Brianna N. Samuels-Week 9

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Purpose

To discuss the findings of this Journal Club 2 paper (Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analysis)

Terms

  • diurnal: the active behavior of an animal or plant during the day (and then inactive at night) (Biology Online Dictionary, 2014)
  • cell wall mannoprotein: The chemical reactions and pathways resulting in the formation of cell wall mannoproteins, any cell wall protein that contains covalently bound mannose residues (Gene Ontology Consortium, n.d)
  • transcriptome: the full range of messenger RNA, or mRNA, molecules expressed by an organism (Nature, n.d)
  • protrophic: A bacterial strain that has the same nutritional requirements as the wild-type strain from which it was derived ((The Free Dictionary by Farlex, n.d)
  • cryostat:A chamber that can maintain very low temperatures (MedicineNet, n.d)
  • specific growth rate: A quantitative measure of cell mass increase per unit of time (Defined Term, n.d)
  • cis-regulatory motifs: a site that is bound by a TF under particular circumstances, and this binding plays a significant role in regulating the transcription initiation rate of the TF-target gene, which is usually located in cis to this TF-target site ("Cis-Regulatory Element", n.d)
  • chemostat: a method to culture a bacterial population at a reduced growth rate for an indefinite period (ScienceDirect, n.d)
  • sphingolipid: Any lipid containing a long-chain base like that of sphingosine (for example, ceramides, cerebrosides, gangliosides, sphingomyelins); a constituent of nerve tissue (The Free Dictionary by Farlex, n.d)
  • Orthologous: Any gene that can be found in two or more different species that can be traced back to the same common [[ ancestor. (Biology Online Dictionary, 2014)

Outline

A.Introduction

  • temperature fluctuations are a crucial role in microbial life in which seasonal changes and diurnal changes are not buffered
    • Saccharomyces cerevisiae displays various responses to temperature changes
      • temperatures below optimal temperature result in slowing of enzyme function and issues with cellular processing
      • they have 2 types of cold responses
        • Early cold response (ECR) within the first 12 hours
        • Late cold response (LCR) after the first 12 hours
  • Transcriptional induction TPS1 and TPS2 are consistently shown in cold responses
  • Other genes are induced as well in heat shock
  • Other cold-induced genes encode three cell-wall mannoproteins (Tip1p, Tir1p, and Tir2p), a fatty-acid desaturase (Ole1p) that influences membrane fluidity, and Nsr1p, a nucleolar protein required for pre-rRNA processing and ribosome biogenesis
  • MSN/MSN4 showed coordinated regulation in cold response
  • Major discrepancies in data
    • inconsistencies observed in the expression ribosomal protein (RP) genes
      • one paper suggests an increased transcription of many RP genes during a temperature downshift to 10°C
      • a different paper suggests a similar temperature downshift resulted in a totally different transcriptional response
    • trehalose accumulation is only indispensable for survival in near-freezing conditions
    • although the Msn2p/Msn4p wee suggested to be involved in cold-shock response, there wasn't any clear low temperature–specific transcriptional network identified
    • the differences in transcriptional response to adaptation and acclimation to low temperature have never been thoroughly investigated
    • batch culture isn't suited for prolonged exposure to low temperatures
      • the specific growth rate (μ) is strongly affected by temperature, which makes it impossible to dissect temperature effects on transcription from effects of specific growth rate
        • relevant because specific growth rate has a strong impact on genome-wide transcript profiles
      • all culture variables evolve over time and result in complex data patterns that obscure data interpretation and make the identification of temperature-specific responses very difficult
    • chemostat cultures enable accurate control of specific growth rate, independent of other culture conditions
      • the dilution rate (D) is the ratio of the flow rate of the ingoing medium and the culture volume
      • concentrations of all metabolites and substrates in steady-state chemostat cultures are constant in time
        • these cultures provide a flexible and reproducible platform for studies on microbial physiology and gene expression in fully acclimatized cultures
      • Evolutionary adaptation phenomena can be minimized by limiting the number of generations of cultivation
  • goal:to investigate steady-state, acclimatized growth of S. cerevisiae at suboptimal temperatures, with emphasis on genome-wide transcriptional regulation
  • experimental outline
    • S. cerevisiae was grown at 12 to 30°C in anaerobic chemostat cultures, at a fixed specific growth rate of 0.03 h−1
      • this was to eliminate interference by specific growth rate
    • transcription was analyzed in both glucose- and ammonium-limited chemostat cultures
    • results were compared with previous studies

B. Materials and Methods

  • Strain and Growth Conditions
    • The prototrophic, haploid reference S. cerevisiae strain CEN.PK113-7D was grown at a dilution rate (D) of 0.03 h−1 at both 12 or 30°C in 2.0 l chemostats with a working volume of 1.0 l
    • A temperature probe connected to a cryostat controlled cultures grown at 12°C
    • Cultures grown in defined synthetic medium limited by carbon or nitrogen with all other growth requirements in excess
    • The dilution rate was set at 0.03 h−1 with pH measured on-line and kept constant at 5.0 by automatic addition of 2 M KOH using an Applikon ADI 1030 Biocontroller
    • stirrer speed set to 600 rpm
    • Anaerobic growth and steady-state conditions were maintained
    • Biomass dry weight, metabolites, dissolved oxygen, and gas profiles were constant for three volume changes before sampling
  • Analytical Method
    • supernatant captured with rapid sampling method
    • high-performance liquid chromatography on an AMINEX HPX-87H ion exchange column using 5 mM H2SO4 as the mobile phase
    • left over ammonium concentrations were determined using cuvette tests from DRLANGE
    • Culture dry weights, whole cell protein contents, trehalose and glycogen measurements were performed as described in previous literature
      • Trehalose determined in triplicate measurements
      • Glycogen determined in duplicate measurements
    • Glucose was determined using the UV method based on Roche kit no. 0716251
    • elemental composition of biomass grown under nitrogen limitation analyzed using the Carlo Erba elemental analyzer following the BN211 protocol from ECN
  • Microarray Analysis
    • RNA quality determined by Agilent 2100 Bioanalyzer
    • average coefficient of variation for the triplicate transcriptome analyses was below 0.20.
    • the level of the ACT1 transcript varied <12%
    • Microsoft Excel was used to run the significance analysis of microarrays add-in used for pair-wise comparisons
    • a threshold fold difference of 2 and a median false discovery rate of 1% was used to see the statistical significance of differences in transcript levels between cultivation conditions
      • Venn diagrams and heat-map visualizations were generated with Expressionist Analyst version 3.2 to display the info above
    • Promoter analysis performed using web-based software Regulatory Sequence Analysis (RSA) Tools
    • statistical assessment of overrepresentation of GO biological processes categories among sets of significantly changed transcripts achieved using the Database for Annotation, Visualization and Integrated Discovery (DAVID) 2006
    • Overrepresentation of transcription-factor binding sites as defined by chromatin immunoprecipitation (ChIP)-on-chip analysis assessed by Fisher's exact test
      • used Bonferroni correction and a p value threshold of 0.01
    • probability calculated with specific equation
  • Comparison with Other S. cerevisiae Low-Temperature Transcriptome Datasets
    • only genes with an average fold difference above 2 were considered to be significantly changed
      • Sahara et al =1609
      • Murata et al = 2339
      • Schade et al = only the statistically significant changed genes were provided, and hence these 634 transcripts were used

C. Results

  • the chemostat cultures grown at a fixed dilution rate of 0.03 h−1 to compare the physiology and transcriptome at 12 and 30°C without interference by the different maximum specific growth rate
  • Biomass yields and fermentation rates were similar at 12 and 30°C in both carbon- and nitrogen-limited chemostat cultures,
    • this implies that growth efficiency was affected much by the growth temperature
  • glucose-limited cultures = 494 genes significantly different transcript level at the two temperatures
  • nitrogen-limited cultures = 806 genes significantly different transcript level at the two temperatures
  • total number of temperature-responsive genes = 1065 (16% of the S. cerevisiae genome)
  • number of consistent up- or down-regulation under both nutrient-limitation regimes = 235 genes
  • concentrations of glucose and ammonia increased at 12 degrees
    • high catabolic repression
  • observed context dependency of the transcriptional responses to low temperature shows the importance of a combinatorial design of transcriptome experiments
  • Chemostat study went against previous studies on cold shock increasing trehalose and glucose
    • acclimation to low temperatures doesn't require accumulation of trehalose and glucose

Figures

Table 1

  • the measurements were made by growing the cultures at 12 and 30 degrees. They observed the physiological characteristics of the aerobic chemostat cultures. They observed the mean and standard deviation from the three cultures as well as the dry weight and biomass yield.

Figure 1

  • A Venn diagram showing the number of significantly different expressed genes between 12 and 30°C in both C and N limitations.

Figure 2

  • displays a heat map representing the transcript level ratio of 1065 differentially expressed genes in anaerobic chemostat cultures (D = 0.03 h−1) grown at 12 and 30°C. The genes indicated in the figure belong to the enriched GO categories. Nineteen NCR-responsive genes are underlined
  • C Lim up regulated for lipid metabolism, carbohydrate transport and rRNA processing, C Lim down regulated for electron transport, AA transport and hexose metabolism. N Lim up regulated for protein synthesis and ribosome biogenesis and assembly. N Lim down regulated for N-compound metabolism, polysaccharide metabolism, M-phase of mitosis, cellular morphogenesis, and response to stimuli. Both up regulate for nuclear export and ribosome biogenesis and assembly. both down regulated for carbohydrate metabolism, response to stimuli, and transport

Table 2

  • displays low glycogen and trehalose concentrations at both temperatures but also high protein concentrations at 12 degrees in specifically the ammonium-limited cultures. The table shows protein and storage biomass yields and dry weights

Table 3

  • A. displays the significantly overrepresented cis-regulatory binding motifs in 5′ upstream regions
  • B. displays the significantly overrepresented promoter elements that bind known transcription factors and pairs according to the ChiP-on-chip analysis in cold up- and down-regulated gene clusters derived from C-Lim and N-Lim chemostat experiments

Figure 3

  • displays the comparison between the different studies of batch cultures. Out of the 259 genes shared between all of them, 91 were up regulated, 48 down regulated, and 120 differentially regulated with a Venn diagram and heat map.

Figure 4

  • displays Venn diagrams and heat maps that shows that out of 29 genes in common, 11 were consistently regulated. They specifically looked at the 259 genes in common from the studies and the chemostat study

Figure 5

  • Compares the genes that are consistently regulated during acclimation and adaptation. overlap of the datasets to low temperature with the growth rate–dependent genes

Figure 6

  • displays the consistently regulated genes with ESR genes. The data suggests that they may not be needed for acclimation but needed for adaptation. They also observed that there was an opposite response in the chemostat study compared to the batch.

D. Discussion

  • Context dependency isn't accounted for in the batch studies which is important in natural environments
  • culture parameters can be affected by cold shock which makes it harder to observe the attribute of the change in gene expression
    • in the chemostat study, fixed specific growth rates offset the this dilemma
  • the results were opposite in the batch studies compared to the chemostat study

Acknowledgments

  • Homework Partner: Leanne Kuwahara
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source

References

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