James P. McDonald Week 3

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Biological Terms

  1. 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." [[1]]
  2. Isomerase: "An enzyme that converts molecules into their positional isomers." [[2]]
  3. Oligonucleotides: "Polymers made up of a few (2-20) nucleotides. In molecular genetics, they refer to a short sequence synthesised to match a region where a mutation is known to occur, and then used as a probe (oligonucleotide probes)." [[3]]
  4. Dehydrogenase: "Enzyme that oxidizes a substrate by transferring hydrogen to an acceptor that is either NAD/NADP or a flavin enzyme. An enzyme that is used to remove hydrogen from its substrate, which is used in the cytochrome (hydrogen carrier) system in respiration to produce a net gain of ATP." [[4]]
  5. Synthetase: "Enzymes of class 6 in the e classification, catalyse synthesis of molecules, their activity being coupled to the breakdown of a nucleotide triphosphate." [[5]]
  6. Biosynthetic: "Relating to or produced by biosynthesis." [[6]]
  7. Glutamate: "Major fast excitatory neurotransmitter in the mammalian central nervous system." [[7]]
  8. Glutamine: "A crystalline amino acid occurring in proteins; important in protein metabolism. One of the 20 amino acids that are commonly found in proteins." [[8]]
  9. GAP1: "General amino acid permease, a gene found in Saccharomyces cerevisiae." [[9]]
  10. PUT4: "Proline permease, a gene found in Saccharomyces cerevisiae." [[10]]

Outline

Introduction

  • Saccharomyces cerevisiae was grown in various ammonia concentrations and the effects on the growth was observed.
    • A single dilution rate was using with a range of different ammonia concentrations.
    • The ammonia concentrations were varied to observe its effects on gene expression and enzyme activities.
  • The main result of the study was that nitrogen metabolism is dependent on ammonia concentration, not its flux.

Significance

  • Ammonia is the prefferred growth source of Saccharomyces cerevisiae as it results in faster growth.
    • Nitrogen metabolism is regulated by gene expression and enzyme activity.
  • Previous research seems to show that ammonia concentration itself is the most important factor in nitrogen metabolism.
    • But, in these previous studies the cultures have differed in ammonium flux, leaving flux as the possible key factor.
    • This experiment uses cultures with the same level flux, only the ammonium concentrations fed in are different.

Methods

Physiological Parameters

  • Saccharomyces cerevisiae SU32 was grown in continuous cultures
    • They were fed with different ammonia concentrations: 29, 44, 61, 66, 78, 90, 96, 114, 118 mM.
    • Contained a fixed glucose concentration at 100 mM.
    • Had a dilution rate of 0.15h-1.
  • Biomass and residual ammonia concentration were measured at the different ammonia concentrations.
  • The ammonium flux was calculated using the biomass, ammonia concentration, and the residual ammonia concentration.
  • The respiratory quotient was calculated using the measured values of CO2 production and O2 consumption at the different ammonia concentrations.
  • Alpha-ketoglutarate, glutamate, and glutamine concentrations were measured at the different ammonia concentrations.

Northern Analyses

  • RNA levels of nitrogen-regulated genes were observed to see if they changed in different ammonia concentrations.
    • Used amino acid permease-encoding genes: GAP1, PUT4 and biosynthetic genes: ILV5, HIS4.
    • P-labelled oligonucleotides were used to detect GDH1, GLN1, GAP1, ILV5, HIS4, ACT1, and H2A-H2B RNA levels and a separate oligonucleotide was used to analyze PUT4 RNA levels.
    • A P-labelled DNA fragment was used to detect GDH2 levels and H2A-H2B was used to detect GLN1 RNA levels.
    • Gene expression levels were detected using x-ray films and plotted to compare expression levels at different ammonia concentrations.

Enzyme Activities

  • Investigated changes in enzyme activity in different ammonia concentrations.
    • Looked at enzymes involved in the conversion of ammonia into glutamate or glutamine: NADPH-GDH, NAD-GDH, and GS.

Results

Figure 1

  • The concentrations were measured using methods from a previous paper
  • In each graph the X-axis is ammonia concentration.
  • Figure 1.A the y-axes are residual ammonia concentration, biomass, and ammonia flux.
    • The results were determined using methods from previous papers.
    • Biomass increased from 29 to 61 mM, indicating that ammonia was limiting and it leveled out above 61 mM indicating that the glucose was limiting.
    • Residual ammonia only appeared after 61 mM and continually increased, showing that above 61 mM there was excess ammonia.
    • The flux was kept constant to analyze the effects of changing ammonia concentrations.
  • Figure 1.B the y-axes are O2 consumption, CO2 production, and respiratory quotient.
    • Above 44 mM the respiratory quotient remained constant as O2 consumption and CO2 production did not vary much.
    • The values only changed below 44 mM when the there was ammonia limitation.
  • Figure 1.C the y-axes are concentrations of alpha-ketoglutarate, glutamate, and glutamine in the three graphs respectively.
    • In the first graph the alpha-ketoglutarate decreased as ammonia concentration increased because the two reacted to produce glutamate and glutamine.
    • In the next two graphs both glutamate and glutamine increased as ammonia concentration increased because both were produced by the ammonia and alpha-ketoglutarate reaction.

Figure 2

  • Gene expression levels were detected using an x-ray film method described in a previous paper.
  • In each graph the X-axis is the ammonia concentration.
  • In each graph the Y-axis is percentage of RNA expression of each gene.
    • GDH1 expression stayed constant until 78 mM when it decreased.
    • GDH2 expression was not detected until 44 mM where it then increased greatly after 61 mM.
    • GAP1 and PUT4 expression were similar, they were both constant until 44 mM where they decreased greatly after that.
    • GAP1 and PUT4 are both impacted by ammonia concentration and not flux.
    • GLN1, HIS4, and ILV5 all follow a similar trend, increasing until about 80 mM and then decreasing there after.

Figure 3

  • Changes in level of activity was investigated in changing ammonia concentrations and averaging three samples of each enzyme.
  • In each graph the X-axis is the ammonia concentration.
  • In each graph the Y-axis is the activity levels of the three enzymes: NADPH-GDH, NAD-GDH, GS transferase, and GS synthetase.
    • NADPH-GDH levels decreased as the ammonia concentration increased, which was due to transcriptional regulation.
    • NAD-GDH increased greatly from 29 to 61 mM and then leveled out, this suggested the enzyme is regulated at the transcriptional level.
    • GS activity levels slightly decreased as ammonia concentration increased, the altered GS activity was found to be post transcriptional.

Conclusion

  • The results showed that nitrogen metabolism in Saccharomyces cerevisiae is regulated by the concentration of ammonia.
  • Since flux remained constant in the experiment, the observed changes were a result of the change in ammonia concentration.
  • The experiment demonstrates that ammonia concentration does have a significant impact on changes on many levels in Saccharomyces cerevisiae
  • An possible future study would be to keep the ammonia concentration constant while changing the flux and see if any similar changes take place as they did in this experiment.
  • Another possible future study would be to try and confirm that Saccharomyces cerevisiae has a two-competent sensing system for nitrogen and compare it to other gram-negative bacteria.

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Journal Entries and Assignments

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