LauraTerada Individual Journal Assignment Week 3

• Laura Terada
• BIOL398-03/S13:Week 3

Term Definitions

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. 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." [[2]]
3. PUT4: A gene for a proline-specific permease, found in Saccharomyces cerevisiae [[3]]
4. GAP1: A gene for a general amino acid permease, found in Saccharomyces cerevisiae [[4]]
5. Histidinol dehydrogenase: Catalyzes the reaction: L-histidinol + NAD+ = L-histidine + NADH + H+; it has a molecular function [[5]]
6. 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)." [[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. Alpha-ketoglutarate: One of the molecules in the 8-step citric acid cycle. Isocitrate undegoes oxidative decarboxylation to get alpha ketoglutarate to form succinyl coA. Source: Nelson, David L., and Michael M. Cox. Lehninger Principles of Biochemistry. 5th ed. New York: W.H. Freeman and Company. Print.
10. Gram-negative bacteria: "A common class of bacteria normally found in the gastrointestinal tract that can be responsible for disease in man (sepsis). Bacteria are considered to be gram-negative because of their characteristic staining properties under the microscope, where they either do not stain or are decolourised by alcohol during grams method of staining. This is a primary characteristic of bacteria that have a cell wall composed of a thin layer of peptidoglycan covered by an outer membrane of lipoprotein and lipopolysaccharide containing endotoxin. The gram staining characteristics of bacteria have resulted in an important classification system for the identification of bacteria."[[9]]

Outline

• Abstract
  • Saccharomyces cerevisiae was grown in different concentrations of ammonia
    • Led to nitrogen limitation or excess and limited glucose
  • Increased intracellular ammonia caused:
    • Increased intracellular glutamate, glutamine, NAD-dependent glutamate dehydrogenase activity and its mRNA
    • Decreased NADPH-dependent glutamate dehydrogenase activity and its mRNA, mRNA for GAP1 and PUT4
  • Main result:
    • Nitrogen metabolism depends on ammonia concentration (not flux)

• Background information
  • S. cerevisiae prefers ammonia for nitrogen source
  • Past experiments have changed both ammonia concentration and flux, but little research on focusing on how the ammonia concentration can affect nitrogen metabolism
  • Ammonia in the cell reacts with alpha-ketoglutarate to get glutamate, which is then turned to glutamine
  • Under decreased amino acid levels, transcription of biosynthetic genes increase
  • Ammonia allows quick growth/metabolism
  • Nitrogen metabolism is regulated by genes and enzyme activity
  • There is a difference between ammonia concentration and ammonia flux that can regulate nitrogen metabolism
  • This study will keep flux constant, but will change the ammonia concentration

• Methods
  • Physiological parameters
    • S. cerevisiae (strain SU32) grown in cultures with differing ammonia concentrations
      • Ammonia concentrations: 29, 44, 61, 66, 78, 90, 96, 114, 118 mM
      • Fixed glucose concentration: 100 mM
      • Fixed dilution rate: 0.15/h
    • Ammonia and biomass concentrations measured at the different concentrations
    • Ammonia flux calculated from biomass, ammonia concentration and residual ammonia concentration
    • Measured concentrations of alpha-ketoglutarate, glutamate and glutamine when ammonia concentrations were changed
  • Northern (RNA) analyses
    • RNA of nitrogen regulated genes will be looked at while changing concentrations of ammonia
    • Amino acid permease-encoding genes: GAP1, PUT4
    • Biosynthetic genes: ILV5, HIS4
    • Phosphorus oligonucleotides used to detect RNA of GDH1, GLN1, GAP1, ILV5, HIS5, ACT1 and H2A-H2B
    • RNA of PUT4 analyzed with another oligonucleotide
    • RNA of GDH2 analyzed with phosphorus labelled DNA fragment (XhoI-BamHI)
    • ACT1 RNA and H2A-H2B were used as internal controls
    • Used X-ray films to detect RNA levels
  • Enzyme activities
    • Tested levels of enzyme activity that contribute to the breakdown of ammonia to glutamate and glutamine
      • NADPH-glutamate dehydrogenase (GDH), NAD-GDH and GS activity

• Results
  • Physiological parameters
    • Fig 1: x axis is NH4+ concentration, y axis is either residual NH4+ concentration, O2 consumption/CO2production, alpha-ketoglutarate concentration, glutamate concentration, or glutamine concentration
      • Measurements were made based on an average of three replicates
    • Increased ammonia concentration (29 to 61 mM) led to:
      • Increased biomass
      • Constant residual ammonia
      • Ammonia limitation
    • Above 61 mM led to:
      • Constant biomass
      • Increased residual ammonia
      • Glucose limitation
    • Ammonia excess led to constant respiratory quotient
    • Ammonia limitation led to increased CO2 and decreased O2 consumption
      • Acetate, acetaldehyde, and ethanol production
    • Ammonia limitation to excess led to:
      • Decreased alpha-ketoglutarate
      • Increased intracellular glutamate and glutamine
  • Northern analyses
    • GDH1, GDH2 and GLN1 gene expression was determined when changing ammonia concentrations
    • Fig 2: x axis is NH4+ concentration, y axis is percent expression of either GDH1, GDH2, GAP1, PUT4, GLN1, HIS4, or ILV5
      • Measurements calculated by using intensity ratios between banding of gene and reference gene
    • Ammonia limitation to excess led to:
      • Decreased GDH1 RNA expression
      • Increased GDH2 RNA expression
      • Increased ILV5 and HIS4 then decreased above 66 mM
    • GLN1 maximum expression at 61 mM
    • GAP1 and PUT4 RNA decreased above 44 mM
    • GAP1 and PUT4 regulated by ammonia concentration
  • Enzyme activities
    • Fig 3: x axis is NH4+ concentration, y axis is either NADPH-GDH concentration, NAD-GDH concentration, or GS transferase concentration
      • Measurements were based on an average of three replicates
    • Ammonia concentration increase led to:
      • Decreased NADPH-GDH
      • Increased NAD-GDH
      • Slight decrease in GS activity

• Conclusion
  • Physiological parameters
    • When going from low levels of ammonia to high levels, carbon metabolism did not significantly change
  • Northern analyses
    • When going from low levels of ammonia to high levels, gene expression levels differed
  • Enzyme activities
    • By comparing NADPH-GDH with GDH1 expression and NAD-GDH with GDH2 expression, it is clear that these two enzymes are regulated at the transcriptional level
    • GS activity proved to have no regulation at the transcriptional level, therefore it must be regulated post-transcriptionally
  • Nitrogen metabolism is regulated at different levels in S. cerevisiae, including intracellular metabolite concentrations and extracellular or intracellular ammonia concentrations. The difference in ammonia concentration affects levels of gene expression and enzyme activity.
  • Importance of this study is to try to understand at what concentration of ammonia do S. cerevisiae grow best and the mechanisms that cause and affect nitrogen metabolism.

• Future research
  • One should research the possible presence of an ammonia sensor in S. cerevesiae
  • Compare the nitrogen metabolism between gram-negative bacteria and S. cerevesiae