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
