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Need-based activation of ammonium uptake in Escherichia coli.

Kim M, Zhang Z, Okano H, Yan D, Groisman A, Hwa T - Mol. Syst. Biol. (2012)

Bottom Line: We find that as the ambient ammonium concentration is reduced, E. coli cells first maximize their ability to assimilate the gaseous NH3 diffusing into the cytoplasm and then abruptly activate ammonium transport.Quantitative modeling of known interactions reveals an integral feedback mechanism by which this need-based uptake strategy is implemented.This novel strategy ensures that the expensive cost of upholding the internal ammonium concentration against back diffusion is kept at a minimum.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, University of California at San Diego, La Jolla, CA, USA.

ABSTRACT
The efficient sequestration of nutrients is vital for the growth and survival of microorganisms. Some nutrients, such as CO2 and NH3, are readily diffusible across the cell membrane. The large membrane permeability of these nutrients obviates the need of transporters when the ambient level is high. When the ambient level is low, however, maintaining a high intracellular nutrient level against passive back diffusion is both challenging and costly. Here, we study the delicate management of ammonium (NH4+/NH3) sequestration by E. coli cells using microfluidic chemostats. We find that as the ambient ammonium concentration is reduced, E. coli cells first maximize their ability to assimilate the gaseous NH3 diffusing into the cytoplasm and then abruptly activate ammonium transport. The onset of transport varies under different growth conditions, but always occurring just as needed to maintain growth. Quantitative modeling of known interactions reveals an integral feedback mechanism by which this need-based uptake strategy is implemented. This novel strategy ensures that the expensive cost of upholding the internal ammonium concentration against back diffusion is kept at a minimum.

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The integral feedback model of AmtB activation control. (A) Intracellular ammonium is assimilated into the biomass in two steps: first, it is captured in the form of glutamate (Glu) using the carbon skeleton α-ketoglutarate (aKG) via the GS/GOGAT pathway; see Supplementary Figure 3 for details. Then, the N-group in Glu is transferred to various carbon precursors to synthesize amino acids and incorporated into the biomass, while recycling the carbon skeleton back to aKG. Importantly, the aKG pool, which integrates imbalances between the ammonium assimilation flux (JGS) and the biomass incorporation flux (Jbiomass) (Equation (6)), is known to activate AmtB strongly (Gruswitz et al, 2007; Radchenko et al, 2010; Truan et al, 2010) via the regulatory GlnK (Coutts et al, 2002; Blauwkamp and Ninfa, 2003; Javelle et al, 2004), as indicated by the dashed cyan and brown lines. These interactions form the integral feedback loop (Equations (6), (7) and (8)). (B) The flux of ammonium assimilation by GS, JGS, plotted against the internal ammonium concentration, [NH4+]int. JGS is obtained from λ × n0, based on the form of  shown in Figure 2C. JGS decreases when [NH4+]int decreases below a certain level, N*int (gray region). (C, D) The steady-state aKG concentration ([aKG], cyan), internal ammonium concentration (purple), and AmtB activity (JAmtB, green) are deduced from Equation (8); see Supplementary Figure 4 for detailed explanation. N*ext is the external ammonium concentration ([NH4+]ext) below which JGS decreases without AmtB. For [NH4+]ext > N*ext, [aKG] remains at its basal level, and [NH4+]ext changes linearly with [NH4+]ext. For [NH4+]ext<N*ext, [aKG] increases and activates AmtB to the level needed to uphold [NH4+]int at N*int.
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f5: The integral feedback model of AmtB activation control. (A) Intracellular ammonium is assimilated into the biomass in two steps: first, it is captured in the form of glutamate (Glu) using the carbon skeleton α-ketoglutarate (aKG) via the GS/GOGAT pathway; see Supplementary Figure 3 for details. Then, the N-group in Glu is transferred to various carbon precursors to synthesize amino acids and incorporated into the biomass, while recycling the carbon skeleton back to aKG. Importantly, the aKG pool, which integrates imbalances between the ammonium assimilation flux (JGS) and the biomass incorporation flux (Jbiomass) (Equation (6)), is known to activate AmtB strongly (Gruswitz et al, 2007; Radchenko et al, 2010; Truan et al, 2010) via the regulatory GlnK (Coutts et al, 2002; Blauwkamp and Ninfa, 2003; Javelle et al, 2004), as indicated by the dashed cyan and brown lines. These interactions form the integral feedback loop (Equations (6), (7) and (8)). (B) The flux of ammonium assimilation by GS, JGS, plotted against the internal ammonium concentration, [NH4+]int. JGS is obtained from λ × n0, based on the form of shown in Figure 2C. JGS decreases when [NH4+]int decreases below a certain level, N*int (gray region). (C, D) The steady-state aKG concentration ([aKG], cyan), internal ammonium concentration (purple), and AmtB activity (JAmtB, green) are deduced from Equation (8); see Supplementary Figure 4 for detailed explanation. N*ext is the external ammonium concentration ([NH4+]ext) below which JGS decreases without AmtB. For [NH4+]ext > N*ext, [aKG] remains at its basal level, and [NH4+]ext changes linearly with [NH4+]ext. For [NH4+]ext<N*ext, [aKG] increases and activates AmtB to the level needed to uphold [NH4+]int at N*int.

Mentions: The known molecular interactions actually suggest an alternative signaling scheme as depicted in Figure 5A. It is known that GlnK binds tightly to AmtB and inhibits its activity in ammonium-replete conditions (brown dashed line) (Coutts et al, 2002; Blauwkamp and Ninfa, 2003; Javelle et al, 2004). Also, GlnK dissociates from AmtB at elevated α-ketoglutarate (aKG) concentrations, as predicted by Gruswitz et al (2007) and later confirmed by Radchenko et al (2010) and Truan et al (2010), thereby setting AmtB free to transport ammonium (cyan dashed line). What ties these two pieces of biochemical interactions together is that the aKG pool can be dramatically affected by the ammonium influx which it controls, as established in the literature (Yuan et al, 2009; Radchenko et al, 2010; Doucette et al, 2011; Yan et al, 2011). At low internal ammonium concentrations, ammonium is assimilated by the GS/GOGAT cycle (Supplementary Figure 3; Reitzer, 2003), producing glutamate (Glu) from aKG (red arrows in Figure 5A). The nitrogen group in Glu is passed on to various precursors for biomass synthesis, turning Glu back to aKG (black arrows). If the internal ammonium level drops, then the rate of ammonium assimilation will drop immediately. This slows aKG drainage (red arrow), resulting in aKG accumulation: see Supplementary Figure 3 for details. Indeed, the rapid accumulation of aKG upon ammonium downshift (>5-folds within 15 s) was reported recently (Yan et al, 2011).


Need-based activation of ammonium uptake in Escherichia coli.

Kim M, Zhang Z, Okano H, Yan D, Groisman A, Hwa T - Mol. Syst. Biol. (2012)

The integral feedback model of AmtB activation control. (A) Intracellular ammonium is assimilated into the biomass in two steps: first, it is captured in the form of glutamate (Glu) using the carbon skeleton α-ketoglutarate (aKG) via the GS/GOGAT pathway; see Supplementary Figure 3 for details. Then, the N-group in Glu is transferred to various carbon precursors to synthesize amino acids and incorporated into the biomass, while recycling the carbon skeleton back to aKG. Importantly, the aKG pool, which integrates imbalances between the ammonium assimilation flux (JGS) and the biomass incorporation flux (Jbiomass) (Equation (6)), is known to activate AmtB strongly (Gruswitz et al, 2007; Radchenko et al, 2010; Truan et al, 2010) via the regulatory GlnK (Coutts et al, 2002; Blauwkamp and Ninfa, 2003; Javelle et al, 2004), as indicated by the dashed cyan and brown lines. These interactions form the integral feedback loop (Equations (6), (7) and (8)). (B) The flux of ammonium assimilation by GS, JGS, plotted against the internal ammonium concentration, [NH4+]int. JGS is obtained from λ × n0, based on the form of  shown in Figure 2C. JGS decreases when [NH4+]int decreases below a certain level, N*int (gray region). (C, D) The steady-state aKG concentration ([aKG], cyan), internal ammonium concentration (purple), and AmtB activity (JAmtB, green) are deduced from Equation (8); see Supplementary Figure 4 for detailed explanation. N*ext is the external ammonium concentration ([NH4+]ext) below which JGS decreases without AmtB. For [NH4+]ext > N*ext, [aKG] remains at its basal level, and [NH4+]ext changes linearly with [NH4+]ext. For [NH4+]ext<N*ext, [aKG] increases and activates AmtB to the level needed to uphold [NH4+]int at N*int.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f5: The integral feedback model of AmtB activation control. (A) Intracellular ammonium is assimilated into the biomass in two steps: first, it is captured in the form of glutamate (Glu) using the carbon skeleton α-ketoglutarate (aKG) via the GS/GOGAT pathway; see Supplementary Figure 3 for details. Then, the N-group in Glu is transferred to various carbon precursors to synthesize amino acids and incorporated into the biomass, while recycling the carbon skeleton back to aKG. Importantly, the aKG pool, which integrates imbalances between the ammonium assimilation flux (JGS) and the biomass incorporation flux (Jbiomass) (Equation (6)), is known to activate AmtB strongly (Gruswitz et al, 2007; Radchenko et al, 2010; Truan et al, 2010) via the regulatory GlnK (Coutts et al, 2002; Blauwkamp and Ninfa, 2003; Javelle et al, 2004), as indicated by the dashed cyan and brown lines. These interactions form the integral feedback loop (Equations (6), (7) and (8)). (B) The flux of ammonium assimilation by GS, JGS, plotted against the internal ammonium concentration, [NH4+]int. JGS is obtained from λ × n0, based on the form of shown in Figure 2C. JGS decreases when [NH4+]int decreases below a certain level, N*int (gray region). (C, D) The steady-state aKG concentration ([aKG], cyan), internal ammonium concentration (purple), and AmtB activity (JAmtB, green) are deduced from Equation (8); see Supplementary Figure 4 for detailed explanation. N*ext is the external ammonium concentration ([NH4+]ext) below which JGS decreases without AmtB. For [NH4+]ext > N*ext, [aKG] remains at its basal level, and [NH4+]ext changes linearly with [NH4+]ext. For [NH4+]ext<N*ext, [aKG] increases and activates AmtB to the level needed to uphold [NH4+]int at N*int.
Mentions: The known molecular interactions actually suggest an alternative signaling scheme as depicted in Figure 5A. It is known that GlnK binds tightly to AmtB and inhibits its activity in ammonium-replete conditions (brown dashed line) (Coutts et al, 2002; Blauwkamp and Ninfa, 2003; Javelle et al, 2004). Also, GlnK dissociates from AmtB at elevated α-ketoglutarate (aKG) concentrations, as predicted by Gruswitz et al (2007) and later confirmed by Radchenko et al (2010) and Truan et al (2010), thereby setting AmtB free to transport ammonium (cyan dashed line). What ties these two pieces of biochemical interactions together is that the aKG pool can be dramatically affected by the ammonium influx which it controls, as established in the literature (Yuan et al, 2009; Radchenko et al, 2010; Doucette et al, 2011; Yan et al, 2011). At low internal ammonium concentrations, ammonium is assimilated by the GS/GOGAT cycle (Supplementary Figure 3; Reitzer, 2003), producing glutamate (Glu) from aKG (red arrows in Figure 5A). The nitrogen group in Glu is passed on to various precursors for biomass synthesis, turning Glu back to aKG (black arrows). If the internal ammonium level drops, then the rate of ammonium assimilation will drop immediately. This slows aKG drainage (red arrow), resulting in aKG accumulation: see Supplementary Figure 3 for details. Indeed, the rapid accumulation of aKG upon ammonium downshift (>5-folds within 15 s) was reported recently (Yan et al, 2011).

Bottom Line: We find that as the ambient ammonium concentration is reduced, E. coli cells first maximize their ability to assimilate the gaseous NH3 diffusing into the cytoplasm and then abruptly activate ammonium transport.Quantitative modeling of known interactions reveals an integral feedback mechanism by which this need-based uptake strategy is implemented.This novel strategy ensures that the expensive cost of upholding the internal ammonium concentration against back diffusion is kept at a minimum.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, University of California at San Diego, La Jolla, CA, USA.

ABSTRACT
The efficient sequestration of nutrients is vital for the growth and survival of microorganisms. Some nutrients, such as CO2 and NH3, are readily diffusible across the cell membrane. The large membrane permeability of these nutrients obviates the need of transporters when the ambient level is high. When the ambient level is low, however, maintaining a high intracellular nutrient level against passive back diffusion is both challenging and costly. Here, we study the delicate management of ammonium (NH4+/NH3) sequestration by E. coli cells using microfluidic chemostats. We find that as the ambient ammonium concentration is reduced, E. coli cells first maximize their ability to assimilate the gaseous NH3 diffusing into the cytoplasm and then abruptly activate ammonium transport. The onset of transport varies under different growth conditions, but always occurring just as needed to maintain growth. Quantitative modeling of known interactions reveals an integral feedback mechanism by which this need-based uptake strategy is implemented. This novel strategy ensures that the expensive cost of upholding the internal ammonium concentration against back diffusion is kept at a minimum.

Show MeSH
Related in: MedlinePlus