Leanne Kuwahara-Week 9

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Purpose

To prepare for Thursday's Journal club and gain an understanding of Chemostat-based transcriptome analysis in comparison to batch-culture analysis.

Unfamiliar Terms

  1. chemostat: growth vessel into which fresh medium is delivered at a constant rate and cells and spent medium overflow at that same rate. Thus, the culture is forced to divide to keep up with the dilution, and the system exists in a steady state where inputs match outputs (Dunham, 2010).
  2. diurnal: happening over a period of a day, or being active or happening during the day rather than at night (Cambridge Dictionary).
  3. specific growth rate: the steepness of a growth curve (sigmoidal), and it is defined as the rate of increase of biomass of a cell population per unit of biomass concentration (Vázquez-Flota & Loyola-Vargas, 2006).
  4. cis-regulatory motif: 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 (Plaza & Payre, 2012).
  5. zero trans-influx transport assay: produces data that are valuable as an indication of trends and magnitudes of kinetic expressions, but are not of the rigor required for detailed mathematical analysis. Generally used to address the nature of the components of a transport system (Coons et al., 1995).
  6. 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 (Biology Online Dictionary, 2005).
  7. prototrophic: Strain's that have the same nutritional requirements as the wild-type strain (Biology Online Dictionary, 2005).
  8. mannoprotein: one of 4 structural components of the yeast cell wall (Kollar et al., 1997).
  9. Monod kinetics: models specific growth rate and is analogous mathematically to the Michaelis–Menten expression for enzyme kinetics (Doran, 2013).
  10. homeoviscous: adaption of membrane lipid composition in order to maintain membrane fluidity in the cold (Ernst et al., 2016).

Outline of Article

Introduction & Background Information
  • decreased temperatures have been associated with decreased enzyme kinetics, decreased growth, decreased respiration, and changes in the lipid composition of membranes
  • previous studies have examined the effects of rapid cold-shock (adaptation), but do not accurately depict the physiological and transcriptional changes that occur over longer periods of time (acclimation)
  • previous studies have found a set of stress response genes that are consistently induced under cold shock conditions, as well as a variety of other stressors (ESR)
    • however, there are many discrepancies between the published cold-shock studies
    • no clear low-temperature gene regulatory network has been ID'd
    • the differences between adaptation and acclimation to cold response has not been well studied.
  • previous studies have been conducted using batch cultures
    • good for studying adaptation to cold-shock (CS), but poor for acclimation studies
      • specific growth rate strongly affected by temperature--cannot differentiate whether response is due to cold shock or changes in specific growth rate
      • culture variables (i.e. pH, O2 availability, etc.) further complicate data interpretation
        • use chemostat-cultures instead
  • chemostat-cultures
    • enable accurate control of specific growth rate (independent of other culture variables)
      • dilution rate: ratio of the flow rate of the ingoing fresh medium and the culture volume
        • culture volume is held constant by an equal outflow rate of cells and spent media
      • RESULTS IN STEADY-STATE SYSTEM (specific growth rate = dilution rate)
    • can choose limiting nutrient

provides a reproducible platform for studying microbial physiology and gene expression, because the concentrations of all metabolites are kept constant over time

  • GOAL: to investigate steady-state, acclimatized growth of S. cerevisiae during cold-shock, emphasizing genome-wide transcriptional regulation, and compare with previous batch studies.
Methods

Strain and Growth Conditions

  • used a prototrophic, haploid S. cerevisiae strain CEN.PK113-7D
    • grown anaerobically in defined synthetic medium limited by glucose (C) or ammonium (N)
    • grown in a 2.0L chemostat at a dilution rate of 0.03h^-1 and a volume of 1.0L at either 12C (cold shock) or 30C (control)
      • controlled temperature with a connected cryostat
      • pH kept at 5.0
      • timespan of cold-shock experiment was not specified
        • likely over the course of days-weeks as the group was studying the acclimation to CS

Analytical Methods

  • analyzed glucose and metabolite concentrations using an HPLC on an AMINEX HPX-87H ion exchange column
    • mobile phase: 5mM sulfuric acid
  • also determined residual ammonium concentrations, culture dry weights, whole cell protein content, and trehalose and glycogen measurements, but methods were not directly stated (only referenced).
    • trehalose measured in triplicate/chemostat
    • glycogen measured in duplicate/chemostat
  • analyzed yeast biomass contents using Carlo Erba elemental analyzer

Microarray Analysis

  • sampled and probed cells, and hybridized to Affymetrix Genechip microarray
  • RNA quality determined via Agilent 2100 Bioanalyzer
    • growth condition analyzed in triplicate
  • used Excel to make pair-wise comparisons
  • used Regulatory Sequence Analysis (RSA0 tools to perform promoter analysis
  • used Database for Annotation, Visualization, and Integrated Discovery (DAVID) for stats on GO terms
  • used a bonferroni p-value and a p-value threshold of 0.01 (very stringent)
  • can access microarray data at Genome Expression Omnibus database (GSE6190)

Comparison with Other s. cerevisiae Low-Temperature Transcriptome Datasets

  • compared with Schade et al. (2004), Murata et al. (2006), Sahara et al. (2002), and Gasch et al. (2000)
    • Sahara: 1609 genes
    • Murata: 2339 genes
    • Schade: 634 genes
    • Gasch: ESR genes
Results

Overview

  • dilution rate chosen based on batch culture experiments to allow for steady-state growth at both cold (12C) and normal temperatures (30C)
  • cultures grown anaerobically to decrease effects of temperature-dependent oxygen availability
    • found growth was not affected by culture temperature
  • used DNA microarrays to determine effect of temperature on gene expression
    • glucose limited: 494 genes significantly changed expression
    • ammonium limited: 806 genes significantly changed expression
      • 235 genes consistently up/down-regulated
        • showed strong context-dependency depending on limiting nutrient

Low-Temperature Chemostat Cultivation Results in Altered Uptake Kinetics for the Limiting Nutrient and Enhanced Catabolite Repression

  • increased concentrations of glucose and ammonium at 12C compared to 30C
    • saw change in transport kinetics at transcript level
    • resulted in higher catabolite repression
      • illustrates importance of context-dependency of transcriptional response to low temperature

Acclimation to Nonfreezing Low Temperature Does Not Require a High Storage Carbohydrate Content

  • previous studies have found that cold shock induced an increase in storage carbohydrates, trehalose and glycogen
    • chemostat study did not observe this increase
      • accumulation of storage carbohydrates not required for yeast acclimation to low temperatures
        • once cells are physiologically adapted to the low temperature, stress response subsides

Up-Regulation of the Translational Machinery at Low Temperature

  • observed an induction of ribosome biogenesis genes at 12C compared to 30C
    • observed an increased protein concentration in –ammonium cultures, but not –glucose cultures at 12C vs. 30C
      • increased protein content may compensate for decreased enzyme kinetics at low temperatures

Transcriptional Responses to Low Temperature: Adaptation versus Acclimation

  • 259 genes changed expression at low temperatures—BUT inconsistent!
    • 91 consistently up-regulated
      • lipid metabolism
      • protein translocation
    • 48 consistently down regulated
      • protein transport

Context Dependency of Temperature Response

  • in batch cultures, specific growth rate decreases with temperature
    • specific growth rate constant in chemostat cultures
      • altered gene expression observed in batch culture studies may be due to changes in specific growth rate as opposed to temperature downshift\
  • in batch cultures, O2 concentration is dependent on temperature\
    • chemostat induced anaerobic respiration by using N2
      • altered gene expression observed in batch cultures may be due to changes in [O2], not temperature

Environmental Stress Response, a Low-Temperature Adaptation-specific Response

  • ESR genes identified in the Gasch study showed opposite trends of what was observed in the chemostat study
      • ESR is not required for growth at low temperatures, occurs during initial, sudden exposure to cold as a part of adaptation
Figures

Table 1

  • Demonstrated that growth was not affected by growth temperature
  • Demonstrated that concentrations of the limiting nutrient were higher at 12c cultures compared to 30C cultures

Figure 1

  • compares the number of up-regulated and down-regulated genes in the C-limited and N-limited cultures
    • showed context dependency of transcriptional regulation

Figure 2

  • GO term clusters for up and down-regulated genes in C-limited and N-limited cultures at 12C
    • C-lim up: lipid metabolism, carbohydrate transport, rRNA processing
    • C-lim down: Electron transport, AA transport, Hexose metabolism
    • N-lim up: protein synthesis, ribosome biogenesis and assembly
    • N-lim down: N-compound metabolism, polysaccharide metabolism, M-phase of mitotic cell cycle and segregation, cellular morphogenesis, response to stimuli
    • C and N-lim up: nuclear export, ribosome biogenesis and assembly
    • C and N-lim down: carbohydrate metabolism, response to stimuli, transport

Table 2

  • significantly lower trehalose and glycogen concentrations at 12c vs 30C
  • protein concentrations higher at 12C vs 30C in ammonium-limited cultures

Table 3

  • found via promoter analysis that there was an over-representation of stress response elements in the upstream regions that showed a decrease in transcript levels at 12C vs 30C

Figure 3

  • comparison of transcriptional response of batch culture studies
  • 259 genes in common
    • 91 genes commonly up-regulated
    • 48 genes commonly down-regulated
    • 120 genes inconsistently regulated

Figure 4

  • comparison of the 259 genes common in batch culture studies to the chemostat study
  • 29 genes found in common
    • 11 consistently regulated

Figure 5

  • Comparison of genes consistently up or down-regulated during adaptation and acclimation
    • altered transcript levels likely due to changes in specific growth

Figure 6

  • Comparison of genes consistently up or down-regulated with ESR genes
    • opposite transcriptional response observed in chemostat study
    • ESR may not be required for acclimation
Discussion

Significance of study:

  • batch studies do not take into account the context dependency of transcriptional response
    • impossible to change a single culture parameter without effecting others
      • context-dependent responses are relevant in natural environments (no control over parameters)
  • because temperature downshift effects other culture parameters, it is difficult to attribute the change in gene expression observed to the change in temperature or to a change in some other parameter (i.e. specific growth rate)
    • chemostat cultures “fix” this issue by having a fixed specific growth rate
  • only consistent changes in gene expression between studies were involved in lipid metabolism
    • opposing transcriptional regulation between batch studies and chemostat studies illustrate the need to study differences in adaptation and acclimation

Future Directions:

  • Apply chemostat studies to analyze posttranscriptional modes of cellular regulation
Critical Evaluation
  • The study fully demonstrated the differences between studying acclimation vs. adaptation by the use of chemostat cultures as opposed to batch cultures. I found that the group did not have a clear control used except for the cells grown at 30C. It may have been beneficial to also conduct a batch study themselves as a control, as well as have a culture grown without a limiting nutrient. Additionally, the groups methods seemed to be somewhat incomplete as they simply referenced a paper in which their method came from. It would have been helpful if they would have gave a brief description of the protocol either in the methods section or in a supplementary section. Finally, I don't think that the statement of acclimation being mutually exclusive from adaptation is completely correct, as adaptation would have to occur first in order for acclimation to be an option. It would be interesting to have a study examining both adaptation and acclimation.

Acknowledgements

Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

References

  • Biology Online Dictionary. (2005). Permease. Accessed from: https://www.biology-online.org/dictionary/Permease
  • Biology Online Dictionary. (2005). Prototrophic strains. Accessed from: https://www.biology-online.org/dictionary/Prototrophic_strains
  • Cambridge Dictionary. Accessed from: https://dictionary.cambridge.org/us/dictionary/english/diurnal
  • Coons, D. M., Boulton, R. B., & Bisson, L. F. (1995). Computer-assisted nonlinear regression analysis of the multicomponent glucose uptake kinetics of Saccharomyces cerevisiae. Journal of bacteriology, 177(11), 3251-3258.
  • Dahlquist, K. & Fitpatrick, B. (2019). "BIOL388/S19: Week 2" Biomathematical Modeling, Loyola Marymount University. Accessed from:Week 9 Assignment Page
  • Doran, P. (2013). Bioprocess Engineering Principles (Second Edition): Chapter 12 - Homogeneous Reactions. PP. 599-703.
  • Dunham, M. J. (2010). Experimental evolution in yeast: a practical guide. In Methods in enzymology (Vol. 470, pp. 487-507). Academic Press.
  • Ernst, R., Ejsing, C. S., & Antonny, B. (2016). Homeoviscous adaptation and the regulation of membrane lipids. Journal of molecular biology, 428(24), 4776-4791.
  • Kollár, R., Reinhold, B. B., Petráková, E., Yeh, H. J., Ashwell, G., Drgonová, J., ... & Cabib, E. (1997). Architecture of the yeast cell wall β (1→ 6)-glucan interconnects mannoprotein, β (1→ 3)-glucan, and chitin. Journal of Biological Chemistry, 272(28), 17762-17775.
  • Plaza, S., & Payre, F. (2012). Transcriptional switches during development (Vol. 98). Academic Press.
  • Tai, S. L., Daran-Lapujade, P., Walsh, M. C., Pronk, J. T., & Daran, J. M. (2007). Acclimation of Saccharomyces cerevisiae to low temperature: a chemostat-based analysis. Molecular Biology of the Cell, 18(2) 5100-5112.
  • Vázquez-Flota, F., & Loyola-Vargas, V. M. (2006). Plant Cell Culture Protocols.

Links

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