thus, the combined data reveal how SlmA derails Z-ring formation at the correct place and time to effect NO. 相似文献
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A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation 总被引:5,自引:0,他引:5
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SeqA is an Escherichia coli DNA-binding protein that acts at replication origins and controls DNA replication. However, binding is not exclusive to origins. Many fragments containing two or more hemi-methylated GATC sequences bind efficiently. Binding was optimal when two such sequences were closely apposed or up to 31 bases apart on the same face of the DNA helix. Binding studies suggest that neighboring bound proteins contact each other to form a complex with the intervening DNA looped out. There are many potential binding sites distributed around the E.coli chromosome. As replication produces a transient wave of hemi-methylation, tracts of SeqA binding are likely to associate with each fork as replication progresses. The number and positions of green fluorescent protein-SeqA foci seen in living cells suggest that they correspond to these tracts, and that the forks are tethered to planes of cell division. SeqA may help to tether the forks or to organize newly replicated DNA into a structure that aids DNA to segregate away from the replication machinery. 相似文献
16.
Preferential binding of the SeqA protein to hemi-methylated GATC sequences functions as a negative regulator for Escherichia coli initiation of chromosomal replication at oriC and is implicated in segregating replicated chromosomes for cell division. We demonstrate that sequential binding of one SeqA tetramer to a set of two hemi-methylated sites mediates formation of higher-order complexes. The absence of cross-binding to separate DNAs suggests that two monomers of a SeqA tetramer bind to two hemi-methylated sites on DNA. The interaction among SeqA proteins bound to at least six adjacent hemi-methylated sites induces aggregation of free proteins to bound proteins. Aggregation might be indicative of SeqA foci, which appear to track replication forks in vivo. Studies of the properties of SeqA binding will contribute to our understanding of the function of SeqA. 相似文献
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MukF, MukE and MukB proteins form a complex that may participate in the organization of folded sister chromosomes in Escherichia coli. We have found that a MukB-GFPuv4 fusion protein is observed as discrete fluorescent foci, which are localized within cellular spaces occupied by nucleoids, but not at the constriction site of cell division in living cells. In contrast, MukB-GFPuv4 is distributed throughout the whole cell when either MukF or MukE is absent. Statistical analysis revealed that most newborn cells have two foci of mukB-gfpUV4 at one-quarter and three-quarter positions in the cell length and one focus of SeqA-bound nascent DNA at or near the middle of the cell. Subsequently, the single SeqA focus divides into two foci, and then these migrate to the one-quarter and three-quarter positions. Before cell division, most long cells have two SeqA foci and four MukB-GFPuv4 foci. In early stationary phase, SeqA foci disappear, but one or two foci of MukB-GFPuv4 remain. We discuss the reorganization and proper arrangement of folded sister chromosome in the cell quarter positions, which are performed after release from the long-time cohesion of sister chromosomes. 相似文献
19.
Brenna Conin Ingrid Billault-Chaumartin Hafez El
Sayyed Nicole QuenechDu Charlotte Cockram Romain Koszul Olivier Espli 《Nucleic acids research》2022,50(5):2635
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. 相似文献
20.
In Escherichia coli, the SeqA protein binds specifically to GATC sequences which are methylated on the A of the old strand but not on the new strand. Such hemimethylated DNA is produced by progression of the replication forks and lasts until Dam methyltransferase methylates the new strand. It is therefore believed that a region of hemimethylated DNA covered by SeqA follows the replication fork. We show that this is, indeed, the case by using global ChIP on Chip analysis of SeqA in cells synchronized regarding DNA replication. To assess hemimethylation, we developed the first genome-wide method for methylation analysis in bacteria. Since loss of the SeqA protein affects growth rate only during rapid growth when cells contain multiple replication forks, a comparison of rapid and slow growth was performed. In cells with six replication forks per chromosome, the two old forks were found to bind surprisingly little SeqA protein. Cell cycle analysis showed that loss of SeqA from the old forks did not occur at initiation of the new forks, but instead occurs at a time point coinciding with the end of SeqA-dependent origin sequestration. The finding suggests simultaneous origin de-sequestration and loss of SeqA from old replication forks. 相似文献