Transcription profiling of E.coli W3110 oxygen downshift
ABSTRACT: Dynamical response to oxygen downshift under fermentation conditions was tested by taking sample before (S1) and after (S2, S3 and S4) the oxygen downshift. The dynamical changes relevant for ongoing research on physiology were applied. Experiment Overall Design: Four microarray chips were analyzed: Experiment Overall Design: S1 was taken 15 min before the oxygen downshift Experiment Overall Design: S2, S3 and S4 were taken 15 min, 45 min and 75 min after the oxygen downshift, respectively
Project description:Dynamical response to oxygen downshift under fermentation conditions was tested by taking sample before (S1) and after (S2, S3 and S4) the oxygen downshift. The dynamical changes relevant for ongoing research on physiology were applied. Overall design: Four microarray chips were analyzed: S1 was taken 15 min before the oxygen downshift S2, S3 and S4 were taken 15 min, 45 min and 75 min after the oxygen downshift, respectively
Project description:The Saccharomyces cerevisiae SFP1 is required for proper regulation of ribosome biogenesis and cell size in response to nutrients. A mutant deleted for SFP1 shows specific traits among which a slow growth phenotype, which is particularly evident during growth on glucose. To assess the effects of nutrients on the activity of Sfp1 independent by growth rate related feedback we grew an sfp1Δ mutant and its isogenic reference strain in chemostat cultures, at the same specific growth rate, under glucose/ethanol-limitation. Our data show that Sfp1 is involved in the modulation of cell size and RiBi gene expression and that these two functions are differently influenced by nutrients. The continuous cultures were then pulsed with a glucose excess generating a situation of batch growth similar to shake flask cultures. The dynamic analysis of the metabolic and transcriptional response following the glucose addition suggested that Sfp1 plays a role at the crossroads of ribosome biogenesis and central carbon metabolism regulation. Finally, we show that the down-regulation of RP genes, which was observed in an sfp1Δ strain during shake flask growth, cannot be directly ascribed to the absence of Sfp1 but is most probably a secondary effect due to the low growth potential of the mutant strain. Experiment Overall Design: After ten volume changes, few seconds after the samples for the steady state analysis were collected, the anaerobic glucose pulse experiments were started by sparging the medium reservoir and the fermenter with pure nitrogen gas (airflow of 0.5 L min-1, Hoek-Loos, Schiedam, <5 ppm O2). NorpreneTM tubing and butyl septa were used to minimize oxygen diffusion into the anaerobic culture. Two minutes after nitrogen sparging and just before the addition of glucose, the medium and the effluent pumps were switched off. At this time point (which we will refer to as time T=0) the 200 mM glucose pulse was injected aseptically through a rubber septum. Experiment Overall Design: Sampling from chemostats, total RNA extraction, probe preparation and hybridization to Affymetrix Genechip® microarrays were performed as previously described (1). Samples were collected at steady state and then at 5, 10, 30, 60 and 120 minutes after the pulse. The results relative to steady state samples were derived from three independent cultures, those relative to the time course analysis were derived from two independent cultures. Experiment Overall Design: 1) Cipollina C., van den Brink J., Daran-Lapujade P., Pronk J.T., Vai M. and de Winde J.H. (2007) Revisiting the role of yeast Sfp1 in ribosome biogenesis and cell size control: A chemostat study. Microbiology. In press.
Project description:The capacity of respiring cultures of Saccharomyces cerevisiae to instantaneously switch to fast alcoholic fermentation upon a transfer to anaerobic sugar-excess conditions is a key characteristic of Saccharomyces cerevisiae in many of its industrial applications. This transition was studied by exposing aerobic glucose-limited chemostat cultures grown at a low specific growth rate to two simultaneous perturbations: oxygen depletion and relief of glucose limitation. This shift towards fully fermentative conditions caused a massive transcriptional response, where one third of all genes within the genome were transcribed differentially. During the first 30 min, most of these changes were driven by relief from glucose limitation. An anaerobic induction response was only observed after the initial response to glucose excess. By comparing this study with public datasets representing dynamic and steady conditions, 14 up-regulated and 11 down-regulated genes were determined to be anaerobiosis specific and can therefore be use as “signature” transcripts for anaerobicity under dynamic as well as under steady state conditions Experiment Overall Design: To invoke rapid and full induction of fermentative capacity, respiratory, aerobic glucose-limited chemostat cultures (D=0.1•h-1) were shifted to fully fermentative conditions by sudden depletion of oxygen and addition of glucose. The glucose was added two min after sparging the continuous culture with pure nitrogen, when the dissolved oxygen concentration had decreased from 75-80% to 10-15% of air saturation. Samples for micro-arrays were taken for each time point after the perturbation (5, 10, 30, 60 and 120 min) from two independently cultured replicates, while steady state data were taken from three independent chemostats. The complete dataset therefore comprised 13 samples.
Project description:This series represents Experiment 3 of the yeast desiccation / rehydration time course analysis. Samples include Control, 50% dry, Dry, 15 min. post rehydration, 45 min. post rehydration, 90 min. post rehydration, and 360 min. post rehydration.
Project description:This series represents Experiment 1 of the yeast desiccation / rehydration time course analysis. Samples include Control, 50% dry, Dry, 15 min. post rehydration, 45 min. post rehydration, 90 min. post rehydration, and 360 min. post rehydration.
Project description:This series represents Experiment 2 of the yeast desiccation / rehydration time course analysis. Samples include Control, 50% dry, Dry, 15 min. post rehydration, 45 min. post rehydration, 90 min. post rehydration, and 360 min. post rehydration.
Project description:Aim of the study was to characterize the transcriptional response of human primary renal proximal tubule endothelial cells (RPTEC) to low oxygen stress. Experiment Overall Design: Passage 4 renal proximal tubule epithelial cells were exposed to a humidified atmosphere consisting of either 5% CO2 and 95% air (20% O2, normoxia) or 5% CO2, 1% oxygen and 95% nitrogen (hypoxia) for 24 hours. Total RNA was extracted immediately after exposure. Three independent biological replicates were performed, resulting in 6 samples (3 control and 3 low oxygen).
Project description:To identify 20E-regulated genes, wandering third instar larvae were dissected and their organs were cultured in the presence of either no hormone, 20E alone, cycloheximide alone, or 20E plus cycloheximide for six hours. Experiment Overall Design: Eight partial blue gut third instar larvae were dissected in each well of a 9 well glass dish (Corning) and cultured in ~100 µl oxygenated Schneiders Drosophila Medium (Gibco) at 25°C. Cultures were incubated in an a styrofoam box under a constant flow of oxygen. Following an initial incubation of 1 hour, the medium was removed and replaced with either fresh Schneiders Drosophila Medium (no hormone), medium plus 8.5x10-5 M cycloheximide (Sigma), medium plus 5x10-6 M 20-hydroxyecdysone (Sigma), or medium plus cycloheximide and 20E, each for 6 hrs at 25°C. Organs were collected and RNA extracted from these samples was analyzed on Affymetrix Drosophila Genome Arrays.
Project description:Commercial brewing yeast strains are exposed to a number of potential stresses including oxidative stress. The aim of this investigation was to measure the physiological and transcriptional changes of yeast cells during full-scale industrial brewing processes with a view to determining the environmental factors influencing the cell’s oxidative stress response. Cellular antioxidant levels were monitored throughout an industrial propagation and fermentation and microarray analysis was employed to determine transcriptional changes in antioxidant-encoding and other stress response genes. The greatest increase in cellular antioxidants and transcription of antioxidant-encoding genes occurred as the rapidly fermentable sugars glucose and fructose were depleted from the growth medium (wort) and the cell population entered the stationary phase. The data suggest that, contrary to expectation, the oxidative stress response is not influenced by changes in the dissolved oxygen concentration of wort but is initiated as part of a general stress response to growth-limiting conditions, even in the absence of oxygen. A mechanism is proposed to explain the changes in antioxidant response observed in yeast during anaerobic fermentation. The results suggest that the yeast cell does not experience oxidative stress, per se, during industrial brewery handling. This information may be taken into consideration when setting parameters for industrial brewery fermentation. Experimenter name: Brian Gibson; Experimenter phone: +44 (0)1159516214 Experiment Overall Design: 17 samples were used in this experiment
Project description:- Background and Aims: Oxygen can fall to low concentrations within plant tissues, either because of environmental factors that decrease the external oxygen concentration or because the movement of oxygen through the plant tissues cannot keep pace with the rate of oxygen consumption. Recent studies document that plants can decrease their oxygen consumption in response to relative small changes in oxygen concentrations to avoid internal anoxia. The molecular mechanisms underlying this response have not been identified yet. The aim of this study was to use transcript and metabolite profiling to investigate the genomic response of Arabidopsis roots to a mild decrease in oxygen concentrations. - Methods: Arabidopsis seedlings were grown on vertical agar plates at 21, 8, 4 and 1% (v/v) external oxygen for 0.5, 2 and 48h. Roots were analyzed for changes in transcript levels using Affymetrix whole genome DNA microarrays, and for changes in metabolite levels using routine GC-MS based metabolite profiling. Root extension rates were monitored in parallel to investigate adaptive changes in growth. - Key results: Results show that root growth was inhibited and transcript and metabolite profiles were significantly altered in response to a moderate decrease in oxygen concentrations. Low oxygen leads to a preferential up-regulation of genes that might be important to trigger adaptive responses in the plant. A small but highly specific set of genes is induced very early in response to a moderate decrease in oxygen concentrations. Genes that were down-regulated mainly encoded proteins involved in energy-consuming processes. In line with this, root extension growth was significantly decreased which will ultimately save ATP and decrease oxygen consumption. This was accompanied by a differential regulation of metabolite levels at short and long term incubation at low oxygen. - Conclusions: Results show that there are adaptive changes in root extension involving large-scale reprogramming of gene expression and metabolism when oxygen concentration is decreased in a very narrow range. Experiment Overall Design: Arabidopsis seedlings were grown on vertical agar plates and treated with various oxygen concentrations (1%, 4%, 8%, and 21% as a control), 350ppm CO2 and N2 (rest) for different time periods (0.5 hours, 2 hours and 2 days. At the end of the experiment, the roots of the seedlings were immediately frozen in liquid nitrogen and used for gene expression analysis.