Tai LT, Daran-Lapujade P, Walsh MC, Pronk JT, Daran JM Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analysis Tai LT, Daran-Lapujade P, Walsh MC, Pronk JT, Daran JM (2007) American Society for Cell Biology 18: 5100-5112 Alex George Bobek Seddighzadeh Journal Club Presentation BIOL 398-01/S10: Bioinformatics Lab April 13, 2010

Outline DNA microarrays determine gene expression Chemostat cultures foster constant growth rates Suboptimal temperatures influence cellular processes Investigate steady state acclimatized growth of suboptimal temperature growth of S. cerevisiae. In-depth analysis of the tables and result presented in the study Discussion on temperature and growth rate changes that illicit a transcriptional response

DNA microarrays are used to determine gene expression levels Each spot contains a probe that correlates to a gene Labeled sample is washed over chip to form complementary bonds Strength of fluorescent signal indicates more binding Our experiment: Only one sample hybridized at a time- relative abundance Benefits of single chip: Poor sample can’t affect the raw data; data is more easily comparable Drawback: Twice as many chips needed

Chemostat cultures maintain a constant specific growth rate Batch culture- limited supply of nutrients provided Chemostat culture- nutrients are continuously provided in order to control specific growth rate Specific growth rate has been shown to impact transcript profiles

Suboptimal Temperatures Affect Various Cellular Processes/Characteristics Optimum growth range for S Cerevisiae: 25-35 C⁰ Below this temperature Enzyme Kinetics slow Cellular processes/characteristics affected: Growth Phase Respiration Membrane lipid composition Trehalose content

Time Exposure is Important to Low-Temp Parameters on Microbial Physiology Cold shock: sudden exposure to environmental changes The response to cold shock triggers adaptation Prolonged exposures leads to acclimation Acclimation: a permanently adapted physiological state in response to environment

Limitations From Previous Studies Led to Further Investigations by Tai et al. Major discrepancies exist amongst low-temperature transcriptome data published Batch cultures do not allow for control of specific growth rate and culture variables The differences between adaptation and acclimation have not been thoroughly investigated The goal of this study: Investigate Steady state acclimatized growth of suboptimal temperature growth of S. cerevisiae. Grow S. cerevisiae in anaerobic chemostat cultures at fixed specific growth grate of 0.03 h ⁻1

Growth efficiency and fermentation rates were not severely affected by growth temperature

The number of significantly different and similar genes between 12º and 30ºC cultures for both N and C limitations

C-Limiting: Down (136 genes) Heat Map representing the level of expression of a total of 1065 genes in Nitrogen- and Carbon-Limiting Cultures C & N-Limiting:Up (96 genes) N-limiting: Up (202 genes) C-Limiting: Up (123 genes) C-lim. 30ºC C-lim 12ºC N-lim 30ºC N-lim 12ºC Different genes along the gradient represent various genes such as “Ribosome biogenesis” or “Amino acid Transport” C & N-Limiting: Down (139 genes) N-Limiting: Down (369 genes) C-Limiting: Down (136 genes)

Trehalose and glycogen levels were not affected by increased gene expression No change in carbohydrates contradicts results from batch culture studies Greater change in Nitrogen content than protein content indicates increase of rRNA in Nitrogen limiting cultures

Few Genes Showed Consistent Responses in Acclimation and Adaption Only 91 genes were consistently up regulated

More Stress Response Elements are Upstream of genes that Were Reduced Once cells become adapted to cold temp, the stress response and up-regulation of carbohydrate storage recedes

Of 29 Genes, only 11 Genes Showed Consistent Patterns of Regulation The only genes that were defined as commonly regulated on low temperature adaptations were involved in lipid metabolism

Negligible Overlap with Growth-rate–responsive Genes was Observed 25% of down-regulated genes and 10% of up-regulated are likely to be only related to specific growth rate.

A Significant Overlap Between Regulated Genes in Batch Cultures Exist

Environmental factors make isolating one variable difficult In study, changing the temperature resulted in a higher residual glucose concentration in 12° C cultures By combining two variables, such as nutrients and temperature, a core set of genes can be identified because the responses are content-independent

Change in specific growth rate may illicit response, not temperature Previous studies indicated increase in synthesis of storage carbohydrates and regulation of carbohydrate storage genes as temperature decreases Chemostat study shows no correlation indicating role of specific growth rate

Slow temperature change elicits different transcriptional response than cold-shock 235 genes responded to low temperature regardless of limiting nutrient Only one gene was in common with batch culture studies Indicates transcription regulation for acclimation is different than that for acclimitization