Alyssa N Gomes Week 10 Journal

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Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analysis


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Outline of "Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analysis"

*Introduction

    • Saccharomyces cerevisiae is a yeast that is exposed to many external and environmental changes that affect processes and chemical structures.
    • This paper will focus on the effect of cold temperatures on the cultures
      • There is a difference in sudden exposure vs gradual exposure because sudden exposure will respond with shock and stress-related responses, whereas gradual exposure will lead to acclimation
    • Previous studies have shown a difference in the early cold response (<1 hour) and late cold response (>12hours)
    • Although this has priorly been studied, some questions remain unanswered:
      • Why did prior studies done by Sahara, Homma , Schade, Murata differentiate in their answers about the growth of expression ribosomal protein genes?
      • Why did cold shock bring out reserve carbohydrate while trehalose only arose in desperate freezing conditions?
      • Is there a way to bring out the Msn2p/Msn4p complex that has been previously suggested to be a transcriptional factor in cold weather?
      • How can we study and describe the difference between acclimation and shock responses in S cerevisiae?
    • Chemostat cultures will allow many factors to remain stable in acclimatized environments
    • Goal: study the steady-state acclimatized growth of this yeast culture and its transcription, under temperatures of 12-30 degrees celsius and at a growth rate of 0.03 h^-1

*Materials and Methods

    • Strain and Growth Conditions
      • Strain: Saccharomyces cerevisiae
      • Growth rate: 0.03 h^-1
      • Volume: 1.0 l
      • Temperature probe set to 12 degrees Celsius initially
      • Anaerobic growth, biomass dry weight, metabolites, dissolved oxygen and steady-state set stable
    • Analytical Methods:
      • Supernatants were collected with rapid sampling
      • Liquid chromotography was used to analyze concentrations of glucose and metabolites on an AMINEX HPX-87H ion exchange column using 5 mM H2SO4
      • Cuvette tests from DRLANGE were used to examine the residual ammonium concentrations
      • Triplicate measurements in the chemostate measured trehalose
      • Roche kit no. 0716251 examined the amount of glucose released from glycogen and trehalose in breakdown methods
    • Microarray Analysis:
      • Results for the listed growth conditions came from three separately cultured replicates.
      • The coefficients of variation for the four growth conditions was 0.20
      • The ACT1 transcript varied by less than 12% for the four growth conditions
      • Microsoft Excel was used in order to run significance analyses on microarray add-ins
      • The analyses used a threshold fold difference of 2 and median false rate discovery of 1%
      • Venn diagrams and heat maps were used with Expressionist Analyst version 3.2
      • Regulatory Sequence Analysis (RSA) Tools were used to examine the promoter values
      • the Database for Annotation, Visualization and Integrated Discovery (DAVID) was used in order to assess overrepresentation
      • Overrepresentation of transcription-factor binding sites as defined by chromatin immunoprecipitation was calculated with Fisher's exact test, a Bonferroni correction, and a p-stat
      • The data has been submitted to the Genome Expression Omnibus database under the number GSE6190
    • Comparison with Other S. cerevisiae Low-Temperature Transcriptome Datasets
      • Batch transcriptome genomes used in this for comparison studies are: Beltran et al., Murata et al., Sahara et al., Schade et al., and Gasch et al.
      • The collection of stress-response genes were found on: http://genome-www.stanford.edu/yeast_stress/

*Results

    • Overview
      • The culture, grown at 12 and 30 degrees celsius was under 0.03 h^-1 growth for its culture, allowing for steady-state growth
      • Cultures were grown anaerobically, in order to decrease external factors
      • In both 12 degree and 30 degree cultures, both carbon and nitrogen limiting cultures had the same biomass, indicating no effect on growth efficiency
      • In glucose limiting cultures, 494 genes showed significantly different transcription levels
      • In nitrogen limiting cultures, 806 genes showed significantly different transcription levels
      • The total number of temperature responsive genes was 1056, 16% of the total genes
      • 235 of the genes showed a consistent increase/decrease in transcript levels, no matter the limiting factor
      • In determining the responses for regulatory networks to temperature acclimation, temperature-responsive genes were evaluated under the screenings in order to seek changes in enrichment of nutrients and promoter regions
    • Low-Temperature Chemostat Cultivation Results in Altered Uptake Kinetics for the Limiting Nutrient and Enhanced Catabolite Repression
      • In the 12 degrees celsius atmosphere, residual nitrogen and ammonium were 7.5-10 fold higher than than the 30 degrees celsius cultures
      • For the lower temperatures, we can assume that higher degrees of substrate concentration and change in transport kinetics is to blame for this difference in residual concentrations
      • In glucose limiting cultures, low-affinity HXT3 and the intermediate-affinity HXT2 and HXT4 hexose-transporter genes had increased transcription levels for 12 degrees celsius, and then decreased for 30 degrees celsius
      • In contrast, HXT5 and HXT16 had decreased transcription rates for 12 degree values and increased for 30 degree values
      • HXT6 and 7 are genes that are used to encode high-affinity glucose transporters, had no significant growth for transcription values
      • In ammonium limiting cultures, the genes that encode ammonia permease showed variance in their growth. High-affinity genes, MEP1 and MEP2, had reduced transcription levels at 12 degrees (-2.8 fold and -4 fold), whereas the low-affinity one, MEP3, was slightly increased (1.8 fold) under 12 degree shock
      • The observed temperature dependency of the residual concentrations of growth-limiting nutrients is consistent with the specific growth rate of 0.03 h^-11 used in this study is much closer to �max at 12 degrees celsius than at 30 degrees celsius
    • Acclimation to Nonfreezing Low Temperature Does Not Require a High Storage Carbohydrate Content
      • Transcriptional values of genes involved in metabolism of storage carbohydrates (especially trehalose) is consistently studied after cold shock
      • Transcription values of trehalose and glycogen metabolism were not affected by temperature shock and increase
      • These values oftentimes have decreased in ammonium limiting cultures at 12 degrees celsius
      • In glucose limiting cultures, trehalose cultures were lower at 12°C, whereas glycogen amounts were 50% higher than in glucose-limited cultures grown at 30°C.
      • Msn2/Msn4 help regulate storage carbohydrate synthesis
      • Once cells are adapted to cold temperature, stress response and the up-regulation of storage carbohydrate synthesis decreases
    • Up-Regulation of the Translation Machinery at Low Temperature
      • At the fixed dilution rate and 30 degrees celsius transcription of protein synthesis genes is constant over a range of external growth factors
      • 16 of the genes that do ribosome biogenesis and assembly showed higher transcription amounts at 12°C than at 30°C in glucose- and ammonium limited cultures
      • The ammonium limited culture showed slightly more than the glucose-limited, 80 genes.
      • The PAC cis-regulatory genes were fed nutrients
      • In the ammonium-limited cultures, cellular protein and nitrogen amounts were higher at 12 than at 30°C. The difference of the nitrogen content was larger than that of the protein content, which may be due to an increased rRNA level
      • At 12°C, the protein content in the ammonium-limited cultures was as high as that in the glucose-limited amounts
    • Transcriptional Responses to Low Temperature: Adaptation versus Acclimation
      • The chemostat transcriptome data were compared with transcriptome datasets from other low-temperature adaptation studies
      • Of 259 genes, only 91 of them were up-regulated during the temperature changes and only 48 were down-regulated.
      • 11 genes showed a consistent pattern of regulation in all situations: PIR3, SFK1, YPC1, YEL073C, YNL024C, and YLR225C were upregulated at low temperature, and PHO84, FUI1, AHA1, FCY2, and YLR413W were down-regulated
      • SFK1, YPC1, YEL073C are all involved in lipid metabolism. NOG1, NOG2, HTM1, NPL3, and NSR1 are all involved in protein translocation across the nuclear envelope.
    • Context Dependency of Temperature Response
      • The small amount of genes transcribed regularly is due to context dependency of transcriptional regulation
      • Comparing to Regenberg and Castrillo showed that altered transcript levels of 25% of the low-temperature down-regulated genes (12 of 48) and 10% of the low-temperature up-regulated genes (9 of 91) are likely to have been primarily related to specific growth rate, instead of temperature experiments
      • Another difference between these experiments is oxygen levels because the chemostats regulated the oxygen amounts much better than the shake-flask.
      • TIP1, TIR1, and TIR2, which encode cell wall mannoproteins, have been characterized as markers for cold shock in batch studies but not in chemostat studies
      • Repression of OLE1 occurred similarly at 30 and 12°C, disqualifying this gene as a low-temperature marker in anaerobic cultures
    • Environmental Stress Response, a Low-Temperature Adaptation-specific Response
      • It has been proposed that a reduction of the growth temperature transcriptionally induces a set of environmental stress response genes
      • When comparing the set of ESR genes to sets of genes that were consistently up- or down-regulated at low temperature in batch culture, Fifty percent of the consistently cold up-regulated genes and 13% of the cold downregulated genes found in the batch-culture studies were also found in the ESR genes. 1/3 of the cold temperature response genes found in the three batch-culture studies can be linked to ESR
      • ESR occurs during adaptation upon a sudden exposure to suboptimal temperatures.

*Discussion

    • Chemostat-based Low-Temperature Transcriptomics: Experimental Design
      • Even in well-controlled chemostat cultures, it is impossible to change a single cultivation parameter without any impact on others. For example, in this experiment, the temperatures and growth rate led to a higher glucose concentration in the 12 degree cultures because a specific growth rate of 0.03 h^-1 represents 75% of �max at 12°C, but only 10% of �max at 30°C.
      • But we could not study only glucose because that would lead to only temperature-responsive gene sets with genes whose transcription is influenced by glucose
      • Processes involved in homeoviscous adaptation of membrane composition may have been masked by inclusion of anaerobic growth factors, such as oleate and ergosterol
    • Specific Growth Rate and ESR
      • For this experiment, at temperatures 12 and 30 degrees celsius the specific growth rate of S. cerevisiae in batch cultures differs by almost an order of magnitude
      • 15% of the genes that showed a consistent transcriptional response in three previous batch-culture studies on cold adaptation were also identified in chemostat studies on growth-rate–dependent transcription performed at 30 degrees celsius
      • Less than 1% of the temperature-responsive genes identified in the chemostat test showed a growth-rate–dependent transcript level
      • Batch cultures showed that low temperatures increased synthesis of storage carbohydrates and transcriptional up-regulation of genes involved in storage carbohydrate metabolism
      • In batch cultures, where low-temperature adaptation has a strong ESR response, but only three ESR-induced genes (YCP1, VPS73, and EMI1) showed a higher transcript at 12 than at 30 degrees in chemostat cultures.
      • Transcription of ESR genes is generally changed and induced by a reduced specific growth rate, rather than by low temperature
    • Transcriptional Responses to Low Temperature: Acclimation versus Adaptation
      • This experiment showed that at 12 and 30 celsius there was a set of 235 genes that showed a consistent transcriptional response to low temperature, irrespective of the growth limiting nutrient
      • The only similarities between low temperature chemostats and batch experiments was the lipid metabolism genes
      • There was some similarities in this experiment with others but discrepancies in others
      • Compensation for the decreased capacity of glycolytic enzymes at low temperature is predominantly accomplished via changes in intracellular metabolite levels


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