Kasey E. O'Connor Week 3 Journal

Vocabulary

1. biomass - Total mass of all living material in a specific area
2. biosynthesis - The production of a complex chemical compound from simpler precursors in a living organism, usually involving enzymes (to catalyze the reaction) and an energy source
3. flux - The total amount of a quantity passing through a given surface per unit time
4. induced - To lead in; to introduce
5. permase - The 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
6. gram-negative bacteria - A bacteria which loses crystal violet stain but are stained pink when treated by Grams method
7. metabolite - Any substance produced by metabolism or by a metabolic process
8. glutamine - A crystalline amino acid occurring in proteins; important in protein metabolism.One of the 20 amino acidsthat are commonly found in proteins
9. glutamate - A major fast excitatory neurotransmitter in the mammalian central nervous system
10. transferase - A suffix to the name of an enzyme indicating that it transfers a specific grouping from onemolecule to another, for example acyl transferases transfer acyl groups

The definitions for these words were found at [Biology Online]

Outline

Introduction and Background Information

• ammonia is the preferred source of nitrogen for Saccharomyces cerevisiae
• nitrogen metabolism components are regulated at the level of enzyme activity and the level of gene expression
• it is thought that the governing parameter of nitrogen metabolism is the ammonia flux, not the ammonia concentration
• this experiment tested effects of different amounts of ammonia on gene expression and enzyme activities

Physiological Parameters

• S. cerevisiae SU32 was grown with feeds containing different concentrations of ammonia
  • 29, 44, 61, 66, 78, 90, 96, 114, and 118 mM
• they were also grown with a fixed glucose concentration of 100 mM at a dilution rate of 0.15 per hour
• Figure 1A showed an increase of the ammonia concentration from 29 to 61 mM caused an increase of the biomass from 4.9 to 8.2 g/liter
  • with ammonia concentrations (x-axis) higher than 61 mM, the residual concentration of ammonia in the culture medium (y-axis) also increased to 62 mM but the biomass (y-axis) remained constant due to the fact that the glucose was now limiting
• Figure 1B showed that an ammonia concentration (x-axis) greater than 44 mM resulted in the CO2 production (y-axis) and O2 consumption (y-axis) to remain constant.
  • input concentrations less than or equal to 44 mM, the values for CO2 produced and O2 consumed differed
    • ex: at 29 mM of ammonia, the CO2 production increased to 7.2 mmol/gh while the O2 consumption decreased to 1.5 mmol/gh
  • this figure shows that there is no real change in the carbon metabolism when the ammonia concentration is increased over 44 mM
• Ammonia within the cell reacts with ketoglutarate to produce glutamate which is then converted to glutamine with another ammonium ion
• Figure 1C showed the ketoglutarate concentration (y-axis) decreased from 10 to about 5 when the culture ammonia concentrations (x-acis) changed from limitation to excess
  • the intracellular glutamate concentration (y-axis) increased from about 75 to 220 and the glutamine concentration (y-axis) increased from approximately 4 to about 27

Northern Analyses

• Northern Analyses were preformed see whether the RNA levels of nitrogen regulated genes changed with increasing ammonia concentrations
• after being analyzed, the RNA data was quantified with X-ray films at different exposure times
• Figure 2 showed that when the ammonia concentrations (x-axis) were increased up to 78 mM, the level of GDH1 RNA (y-axis) remained constant, but when it kept being increased the GDH1 RNA level decreased
  • no GDH2 RNA (y-axis) could be detected at ammonia concentrations of 29 and 44 mM
  • this figure showed the concentration of ammonia both repressed and induced the RNA expression of nitrogen-regulated genes.
• GAP1 expression has been shown to be dependent on the ammonia concentration or flux
• Put4p has also been shown to be regulated in the same way
• also in figure 2, at 29 and 44 mM ammonia the levels of GAP1 and PUT4 RNA remained constant
  • after, 44 mM the amounts of GAP1 and PUT4 RNA decreased and there was practically no GAP1 RNA at 118 mM ammonia, but there was still a small amount of PUT4 RNA
  • the figure showed that GAP1 and PUT4 is regulated by the ammonia concentration
• Figure 2 also shows that the RNA in ILV5 and HIS4 increased and maximized at 66 mM of ammonia, but as the concentration continued to increase, the RNA expression decreased

Enzyme Activities

• to look at the effect of changed ammonia concentrations on the levels of enzyme activity, the levels of NADPH-GDH were determined and measured under Vmax conditions
• Figure 3 showed that the ammonia concentration (x-axis) being increased from 29 to 118 mM decreased the activity level of NADPH-GDH (y-axis)
  • there was also a decrease in the level of GDH1 expression (y-axis)
  • there was a small decrease in the levels of GS activity (y-axis) with increasing ammonia concentrations until 61 mMS, but as the concentrations kept increasing, there were no more changes in GS activity

Conclusion

• the data in this study showed that the concentration of ammonia is regulatory for the nitrogen metabolism because the flux remained constant during all of the changes in concentration
• this could imply that S. cerevisiae may have an ammonia sensor for the system for nitrogen, which has been found in other gram-negative bacteria

• Template: Kasey E. O'Connor