Transcription profiling of E.coli overexpressing sigma32
Ontology highlight
ABSTRACT:
ORGANISM(S): Escherichia coli
SUBMITTER: Kai Zhao
PROVIDER: E-GEOD-2140 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
The global transcriptional response of Escherichia coli to induced sigma 32 protein involves sigma 32 regulon activation followed by inactivation and degradation of sigma 32 in vivo.
Zhao Kai K Liu Mingzhu M Burgess Richard R RR
The Journal of biological chemistry 20050309 18
sigma(32) is the first alternative sigma factor discovered in Escherichia coli and can direct transcription of many genes in response to heat shock stress. To define the physiological role of sigma(32), we have used transcription profiling experiments to identify, on a genome-wide basis, genes under the control of sigma(32) in E. coli by moderate induction of a plasmid-borne rpoH gene under defined, steady-state growth conditions. Together with a bioinformatics approach, we successfully confirme ...[more]
Similar Datasets
Project description:This study examined the genes under the control of sigma32 in E. coli by moderate induction of a plasmid-borne rpoH gene under defined steady-state growth condition. Samples were taken from culture at mid log phase (OD=0.2) before or 5 minutes, 10 minutes or 15 minutes after induction. Samples were then RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChip E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com). Meanwhile, we measured the protein level of sigma32 during the time course. The changes of some sigma32 –dependent genes were also determined in a DnaK null mutant to examine the anti- sigma32 function of DnaK. Keywords = sigma factor Keywords = regulon Keywords = microarray Keywords = Western blot Keywords = E. coli Keywords = anti-sigma factor Keywords: time-course
2007-08-01 | GSE2140 | GEO
Project description:The study investigated the effect of different carbon sources on E. coli global gene expression. We grew MG1655 cells aerobically in MOPS minimal medium with either glucose, glycerol, succinate, L-alanine, acetate, or L-proline as the carbon supply. Samples were taken from each culture at mid log phase and were RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChipÒ E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com). Keywords: parallel sample
2005-03-11 | GSE2037 | GEO
Project description:This study examined the genes under the control of FlhDC and sigmaF in E. coli. Keywords: wild-type, deletion and overexepression strains Under defined steady-state growth condition, we used two different genetic approaches to alter the modulator concentration in cells; (1) moderately expressing FlhDC or sigmaF from anhydrotetracycline (aTc) inducible and Tet repressor-controlled PLtet promoter in a plasmid-borne flhDC or fliA gene; (2) disrupting the expression of FlhDC or sigmaF in flhDC or fliA deletion mutant strains. Samples were taken from culture with wild-type or deletion strains at mid log phase (OD=0.2) or from overexpression strains at mid log phase (OD=0.2) before or 5 minutes after moderate induction. Samples were then RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChip E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com).
2009-05-01 | E-GEOD-5818 | biostudies-arrayexpress
Project description:The study investigated the effect of different carbon sources on E. coli global gene expression. We grew MG1655 cells aerobically in MOPS minimal medium with either glucose, glycerol, succinate, L-alanine, acetate, or L-proline as the carbon supply. Samples were taken from each culture at mid log phase and were RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChipà E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com).
2007-07-19 | E-GEOD-2037 | biostudies-arrayexpress
Project description:The purpose of this study is to investigate the changes of global gene expression in E. coli during an oxygen shift. All cultures were grown under aerobic or anaerobic conditions in M9 minimal media supplemented with glucose. Samples were RNA-stabilized using Qiagen RNAProtect Bacterial Reagent, and total RNA was isolated from exponentially growing cells using a Qiagen RNeasy mini kit (protocols available at www1.qiagen.com). The RNA (10 µg) was then used as the template for cDNA synthesis, the product of which was fragmented, labelled, and hybridized to an Affymetrix E. coli Antisense Genome Array, which was washed and scanned to obtain an image. All of these steps were performed according to Affymetrix protocols (available at www.affymetrix.com). This SuperSeries is composed of the SubSeries listed below.
2004-03-10 | GSE1121 | GEO
Project description:We report the different expression level of in vivo transcriptome and DMEM cultured bacteria E.coli O157:H7 samples. After obtaining RNA from mice colon and LB cultured free living cells, RNA samples were purified using an RNeasy Mini Kit (Qiagen) and bacterial rRNAs were depleted using the Ribo-Zero rRNA Removal Kit (Epicentre; #RZNB1056). Library construction of RNA samples was carried out using a NEBNext R UltraTM Directional RNA Library Prep Kit for Illumina R (NEB, USA), following the manufacturer’s recommendations. After cluster generation, library preparations were sequenced on an Illumina Hiseq platform to generate paired-end reads.We find that a novel sRNA s77 significantly upregulated compared in vivo samples and LB-cultured sample. This study provides a novel sRNA for further identifications.
2022-06-23 | GSE133193 | GEO
Project description:We determined the global gene expression profiles of wildtype, dam, dam mutS, and mutS mutant E. coli strains. Experiment Overall Design: Overnight cultures were diluted 1000-fold with fresh LB medium and cultured further. Log phase cultures were diluted to a cell density of 2 X 108 cells/ml in M9 salts and incubated at 37°C for two hours, after which they were resuspended in LB broth for 90 minutes. This treatment served as a mock treatment for cultures incubated with a chemical (cisplatin) in a parallel study (Robbins et al., unpublished results). Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) followed by DNAse digestion according to the manufacturerâs protocol. The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm. RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20 mM 3-[N-morpholino]propanesulfonic acid, 5 mM sodium acetate, 1 mM ethylenadiaminetetraacetic acide (EDTA))) or by analysis on an Agilent 2100 Bioanalyzer. All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure. mRNA was enriched from total RNA as described in the Affymetrix GeneChip Expression Analysis Technical Manual for GeneChip E. coli Sense Genome Arrays.
2007-07-20 | E-GEOD-2928 | biostudies-arrayexpress
Project description:Metals at high concentrations can exert toxic effects on microorganisms. It has been widely reported that lowering environmental pH reduces effects of cadmium toxicity in bacteria. Understanding the effects of pH-mediated cadmium toxicity on bacteria would be useful for minimizing cadmium toxicity in the environment and gaining insight into the interactions between organic and inorganic components of life. Growth curve analysis confirmed that cadmium was less toxic to Escherichia coli at pH 5 than at pH 7 in M9 minimal salts medium. To better understand the cellular mechanisms by which lowering pH decreases cadmium toxicity, we used DNA microarrays to characterize global gene expression patterns in E. coli in response to cadmium exposure at moderately acidic (5) and neutral (7) values of pH. Higher expression of several stress response genes including hdeA, otsA, and yjbJ at pH 5 after only 5 minutes was observed and may suggest that acidic pH more rapidly induces genes that confer cadmium resistance. Genes involved in transport were more highly expressed at pH 7 than at pH 5 in the presence of cadmium. Of the genes that showed an interaction between pH and cadmium effects, 46% encoded hypothetical proteins, which may have novel functions involved in mitigating cadmium toxicity. GROWTH CONDITIONS FOR MICROARRAY EXPERIMENTS: Two overnight cultures of E. coli K-12 were started in M9 medium. A 250 mL Erlenmeyer flask containing 100 mL of M9 medium was inoculated with 0.5 mL for each of the two overnight cultures, each of which was considered a biological replicate. The cultures were grown on a rotary shaker (200 rpm) at 37 °C until the contents of the flask reached an OD600 of 0.3 (mid-log phase of growth). Each culture was divided into four 25 mL aliquots, transferred to 50 mL conical tubes (Corning), and centrifuged at 2540 x g for 12 minutes. The supernatant was decanted, and the cells were resuspended in 25 mL of M9 medium at pH 7 or pH 5 in the presence or absence (two cultures of each) of 5.4 µM (1 µg/mL) total cadmium, added as CdCl2. The cultures were incubated at 25 °C for either 5 or 15 minutes with manual rotation of the flasks once per minute. After the appropriate amount of time, 15 mL of RNAProtect Bacteria Reagent (Qiagen) was added to each culture to immediately halt all metabolic processes. The solutions were vortexed, incubated at 25 °C for 5 minutes, and centrifuged for 12 minutes at 3750 x g. RNA was extracted from the cell pellets immediately following centrifugation. RNA EXTRACTION AND HYBRIDIZATION PROCEDURES: RNA was extracted and purified using a Masterpure RNA purification kit (Epicentre Technologies). The quantity and quality of the RNA samples were determined spectrophotometrically. Preparation of the cDNA, labeling with Cy3 and Cy5, and successive hybridizations were accomplished using a 3DNA Array 900MPX kit following the manufacturer’s protocols (Genisphere) with the following modifications. 3DNA reverse transcriptase enzyme (Genisphere # RT300320) rather than SuperScript II was added to 1 µg of RNA and 2 µL of a random primer (1 µg/µL). The final cDNA hybridization mix contained 29 µL 2X enhanced cDNA hybridization buffer rather than 2X SDS-based hybridization buffer or 2X formamide-based hybridization buffer. The cDNA mix was hybridized to a cDNA microarray printed by the Microarray and Proteomics Facility at the University of Alberta (Operon version 1.0 oligonucleotides). The arrays were scanned with a Versarray ChipReader (BioRad) with laser power at 75%, photomultiplier tube (PMT) sensitivity at 800 V, and detector gain at 1. DATA ANALYSIS: Each array directly compared transcription at pH 5 and pH 7 for a given cadmium treatment (0 or 5.4 µM cadmium) and exposure time. Dye swaps were performed for each biological replicate for each of the following treatments (8 total arrays): 5 minutes with cadmium exposure, 5 minutes without cadmium exposure, 15 minutes with cadmium exposure, and 15 minutes without cadmium exposure. The 0-minute exposure to cadmium treatment was obtained from the 5-minute microarray without cadmium exposure. Spot intensities and locations were determined using TIGR Spotfinder, Version 3.1.1. All subsequent analyses were performed using the ma-anova package in the open-source statistical software package, R (www.r-project.org), Version 2.4.1. The data were normalized using the regional lowess method. Following normalization the median expression values of genes represented in triplicate on each array were determined for each gene. A mixed model two-way ANOVA for the main fixed effects of pH, cadmium, and their interaction (array and spot were the random effects) were performed (using Type III F-tests) separately for each time point to identify genes for which pH and cadmium interacted to significantly affect expression (FDR-adjusted p ? 0.05).
2008-08-21 | E-GEOD-12373 | biostudies-arrayexpress
Project description:DNA microarray analysis of genes regulated by the alternative sigma factor, sigma 32 (rpoH). Sigma 32-dependent genes were initially identified by comparing a wild type (wt) E. coli K-12 strain that has a low level of sigma 32, with a strain over-expressing sigma 32 (following induction of rpoH from an inducible promoter by IPTG). We monitored changes in gene expression in 4 separate time-courses. Because sigma 32 is negatively regulated, its activity decreases 10 min after overexpression. Consequently, to identify genes regulated by sigma 32 we analyzed the expression data for 10 min after overexpression using SAM (Statistical Analysis of Microarrays). We identified 105 genes organized in 66 separate transcription units. This study is detailed in Nonaka et al 2006 (submitted). Keywords: time course
2006-03-06 | GSE4321 | GEO
Project description:The bacterial heat-shock response is regulated by the alternative sigma factor sigma 32 (RpoH), which responds to misfolded protein stress and directs the RNA polymerase to the promoterss for genes required for protein refolding or degradation. In P. aeruginosa, RpoH is essential for viability under laboratory growth conditions. Here, we used a transcriptomics approach to identify the genes of the RpoH regulon, including RpoH-regulated genes that are essential for P. aeruginosa. We placed the rpoH gene under control of the arabinose inducible PBAD promoter, then deleted the chromosomal rpoH allele. This allowed transcriptomic analysis of the RpoH regulon following a short up-shift in the cellular concentration of RpoH by arabinose addition, in the absence of a sudden increase in temperature. The P. aeruginosa ∆rpoH (PBAD-rpoH) strain grew in the absence of arabinose, indicating that some rpoH expression occurs without arabinose-induction. When arabinose was added, the rpoH mRNA abundance of P. aeruginosa ∆rpoH (PBAD-rpoH) measured by RT-qPCR increased fivefold within 15 min of arabinose addition. Whole genome transcriptome results showed that P. aeruginosa genes required for protein repair or degradation are induced by increased RpoH levels, and that many of the genes induced by RpoH are essential for P. aeruginosa growth. Other stress response genes induced by RpoH are involved in nucleic acid damage and repair and in amino acid metabolism. Annotation of the hypothetical proteins under RpoH control included proteins that may play a role in antibiotic resistances and in non-ribosomal peptide synthesis. The P. aeruginosa ∆rpoH (PBAD-rpoH) strain is impaired in its ability to survive during starvation compared to the wild-type strain. P. aeruginosa ∆rpoH (PBAD-rpoH) also has increased sensitivity to aminoglycoside antibiotics, but not to other classes of antibiotics, whether cultured planktonically or in biofilms. The enhanced aminoglycoside resistance of the mutant strain may be due to indirect effects, such as the build-up of toxic misfolded proteins, or to the direct effect of genes such as aminoglycoside acetyl transferases that are regulated by RpoH. Overall, the results demonstrate that RpoH regulates genes that are essential for viability of P. aeruginosa, that it protects P. aeruginosa from damage from aminoglycoside antibiotics, and that it is required for survival during nutrient limiting conditions. We used Affymetrix microarrays to characterize the RpoH regulon in P. aeruginosa. Using the P. aeruginosa ∆rpoH strain with rpoH under control of the PBAD promoter, we were able to perform transcriptomic analysis of genes induced by a sudden increase (15 min) in the cellular concentration of RpoH, independent from a sudden increase in temperature.
2022-11-06 | GSE217157 | GEO