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MatP local enrichment delays segregation independently of tetramer formation and septal anchoring in Vibrio cholerae [HiSC2]

ABSTRACT: Vibrio cholerae harbours a primary chromosome derived from the monochromosomal ancestor of the Vibrionales (ChrI) and a secondary chromosome derived from a megaplasmid (ChrII). Both carry a single origin of replication with replication terminating in a diametrically opposite zone, TerI and TerII, respectively. The choreography of TerI and TerII segregation determines when and where cell division occurs. Using a combination of deep sequencing and fluorescence microscopy techniques, we show that TerI and TerII both carry a very high density of MatP binding sites, are both structured into macrodomains, and are both targeted to mid-cell during replication by MatP, demonstrating that ChrII behaves as a bona fide chromosome. We further show that MatP-mediated cohesion and cellular positioning are independent of cell division and tetramer formation, but are linked to the local density of matS sites, in apparent contradiction to the mechanism of action of MatP in Escherichia coli.

ORGANISM(S): Vibrio cholerae

PROVIDER: GSE273190 | GEO | 2024/10/21

REPOSITORIES: GEO

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MatP local enrichment delays segregation independently of tetramer formation and septal anchoring in Vibrio cholerae

Project description:Vibrio cholerae harbours a primary chromosome derived from the monochromosomal ancestor of the Vibrionales (ChrI) and a secondary chromosome derived from a megaplasmid (ChrII). Both carry a single origin of replication with replication terminating in a diametrically opposite zone, TerI and TerII, respectively. The choreography of TerI and TerII segregation determines when and where cell division occurs. Using a combination of deep sequencing and fluorescence microscopy techniques, we show that TerI and TerII both carry a very high density of MatP binding sites, are both structured into macrodomains, and are both targeted to mid-cell during replication by MatP, demonstrating that ChrII behaves as a bona fide chromosome. We further show that MatP-mediated cohesion and cellular positioning are independent of cell division and tetramer formation, but are linked to the local density of matS sites, in apparent contradiction to the mechanism of action of MatP in Escherichia coli.

2024-10-21 | GSE273189 | GEO

MatP local enrichment delays segregation independently of tetramer formation and septal anchoring in Vibrio cholerae [3C]

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Genome-wide analysis of E. coli MukB distribution using ChIP-sequencing

Project description:Paper abstract : The Escherichia coli SMC complex, MukBEF, interacts with the ParC subunit of topoisomerase IV (TopoIV), consistent with sequential roles in chromosome unlinking and segregation. Although clusters of MukBEF molecules are normally associated with the chromosome replication origin region (ori), we demonstrate their association with the replication termination region (ter), which is enhanced when ATP hydrolysis by MukB is impaired. We show that MukBEF interacts in vivo and in vitro with MatP, which binds matS sites in ter. The MatP-MukBEF interaction limits the stable association of MukBEF complexes with ter and the availability of functional TopoIV during decatenation of sister ters. The MukBEF-TopoIV interaction is influenced by the nucleotide state of MukBEF. The combined action of MukBEF and MatP helps position MukBEF clusters in relation to the chromosome, thereby, influencing chromosome organization and segregation. Finally, we demonstrate that the regulated sequential action of TopoIV and MukBEF promotes chromosome decatenation and segregation.

2016-01-28 | GSE67221 | GEO

Genome-wide ParB2-DNA and RctB-DNA interaction analyses in Vibrio cholerae

Project description:Replication and segregation are the two main processes that maintain chromosomes in growing cells. In bacteria, replication and transcription have been proposed to provide motive force in chromosome segregation. Recently, ParB2, a segregation protein, encoded by V. cholerae chromosome II (chrII) was also found to influence chrII replication. V. cholerae chrII replication is primarily controlled by its specific initiator protein, RctB. Here, we have screened for ParB2 and RctB binding sites using a genome-wide DNA binding analysis (ChIP-chip). We report the identification of a new region containing additional RctB and ParB2 binding sites, and suggest the mechanisms to coordinate replication initiation with segregation of chromosomes.

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Genome-wide ParB2-DNA and RctB-DNA interaction analyses in Vibrio cholerae

2015-09-14 | E-GEOD-72978 | biostudies-arrayexpress

Loss of the SUMO Protease Ulp2 Triggers a Specific Multi-Chromosome Aneuploidy

Project description:Post-translational protein modification by the small ubiquitin-related modifier (SUMO) protein regulates numerous cellular pathways, including transcription, cell division and genome maintenance. The SUMO protease Ulp2 modulates many of these SUMO-dependent processes in budding yeast. To investigate changes to the transcriptome of cells lacking Ulp2, whole-genome RNA sequencing (RNA-seq) was employed. Unexpectedly, ulp2Δ cells display a general two-fold increase in transcript levels across two particular chromosomes, Chromosome I (ChrI) and Chromosome XII (ChrXII). Quantification of relative DNA levels showed that ChrI and ChrXII are present at twice their normal copy number in ulp2Δ cells. This double disomy occurs in mutants in multiple genetic backgrounds and does not require passage through meiosis. An abnormal number of chromosomes in a cell, termed aneuploidy, is usually deleterious. However, development of specific aneuploidies allows rapid adaptation to cellular stresses in yeast and other species, and aneuploidy characterizes most tumors. Extra copies of ChrI and ChrXII appear quickly following loss of active Ulp2, suggesting aneuploidy is an adaptive mechanism in cells lacking the functional SUMO protease. The specific aneuploidy in ulp2Δ cells highlights a unique example of a homogeneous, multi-chromosome aneuploidy in response to mutation of a single gene.

2017-08-11 | GSE85501 | GEO

RNA sequence (RNA-seq) profiling of synthesized chromosome SynI in Saccharomyces cerevisiae

Project description:We designed and synthesized synI, which is ~21.4% shorter than native chrI, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns surrounding potential instability and karyotype imbalance, and is now attached to synIII, yielding the first synthetic yeast fusion chromosome. We constructed additional fusion chromosomes to investigate effects of fusions on nuclear function. We observed unexpected loops and twisted structures in chrIII-I and chrIX-III-I fusion chromosomes dependent on silencing protein Sir3. ChrI faces special challenges in assuring meiotic crossovers required for efficient homolog disjunction. Centromere deletions engineered into fusion chromosomes revealed opposing effects of core centromeres and pericentromeres in modulating deposition of meiotic recombination protein Red1. These effects extended over >100kb, to disproportionally promote meiotic recombination of small chromosomes like chrI. These findings reveal the power of synthetic genomics to uncover new biology and deconvolute complex biological systems.

2023-09-15 | GSE242509 | GEO

Multiscale structuring of the E. coli chromosome by nucleoid-associated and condensin proteins

Project description:As in Eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic information is generally carried on a circular chromosome with a single origin of replication from which two replication forks proceed bidirectionaly towards the opposite terminus region. Here we investigate the higher-order architecture of the Escherichia coli genome, showing its partition into two structuraly distinct entities by a complex and intertwined network of contacts: the replication terminus (ter) region and the rest of the chromosome. Outside ter, the condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF activity and contacts are restricted to ~280 kb creating a domain with distinct structural properties. We also show how other NAPs contribute to nucleoid organization, such as H-NS that restricts short-range interactions. Combined, these results reveal the contributions of major, evolutionary conserved proteins in a bacterial chromosome organization.

2017-12-07 | GSE107301 | GEO

Chromatin immunoprecipitation of Bacillus subtilis to identify Noc protein binding sites

Project description:Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis the nucleoid occlusion protein Noc protects chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP-on-Chip and bioinformatics, we have identified a consensus Noc-binding DNA sequence (NBS), and show that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus. Purified Noc bound specifically to an NBS in vitro. NBSs inserted near the replication terminus bound Noc-YFP and caused a delay in cell division. An autonomous plasmid carrying an NBS recruited Noc-YFP and conferred a severe Noc-dependent inhibition of cell division. This shows that Noc is a potent inhibitor of division but that its activity is strictly localized by interaction with NBS sites in vivo. We propose that Noc not only serves as a spatial regulator of cell division to protect the nucleoid, but also a timing device with an important role in the co-ordination of chromosome segregation and cell division.

2009-07-01 | E-MEXP-2133 | biostudies-arrayexpress

Terminus DNA loss in the Escherichia coli

Project description:Study of the mechanisms of RecB mutant terminus DNA loss in Escherichia coli. FX158: WT MG1655 FX35: recB- FX37: ruvAB- FX51: matP- MIC18: recB- sbcD- sbcC- MIC20: recB- ruvAB- MIC24: matP- recB- MIC25: recA- recB- MIC31: sbcB- sbcD- MIC34: recA- recD- MIC40: linear chromosome MIC41: linear chromosome recB- MIC42: matP- ftsKC- MIC43: matP- ftsKC- recB- MIC48: recA- Cells were grown in M9 minimal medium supplemented with 0.4 % glucose to exponential phase (0.2 OD 650 nm). Chromosomal DNA was extracted using the Sigma GenElute bacterial genomic DNA kit. 5 μg of DNA were used to generate a genomic library according to Illumina's protocol. The libraries and the sequencing were performed by the High-throughput Sequencing facility of the I2BC (http://www.i2bc.paris-saclay.fr/spip.php?article399&lang=en, CNRS, Gif-sur-Yvette, France). Genomic DNA libraries were made with the ‘Nextera DNA library preparation kit’ (Illumina) following the manufacturer’s recommendations. Library quality was assessed on an Agilent Bioanalyzer 2100, using an Agilent High Sensitivity DNA Kit (Agilent technologies). Libraries were pooled in equimolar proportions. 75 bp single reads were generated on an Illumina MiSeq instrument, using a MiSeq Reagent kit V2 (500 cycles) (Illumina), with an expected depth of 217X. An in-lab written MATLAB-based script was used to perform marker frequency analysis. Reads were aligned on the Escherichia coli K12 MG1655 genome using BWA software. Data were normalized by dividing uniquely mapping sequence reads by the total number of reads. Enrichment of uniquely mapping sequence reads in 1 kb non-overlapping windows were calculated and plotted against the chromosomal coordinates.

2017-11-10 | E-MTAB-6122 | biostudies-arrayexpress

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