Acid-Shifted Time Course of Gene Expression Profiles of Aerobically Grown Escherichia coli K-12
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ABSTRACT:
ORGANISM(S): Escherichia coli K-12 Escherichia coli
PROVIDER: GSE4778 | GEO | 2008/02/20
SECONDARY ACCESSION(S): PRJNA95551
REPOSITORIES: GEO
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Project description:The effect of an extracellular acid shift on gene expression profiles of Escherichia coli K-12 W3110 was observed using Affymetrix E. coli arrays. In order to maximize aeration and maintain logarithmic growth, the overnight culture (LBK broth medium) was diluted 500-fold into a 250-ml baffled flask containing 55 ml of 20mM HOMOPIPES buffered medium (pH 7.6). Cultures were grown to OD600=0.2. A shift to acid external pH was conducted by rapid addition of 840 µl 1M HCl, which lowered the pH of the medium to pH 5.5. For each of five biological replicates, 10-ml samples were taken at times 0, 1, 5, and 10 min post addition of HCl. Each sample was added to 1 ml 10% phenol-ethanol stop solution in <5 sec. For each sample, cDNA synthesized from total RNA was hybridized onto Affymetrix E. coli arrays. Model-based gene expression intensities were determined using GCOS software. Gene-by-gene temporal differential expression was analyzed as a mixed-effects model using polynomial time functions as fixed effects and flask variation as a random effect. Experiment Overall Design: Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of time versus change in external pH. Overnight cultures were diluted 1:500 in potassium-modified Luria-Bertani medium (LBK) buffered with 20 mM HOMOPIPES at 7.6. Bacteria were cultured in baffled flasks (less than 10% volume) with rotation at 225 rpm, incubated at 37°C to an optical density at 600 nm of 0.2. External pH was lowered to pH 5.5 by adding 840 µl of 1M HCl to 40 mls of culture within 5 seconds. Four samples were taken from each of five replicate cultures, corresponding to time 0 (before HCl addition),1, 5, and 10 min post HCl addition.
2008-04-07 | E-GEOD-4778 | biostudies-arrayexpress
Project description:Escherichia coli O157 presents a number of specific problems in terms of food safety and public health. It has been found that E. coli O157 is more resistant to a number of the stresses encountered during food production such as heat, pH and osmotic shock. This greater resistance is thought to contribute to the low infectious dose of E. coli O157 (<100 organisms). Moreover, E. coli O157 is associated with debilitating conditions such as haemorrhagic colitis and haemoytic uraemic syndrome, particularly in children and the elderly. We have been studying the stress responses of E. coli O157:H7 (Sakai) and comparing with a commensal strain of E. coli K-12, MG1655. We found that E. coli O157 (Sakai) is more resistant to heat stress than MG1655. A microarray study of these strains subjected to sub-lethal heat-stress at 45M-BM-0C was carried out. In E. coli O157 (Sakai), 380 genes responded significantly to the treatment compared to 410 genes in MG1655. Overnight cultures of E. coli O157 (Sakai) and E. coli K-12 MG1655 were grown in Neidhardt's EZ Rich Defined Medium and diluted 1:100 in 50 ml fresh medium in 125 ml Ehrlenmeyer flasks. The cultures were shaken at 37M-BM-0C until the optical density (OD600) reached 0.4. Each culture was divided into 2 equal parts in identical flasks. One flask flask was transferred to a shaking water bath and incubated at 45M-BM-0C for 10 min; the other flask was incubated at 37M-BM-0C for 10 min. After incubation, the cultures were transferred to 50 mL centrifuge tubes and treated with RNAprotectM-bM-^DM-" to stabilise the mRNA. The experiment was performed 3 times on different days. Six custom-made microarray slides were used in this study; each slide was hybridised with labelled cDNA made from untreated and heated E. coli O157 (Sakai) or MG1655.
2018-03-20 | E-MTAB-5912 | biostudies-arrayexpress
Project description:Each colony of A. hydrophila and the OXY-R strain were incubated in 5 mL LB medium overnight, and then diluted in 100 mL fresh LB medium at a ratio of 1:100 and subsequently cultured until the optical density at 600 nm (OD600) reached 1.0. The cultures were harvested via centrifuging for 20 min at 10,000 x g, 4°C, and then the bacterial cells were washed with cold phosphate buffered saline (PBS, pH 7.4) for three times. The cell pellets were resuspending in the 10 mL ultrasonic buffer (50 mM Tris-HCl, pH 7.4, 1 mM PMSF) and disrupted with intermittent sonic oscillation for a total of 30 minutes at 9 seconds intervals on ice. Subsequently, the cell debris and unbroken cells were separated by centrifugation at 8,000 x g for 20 min at 4°C. Then the supernatant was centrifuged at 100,000 x g for 1 h at 4°C in the Optima LE-80 K Ultracentrifuge (Beckman, Palo Alto, CA, USA). After the pellet was dissolved with 2% sodium lauroyl sarcosine (in 50 mM Tris-HCl, pH 7.5) for 40 min at room temperature (RT), the pellets were ultracentrifuged again at 100,000 x g for 1 h at 4°C. Finally, the precipitate was dissolved in an appropriate volume of SDT buffer (4 % SDS, 0.1 M DTT [dithiothreitol], and 0.5 M triethylammonium bicarbonate buffer [TEAB, pH 8.5]). The protein concentration was determined using the Bradford method and then stored at -20°C until subsequent use.The proteins were digested with trypsin after being reduced with DTT and alkylated with Iodoacetamide using a filter-aided sample preparation method (Tanca et al., 2013; Li et al., 2016b). After washing three times with 0.5 M TEAB followed by fractionation using the 10 kDa ultrafiltration system (Millipore, Billerica, MA, USA), about 100 μg digested peptide from each group, including two biological replicates, was taken out for further labeled using TMT isobaric and isotopic mass-tagging kits (Thermo Fisher Scientific, MA, USA), which was performed according to instructions from the kit.
2018-11-23 | PXD009622 | Pride
Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.
2021-09-05 | GSE152862 | GEO
Project description:Flag-YBX1 overexpressed T24 cells pellets were resuspended with 2 volume of lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U/ml RNase inhibitor), and incubated at 4 °C for 30 min with rotation. Then the lysate was centrifuged at 15 000 g for 20 min. Before incubating the lysate with Flag beads, 100ul were taken as input. The anti-Flag M2 magnetic beads (Sigma, 10 μl per mg lysate) were washed with NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U/ml RNase inhibitor) four times. Cell lysate was mixed with M2 beads and incubated at 4 °C for 4 h with rotation. The beads were washed two times with 1 ml ice-cold NT2 buffer. Then the beads were subject to Micrococal nuclease (NEB) digestion (1:1 000 000 dilution) for 8 min at 37 °C. The beads were cooled on ice immediately for 5 min and washed two times with 1 ml ice-cold 1× PNK+EGTA buffer (50 mM Tris-HCl pH 7.5, 20 mM EDTA, 0.05% NP-40, 200 U/ml RNase inhibitor) and two times with 1 ml ice-cold 1× PK buffer (50 mM NaCl, 100 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.2% SDS, 200 U/ml RNase inhibitor). Then the beads were digested with 200 μl pre-heated (20 min at 50 °C) Proteinase K and RNAs were extracted with an equal volume of Acid-Phenol: Chloroform, pH 4.5 (Ambion). The RNAs were subjected to rRNA removal and Bisseq. The libraries were sequenced on the Illumina HiSeq X-Ten platform at Novogene (Tianjin, CA) with paired-end 150 bp read length.The m5C sites were called using meRanCall from meRanTK (FDR < 0.01).
2019-07-02 | GSE133625 | GEO
Project description:RNA-seq libraries were constructed for three samples, including (I) ΔURA3 strain grown in SD medium containing 2% w/v glucose(pH=5.0);(II)ΔURA3 strain grown in SD medium containing 2%w/v glucose and 2% w/v succinic acid(pH=5.0);(III)ΔURA3 strain grown in SD medium containing 2% w/v succinic acid(pH=5.0). For preparation of RNA samples, ΔURA3 cells grown overnight were inoculated into 50 ml liquid Synthetic Dextrose (SD) medium with the initial inoculation amount of OD600= 0.1, and cultured for 24 hours to the logarithmic growth phase (OD600= 2-3). The cells were collected by centrifugation at 6000g for 5 minutes. After washing twice with phosphate buffer saline (PBS), they were inoculated into new 50 mL SD medium containing carbon sources of different combinations of glucose and succinic acid .The pH of the culture sample is adjusted to 5.0 with NaOH. After flask culturing at 30°C and 250 rpm for an additional three hours, the yeast cells were collected by centrifugation for total RNA isolation and Illumina RNA-seq library construction. Total RNA for samples were isolated using TRIzol reagent (Invitrogen, Grand Island, USA), then used for high-throughput RNA sequencing. The 150-nt paired-end RNA-seq libraries were generated commercially at Novogene Biotechnology Co. Ltd (Tianjin, China) by using Illumina’s Hiseq X Ten platform (Illumina, San Diego, USA). Each sample contains two biological replicates.
2021-05-05 | GSE159457 | GEO
Project description:Escherichia coli O157 presents a number of specific problems in terms of food safety and public health. It has been found that E. coli O157 is more resistant to a number of the stresses encountered during food production such as heat, pH and osmotic shock. This greater resistance is thought to contribute to the low infectious dose of E. coli O157 (<100 organisms). Moreover, E. coli O157 is associated with debilitating conditions such as haemorrhagic colitis and haemoytic uraemic syndrome, particularly in children and the elderly. We have been studying the stress responses of E. coli O157:H7 (Sakai) and comparing with a commensal strain of E. coli K-12, MG1655. We found that E. coli O157 (Sakai) is more sensitive to oxidative stress than MG1655. A microarray study of these strains treated with sub-lethal concentrations (0.5mg/ml) of menadione revealed big differences in their responses. In E. coli O157 (Sakai), 540 genes responded significantly to the treatment compared to 121 genes in MG1655. One surprising finding from the microarray data was the observation that many iron-transport genes were up-regulated in E. coli O157 (Sakai) whereas relatively few were induced in MG1655 despite the fact that the bacteria were grown in a medium containing ample iron. We speculated that the induction of iron transport genes in an iron-rich medium might have contributed to the enhanced killing of E. coli O157 (Sakai) through triggering of a Fenton reaction. We speculated that the difference in sensitivity to oxidative stress might be due to differences in the intracellular iron content of E. coli O157 and MG1655. We found that E. coli O157 contains ~50% more iron than MG1655 and believe that during oxidative stress, this iron is released by damaged proteins. The greater levels of free iron in E. coli O157 will trigger a greater Fenton reaction that can damage the ferric uptake regulator (Fur), resulting in unregulated iron transport. In MG1655, the lower iron content results in a smaller Fenton reaction, enabling the cellular protection systems to limit damage and protect Fur. Overnight cultures of E. coli O157 (Sakai) and E. coli K-12 MG1655 were grown in Neidhardt's EZ Rich Defined Medium and diluted 1:100 in 50 ml fresh medium in 125 ml Ehrlenmeyer flasks. The cultures were shaken at 37C until the optical density (OD600) reached 0.4. Each culture was divided into 2 equal parts in identical flasks. One flask contained menadione bisulphite to a final concentration of 0.5 mg/ml; the other flask contained an equivalent volume of distilled water. The flasks were shaken for a further 10 mins and then treated with RNAprotect™ to stabilise the mRNA. The experiment was performed 3 times on different days. Six custom-made microarray slides were used in this study; each slide was hybridised with labelled cDNA made from mRNA taken from untreated and treated E. coli O157 (Sakai) or MG1655.
2015-02-22 | E-GEOD-36130 | biostudies-arrayexpress
Project description:Liver samples were lysed (15 mM Tris-HCl (pH 8.0), 300 mM NaCl and 15 mM MgCl2, plus inhibitors (1 mg/ml heparin 100 µg/ml cycloheximide and 80 U RNAsin)), added to a 10-50% sucrose gradient and ultracentrifuged at 182,000x g for 2 hours. The gradient was aliquotted into 16 1ml fractions and added to Tri reagent (Sigma). RNA was extracted as per the manufacturer's instructions and then sub-pooled into fractions corresponding to monosomes, light polysomes, medium polysomes and heavy polysomes, according to ribosomal density (more ribosomal occupancy = higher translational activity = heavier, and therefore, located in the more dense fractions). Microarray analysis was performed by hybridising control (vehicle treated) monosomes against test (high-dose treated) monosomes on one microarray, control light polysomes against test light polysomes on a second microarray, and so forth for each sub-pool of fractions. Following microarray analysis there were a maximum of four values for each mRNA, corresponding to the proportional representation of that mRNA within each sub-pool of fractions. By calculating the change in values, i.e. degree of slope, across the monosomal region, the light polysomal region, the medium polysomal region and the dense polysomal region it was possible to determine any translational change in activity. In addition, the overall transcriptional change could be analysed for each mRNA.
2012-06-21 | GSE38807 | GEO
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:JN54 (wild-type) cells were incubated in YPD medium at 30 degrees C to a logarithmic phase (OD660=1), followed by treatment with mild heat-shock at 43 degrees C for 30 min in pre-warmed (43 degrees C) 100ml of YPD medium using 500 ml Erlenmeyer flask. JN54 (wild-type) cells were incubated in YPD medium at 30 degrees C to a logarithmic phase (OD660=1), followed by treatment with mild heat-shock at 43 degrees C for 60 min in pre-warmed (43 degrees C) 100ml of YPD medium using 500 ml Erlenmeyer flask. Keywords: Stress response
2005-09-21 | GSE3316 | GEO