![Page 1: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/1.jpg)
Quantitative Studies ofNitrogen Assimilation in E. coli
Peter Lenz
Collaboration with T. Hwa, S. Kustu, Y. Wang and D. Yan
![Page 2: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/2.jpg)
Bacterium able to produce exact copy of itselfdoubling time: T=20-200min (depending onenvironment/media)
The life of a bacterium
life of a bacterium:matter + energy → biomass
E. coli (minimal medium):glucose + NH3 → biomass
Only produced what'snot available→bacteria can sense the
environment and adjustits “growth program”according to nutrientsprovided by the medium
![Page 3: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/3.jpg)
Bacterium able to produce exact copy of itselfdoubling time: T=20-200min (depending onenvironment/media)
The life of a bacterium
life of a bacterium:matter + energy → biomass
E. coli (minimal medium):glucose + NH3 → biomass
DNA-replication:
T<60min: several replication forks
DNA for grand-children is replicated
![Page 4: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/4.jpg)
Bacterium able to produce exact copy of itselfdoubling time: T=20-200min (depending onenvironment/media)
The life of a bacterium
life of a bacterium:matter + energy → biomass
E. coli (minimal medium):glucose + NH3 → biomass
→bacteria can sense theenvironment and adjustits “growth program”according to nutrientsprovided by the medium
Adjustment of growth program:
Faster growing bacteria
•have more ribosomes
•have more DNA
•are bigger
![Page 5: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/5.jpg)
Optimal growth
Central questions:• What’s the optimal way of partitioning the cell’s activity in producing
ribosomes, proteins, DNA ...→What's the optimal way for setting up a bacterium?
• What sets/determines growth rate?• Which regulation guarantees optimal growth under varying conditions?
Bacteria need to optimize their growth rate:evolutionary pressure
97%50%%bacteria with µ=60min3%50%%bacteria with µ=66min
t=48ht=0
![Page 6: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/6.jpg)
Molecular view of growth: Metabolism
‘Modules’=production units.
E.coli metabolic network:
~250 pathways
~1500 enzymes
~2000 reactions
~1200 compounds
![Page 7: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/7.jpg)
Molecular view of growth: Metabolism
Nitrogen assimilation ‘module’
Coupled to carbon, energyand amino acids ‘modules’
![Page 8: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/8.jpg)
Nitrogen assimilation
•GDH-GS pathway: high ammonia
energetically preferred, but GDH has low affinity for NH3 (large KM)
•GS-GOGAT pathway: dominant for energy rich conditions at low [NH3]
Well defined module: 3 enzymes, 3 metabolites
Common view:
![Page 9: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/9.jpg)
Coupling to growth
High AmmoniaLow Ammonia
![Page 10: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/10.jpg)
Coupling to growth
High AmmoniaLow Ammonia
transaminase current
![Page 11: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/11.jpg)
Coupling to growth
High AmmoniaLow Ammonia
Precursor Amino acid
![Page 12: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/12.jpg)
Proteins
Coupling to growth
High Ammonia
N-Demand: Gln,Glu→amino acids
Balance of currents Cycles run 109 times/doubling time!
Low Ammonia
N-demand:transaminase current
N-supply
![Page 13: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/13.jpg)
Coupling to carbon
JaKG
aKG is gatekeeper
Energy-production: dependson carbon source
Sugar from medium
aKG regulates fluxinto bypass
![Page 14: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/14.jpg)
Coupling to carbon
Optimal growth: balance of all components (N, C, Energy) Centrale role: aKG
JaKG
![Page 15: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/15.jpg)
Allosteric and transcriptional regulation of GS
high Gln/aKG ratio deactivates GS, low Gln/akG activates GS
12 active sitesturned on/off
transcription on
transcription off
GS-activity can be fine-tuned over several orders of magnitude!
bifunctionalprotein
![Page 16: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/16.jpg)
Allosteric and transcriptional regulation of GS
high Gln/aKG ratio deactivates GS, low Gln/akG activates GS
transcription on
transcription off
GS-activity can be fine-tuned over several orders of magnitude!
GlnAp1: σ70
+ : CRP
- : NtrC-P
GlnAp2: σ54
+ : NtrC-P
- : CRP
![Page 17: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/17.jpg)
Allosteric and transcriptional regulation of GS
high Gln/aKG ratio deactivates GS, low Gln/akG activates GS
transcription on
transcription off
GS-activity can be fine-tuned over several orders of magnitude!
Why? What’s theadvantage of thisregulation?
![Page 18: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/18.jpg)
Steady state analysis
![Page 19: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/19.jpg)
Steady state analysis
measured dependence of #a.aper cell, cell mass on DT[Bremer & Dennis, 1996]
![Page 20: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/20.jpg)
Steady state analysis
parameters known from invitro assays
First model: only transcriptional regulation of GS taken into account:
CS, GDH, GG: constant #/cell
Only free parameters
![Page 21: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/21.jpg)
Steady state analysis
![Page 22: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/22.jpg)
Feedback regulation of JaKG
CS and aKGDH-levels depend oncarbon source and energy status.For fixed carbon source, [aKG]directly depends on growth rate andis independent of the details of theGS/GG/GDH system
![Page 23: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/23.jpg)
Steady state analysis
3 equations for 4 unknowns([aKG],[Glu],[Gln],JAA)
want to solve for varying [NH3]
![Page 24: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/24.jpg)
A Physiological constraint
High glutamate level crucial for growth
NH4 upshift Glu is main counterion for K+
[K+] can be changed by osmolarity ofmedium later
Yan & Kustu (1996)
![Page 25: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/25.jpg)
1st attempt: GS-GG cycle (low NH3)
•no GDH
•assume that Glu-level is set externally
3 equations for 3 unknowns([aKG],[Gln],JAA)
Steady state: nosignificant differencesbetween different GS-regulation strategies
![Page 26: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/26.jpg)
Back to full model
3 equations for 4 unknowns([aKG],[Glu],[Gln],JAA)
want to solve for varying [NH3]
![Page 27: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/27.jpg)
GOGAT-
070927 WT (FG1047) & GOGAT- (FG1195)
0 50 100 150 200 250 300 350 400
0.1
1
WT_10mM NH4+
GG-_10mM NH4+
GG-_8mM NH4+
GG-_6mM NH4+
GG-_4mM NH4+
GG-_2mM NH4+
Time (min)
OD600
WT: maintains exponential growthuntil hardly any NH4 left
GG: slows down as NH4 used up
GOGAT-: Linear Pathway: aKG Glu GlnGDH GS
![Page 28: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/28.jpg)
GOGAT-
Dalai Yan, PNAS 2007
Change in osmolarity leads tochange in Glu-level
WT and suppressor mutants
⇒GOGAT-: direct correlation between growth rate and Glu-level
![Page 29: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/29.jpg)
Theoretical proposition
Ribosomal activity =
Propose: relation from the control of a.a. level on growth rate
Mediated via stringentresponse (ppGpp)
Stringent responsedue to uncharging oftRNA-Gln
hypothesizeddependence ofribosome activiy onthe Glu (or K+) level
depends on osmolarity(1 more free parameter)
![Page 30: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/30.jpg)
Full model: steady state
3 equations for 3 unknowns([aKG],[Glu],[Gln])
![Page 31: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/31.jpg)
Full model: steady state
GDH sets growth rate
![Page 32: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/32.jpg)
Comparison with experimental data: GG-
key parameters:KNH3 =1.1mMk+ = 825/sec (>> k-)[Sakamoto et al(1975)]
Best fit: 350 GDH/cell
Only weak influence
Growth rate setby N-supplyfrom GDH
suggests constant GDH level
![Page 33: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/33.jpg)
• best-fit: 560 CS, 30 aKGDH• measurement: 600 CS (Robinson et al, 1983) 70 aKGDH (Waskiewica & Hammes,1984)
Comparison with experimental data: GG-
![Page 34: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/34.jpg)
Comparison with experimental data: GG-
Gln produced by GS: supply>demand → minimal supply: basal GS activity
basal GS activity
![Page 35: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/35.jpg)
Comparison with experimental data: GG-
Gln produced by GS: supply>demand → minimal supply: basal GS activity
basal GS activity basal GS activity + Gln leakage/ degradation
best fit: ~150 GS/cell+ 30% Gln/hr leakage
![Page 36: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/36.jpg)
Comparison with experimental data: GG-
Gln produced by GS: supply>demand → minimal supply: basal GS activity
basal GS activity basal GS activity + Gln leakage/ degradation
best fit: ~150 GS/cell+ 30% Gln/hr leakage
Glutaminase B?
![Page 37: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/37.jpg)
Comparison with experimental data: GG-
Glu produced by GDH
Result of ‘ribosome model’: single fit parameter J0 works for GG- and WT
![Page 38: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/38.jpg)
Comparison with experimental data: GG-
Glu produced by GDH
![Page 39: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/39.jpg)
High osmolarity: lower J0
Comparison with experimental data: GG-
![Page 40: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/40.jpg)
Comparison with experimental data: GG- (high osm.)
Increase in Glu-pool → increased Gln-pool?
![Page 41: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/41.jpg)
Comparison with experimental data: GG- (high osm.)
aKG pool: data describedby the same model
originalGDH model
with Gln-inhibition(Sakamoto)
Best fit: 40% reduction inGDH-level
![Page 42: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/42.jpg)
Theoretical predictions: mutants
Other mutants: glnE, inducible GSmin, inducible CS
![Page 43: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/43.jpg)
GDH kinetic parameters
Km depend ontemperature, salt…
⇒wrong Km used!
KNH4=8.3 mM and notKNH4=1.1 mM
GDH assay: direct measurement ofGDH activity
GG- data converted in GDH activity(with theoretical model)
![Page 44: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/44.jpg)
GDH kinetic parameters
Km depend ontemperature, salt…
⇒wrong Km used!
KNH4=8.3 mM and notKNH4=1.1 mM
→ GDH isreversible
![Page 45: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/45.jpg)
GDH kinetic parameters
Km depend ontemperature, salt…
⇒wrong Km used!
KNH4=8.3 mM and notKNH4=1.1 mM
constantGDH level
increase in GDH levelwith decrease in N
![Page 46: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/46.jpg)
Reversible GDH reaction
New parameters not compatible with constant GDH level
![Page 47: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/47.jpg)
Reversible GDH reaction
New parameters not compatible with constant GDH level
Single cell: volume doubles during growth
![Page 48: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/48.jpg)
Test new GDH parameters in vivo
gdhrbs
gdhrbs
Pgdh
Ptet
Construct strain (NQ5) with weak constitutive gdh expression in GG- background removes possible regulation
• qualitatively consistent with large KNH4• cannot directly fit GDH kinetics because poolsand GDH amount all depend on growth rate
![Page 49: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/49.jpg)
Ensemble measurements
Measurements per OD (~cell mass):
•GDH amount (from quantitativeWestern)
•Pool sizes
NH4 consumption ~ 3mM per OD600Compulsory binding:
! =[NADP]
KNADP
[NADPH ]
KNADPH
Unknown parameter:
(E. coli in glucose: η≈0.3)
![Page 50: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/50.jpg)
Ensemble measurements
Measurements per OD (~cell mass):
•GDH amount (from quantitativeWestern)
•Pool sizes
NH4 consumption ~ 3mM per OD600Compulsory binding:
! =[NADP]
KNADP
[NADPH ]
KNADPH
Unknown parameter:
(E. coli in glucose: η≈0.3)
Test: in vitro
![Page 51: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/51.jpg)
Ensemble measurements
NH4 consumption ~ 3mM per OD600Compulsory binding:
! =[NADP]
KNADP
[NADPH ]
KNADPH
Unknown parameter:
(E. coli in glucose: η≈0.3)
Ultimate test in WT: GDH runs in reverse for NH4<2mM: stronglyoverexpressed GDH should lead to growth defect
![Page 52: Quantitative Studies of Nitrogen Assimilation in E. coli · Nitrogen assimilation •GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large](https://reader034.vdocument.in/reader034/viewer/2022050209/5f5b867d6eaf4e50f43f3ab2/html5/thumbnails/52.jpg)
Key findings and outlook
Microscopic analysis of cell growth
Theoretical model based on•biochemical characterization of enzymes
•coupling to transamination current
Comparison with exp. measured pool sizes/enzyme levels•Gln leakage/degradation
•Gln-inhibition of GDH
•reversible GDH reaction
•GS basal level
General lessons•mutants elucidate specific aspects of regulation
•every detail matters!
•single cell vs. ensemble measurement
Outlook:•WT: aKG/Gln regulation of GS