Paul Magnano:Week 3 Individual Journal

Paul Magnano

Outline

Experiment

• sacchromyces cerevisiae was grown in continuous culture, with varying ammonia input concentrations, while ammonia assiilation rates remained constant
• ammonia is a preffered nitrogen source for s. cerevisiae, because it results in faster growth than other sources
• in this study, cultures were grown with same flux (relationship b/w external ammonia concentration and rte of assimilation) but different inflow ammonia concentrations
• Physiological parameters:
  • S. cerevisiae SU32 grown in continuous cultures with ammonia inflow concentrations ranging from 29 to 118mM
  • fixed glucose concentration of 100mM
• Fig 1A
  • increase of ammonia concentration from 29 to 61mM resulted in increase of biomass from 4.9 to 8.2 g/liter
  • ammonia concentration above 61mM biomass remained at 8.2 g/liter
  • over trhe entire range of ammonia concentrations the ammonia flux into biomass was 1.1 mmol/gh
• Fig 1B
  • input ammonia concentration above 44mM resulted in (c02 production/02 consumption) remained constant
  • with ammonia limitation (ammonia input below 44mM) c02 production and 02 consumption values differed
  • no changes in the residual glucose concentration were seen
  • no significant changes in carbon metabolism occured when the culture was switched from ammonia excess to ammonia limitation
• Fig 1C
  • ammonia within the cells reacts with ketoglutarate to produce glutamate which is converted to glutamine
  • ketoglutarate concentration decreased from 10 to 5 umol/g when the ammonia concentration changed from limited to excess
  • glutamate concentration increased within the cell, linearly
  • increaing ammonia concentrations caused the concentrations of glutamate and glutamine within the cell to increase

Northern Analyses

• RNA analyses were done to see if RNA levels of nitrogen regulating genes changed with increased ammonia concentrations
• genes GDH1, GDH2,GLN1 were determined to study responses to ammonia levels
• GAP1 and PUT4 are amino acid permease genes
• biosynthetic genes were ILV5 and HIS4
• Fig 2
  • increasing ammonia levels resulted in GDH1 levels staying the same
  • ammonia concentrations of 29-44mM there was no GDH2, but with increase to 61mM GDH2 level increased
  • maximum GLN1 expression was observed at 61mM
  • overall it was seen that ammonia concentration both repressed GDH1 and caused the expression of GDH2
  • GAP1 was shown to be regulated in response to ammonia concentration
  • the influence of ammonia concentration on GAP1 and PUT4 was analzyed in this study
  • when ammonia concentration was above 44mM, GAP1 and PUT4 decreased
  • in ammonia limited culture a relationship between ammonia flux GAP1 expression wa observed
  • GAP1 and PUT4 expression are regulated by ammonia flux not by ammonia concentration
  • genes ILV5 and HIS4 increased with increasing ammonia concentration outside the cell, it was highest at 66mM

Enzyme Activities

• levels of NADPH-glutamate dehydrogenase(GDH), NAD-GDH and Gs activity were meaured to see if ammonia concentrations affected them
• when input ammonia concentrationincreased, levels of NADPH-GDH decreased
• Fig 3.
  • the decrease in NADPH-GDH was coupled with GDH1 decrease
  • NAD-GDH activity increased dratically as ammonia concentration increased from 29-61mM
  • decreases in the levels of GS transferase activity were seen as ammonia concentration levels increased
• overall the data shows that the concentration of ammonia is thekey factor that controls how S. cerevisiae regulates its nitrogen metabolism
• changes in nitrogen metabolism are controlled by the concentrations of ammonia inside or outside the cell

Ten New Terms

1. biomass:the amount of living matter in a given habitat, expressed either as the weight of organisms per unit area or as the volume of organisms per unit volume of habitat
2. flux:continuous change, passage, or movement
3. acetate:a salt or ester of acetic acid
4. acetaldehyde:volatile, colorless, water-soluble liquid, C 2 H 4 O, having a pungent, fruitlike odor: used chiefly in the silvering of mirrors and in organic synthesis.
5. quantification:to determine, indicate, or express the quantity o
6. blotted:to darken; make dim; obscure or eclipse
7. biosynthetic:of or pertaining to biosynthesis
8. glutamine:crystalline amino acid, HOOCCH(NH 2 )CH 2 CH 2 CONH 2 , related to glutamic acid
9. glutamate:salt or ester of glutamic acid
10. metabolites:product of metabolic action