Helena Olivieri Individual Journal Assignment Week 3

The Concentration of Ammonia Regulates Nitrogen Metabolism in Saccharomyces cerevisiase

Important Terms

• Flux: "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."[1]
• Glutamate: "major fast excitatory neurotransmitter in the mammalian central nervous system."[2]
• Northern blot analysis: "A procedure similar to the southern blot analysis, used mostly to separate and identify rNA fragments; typically via transferring RNA fragments from an agarose gel to a nitrocellulose filter followed by detection with a suitable probe."[3]
• 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."[4]
• Biosynthesis: "The production of a complex chemical compound from simpler precursors in a living organism, usually involving enzymes (to catalyze the reaction) and energy source (such as ATP)"[5]
• Ammonia: "The common Name for NH3, a strongly basic, irritating, colourlessgas which is lighter than air and readily soluble in water. It is formed in nature as a by-product of protein metabolism in animals.Industrially, it is used in explosives, fertiliser, refrigerants, household cleaningsolutions, etc."[6]
• Metabolite: "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." [7]
• Gram-negative bacteria: "bacteria which lose crystal violet stain but are stained pink when treated by grams method"[8]
• 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). [9]
• Glutamine: "A crystalline amino acid occurring in proteins; important in protein metabolism.One of the 20 amino acids that are commonly found in proteins." [10]

Abstract:

• S. cerevisiase cultured with various concentrations of ammonia, effects observed
• Relationship between extracellular ammonia and intracellular glutamate and glutamine concentrations, levels of NAD-dependent glutamate dehydrogenase activity and mRNA studied

Introduction:

• Ammonia is nitrogenous source that encourages most rapid growth in Saccharomyces cervisiae
• Nitrogen metabolism regulated at genetic and enzymatic levels
• Concentration of ammonia relevant to growth activity
• The study emphasizes the significance of having constant flux levels between cultures in order to study the effects of differing ammonia concentration.

Physiological Parameters:

• While glucose concentration remained constant at 100 mM, S. cerevisiae concentration was studied at concentrations of 29, 44, 61, 66, 78, 90, 96, 114, and 118 mM
• Figure 1A displays a direct correlation between ammonia concentration and biomass between 29 and 61 mM. Limited by glucose levels, concentrations higher than 61 mM did not alter biomass.
• The overall ammonia flux into biomass was approximately 1.1 mmol/gh
• Figure 1B displays that at ammonia concentrations above 44 mM, CO2 production and O2 consumptions remain constant.
• Below concentrations of 44 mM, O2 consumption greatly decreased, while CO2 production increased. Residual glucose levels did not change.
• After 29 mM, increase in ammonia concentration, therefore, did not cause relevant change to carbon metabolism.
• Figure 1C displays the relationship between ketoglutarate, glutamate, and glutamine
• Given incorporation of ammonia, glutamate is converted to glutamine
• Ketoglutarate concentration is cut in half when excess of ammonia is present
• Intracellular glutamate concentration tripled given excess ammonia
• Glutamine concentration increases linearly

Northern Analyses:

• Northern Blot technique used to analyze if changes were made to RNA levels of nitrogen-regulated genes
• Figure 2 displays gene expression in relation to ammonia concentration
• Amino acid permease-encoding genes: GAP1 and PUT4
• Biosynthetic genes: ILV5 and HIS4
• GDH1, GLN1, HIS4, ACT1, and H2A-H2B genes also detected through Northern Blot
• Most GLN1, a gene that utilized ammonia, was maximized at 61 mM.
• Ammonia concentration repressed, through gene GDH1, and induced, through GDH2, RNA expression of nitrogen-regulated genes.
• Proline permease Put4p and Gap1p genes regulated in response to ammonia concentration
• GAP1 and PUT4 gene RNA decreased with increase of ammonia concentration past 44 mM
• Biosynthetic genes ILV5 and HIS4 RNA quantities increased with higher ammonia concentrations at a max of 66 mM, after 66 mM, amounts decrease

Enzyme Activities:

• Do changes in ammonia concentrations affect enzyme activity involved in the conversion of ammonia to glutamate or glutamine? (Figure 3)
• NADPH-glutamate dehydrogenase, NAD-GDH, and GS activity studied
• Between 29-118 mM NADPH-GDH decreased, less GDH1 expressed
• NADPH-GDH increased between 20 and 61 mM
• After 61 mM, no changes
• Ultimately, article suggestes that S. cerevisiae likely may have a mechanism to detect nitrogen