Computer experimentation reveals mechanisms for signaling and coupled transport that trade efficiency for robust growth

Abstract Nitrogen is essential for life, 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, but the mechanism by which membrane potential drives ammonium accumulation is unresolved. We compare six kinetic models of E. coli ammonium transport and assimilation against published experimental data. Three models assume that membrane potential affects AmtB–NH 4 + binding (electro-binding). Three others assume that it drives the conformational flip of the transporter (electro-flipping). Computer experimentation reveals that the electro-binding models are 28-fold more plausible than the electro-flipping models and suggests that the membrane potential affects AmtB–NH 4 + binding from the cytoplasmic side. Integrating these kinetic and thermodynamic features with existing structural information suggests a new spatiotemporal mechanism for coupling ammonia and proton flows in AmtB. Simulations further show that coordinated regulation of GS and AmtB minimizes futile cycling while maintaining rapid growth, even as transport-related Gibbs energy dissipation becomes substantial under ammonium limitation. These findings provide new insights into the energetic trade-offs underlying bacterial ammonium acquisition.