Global spatial organization of γ proteobacterial nucleoids is determined by specific long-range interactions.
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ABSTRACT:
ORGANISM(S): Pseudomonas aeruginosa PAO1
PROVIDER: GSE40353 | GEO | 2016/11/02
SECONDARY ACCESSION(S): PRJNA173653
REPOSITORIES: GEO
Similar Datasets
Project description:GCC was used to determine the structure of E. coli grown in LB or treated with SHX. The bacterial genome is highly condensed into a nucleoid structure. Here we present global analyses of the genome spatial organization for two γ-proteobacteria: Escherichia coli and Pseudomonas aeruginosa by Genome Conformation Capture. Long distance interactions occurred within the E. coli and P. aeruginosa nucleoids with frequencies that were affected by growth condition and gene dosage. Spatial clustering of genes that are either up or down-regulated depended on the environmental signals, indicating a non-random functional organization of the nucleoid. The largest changes in gene expression upon amino acid starvation occurred in genes that participate in long-range interactions. These genes remained highly spatially clustered when transcript levels decreased. Environment specific interactions were related to DNA motifs but did not correlate with binding sites for nucleoid associated proteins. Overall we identify spatial organization as a significant factor in bacterial gene regulation and suggest that the prokaryotic operon is not simply a linear entity.
2013-05-02 | GSE40603 | GEO
Project description:GCC was used to determine the structure of E. coli grown in LB or treated with SHX. The bacterial genome is highly condensed into a nucleoid structure. Here we present global analyses of the genome spatial organization for two M-NM-3-proteobacteria: Escherichia coli and Pseudomonas aeruginosa by Genome Conformation Capture. Long distance interactions occurred within the E. coli and P. aeruginosa nucleoids with frequencies that were affected by growth condition and gene dosage. Spatial clustering of genes that are either up or down-regulated depended on the environmental signals, indicating a non-random functional organization of the nucleoid. The largest changes in gene expression upon amino acid starvation occurred in genes that participate in long-range interactions. These genes remained highly spatially clustered when transcript levels decreased. Environment specific interactions were related to DNA motifs but did not correlate with binding sites for nucleoid associated proteins. Overall we identify spatial organization as a significant factor in bacterial gene regulation and suggest that the prokaryotic operon is not simply a linear entity. 4 samples grown in LB; 4 samples treated with SHX. For Genome Conformation Capture (GCC) analyses, E. coli strains were recovered from -80M-BM-0C on LB agar (2%) plates for 24 hours. LB medium (3ml) starter cultures were inoculated and grown (37M-BM-0C, 220rpm, 16h). The Optical Density (OD600) of cultures was measured and used to inoculate LB test cultures to an OD600 of ~0.02. The test cultures were grown (37M-BM-0C, 220rpm) until the OD600 reached ~0.25 and then harvested. For the serine hydroximate (SHX) treated samples the cultures were treated with SHX (500 M-BM-5g/ml, 30 min) before harvesting. E. coli chromatin was prepared according to Rodley et al. (2009)with the following modifications. A total of 5*109 cells were cross-linked with formaldehyde (1% final v/v, 20min, RT) and then quenched with glycine (125mM final, 10min). Cells were collected by centrifugation (4000rpm, 15min, 4M-BM-0C), washed twice (1% PBS, 1% TritonX-100, 5ml/50ml culture) and pelleted (4000rpm, 15min, 4M-BM-0C). Cell pellets were suspended in 800M-NM-<l of B1 lysis buffer (10mM Tris pH 8.0, 50mM NaCl, 10mM EDTA, 20% (w/v) sucrose, 1mg/ml lysozyme) and incubated (37M-BM-0C, 30min). 800M-NM-<l of B2 lysis buffer (200mM Tris pH 8.0, 600mM NaCl, 4% TritonX-100, 1 protease inhibitor tablet (Roche) per 10ml of buffer added just before use) was gently added, mixed by inversion 3-4 times and incubated (37M-BM-0C, 10min). The cell lysate was centrifuged (21,500g, 20min, 4M-BM-0C) and the supernatant decanted. The chromatin was washed once with 1ml of chromatin digestion buffer (10mM Tris-HCl pH 8.0, 5mM MgCl2, 0.1% TritonX-100) by inverting the tube 3-4 times and centrifuged (21,500g, 20min, 4M-BM-0C). The supernatant was decanted and the chromatin pellet was suspended in 500M-NM-<l chromatin digestion buffer. Chromatin samples were aliquoted into 10 sets of 5*108 cells. Samples were digested with HhaI (100U, New England Biolabs). Following digestion a ligation control was added and the samples were ligated with T4 DNA ligase (20U, Invitrogen). Following ligation, cross-links were removed in the presence of proteinase K (0.45U, Fermentas). RNA was removed and pUC19 plasmid (27.4pg/2ml) was added as a sequencing control prior to three extractions with 1:1 Phenol:Chloroform. DNA was column purified (Zymo, DNA clean and concentratorTM-5 kit) according to the manufacturerM-bM-^@M-^Ys instructions and eluted in milliQ H2O before combining for sequencing. 3M-NM-<g of purified DNA was sent for pared-end sequencing at the ATC sequencing facility (Rockville, MD, USA) on an Illumina Hi-Seq. External ligation controls were produced by PCR amplification of short regions from the Lambda phage genome and the pRS426. Primers (Table S4 were designed to include an HhaI site at one end of the final product. PCR products were purified using a PCR purification kit (Qiagen), digested with HhaI (4U, 37M-BM-0C, 2h) and purified again. Purified, digested PCR products were introduced into the GCC samples at a 1:1 ratio with the number of genomes prior to the ligation step during GCC preparation. The pRS426 fragment was introduced into the exponential phase (LB grown) samples and resulted in 220 separate ligation events with HhaI restriction fragments on the genome. The Lambda phage fragment was introduced into the SHX treated samples and resulted in 2 ligation events with HhaI fragments on the genome. Network assembly was performed using the Topography suite v1.19. GCC networks were constructed from 100bp (E. coli) paired end Illumina Genome Analyser sequence reads. Topography uses the SOAP algorithm to position PE tags and single ends which contain a HhaI (E. coli) restriction site onto the E. coli (NC_000913) reference genomes, respectively. Each reference genome also contained the pUC19 (SYNPUC19CV) sequence. For the E. coli alignment the sequences of the pRS426 plasmid and Lambda phage ligation controls were also included in the file to which the sequences were aligned. No mismatches or unassigned bases (N) were allowed during positioning. Except where indicated, bioinformatics and statistical analyses were performed on interactions in which the sequence reads were able to be mapped uniquely onto the reference genome and were above the FDR cut-off value (5). All bioinformatics analysis was perfumed using in house Perl scripts.
2013-05-02 | E-GEOD-40603 | biostudies-arrayexpress
Project description:A fundamental objective in molecular biology is to understand how DNA is organized in concert with various proteins, RNA, and biological membranes. Mitochondria maintain and express their own DNA (mtDNA), which is arranged within structures called nucleoids. Their functions, dimensions, composition, and precise locations relative to other mitochondrial structures are poorly defined. Superresolution fluorescence microscopy techniques that exceed the previous limits of imaging within the small and highly compartmentalized mitochondria have been recently developed. We have improved and employed both two- and three-dimensional applications of photoactivated localization microscopy (PALM and iPALM, respectively) to visualize the core dimensions and relative locations of mitochondrial nucleoids at an unprecedented resolution. PALM reveals that nucleoids differ greatly in size and shape. Three-dimensional volumetric analysis indicates that, on average, the mtDNA within ellipsoidal nucleoids is extraordinarily condensed. Two-color PALM shows that the freely diffusible mitochondrial matrix protein is largely excluded from the nucleoid. In contrast, nucleoids are closely associated with the inner membrane and often appear to be wrapped around cristae or crista-like inner membrane invaginations. Determinations revealing high packing density, separation from the matrix, and tight association with the inner membrane underscore the role of mechanisms that regulate access to mtDNA and that remain largely unknown.
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Project description:Dynamic functional connectivity (dFC) derived from resting-state functional magnetic resonance imaging sheds light onto moment-to-moment reconfigurations of large-scale functional brain networks. Due to computational limits, connectivity is typically computed using pre-defined atlases, a non-trivial choice that might influence results. Here, we leverage new computational methods to retrieve dFC at the voxel level in terms of dominant patterns of fluctuations, and demonstrate that this new representation is informative to derive meaningful brain parcellations, capturing both long-range interactions and fine-scale local organization. Specifically, voxelwise dFC dominant patterns were captured through eigenvector centrality followed by clustering across time/subjects to yield most representative dominant patterns (RDPs). Voxel-wise labeling according to positive/negative contributions to RDPs, led to 37 unique labels identifying strikingly symmetric dFC long-range patterns. These included 449 contiguous regions, defining a fine-scale parcellation consistent with known cortical/subcortical subdivisions. Our contribution provides an alternative to obtain a whole-brain parcellation that is for the first time driven by voxel-level dFC and bridges the gap between voxel-based approaches and graph theoretical analysis.
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Project description:Mitochondrial DNA (mtDNA) is packaged by association with specific proteins in compact DNA-protein complexes named mitochondrial nucleoids (mt-nucleoids). The budding yeast Saccharomyces cerevisiae is able to grow either aerobically or anaerobically. Due to this characteristic, S. cerevisiae has been extensively used as a model organism to study genetics, morphology and biochemistry of mitochondria for a long time. Mitochondria of S. cerevisiae frequently fuse and divide, and perform dynamic morphological changes depending on the culture conditions and the stage of life cycle of the yeast cells. The mt-nucleoids also dynamically change their morphology, accompanying morphological changes of mitochondria. The mt-nucleoids have been isolated morphologically intact and functional analyses of mt-nucleoid proteins have been extensively performed. These studies have revealed that the functions of mt-nucleoid proteins are essential for maintenance of mtDNA. The aims of this review are to summarize the history on the research of yeast mt-nucleoids as well as recent findings on the organization of the mt-nucleoids and mitochondrial dynamics.
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Project description:Nucleoid remodeling facilitated by DNA supercoiling results in changes in nucleoid configuration and involves nucleoid-associated proteins (NAPs) and structural maintenance of chromosomes (SMC) proteins among others. Changes in nucleoid configurations regulated by NAPs are synchronized with cellular adaptation and influence the simultaneous expression of several genes. HU, a ubiquitous bacterial histone-like protein, is among the most conserved and abundant NAPs in eubacteria. In Escherichia coli, HU forms dimers by HUα self-association (HUαα) or by HUα-HUβ interactions (HUαβ). HUα is mostly expressed during lag and early exponential growth phase and HUβ is expressed only during the later exponential and stationary phase, pointing to distinct HUαα/DNA and HUαβ/DNA packaging of the nucleoid in regulating expression patterns during growth and stasis. Mutations or the deletion of HU transform the E. coli nucleoid to a different form and alter overall transcription program; thus, HU interactions with DNA directly affect global gene regulation. Recently, analysis of high-resolution contact maps of the E. coli nucleoid revealed an important role of HU to promote long-range DNA-DNA contacts within the nucleoid. Yet, the molecular connections between HU-DNA interactions and changes in nucleoid architecture that regulate gene expression globally remain unknown. Here, we explored the higher-order E. coli nucleoid organization by soft x-ray tomography (SXT) and revealed an effect of HU surface charges in overall nucleoid organization and rearrangements. We also studied global transcription by next-generation RNA sequencing (RNA-Seq) and found a link between nucleoid rearrangement and changes in global transcription. To determine the functional relationships of observed nucleoid rearrangement and HU-DNA interactions, we also characterized the overall organization of HU nucleoprotein complexes in solution by small angle x-ray scattering (SAXS). We found that HUαα-mediated DNA networks are different at different ionic strengths and pHs. By means of macromolecular crystallography, we additionally elucidate HUαα dependent molecular switches that modulate DNA networking. This integrative structural study explains how HUαα regulates dynamic transformations of the nucleoid by DNA bridging to control nucleoid rearrangement and global gene regulation.
2020-04-01 | GSE134667 | GEO
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