Quantitative Studies of Nitrogen Assimilation in E. coli. Peter Lenz Collaboration with T. Hwa, S. Kustu, Y. Wang and D. Yan

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1 Quantitative Studies of Nitrogen Assimilation in E. coli Peter Lenz Collaboration with T. Hwa, S. Kustu, Y. Wang and D. Yan

2 The life of a bacterium life of a bacterium: matter + energy biomass E. coli (minimal medium): glucose + NH 3 biomass Bacterium able to produce exact copy of itself doubling time: T=20-200min (depending on environment/media) bacteria can sense the environment and adjust its growth program according to nutrients provided by the medium Only produced what's not available

3 The life of a bacterium life of a bacterium: matter + energy biomass E. coli (minimal medium): glucose + NH 3 biomass Bacterium able to produce exact copy of itself doubling time: T=20-200min (depending on environment/media) DNA-replication: T<60min: several replication forks DNA for grand-children is replicated

4 The life of a bacterium life of a bacterium: matter + energy biomass E. coli (minimal medium): glucose + NH 3 biomass Bacterium able to produce exact copy of itself doubling time: T=20-200min (depending on environment/media) bacteria can sense the environment and adjust its growth program according to nutrients provided by the medium Adjustment of growth program: Faster growing bacteria have more ribosomes have more DNA are bigger

5 Optimal growth Bacteria need to optimize their growth rate: evolutionary pressure %bacteria with µ=66min %bacteria with µ=60min t=0 50% 50% t=48h 3% 97% 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?

6 Molecular view of growth: Metabolism Modules =production units. E.coli metabolic network: ~250 pathways ~1500 enzymes ~2000 reactions ~1200 compounds

7 Molecular view of growth: Metabolism Nitrogen assimilation module Coupled to carbon, energy and amino acids modules

8 Nitrogen assimilation Well defined module: 3 enzymes, 3 metabolites Common view: GDH-GS pathway: high ammonia energetically preferred, but GDH has low affinity for NH 3 (large K M ) GS-GOGAT pathway: dominant for energy rich conditions at low [NH 3 ]

9 Coupling to growth Low Ammonia High Ammonia

10 Coupling to growth Low Ammonia High Ammonia transaminase current

11 Coupling to growth Low Ammonia High Ammonia Precursor Amino acid

12 Coupling to growth Low Ammonia High Ammonia N-supply N-demand: transaminase current N-Demand: Gln,Glu amino acids Proteins Balance of currents Cycles run 10 9 times/doubling time!

13 Coupling to carbon Sugar from medium akg is gatekeeper J akg Energy-production: depends on carbon source akg regulates flux into bypass

14 Coupling to carbon J akg Optimal growth: balance of all components (N, C, Energy) Centrale role: akg

15 Allosteric and transcriptional regulation of GS 12 active sites turned on/off transcription on bifunctional protein transcription off high Gln/aKG ratio deactivates GS, low Gln/akG activates GS GS-activity can be fine-tuned over several orders of magnitude!

16 Allosteric and transcriptional regulation of GS GlnAp1: σ 70 + : CRP - : NtrC-P transcription on GlnAp2: σ 54 + : NtrC-P - : CRP transcription off high Gln/aKG ratio deactivates GS, low Gln/akG activates GS GS-activity can be fine-tuned over several orders of magnitude!

17 Allosteric and transcriptional regulation of GS transcription on Why? What s the advantage of this regulation? transcription off high Gln/aKG ratio deactivates GS, low Gln/akG activates GS GS-activity can be fine-tuned over several orders of magnitude!

18 Steady state analysis

19 Steady state analysis measured dependence of #a.a per cell, cell mass on DT [Bremer & Dennis, 1996]

20 Steady state analysis First model: only transcriptional regulation of GS taken into account: CS, GDH, GG: constant #/cell Only free parameters parameters known from in vitro assays

21 Steady state analysis

22 CS and akgdh-levels depend on carbon source and energy status. For fixed carbon source, [akg] directly depends on growth rate and is independent of the details of the GS/GG/GDH system Feedback regulation of J akg

23 Steady state analysis 3 equations for 4 unknowns ([akg],[glu],[gln],j AA ) want to solve for varying [NH 3 ]

24 A Physiological constraint High glutamate level crucial for growth NH 4 upshift Glu is main counterion for K + [K + ] can be changed by osmolarity of medium later Yan & Kustu (1996)

25 1st attempt: GS-GG cycle (low NH 3 ) no GDH assume that Glulevel is set externally Steady state: no significant differences between different GSregulation strategies 3 equations for 3 unknowns ([akg],[gln],j AA )

26 Back to full model 3 equations for 4 unknowns ([akg],[glu],[gln],j AA ) want to solve for varying [NH 3 ]

27 GOGAT - GDH GS GOGAT - : Linear Pathway: akg Glu Gln WT (FG1047) & GOGAT - (FG1195) 1 WT_10mM NH4+ GG-_10mM NH4+ GG-_8mM NH4+ GG-_6mM NH4+ GG-_4mM NH4+ GG-_2mM NH4+ WT: maintains exponential growth until hardly any NH 4 left 0 0 D 6 O GG: slows down as NH 4 used up Time (min)

28 GOGAT - WT and suppressor mutants Change in osmolarity leads to change in Glu-level GOGAT - : direct correlation between growth rate and Glu-level Dalai Yan, PNAS 2007

29 Theoretical proposition Mediated via stringent response (ppgpp) Propose: relation from the control of a.a. level on growth rate Ribosomal activity = depends on osmolarity (1 more free parameter) Stringent response due to uncharging of trna-gln hypothesized dependence of ribosome activiy on the Glu (or K + ) level

30 Full model: steady state 3 equations for 3 unknowns ([akg],[glu],[gln])

31 Full model: steady state GDH sets growth rate

32 Comparison with experimental data: GG - Growth rate set by N-supply from GDH key parameters: K NH3 =1.1mM k + = 825/sec (>> k - ) [Sakamoto et al (1975)] Best fit: 350 GDH/cell suggests constant GDH level Only weak influence

33 Comparison with experimental data: GG - best-fit: 560 CS, 30 akgdh measurement: 600 CS (Robinson et al, 1983) 70 akgdh (Waskiewica & Hammes, 1984)

34 Comparison with experimental data: GG - Gln produced by GS: supply>demand minimal supply: basal GS activity basal GS activity

35 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

36 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?

37 Comparison with experimental data: GG - Glu produced by GDH Result of ribosome model : single fit parameter J 0 works for GG - and WT

38 Comparison with experimental data: GG - Glu produced by GDH

39 Comparison with experimental data: GG - High osmolarity: lower J 0

40 Comparison with experimental data: GG - (high osm.) Increase in Glu-pool increased Gln-pool?

41 Comparison with experimental data: GG - (high osm.) original GDH model akg pool: data described by the same model with Gln-inhibition (Sakamoto) Best fit: 40% reduction in GDH-level

42 Theoretical predictions: mutants Other mutants: glne, inducible GS min, inducible CS

43 GDH kinetic parameters Km depend on temperature, salt wrong Km used! K NH4 =8.3 mm and not K NH4 =1.1 mm GDH assay: direct measurement of GDH activity GG- data converted in GDH activity (with theoretical model)

44 GDH kinetic parameters Km depend on temperature, salt wrong Km used! K NH4 =8.3 mm and not K NH4 =1.1 mm GDH is reversible

45 GDH kinetic parameters Km depend on temperature, salt wrong Km used! K NH4 =8.3 mm and not K NH4 =1.1 mm constant GDH level increase in GDH level with decrease in N

46 Reversible GDH reaction New parameters not compatible with constant GDH level

47 Reversible GDH reaction New parameters not compatible with constant GDH level Single cell: volume doubles during growth

48 Test new GDH parameters in vivo P gdh rbs gdh P tet rbs gdh Construct strain (NQ5) with weak constitutive gdh expression in GG - background removes possible regulation qualitatively consistent with large K NH4 cannot directly fit GDH kinetics because pools and GDH amount all depend on growth rate

49 Ensemble measurements Measurements per OD (~cell mass): GDH amount (from quantitative Western) Pool sizes NH 4 consumption ~ 3mM per OD600 Compulsory binding: Unknown parameter:! = [NADP] K NADP [NADPH ] K NADPH (E. coli in glucose: η 0.3)

50 Ensemble measurements Measurements per OD (~cell mass): GDH amount (from quantitative Western) Pool sizes Test: in vitro NH 4 consumption ~ 3mM per OD600 Compulsory binding: Unknown parameter:! = [NADP] K NADP [NADPH ] K NADPH (E. coli in glucose: η 0.3)

51 Ensemble measurements Ultimate test in WT: GDH runs in reverse for NH4<2mM: strongly overexpressed GDH should lead to growth defect NH 4 consumption ~ 3mM per OD600 Compulsory binding: Unknown parameter:! = [NADP] K NADP [NADPH ] K NADPH (E. coli in glucose: η 0.3)

52 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