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Matthew E. Jurek BIOL398-03/S13

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The Concentration of Ammonia Regulates Nitrogen Metabolism in Saccharomyces cerevisiae

Eelko G. ter Shure, Herman H.W. Sillje, Arie J. Verkleij, Johannes Boonstra, and C. Theo Verrips

Defining Terms

All definitions from biology-online.org [1]

• Biosynthetic- Relating to or produced by biosynthesis.

• Continuous Culture-A large-scale closed system culture in which cells are grown in a fixed volume of nutrient culture medium under specific environmental conditions (e.g. nutrient type, temperature, pressure, aeration, etc.) up to a certain density in a tank or airlift fermentor, harvested and processed as a batch, especially before all nutrients are used up.

• Dehydrogenase-(Science: enzyme) 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.

• Flux-(Science: radiobiology) The total amount of a quantity passing through a given surface per unit time. Typical quantities include (magnetic) field lines, particles, heat, energy, mass of fluid, etc. Common usage in plasma physics is for flux by itself to mean magnetic field flux, unless specified otherwise.

• Glutamate-(Science: biochemistry, physiology) major fast excitatory neurotransmitter in the mammalian central nervous system.

• Metabolite-(Science: biochemistry) Any substance produced by metabolism or by a metabolic process. Any substance involved in metabolism (either as a product of metabolism or as necessary for metabolism). An end product as a result of metabolism.

• 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).

• Permease-(Science: enzyme) 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.

• Physiological- Of, or pertaining to physiology or normal functioning of an organism.

• Proline- (Science: amino acid) One of the 20 amino acids directly coded for in proteins. Structure differs from all the others, in that its side chain is bonded to the nitrogen of the amino group, as well as the carbon. This makes the amino group a secondary amine and so proline is described as an amino acid. Has strong influence on secondary structure of proteins and is much more abundant in collagens than in other proteins, occurring especially in the sequence glycine proline hydroxyproline. A proline rich region seems to characterise the binding site of SH3 domains. An amino acid that is found in many proteins (especially collagen).One of 20 amino acids commonly found as part of a protein.

Outline

Introduction

• Ammonia is the preferred nitrogen source of yeast,Saccharomyces cerevisiae, as noted by rapid growth
• The breakdown, or metabolism, of nitrogen is regulated at two levels: gene expression and enzyme activity
• Continuous cultures differ in external ammonia concentration and ammonia assimilation
• It is thought the flux of ammonia is more critical in nitrogen breakdown than ammonia concentration

Physiological parameters

• A strain of yeast (SU32) was grown under the following conditions in continuous culture in a fermenter:
  • Different ammonia concentrations(mM): 29,44,61,66,78,90,96,114, and 118
  • Fixed glucose concentration (mM): 100
• Such parameters allowed for an investigation of ammonia's influence on both gene expression and regulation
  • Fig 1A
    • X-axis shows ammonia concentration within the feed
    • Y-axis on the left shows residual ammonia concentration, or ammonia left in culture
    • Y-axis on the right shows biomass/ flux or the product of ammonia metabolism
    • Feed with 61mM ammonia or lower showed ammonia limitation
    • Feed with more than 61 mM ammonia resulted in glucose acting as the limiting factor
  • Fig 1B
    • X-axis shows which feed is used
    • Y-axis on the left shows the oxygen consumed and resulting carbon dioxide produced
    • Y-axis on the right shows the ratio of carbon dioxide produced over oxygen consumed
    • Feed with an ammonia concentration of 44mM or lower resulted in ammonia limitation
    • Input ammonia concentration above 44mM produced a constant respiratory quotient
    • No significant changes in carbon metabolism (aside from 29mM ammonia feed) occurred as excess of ammonia was introduced
• The different steps of glutamine production were observed as ammonia concentrations were increased
  • Fig 1C
    • X-axis shows the ammonia concentration in the feed
    • Y-axis shows concentration of listed substance based on ammonia
    • Ammonia excess increased the levels of glutamate and glutamine intracellularly

Northern analyses

• Observed if RNA levels of nitrogen-regulated genes change as ammonia concentrations increased
• RNA levels were monitored using labelled oligonucleotides, which can find certain sequences
• DNA fragments were also used to observe RNA levels
• A quantification of RNA levels was performed using X-ray and varying exposure times
• The utilization of ammonia relies on three genes (GDH1, GDH2, and GLN1)
  • Fig 2
    • X-axis shows the ammonia concentration
    • Y-axis shows the expression of RNA of the three genes as a percentage
    • Increasing ammonia resulted in a steady expression of GDH1 RNA for a while, followed by a decrease
    • Low levels of ammonia produced undetectable amounts of GDH2 RNA
    • Increasing ammonia concentrations produced more GDH2 RNA
    • Ammonia concentration repressed GDH1 RNA and induced GDH2 RNA
• GAP1 and PUT4 RNA was reduced as ammonia concentration increased
• GAP1 and PUT4 are regulated by ammonia concentration, as opposed to ammonia flux
• Amino acid starvation increased the activity of the Gcn4p regulator
• Ultimately, Gcn4p-regulated genes behaved similarly to elevated amino acid concentrations

Enzyme activities

• Enzyme activity in response to changed ammonia concentrations was also observed
• It appears enzymatic activity is controlled at the transcriptional level as increased ammonia concentration did not increase enzymatic activity in NADPH-GDH and NAD-GDH
• GS activity, on the other hand, must be altered following transcription
  • Fig 3
    • • X-axis shows the ammonia concentration
      • Y-axis shows the activity of the listed enzymatic functions
      • All results illustrate in vitro experimentation
      • NADPH-GDH activity decreases as ammonia increases
      • NAD-GDH activity parallels ammonia concentration
      • GS transferase activity decreases as ammonia increases

Conclusion

• The concentration of ammonia impacts the metabolism of nitrogen within yeast
• This is true because the concentration of ammonia was changed while the flux remained the same
• Regulation of nitrogen metabolism within yeast occurs in the following ways:
  • Extracellular concentrations of ammonia
  • Intracellular concentrations of ammonia
  • Amount of intracellular metabolites
• Yeast may have a sensor for nitrogen
• Previous work has shown that gram-negative bacteria has a two-component nitrogen-sensing system