Paul Magnano: Week 11 Individual Journal

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Paul Magnano BIOL398-03/S13 User: Paul Magnano

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Outline of article

  • effects of specific growth rates inherent to batch cultivation studies were eliminated by analyzing the transcriptional responses to low temperatures in steady state chemostats grown at fixed growth rate
  • transcriptional responses to low temperature showed large differences between long term low temp. acclimation vs. rapid transition to low temp.

Introduction

  • s. cerv. exhibits an array of responses to temp change
  • temps below ideal range (25-35 C) slow cellular processes
  • time scale of exposure is essential, sudden exposure results in adaptation (quick stress response) while prolonged exposure results in acclimation (adapts to function better in the suboptimal environment)
  • most studies focus on cold shock (adaptation) identifying two phases; early cold respose (within 12 hr)and late cold response (after 12 hr)
  • genes associated with cold shock: TPS1, TPS2, HSP12, HSP26, HSP42, HSP104,YRO2, SSE2
  • data already published reveals discrepencies:
    • inconsistencies in ribosomal protein genes
    • genes involved in reserve carbohydrate seem consistent in cold shock, trehalose accumulation only useful in near freezing
    • although Msn2p/Msn4p complex suggested to be involved in cold shock, no transcriptional network that is low temp specific has been identified
    • adaption response has never been fully explored
  • benefit of chemostat, it allows accurate control of specific growth rate
  • study goal is to investigate steady state growth of s. cerv. at low temp

Materials and Methods

  • strain and growth conditions: strain CEN.PK113-7D, dilution rate of 0.03/h, growth at 12 or 30 deg. C in 2.0 L chemostat
  • analytical methods: glucose and metabolite concentrations analyzed by high performance liquid chromatography (AMINEX HPX-87H), residual ammonium determined with curvette tests from DRLANGE
  • microarray analysis: Affymetrix Genechip Microarrays, Microsoft excel used for pairwise comparisons, Expressionist Analyst used to produce Venn diagrams and heat map visualizations of data, promotor analysis performed using Regulatory Sequence Analysis software
  • Comparison with other S. Cerv. low temp transcriptome datasets: only genes with an average fold difference above 2 were considered to be significantly changed

Results

  • DNA microarray analysis used to analyze effects of growth temp on gene expression in glucose and ammonium limited cultures
    • glucose limited, 494 genes yielded significantly diff. transcript level
    • ammonium limited, 806 genes
  • only 235 genes showed consistent up/down regulation under both nutrient limited regimes (fig 1)
  • temp responsive genes were screened for enrichment of specific functional categories/promotor regions searched for cis-regulatory motifs, to identify regulatory networks (fig 2)
  • Low Temp chemostat cultivation results:
    • at 12 C growth limiting nutrient concentrations were 7.5-10 fold higher than 30C
    • change in transport kinetics also reflected in level of transcript in genes involved in nutrient uptake
  • Acclimation to Nonfreezing low temp does not require a high storage carbohydrate content:
    • transcriptional induction of carb storage genes typically seen in cold shock
    • this was not observed in steady state conditions at 12 C
    • genes encoding enzymes in trehalose metabolism and glycogen metabolism show to be -2.0 to -4.1 fold lower transcript level in 12C cultures vs. those grown in 30C
    • trehalose and glycogen genes are stress response to the rapid temp. change
    • once cells adapt to low temp, no stress response therefore recession of upregulation of these genes
  • Upregulation of the translation machinery at low temp:
    • at 30C transcription of genes involved in protein synthesis is virtually constant over wide range of growth conditions
    • 16 genes involved in ribosome biogenesis showed higher transcript levels at 12 C
    • low temp. affects the stability of RNA secondary structure, thus kinetics of translation
    • increase in genes involved in protein synthesis did not show effect on biomass comp. in 12 C cultures
    • increased protein content may contribute to compensation for impaired enzyme kinetics at low temp
  • Transcriptional Responses to low temp: adaptation vs. acclimation:
    • group of 259 genes that responded to temp downshifts in all 3 studies (fig 3)
    • of these as few as 91 were consistently upregulated at low temp, and 48 consistently down regulated
    • 11 genes showed a consistent pattern of regulation: PIR3,SFK1,YPC1,YEL073C,YNL024C,YLR225C upregulated at low temp; PHO84,FUI1,AHA1,FCY2,YLR413W downregulated at low temp
    • 29 genes transcriptionally reg. during acclimation and adaptation (fig 4)
    • 3 genes encoding transporters were found among the 5 consistently down regulated genes
  • Context dependency of temperature response
    • genes that showed consistent transcriptional response to low temp (acclimation and adaptation) can be partially attributed to context-dependancy of transcriptional regulation
    • investigated whether previous batch culture studies may in fact have been related to differences in specific growth rates, the 365 genes showing consistent transcriptional response were compared with set of 139 genes that consistently showed response to low temp in the batch culture studies (fig 3&5)
    • oxygen availability is another possible explanation for discrepencies
  • environmental stress response
    • general environmental stress response mechanism has been implicated in regulation of genes
    • 50% of genes consistently regulated and 13% of genes consistently regulated in batch culture studies found in environmental stress response genes that were already identified

Discussion

  • transcriptional response to a stimulus may be strongly dependent on other environmental parameters
  • changes in experimental conditions may affect specific growth rates
  • chemostat based analysis yielded 235 genes that showed a consistent transcriptional response to low temp
  • only clearly defined group of genes in this study and batch studies was involved in lipid metabolism
  • this study demonstrates that transcriptional responses to low temp and low specific growth rate (linked in batch cultures) can be dissected (analyzed separately) with chemostat use
  • low temp acclimation in S. Cerv. does not solely involve transcriptional reprogramming
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