Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Anaerobic transcriptional responses of Escherichia coli to NO under defined chemostat conditions.

ABSTRACT: Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions. Cells were grown in defined media containing 54 mM glycerol as the sole and limiting source of energy and carbon. The working volume was 1 litre, and the dilution rate 0.1 h-1. In order to establish anaerobic growth, nitrogen was sparged through the chemostat medium prior to inoculation and throughout the course of the experiment at a rate of 0.2 l/min. No dissolved oxygen was detectable using the OxyProbe. Sodium fumarate was added to anaerobic medium at a final concentration of 50 mM to act as a terminal electron acceptor. Cells were grown as above to steady-state, At steady-state, NOC-5 and NOC-7 were added to the chemostat culture and to the nutrient feed at a final concentration of 10 uM of each, unless otherwise stated. Samples were taken immediately prior to the addition of NOCs and after a period of 5 min exposure to NOC for subsequent analysis using microarrays. Cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA, and total RNA was purified using Qiagenâ??s RNeasy Mini kit as recommended by the suppliers. Equal quantities of RNA from control and NOC-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. Two biological repeats (ie. two chemostat runs) were grown. Control and exposed samples were taken as described in summary. For each chemostat run 2 hybridisations were performed. One in which the control was Cy3 labelled whilst the exposed was Cy5 labelled, and a dye swap.

ORGANISM(S): Escherichia coli

SUBMITTER: Robert Poole

PROVIDER: E-GEOD-5076 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Nitric oxide in chemostat-cultured Escherichia coli is sensed by Fnr and other global regulators: unaltered methionine biosynthesis indicates lack of S nitrosation.

Pullan Steven T ST Gidley Mark D MD Jones Richard A RA Barrett Jason J Stevanin Tania M TM Read Robert C RC Green Jeffrey J Poole Robert K RK

Journal of bacteriology 20061222 5

We previously elucidated the global transcriptional responses of Escherichia coli to the nitrosating agent S-nitrosoglutathione (GSNO) in both aerobic and anaerobic chemostats, demonstrated the expression of nitric oxide (NO)-protective mechanisms, and obtained evidence of critical thiol nitrosation. The present study was the first to examine the transcriptome of NO-exposed E. coli in a chemostat. Using identical conditions, we compared the GSNO stimulon with the stimulon of NO released from two ...[more]

PMID: 17189370

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Project description:Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions. Cells were grown in defined media containing 8 mM glycerol as the sole and limiting source of energy and carbon. The working volume was 1 litre, and the dilution rate 0.1 h-1. For aerobic growth, the air-flow rate was 1 l/min, and the dissolved oxygen tension was maintained at 40% air saturation by measuring oxygen dissolved in the culture using a Broadley James D140 OxyProbeÂ® electrode and automated adjustment of stirring rate. Cells were grown as above to steady-state, At steady-state, NOC-5 and NOC-7 was added to the chemostat culture and to the nutrient feed at a final concentration of 10 uM of each unless stated otherwise. Samples were taken immediately prior to the addition of NOCs and after a period of 5 min exposure to NOCs for subsequent analysis using microarrays. Cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by the suppliers. Equal quantities of RNA from control and NOC-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. Two biological repeats (ie. two chemostat runs) were grown. Control and exposed samples were taken as described in summary. For each chemostat run 2 hybridisations were performed. One in which the control was Cy3 labelled whilst the exposed was Cy5 labelled, and a dye swap.

Project description:Escherichia coli strain MG1655 was grown in parallel chemostat cultures in GGM. In one chemostat the culture was fed adequate Zn; in the other, measures were taken to eliminate Zn from the chemostat vessel and culture medium. Culture volume (120 ml), temperature (37 oC) and stirring speed (437 rpm) were maintained. Steady state values for pH and OD600 were 6.9 and 0.6, respectively. After 50 hours, samples from the adequate Zn and Zn-limited chemostats were harvested into RNAprotect and total RNA was purified using Qiagenâ??s RNeasy Mini kit (using the supplierâ??s protocol) prior to use in microarray analysis. Biological experiments (i.e. a comparison of adequate versus low Zn in chemostat culture) were carried out three times, and a dye swap performed for each experiment, providing two technical repeats for each of the three biological repeats. Cells were grown in glycerol-glycerophosphate medium (GGM). Final concentrations in GGM are: MES (40.0 mM), NH4Cl (18.7 mM), KCl (13.4 mM),beta-glycerophosphate (7.64 mM), glycerol (5.00 mM), K2SO4 (4.99 mM), MgCl2 (1.00 mM), EDTA (134 microM), CaCl2.2H2O (68.0 microM), FeCl3.6H2O (18.5 microM), ZnO (6.14 microM), H3BO3 (1.62 microM), CuCl2.2H2O (587 nM), CoNO3.6H2O (344 nM), and (NH4)6Mo7O24.4H2O (80.9 nM) in MilliQ water (Millipore). Bulk elements (MES, NH4Cl, KCl, K2SO4 and glycerol in MilliQ water at pH 7.4 (batch growth) or 7.6 (continuous culture)) were passed through a column containing Chelex-100 ion exchange resin (Bio-Rad) to remove contaminating cations. Trace elements (with or without Zn as necessary) and a CaCl2 solution were then added to give the final concentrations shown above prior to autoclaving. After autoclaving, MgCl2 and beta-glycerophosphate were added at the final concentrations shown above. All chemicals were of AnalaR grade of purity or higher. Chelex-100 was packed into a Bio-Rad Glass Econo-column (approximately 120 mm Ã? 25 mm) that had previously been soaked in 3.5% nitric acid for 5 d. E. coli strain MG1655 was grown in parallel custom-built chemostats made entirely of non-metal parts. One chemostat was fed medium that contained â??adequateâ?? Zn (that normally found in GGM), whilst the other contained no added Zn and had been actively depleted of Zn by use of the special precautions listed here. Glass growth vessels and flow-back traps were soaked extensively (approximately two months) in 10% nitric acid before being rinsed thoroughly in MilliQ water. Vent filters (Vent Acro 50 from VWR) were connected to the vessel using PTFE tubing. Metal-free pipette tips were used (MAXYMum Recovery Filter Tips from Axygen). Culture volume was maintained at 120 ml using an overflow weir in the chemostat vessel. The dilution rate (and hence the specific growth rate) was 0.1 h-1. The vessel was inoculated using one of the side-arms. Flasks were placed on KMO 2 Basic IKA-Werke stirrers (arbitrary setting: 400). Stirring speed was calibrated (437 rpm) and matched between vessels using a handheld laser tachometer (Compact Instruments Ltd). Temperature was kept constant at 37Â°C. Twice daily, a 2-ml sample was taken and used to test the pH (which stayed constant at pH 6.9), OD600 (0.6 at steady state), glycerol limitation, and contamination on nutrient agar plates. The â??Zn-freeâ?? chemostat was inoculated with cells that had been sub-cultured in Zn-free medium to deplete internal stores of Zn. A 0.25 ml aliquot of a saturated culture of strain MG1655 grown in LB was centrifuged and the pellet used to inoculate 5 ml of GGM that was incubated overnight at 37Â°C with shaking. A 2.4 ml (i.e. 2% of chemostat volume) aliquot of this was then used to inoculate the chemostat. The â??adequate Znâ?? chemostat was inoculated with cells treated in essentially the same way but grown in GGM containing an â??adequateâ?? level of Zn. The two cultures (+/- Zn) used to inoculate the chemostats had OD600 readings within 2.5% of each other. Chemostats were grown for 50 h to allow five culture volumes to pass through the vessel and allow an apparent (pseudo-)steady state to be reached. At this point, 10 ml samples were removed from the chemostats and total RNA was stabilised using RNAprotect (Qiagen). Total RNA was purified using Qiagenâ??s RNeasy mini kit. Equal quantities of RNA from adequate Zn and Zn-limited chemostats were labeld by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer). For each microarray slide, one sample was labeld with Cy3-dCTP, while the other sample was labeld with Cy5-dCTP. RNA (15-20 micrograms) was annealed to 9 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65Â°C for 10 min, followed by 10 min at 22Â°C. This was supplemented with 4 microlitres of 5Ã? First-strand buffer (Invitrogen), 2 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 2 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1 microlitre Superscript II reverse transcriptase (200 U) (Invitrogen). This was incubated at 42Â°C for 2 hours. The reaction was terminated by the addition of NaOH (5 microlitres of 1M) and HCl (5 microlitres of 1M) before the addition of TE buffer (200 microlitres). Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen). The Cy3-dCTP-labeld sample was mixed with the Cy5-dCTP-labeld sample and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95Â°C for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 Â°C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 Â°C) SSC buffers for 5 min each at 37Â°C: 1Ã? SSC/0.1 % SDS, 1Ã? SSC, 0.2Ã? SSC and 0.01Ã? SSC. Microarray slides were dried by centrifugation at 500 Ã? g for 2 min before scanning. Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 or Cy5/Cy3 (test/reference) fluorescent ratios were calculated from the normalized values. Biological experiments (i.e. a comparison of adequate versus low Zn in chemostat culture) were carried out three times, and a dye swap performed for each experiment, providing two technical repeats for each of the three biological repeats. Data from the independent experiments were combined. Data from the independent experiments were combined. Genes that were differentially expressed â?¥ twofold and displayed and P value of < 0.05 (as determined by a t-test) were defined as being statistically significantly differentially transcribed.

Project description:Wild-type Campylobacter jejuni NCTC11168 was grown in 2 homemade, parallel chemostats. Cells were grown in 125 ml volumes of Mueller-Hinton Broth where the growth rate was controlled by the dilution rate (0.1 h-1). A gas mix of 80% nitrogen, 10% carbon dioxide and 10% oxygen was pumped into the head-space of the vessels at a rate of 0.25 l/min to obtain a microaerobic environment and the culture was maintained at 42 ÂºC via a water jacket. Cells were grown as above until steady-state had been reached. At steady-state one of the cultures was exposed to 10 uM NOC-5 & -7 (final concentration) for a period of 10 min. Samples of both the control and stressed cultures were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagenâ??s RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. Equal quantities of RNA from control and GSNO-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 4). The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. C. jejuni NCTC11168 was grown in two parallel custom-built chemostats based on Sigma Proculture Dynalift spinner flasks with a working volume of 125 ml, and the dilution rate (which at steady state is equal to the specific growth rate) of 0.1 h-1. A gas mix of 10% O2, 10% CO2 and 80% N2 was passed into the head space of the culture vessel at a rate of 0.25 l min-1 to obtain a microaerobic atmosphere, and silicone water jackets maintained the temperature at 42 ÂºC. The flasks were placed on KMO 2 basic IKA-Werke stirrers (arbitrary setting, 260; Scientific Laboratory Supplies), which gaveeffective transfer of O2 from the gaseous atmosphere to the culture by virtue of a stable vortex. A fresh overflow sample was used to check the pH at the time of harvest (which was consistently between 6.6 and 7) and to ensure that there was no contamination by plating a 20 Î¼l sample onto both nutrient agar plates incubated at 37 Â°C at atmospheric oxygen, and Mueller-Hinton plates incubated at 42 Â°C in the microaerophilic work station. When staedy-state was reached, as verified by collecting at least 5 culture volumes, NOC-5 & -7 (final concentration 10 uM) was added to one culture. After a further 10 min the 125 ml volume was separated into 3 equal volumes and each was mixed immediately on ice with 3.56 ml 100% ethanol and 185 ml phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. cDNA synthesis was carried out using 12 Âµg of starting material, which was primed with 9 Âµg of pd(N)6 random hexamers (Amersham Biosciences). Reaction mixtures (20 Âµl) containing 0.5 mM ATP, 0.5 mM TTP, 0.5 mM GTP, 0.2 mM CTP, and 1 mM Cy3- or 1 mM Cy5-dCTP were incubated for 2 h at 42 Â°C with 200 U of Superscript II RNase H reverse transcriptase (Invitrogen). Following synthesis, cDNA was purified by using a PCR purification kit (Qiagen) to remove the unincorporated deoxynucloside triphosphates, fluorescent dye, and primers. Equal volumes of cDNA were combined and evaporated to near dryness with an SPD121P Speed Vac (Thermon Savant). For hybridisation to the microarray slides, cDNA was resuspended in an appropriate volume of salt-based hybridization buffer (provided by Ocimum Biosolutions). Prior to addition to the slides, cDNA samples were heated to 95 Â°C for 3 min, and then placed on ice for no more than 3 min. The slides were placed in MWG Biotech hybridization chambers and incubated for 16 to 24 h in a shaking water bath at 110 rpm and 42 Â°C. Following incubation , the slides were washed sequentially for 5 min in 2x SSC-1 % sodium dodecyl sulphate, 1x SSC, 0.5x SSC and 0.1x SSC at 37 Â°C with gentle agitation (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The slides were dried by centrifugation at 254 xg for 5 min and subsequently scanned with an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained by using commercially available software from Biodiscovery, Inc. (Imagine version 4.0, and Genesight, version 3.5). The mean values from each channel were log2 transformed and normalized by using the subtract-by-mean method to remove intensity-dependent effects in the log2 (ratio) values. The Cy3/Cy5 fluorescence ratios were calculated from the normalized values. Biological experiments (i.e. chemostat growth with and without 0.25 mM GSNO addition) were carried out three times and a dye swap was performed for two of the three experiments which provided two technical repeats, one each for two of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed â?¥ twofold and displayed and P value of â?¤ 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.

Project description:Escherichia coli strain MG1655 was grown in a Zn-depleted custom-built chemostat. Culture volume (120 ml), temperature (37 oC) and stirring speed (440 rpm) were maintained. Steady state values for pH and OD600 were 6.9 and 0.6, respectively. Chemostats were grown for 50 h to allow five culture volumes to pass through the vessel and allow an apparent (pseudo-)steady state to be reached. At this point, ZnSO4.7H2O in water was added to a final concentration of 0.2 M in the chemostat. A 10 ml sample of culture was taken using a polypropylene pipette tip immediately prior to Zn addition and 2.5, 7, 10 and 30 min after addition. Samples were harvested into RNAprotect and total RNA was purified using QiagenM-bM-^@M-^Ys RNeasy Mini kit (using the supplierM-bM-^@M-^Ys protocol) prior to use in microarray analysis. A control experiment was also carried out in which water was added. Biological experiments were carried out twice (i.e. control (water added) and experiment (ZnSO4 added) chemostats were grown separately twice), and a dye swap performed for each experiment, providing at least two technical repeats for each of the two biological repeats at all five time points. Slides were analysed so that time points M-bM-^@M-^\with ZnM-bM-^@M-^] were compared to the same time point M-bM-^@M-^\without ZnM-bM-^@M-^] (e.g. 30 min after Zn was added was compared to 30 min after water being added). Cells were grown in glycerol-glycerophosphate medium (GGM) lacking Zn. Final concentrations in GGM are: MES (40.0 mM), NH4Cl (18.7 mM), KCl (13.4 mM), beta-glycerophosphate (7.64 mM), glycerol (5.00 mM), K2SO4 (4.99 mM), MgCl2 (1.00 mM), EDTA (134 microM), CaCl2.2H2O (68.0 microM), FeCl3.6H2O (18.5 microM), H3BO3 (1.62 microM), CuCl2.2H2O (587 nM), CoNO3.6H2O (344 nM), and (NH4)6Mo7O24.4H2O (80.9 nM) in MilliQ water (Millipore). Bulk elements (MES, NH4Cl, KCl, K2SO4 and glycerol in MilliQ water at pH 7.4 (batch growth) or 7.6 (continuous culture)) were passed through a column containing Chelex-100 ion exchange resin (Bio-Rad) to remove contaminating cations. Trace elements (with no added Zn) and a CaCl2 solution were then added to give the final concentrations shown above prior to autoclaving. After autoclaving, MgCl2 and beta-glycerophosphate were added at the final concentrations shown above. All chemicals were of AnalaR grade of purity or higher. Chelex-100 was packed into a Bio-Rad Glass Econo-column (approximately 120 mm M-CM-^W 25 mm) that had previously been soaked in 3.5% nitric acid for 5 d. E. coli strain MG1655 was grown a custom-built chemostat made entirely of non-metal parts. Glass growth vessels and flow-back traps were soaked extensively (approximately two months) in 10% nitric acid before being rinsed thoroughly in MilliQ water. Vent filters (Vent Acro 50 from VWR) were connected to the vessel using PTFE tubing. Metal-free pipette tips were used (MAXYMum Recovery Filter Tips from Axygen). Culture volume was maintained at 120 ml using an overflow weir in the chemostat vessel. The dilution rate (and hence the specific growth rate) was 0.1 h-1. The vessel was inoculated using one of the side-arms. Flasks were placed on KMO 2 Basic IKA-Werke stirrers (arbitrary setting: 400). Stirring speed was calibrated (440 rpm) and matched between vessels using a handheld laser tachometer (Compact Instruments Ltd). Temperature was kept constant at 37 oC. Twice daily, a 2-ml sample was taken and used to test the pH (which stayed constant at pH 6.9), OD600 (0.6 at steady state), glycerol limitation, and contamination on nutrient agar plates. E. coli strain MG1655 was grown in a M-bM-^@M-^\Zn-freeM-bM-^@M-^] chemostat. Chemostats were grown for 50 h to allow five culture volumes to pass through the vessel and allow an apparent (pseudo-)steady state to be reached. At this point, ZnSO4.7H2O in water was added to a final concentration of 0.2 M in the chemostat. A 10 ml sample of culture was taken using a polypropylene pipette tip immediately prior to Zn addition and 2.5, 7, 10 and 30 min after addition. Total RNA was stabilised using RNAprotect (Qiagen). Total RNA was purified using QiagenM-bM-^@M-^Ys RNeasy mini kit. A control experiment was carried out in which water was added. Microarray slides were set up so that time points M-bM-^@M-^\with ZnM-bM-^@M-^] were compared to the same time point M-bM-^@M-^\without ZnM-bM-^@M-^] (e.g. 30 min after Zn was added was compared to 30 min after water being added). For each time point, equal quantities of RNA obtained after addition of zinc and addition of water were labelled by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer). For each microarray slide, one sample was labelled with Cy3-dCTP, while the other sample was labelled with Cy5-dCTP. RNA (15-20 micrograms) was annealed to 9 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC. This was supplemented with 4 microlitres of 5M-CM-^W First-strand buffer (Invitrogen), 2 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 2 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1 microlitre Superscript II reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 2 hours. The reaction was terminated by the addition of NaOH (5 microlitres of 1M) and HCl (5 microlitres of 1M) before the addition of TE buffer (200 microlitres). Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen). The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95 oC for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 M-BM-0C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 M-BM-0C) SSC buffers for 5 min each at 37oC: 1M-CM-^W SSC/0.1 % SDS, 1M-CM-^W SSC, 0.2M-CM-^W SSC and 0.01M-CM-^W SSC. Microarray slides were dried by centrifugation at 500 M-CM-^W g for 2 min before scanning. Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. Biological experiments were carried out twice times, and a dye swap performed for each experiment, providing at least two technical repeats for each of the two biological repeats at all five time points. Data from the independent experiments were combined. Genes that were differentially expressed M-bM-^IM-% twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.

Dataset Information

Anaerobic transcriptional responses of Escherichia coli to NO under defined chemostat conditions.

Publications

Nitric oxide in chemostat-cultured Escherichia coli is sensed by Fnr and other global regulators: unaltered methionine biosynthesis indicates lack of S nitrosation.

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