Kevin Matthew McKay Week 11
- Batch Culture-A large-scale closed system culture in which cells are grown in a fixed volume of nutrient culture medium under specific environmental conditions (e.g. nutrient type, temperature, pressure, aeration, etc.) up to a certain density in a tank or airlift fermentor, harvested and processed as a batch, especially before all nutrients are used up.-http://www.biology-online.org/dictionary/Batch_culture
- Biogenesis-The process in which life forms arise from similar life forms. or It asserts that living things can only be produced by another living thing, and not by a non-living thing.-http://www.biology-online.org/dictionary/Biogenesis
- Diurnal-occuring during the day-http://www.biology-online.org/dictionary/Diurnal
- Prototrophic-Strain's that have the same nutritional requirements as the wild-type strain.-http://www.biology-online.org/dictionary/Prototrophic_strains
- Supernatant-The soluble liquid fraction of a sample after centrifugation or precipitation of insoluble solids.-http://www.biology-online.org/dictionary/Supernatant
- Chromotography-The process or technique of separating molecules or components in a mixture according to the differential absorption and elution-http://www.biology-online.org/dictionary/Chromatography
- Catabolite- product of catabolism, the breakdown of complex molecules into simpler ones.-http://www.biology-online.org/dictionary/Catabolite
- Cuvette-a transparent or translucent box-shaped container with precisely-measured dimensions for holding liquid samples to be put into a spectrophotometer.-http://www.biology-online.org/dictionary/Cuvette
- Gene regulatory protein- Any protein that interacts with dna sequences of a gene and controls its transcription.-http://www.biology-online.org/dictionary/Gene_regulatory_protein
- Permease- general term for a membrane protein that increases the permeability of the plasma membrane to a particular molecule, by a process not requiring metabolic energy.-http://www.biology-online.org/dictionary/Permease
“Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analysis”
Experiment and Parameters
- Analyzed genome-wide transcriptional responses at low temperatures*
- Used Steady state chemostats instead of batch cultures (better suited for study of prolonged exposure to cold temperature because chemostat enables better control of specific growth rate)
- Growth rate fixed at (0.03/h)
- 12 and 30 degrees Celsius
- anaerobic conditions
- glucose and ammonia-limited chemostat cultures of synthetic medium containing all other growth requirements, pH of 5, stirrer speed of 600 rpm
- between the temperatures and nutrient limitations there were 4 total growth conditions
- compared results with those of yeast grown in batch cultures
- haploid yeast strain CEN.PK113-7D (MATa)
- biomass dry weight, metabolites, dissolved oxygen, and gas profiles were constant for three volume changes before sampling
- three independent steady-state chemostat cultivations were performed per condition (ie three for glucose limiting and 12 degrees, 3 for ammonium limiting and 12 degrees ect…)
- results were compared with those of the previously done batch culture data sets
- statistical analysis was done using Fisher’s exact test, using hypergeometric distribution with a Bonferroni correction and a p value threshold of 0.01
- In-vivo metabolic fluxes were the same in both cultures (one grown at 12 and the other at 30 degrees Celsius)
- residual concentration of limiting nutrient glucose or ammonia was higher at 12 degrees -Celsius
- This difference was measured through transcript levels of genes that encode transporters for the nutrient
- There was an increase in transcription of ribosome-biogenesis genes
- Transcription of trehalose-biosynthesis genes and change in intracellular trehalose levels was not observed in chemostat cultures as opposed to batch cultures
- big difference between transcriptional reprogramming during long-term low temp acclimation vs. rapid transition to low temp.
- low temp acclimated growth does not involve a Msn2/Msn4 complex regulatory role
- 235 genes showed a consistent transcriptional response to low temp, regardless of the limiting nutrient and these genes are used in acclimation
- genes involved in lipid metabolism were commonly regulated in chemostat and batch culture runs
- using chemostat, transcriptional responses to low temp and low specific growth rate can be studied separately
- low temp acclimation in S. cerevisiae is not just at the transcriptional level.
- Yeast undergo temperature fluctuations living in nature
- they respond in multiple ways to temperature changes and grow most efficiently in temperature of 25-35 degrees Celsius
- drop in temperature causes the slowing of enzyme kinetics and cellular processes
- also affected are their growth phase, respiration, lipid composition of membranes and trehalose content
- how rapid or gradual the temperature change comes on is important
- sudden change or cold shock leads to a stress response which is different from the acclimation which occurs in response to a gradual temperature change which allows the yeast to adapt fully to its new suboptimal environment (on the genomic level)
- most focus on “cold shock”
- include discrepancies
- inconsistencies observed in expression of ribosomal protein genes
- trehalose accumulation only absolutely necessary in near freezing conditions (below 10 degrees Celsius)
- no identification yet of a low temperature-specific transcriptional network
- no real investigation of differences between cold adaptation and acclimation
- no study of cold acclimation in a chemostat yet
- Msn2/4, Mbp1p, Hap2-Hap1, Hap3-Hap1, Fhl1p, Sfp1p, Gln3p, Gln3-Dal82, Hap2-Dal82, Aft2p, Hsf1p, Nrg1p, Phd1p, Rsc1p, Rox1p, Sok2p, Nrg1-Aft2, Phd1-Nrg1, Rox1-Phd1, Sok2-Nrg1
Tables and Figures
- Table 1: Shows biomass yields and fermentation rates at 12 and 30 degrees Celsius in both nutrient limited cultures were similar: means growth efficiency was not severely affected by temperature.
- Table 2: Protein and storage carbohydrates contents of S. cerevisiae biomass in the nutrient limited cultures. In chemostat cultures where ammonium was limited, trehalose and glycogen contents were lower in 12 than 30 degrees Celsius.
- Table 3: A) Overrepresented cis-regulatory binding motifs in 5’ upstream regions in low temperature differently regulated gene clusters
B) Overrepresented promoter elements that bind TF’s in low temperature differently regulated gene clusters So overrepresentation of STRE elements in genes that had reduced transcript level at 12 degrees Celsius in nitrogen limited cultures.
- Figure 1: Shows in glucose limitation, 494 genes transcribed in different amount compared with 806 genes in nitrogen limitation
- Figure 2: Shows a heat map representing the 1065 total genes expressed differently in the nutrient limited cultures at 12 and 30 degrees Celsius. 16 genes involved in ribosome biogenesis had higher transcript levels at 12 than 30 degrees Celsius in both limited nutrient cultures. Although in nitrogen limited cultures, an additional 80 genes involved in protein synthesis had increased transcript levels at 12 degrees.
- Figure 3: Shows genes from batch culture experiments that were differently expressed during “adaptation” instead of acclimation. 259 genes responded to temperature drop in all three batch culture studies.
- Figure 4: Compares transcript ratio of the 259 genes that three batch culture low temp transcriptome datasets shared, venn diagrams show low temperature responsive genes common to batch culture and chemostat based data. 3 genes encoding transporters were found with the 5 consistently down regulated genes.
- Figure 5: Compares genes that were up or down regulated in this study or in adaptation studies to low temp with “growth rate dependent” genes. Little overlap with growth-rate-responsive genes was observed when temperature responsive genes were compared between 139 common temperature responsive genes from batch cultures and those of Castrillo and Regenberg.
- Figure 6: Compares ESR genes that were up or down regulated during adaptation experiments and acclimation. One third of low temp responsive genes of the 3 batch cultures could be linked to ESR genes. 233 genes regulated in Gasch study had opposing effect, hinting at an alleviation of environmental stress at low temp.