Computer experimentation on E. coli ammonium transport and assimilation reveals mechanisms for energy coupling, balanced futile cycling, and robust growth

Le résumé fourni par la source

Abstract Nitrogen is essential for all life forms, and microorganisms prefer ammonium as a nitrogen source. Due to the low affinity of glutamine synthetase (GS) for ammonium, E. coli must maintain high intracellular ammonium (NH 4 + ) concentrations to sustain its rapid growth. Under ammonium limitation, E. coli imports ammonium through the transporter AmtB and incorporates it into glutamine by using GS. On the basis of structural and mutagenesis information, mechanisms have been proposed for the transport of ammonia (NH 3 ) and protons by AmtB through spatially (partly) separate routes. These mechanisms do not explain the required coupling between proton and ammonia transports. How does the membrane potential push the ammonia inward so as to attain high concentrations near GS? We here compare six candidate kinetic models of E. coli ammonium transport and assimilation in terms of how they reproduce experimental data from the literature: three variants of the ‘electro-binding model’ in which the membrane potential affects AmtB–NH 4 + binding, and three variants of the ‘electro-flipping model’ in which it influences the conformational flip of the transporter. The computer simulations decide that the electro-binding models are 28 times more plausible than the electro-flipping models and suggest that the transmembrane electric potential affects AmtB–NH 4 + binding from the cytoplasmic side. The addition of kinetic and thermodynamic features to existing structural information plus our requirement of an explanation of the coupling, suggest a new spatiotemporal mechanism of coupling of ammonia and proton flows in AmtB. Further simulations show that GS and AmtB regulation is coordinated via both the uridylyltransferase/uridylyl-removing enzyme (UTase) and 2-oxoglutarate binding, allowing the cell to minimize futile cycling while maintaining rapid growth. The free energy cost of transport-related futile cycling exceeded that of the GS reaction itself. Moreover, AmtB enabled robust growth under varying ammonium concentrations and pH levels, albeit at a cost of futile cycling that became substantial at low ammonium. These findings highlight the crucial roles of GS and AmtB in E. coli ’s adaptations and provide new insights into the trade-off mechanism between nutrient acquisition and energy efficiency.

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