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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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Ammonium sequestration by AmtB. (A) NH3 is in equilibrium with NH4+ and diffuses across the cell membrane rapidly (blue arrow) (Walter and Gutknecht, 1986). The passive diffusion of NH3 alone can support rapid cell growth when the ambient ammonium concentration is high (Soupene et al, 1998; Andrade and Einsle, 2007; Fong et al, 2007). Intracellular ammonium is assimilated into biomass through glutamine synthetase (GS) (Reitzer, 2003). (B) When the ambient ammonium concentration is low, AmtB concentrates it internally (green arrow) (Boussiba et al, 1984; Andrade and Einsle, 2007; Fong et al, 2007). Higher internal concentrations of NH4+ and NH3 lead to the outward diffusion of NH3 (blue arrow), forming an energetically costly futile cycle; see Supplementary Figure 5 for the estimate of the energetic cost. (C) Time-lapse phase-contrast images of E. coli cells growing in microfluidic chambers with minimal medium containing a very low concentration (12 μM) of NH4Cl as the sole nitrogen source and saturating amounts of glycerol as the sole carbon source. The ΔamtB strain (EQ130, right) grew more slowly than the control (EQ66, left); see Supplementary Table 1 for strain details. From these images, growth rates were determined during the first three generations when the increase is clearly exponential (Supplementary Figure 1C). Here and elsewhere, the reported external NH4+ concentration includes a residual concentration of ∼4 μM estimated in the medium (Supplementary Figure 6). (D) mCherry (red) and GFP (green) intensities reflect the GS and amtB promoter activities, respectively. (E) While the wild type (solid circles) maintained its growth rate, the ΔamtB strain (open circles) grew more slowly (gray zone) below ∼20 μM of external NH4+ (black arrow), in agreement with previous findings obtained in low pH medium (Soupene et al, 1998, 2002; Fong et al, 2007); see also Supplementary Figure 7. (F, G) The promoter activities of GS (reported by mCherry, red) and AmtB (reported by GFP, green) for the wild-type (solid circle) and ΔamtB strain (open circle) increase as the ambient ammonium concentration is reduced. Below a characteristic NH4+ concentration, N*ext≈30μM (green arrow), the GFP intensities of the two strains deviate, indicating differences in the internal nitrogen status; see text. All the data plotted here are provided in Supplementary Tables 6 and 7. a.u., arbitrary units.
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f1: Ammonium sequestration by AmtB. (A) NH3 is in equilibrium with NH4+ and diffuses across the cell membrane rapidly (blue arrow) (Walter and Gutknecht, 1986). The passive diffusion of NH3 alone can support rapid cell growth when the ambient ammonium concentration is high (Soupene et al, 1998; Andrade and Einsle, 2007; Fong et al, 2007). Intracellular ammonium is assimilated into biomass through glutamine synthetase (GS) (Reitzer, 2003). (B) When the ambient ammonium concentration is low, AmtB concentrates it internally (green arrow) (Boussiba et al, 1984; Andrade and Einsle, 2007; Fong et al, 2007). Higher internal concentrations of NH4+ and NH3 lead to the outward diffusion of NH3 (blue arrow), forming an energetically costly futile cycle; see Supplementary Figure 5 for the estimate of the energetic cost. (C) Time-lapse phase-contrast images of E. coli cells growing in microfluidic chambers with minimal medium containing a very low concentration (12 μM) of NH4Cl as the sole nitrogen source and saturating amounts of glycerol as the sole carbon source. The ΔamtB strain (EQ130, right) grew more slowly than the control (EQ66, left); see Supplementary Table 1 for strain details. From these images, growth rates were determined during the first three generations when the increase is clearly exponential (Supplementary Figure 1C). Here and elsewhere, the reported external NH4+ concentration includes a residual concentration of ∼4 μM estimated in the medium (Supplementary Figure 6). (D) mCherry (red) and GFP (green) intensities reflect the GS and amtB promoter activities, respectively. (E) While the wild type (solid circles) maintained its growth rate, the ΔamtB strain (open circles) grew more slowly (gray zone) below ∼20 μM of external NH4+ (black arrow), in agreement with previous findings obtained in low pH medium (Soupene et al, 1998, 2002; Fong et al, 2007); see also Supplementary Figure 7. (F, G) The promoter activities of GS (reported by mCherry, red) and AmtB (reported by GFP, green) for the wild-type (solid circle) and ΔamtB strain (open circle) increase as the ambient ammonium concentration is reduced. Below a characteristic NH4+ concentration, N*ext≈30μM (green arrow), the GFP intensities of the two strains deviate, indicating differences in the internal nitrogen status; see text. All the data plotted here are provided in Supplementary Tables 6 and 7. a.u., arbitrary units.

Mentions: Ammonium exists predominantly in the ionic form (NH4+) at neutral pH, and the minor gaseous species (NH3) can diffuse rapidly through the cell membrane (Walter and Gutknecht, 1986). At high ambient ammonium concentrations, the passive diffusion of NH3 can provide enough nitrogen for optimal cell growth (blue arrow, Figure 1A; Soupene et al, 1998; Andrade and Einsle, 2007; Fong et al, 2007). As the ambient ammonium concentration is reduced and active ammonium transport is needed to sustain cell growth, a wide range of organisms expresses the Amt family proteins (Boussiba et al, 1984; Loque and von Wiren, 2004; Andrade and Einsle, 2007) which concentrate ammonium inside cells (green arrow, Figure 1B) (Boussiba et al, 1984; Andrade and Einsle, 2007; Fong et al, 2007). However, the higher internal concentration of NH4+/NH3 imposes a dangerous futile cycle on the organism (Kleiner, 1985), as NH3 unavoidably diffuses outward down the gradient (blue arrow), forcing a much larger ammonium uptake than the nitrogen flux needed for biosynthesis (red arrow).


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)

Ammonium sequestration by AmtB. (A) NH3 is in equilibrium with NH4+ and diffuses across the cell membrane rapidly (blue arrow) (Walter and Gutknecht, 1986). The passive diffusion of NH3 alone can support rapid cell growth when the ambient ammonium concentration is high (Soupene et al, 1998; Andrade and Einsle, 2007; Fong et al, 2007). Intracellular ammonium is assimilated into biomass through glutamine synthetase (GS) (Reitzer, 2003). (B) When the ambient ammonium concentration is low, AmtB concentrates it internally (green arrow) (Boussiba et al, 1984; Andrade and Einsle, 2007; Fong et al, 2007). Higher internal concentrations of NH4+ and NH3 lead to the outward diffusion of NH3 (blue arrow), forming an energetically costly futile cycle; see Supplementary Figure 5 for the estimate of the energetic cost. (C) Time-lapse phase-contrast images of E. coli cells growing in microfluidic chambers with minimal medium containing a very low concentration (12 μM) of NH4Cl as the sole nitrogen source and saturating amounts of glycerol as the sole carbon source. The ΔamtB strain (EQ130, right) grew more slowly than the control (EQ66, left); see Supplementary Table 1 for strain details. From these images, growth rates were determined during the first three generations when the increase is clearly exponential (Supplementary Figure 1C). Here and elsewhere, the reported external NH4+ concentration includes a residual concentration of ∼4 μM estimated in the medium (Supplementary Figure 6). (D) mCherry (red) and GFP (green) intensities reflect the GS and amtB promoter activities, respectively. (E) While the wild type (solid circles) maintained its growth rate, the ΔamtB strain (open circles) grew more slowly (gray zone) below ∼20 μM of external NH4+ (black arrow), in agreement with previous findings obtained in low pH medium (Soupene et al, 1998, 2002; Fong et al, 2007); see also Supplementary Figure 7. (F, G) The promoter activities of GS (reported by mCherry, red) and AmtB (reported by GFP, green) for the wild-type (solid circle) and ΔamtB strain (open circle) increase as the ambient ammonium concentration is reduced. Below a characteristic NH4+ concentration, N*ext≈30μM (green arrow), the GFP intensities of the two strains deviate, indicating differences in the internal nitrogen status; see text. All the data plotted here are provided in Supplementary Tables 6 and 7. a.u., arbitrary units.
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Related In: Results  -  Collection

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f1: Ammonium sequestration by AmtB. (A) NH3 is in equilibrium with NH4+ and diffuses across the cell membrane rapidly (blue arrow) (Walter and Gutknecht, 1986). The passive diffusion of NH3 alone can support rapid cell growth when the ambient ammonium concentration is high (Soupene et al, 1998; Andrade and Einsle, 2007; Fong et al, 2007). Intracellular ammonium is assimilated into biomass through glutamine synthetase (GS) (Reitzer, 2003). (B) When the ambient ammonium concentration is low, AmtB concentrates it internally (green arrow) (Boussiba et al, 1984; Andrade and Einsle, 2007; Fong et al, 2007). Higher internal concentrations of NH4+ and NH3 lead to the outward diffusion of NH3 (blue arrow), forming an energetically costly futile cycle; see Supplementary Figure 5 for the estimate of the energetic cost. (C) Time-lapse phase-contrast images of E. coli cells growing in microfluidic chambers with minimal medium containing a very low concentration (12 μM) of NH4Cl as the sole nitrogen source and saturating amounts of glycerol as the sole carbon source. The ΔamtB strain (EQ130, right) grew more slowly than the control (EQ66, left); see Supplementary Table 1 for strain details. From these images, growth rates were determined during the first three generations when the increase is clearly exponential (Supplementary Figure 1C). Here and elsewhere, the reported external NH4+ concentration includes a residual concentration of ∼4 μM estimated in the medium (Supplementary Figure 6). (D) mCherry (red) and GFP (green) intensities reflect the GS and amtB promoter activities, respectively. (E) While the wild type (solid circles) maintained its growth rate, the ΔamtB strain (open circles) grew more slowly (gray zone) below ∼20 μM of external NH4+ (black arrow), in agreement with previous findings obtained in low pH medium (Soupene et al, 1998, 2002; Fong et al, 2007); see also Supplementary Figure 7. (F, G) The promoter activities of GS (reported by mCherry, red) and AmtB (reported by GFP, green) for the wild-type (solid circle) and ΔamtB strain (open circle) increase as the ambient ammonium concentration is reduced. Below a characteristic NH4+ concentration, N*ext≈30μM (green arrow), the GFP intensities of the two strains deviate, indicating differences in the internal nitrogen status; see text. All the data plotted here are provided in Supplementary Tables 6 and 7. a.u., arbitrary units.
Mentions: Ammonium exists predominantly in the ionic form (NH4+) at neutral pH, and the minor gaseous species (NH3) can diffuse rapidly through the cell membrane (Walter and Gutknecht, 1986). At high ambient ammonium concentrations, the passive diffusion of NH3 can provide enough nitrogen for optimal cell growth (blue arrow, Figure 1A; Soupene et al, 1998; Andrade and Einsle, 2007; Fong et al, 2007). As the ambient ammonium concentration is reduced and active ammonium transport is needed to sustain cell growth, a wide range of organisms expresses the Amt family proteins (Boussiba et al, 1984; Loque and von Wiren, 2004; Andrade and Einsle, 2007) which concentrate ammonium inside cells (green arrow, Figure 1B) (Boussiba et al, 1984; Andrade and Einsle, 2007; Fong et al, 2007). However, the higher internal concentration of NH4+/NH3 imposes a dangerous futile cycle on the organism (Kleiner, 1985), as NH3 unavoidably diffuses outward down the gradient (blue arrow), forcing a much larger ammonium uptake than the nitrogen flux needed for biosynthesis (red arrow).

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