Unknown,Transcriptomics,Genomics,Proteomics

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ADH1 deletion cells upon growth on alternative carbon sources; Steady-state grown cells upon environmental perturbations

ABSTRACT: Cells must adjust their gene expression in order to compete in a constantly changing environment. Two alternative strategies could in principle ensure optimal coordination of gene expression with physiological requirement. First, the internal physiological state itself could feedback to regulated gene expression. Second, the expected physiological state could be inferred from the external environment, using evolutionary-tuned signaling pathways. Coordination of ribosomal biogenesis with the requirement for protein synthesis appears to be particularly important, since cells devote a large fraction of their biosynthetic capacity for ribosomal biogenesis. To define the relative importance of internal vs. external sensing to the regulation of ribosomal biogenesis gene expression, we subjected S. cerevisiae cells to conditions which decoupled the actual vs. environmentally-expected growth rate. Gene expression followed the environmental signal according to the expected, but not the actual, growth rate. Simultaneous monitoring of gene expression and growth rate in chemostat-grown cultures further confirmed that ribosome biogenesis genes responded rapidly to changes in the environments but were oblivious to longer-term changes in growth rate. Our results suggest that the capacity to anticipate and prepare for environmental changes presented a major selection force during yeast evolution. Keywords: Saccharomyces_Cerevisiae, Stress response, ADH1 deletion, time courses, chemostat Experiment 1: ADH1 deletion cells, which grow faster on glycerol rather then glucose medium, were grown in both glucose and glycerol medium. The cells expression on glycerol is compared to their expression glucose (5 microarrays). Experiment 2: Time course of ADH1 deletion cells upon growth on glycerol and on glucose (7 microarrays). Experiment 3: Response of steady state grown cells to environmental perturbations. Cells were grown in glucose or histidine limited chemostats and reached steady state. The cells were then subjected to perturbations such as DTT, heat shock, NaCl, Clotrimazole, H2O2, and addition of limiting factors (histidine and glucose, respectively). Overall we have examined 10 timecourses with 83 microarrays.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: sagi levy

PROVIDER: E-GEOD-6302 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Strategy of transcription regulation in the budding yeast.

Levy Sagi S Ihmels Jan J Carmi Miri M Weinberger Adina A Friedlander Gilgi G Barkai Naama N

PloS one 20070228 2

Cells must adjust their gene expression in order to compete in a constantly changing environment. Two alternative strategies could in principle ensure optimal coordination of gene expression with physiological requirements. First, characters of the internal physiological state, such as growth rate, metabolite levels, or energy availability, could be feedback to tune gene expression. Second, internal needs could be inferred from the external environment, using evolutionary-tuned signaling pathway ...[more]

PMID: 17327914

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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 sfp1Î 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 sfp1Î 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 GenechipÂ® 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:Previously, it has been demonstrated that formate can be utilized by Saccharomyces cerevisiae as additional energy source using cells grown in a glucose-limited chemostat. Here, we investigated utilization of formaldehyde as co-substrate. Since endogenous formaldehyde dehydrogenase activities were insufficient to allow co-feeding of formaldehyde, the Hansenula polymorpha FLD1, encoding formaldehyde dehydrogenase, was introduced in S. cerevisiae. Chemostat cultivations revealed that formaldehyde was co-utilized with glucose, but the yield was lower than predicted. Moreover, formate was secreted by the cells. Upon co-expression of the H. polymorpha gene encoding formate dehydrogenase, FMD, the levels of secreted formate decreased, but the biomass yield was still lower than anticipated. Transcriptome comparisons of cells of the engineered FLD1/FMD-expressing S. cerevisiae strain grown with or without formaldehyde feed, suggested that the cells experienced biotin limitation, possibly due to inactivation of biotin by formaldehyde in the feed. When separate feeds were used for formaldehyde and biotin, the engineered S. cerevisiae strain was able to efficiently utilize formaldehyde as additional energy source. Experiment Overall Design: In industrial biotechnology, the cost of feedstocks (often sugars) are crucial for production of both biomass related products, such as proteins, or for the production of commodity chemicals. In many such processes a large fraction of the sugars is dissimilated to either provide free-energy (in the form of ATP) or to provide electrons (in the form of NADH). Co-consumption of methanol, which can be derived from fossil sources (via natural gas) or from biomass (via syngas) , via the above mentioned route can provide NADH and/or ATP, thereby creating the possibility to utilise the sugars more efficiently. It would therefore be advantageous to co-feed S. cerevisiae with methanol during fermentations since it is a cheap alternative to sugars as energy source. Experiment Overall Design: Efficient dissimilation of the intermediate formaldehyde is a crucial first step for utilisation of methanol by S. cerevisiae. Therefore, his study analyses the effect of co-expression of H. polymorpha FLD1 encoding NAD+-dependent formaldehyde and FMD encoding formate dehydrogenases, on tolerance to formaldehyde and co-consumption of formaldehyde by S. cerevisiae.

Dataset Information

ADH1 deletion cells upon growth on alternative carbon sources; Steady-state grown cells upon environmental perturbations

Publications

Strategy of transcription regulation in the budding yeast.

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