Anthony J. Wavrin Week 3

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Contents

Introduction

This article is exploring one of the possible explanations of how nitrogen, used in the form of ammonia in this study, can effect Saccharomyces cerevisiae. It is well known that nitrogen is an essential nutrient that can increase growth, as utilized in fertilizer. It is hypothesized that the actual influx of nitrogen may cause growth instead of the concentration. This study tests explores if increasing concentration of ammonia while keeping a constant influx will cause nitrogen related responses. The concentrations used resulted in testing from nitrogen limitation to nitrogen excess. Overall, they analyze effects of physiological parameters, RNA expression, and enzyme activites.

Physiological parameters

  • The concentrations of ammonia used were 29, 44, 61, 66, 78, 90, 96, 114, and 118mM.
    • It is interesting to note that at the concentration of 61mM of ammonia, glucose becomes the limiting nutrient.

Figure 1

A

  • The X-axis represents the increase in the concentration of the ammonia.
  • The Y-axis on the left represents the residual ammonia concentration.
  • The Y-axis on the right represents the biomass (dry weight).
  • The Y-axis on the far right represents the ammonia flux, which is calculated using ammonia concentration, residual ammonia concentration, and biomass.
  • As ammonia increased to ammonia saturation, there was an increase in biomass, but stayed relatively constant after ammonia excess (>61mM).
  • The residual ammonia concentration sky rockets after 61mM which is expected due to nitrogen excess.

B

  • The X-axis represents the increase in the concentration of the ammonia.
  • The Y-axis on the left represents the CO2 production.
  • The Y-axis on the far left represents the O2 usage.
  • The Y-axis on the right represents the respiratory quotient, which is CO2 production/ O2 usage.
  • Concentrations above 44mM of ammonia have a relatively flat respiratory quotient, indicating carbon is the limiting nutrient.

C

Left Panel
  • The X-axis represents the increase in the concentration of the ammonia.
  • The Y-axis on the left represents the concentration of α-ketogluterate present.
  • As the ammonia concentration increases, α-ketogluterate concentration decreases until 61mM. This represents the conversion of α-ketogluterate with nitrogen to form glutamate and eventually glutamine.
Middle Panel
  • The X-axis represents the increase in the concentration of the ammonia.
  • The Y-axis on the left represents the concentration of glutamate present.
  • As the ammonia concentration increases, glutamate concentration increases until 61mM.
Right Panel
  • The X-axis represents the increase in the concentration of the ammonia.
  • The Y-axis on the left represents the concentration of glutamine present.
  • As the ammonia concentration increases, glutamine concentration increases continually.

RNA Expression

  • To measure the levels of RNA of nitrogen related genes, northern analysis was performed.
  • RNA was detected using labeled phosphate oligonucleotides or 2.5kB Xho1-Bam1 DNA fragments.
  • X-ray films were utilized to quantify the RNA Detected.

Figure 2

Left Panel
  • The X-axis represents the increase in the concentration of ammonia.
  • The Y-axis represents the % expression of a given RNA, using ACT1 and H2A-H2B as internal controls.
  • As the ammonia concentration increases past 61mM, GDH1 decreases while GDH2 increases, indicating that GDH1 is repressed by excess nitrogen while GDH2 is induced by excess nitrogen.
Middle Panel
  • The X-axis represents the increase in the concentration of ammonia.
  • The Y-axis represents the % expression of a given RNA, using ACT1 and H2A-H2B as internal controls.
  • Both GAP1 and PUT4 "peak" and 44mM and decrease til 78mM then become relatively stable, indicating that they are repressed by excess nitrogen and are concentration regulated.
Right Panel
  • The X-axis represents the increase in the concentration of ammonia.
  • The Y-axis represents the % expression of a given RNA, using ACT1 and H2A-H2B as internal controls.
  • GLN1, HIS4, and ILV5 all peak around 66mM and 78mM and stay at a high expression until the highest concentration (118mM), in which the expression decreases drastically.

Enzyme Activities

  • The enzymes focused on were the enzymes that assisted in the conversion of α-ketogluterate to glutatmate or glutamine.
  • The four enzymes measured were NADPH-GDH, NADH-GDH, GS transferase and GS synthetase.

Figure 3

Top Panel
  • The X-axis represents the increase in the concentration of ammonia.
  • The Y-axis represents the concentration of NADPH-GDH.
  • The NADPH-GDH concentration decreases in a linear like fashion until an ammonia concentration of 78mM, in which is levels out. This indicates a decrease in the activity of NADPH-GDH as nitrogen concentration increases
Middle Panel
  • The X-axis represents the increase in the concentration of ammonia.
  • The Y-axis represents the concentration of NADH-GDH.
  • The NADH-GDH concentration increases consistently with the exception of a peak at 61mM, which may be indicative of a mechanisms associated with nitrogen saturation.
Bottom Panel
  • The X-axis represents the increase in the concentration of ammonia.
  • The Y-axis on the left represents the concentration of GS transferase.
  • The Y-axis on the right represents the concentration of GS synthetase.
  • The concentrations of GS transferase and GS synthetase have a parallel appearance indicating their use may be coupled.

Conclusion

The main question being explored in this study was if the concentration of ammonia (nitrogen) would effect bacteria even if the influx of the ammonia was constant. This study proves thoroughly that the concentration of ammonia does infact effect the growth of S. cerevisiae. They show that at the mass, RNA, and enzyme level there are changes that are directly due to the increase in ammonia present. This is significant because it proves that increasing concentrations of ammonia can increase growth however, at higher concentrations there are other limiting nutrients that can negate any more increase that could result from the increased nitrogen. And interesting future study would be to conduct the same measurements but with changing both the rate of influx and concentration of ammonia.

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