NH4+ Transport Systems in Escherichia Coli

As holds true for other microbial ammonium transport systems, most of the information concerning the Escherichia coli system has been derived from studying the uptake of the [14C]-methylammonium ion. Only cells grown on nitrogen sources other than ammonia are capable of significant accumulation of this tracer. Stevenson and Silver 1 originally described energy-dependent uptake of 14C-methylammonium by E. coli. Accumulation of the radiolabel was inhibited by NH4 +. Servin-Gonzalez and Bastarracha 2 also found an energy-linked process for 14C-methylammonium incorporation which was repressed by growth of E. coli on excess ammonium. However, the assay method used in these earlier studies involved washing the cells following the 14C-methylammonium uptake period. As a result, the highly permanent intracellular pool of chemically unaltered 14C-methylammonium was washed out and the impermeant metabolite, [14C)-γ-glutamy1methylamide. remained inside the cells. This led to very large underestimates of the actual rates of 14C-methylammonium translocation. Furthermore, such procedures rely on the metabolic trap provided by glutamine synthetase. Our approach for determining rates of l4C-methylammonium transport utilizes filtration of cells on porous polycarbonate membranes which need not be washed. 3 Alternatively, the cells may be separated from the medium by centrifugation through a layer of oil. 4 Using the filtration method with E. coli, accumulation of chemically unaltered 14C-methylammonium against 100-fold concentration gradients was demonstrated. 5 If rapid pulses of 14C-methylammonium were utilized, the kinetic parameters of the translocation process could be estimated (Km = 36 μM methylammonium; Vmax = 4.0 μmol • sec-1 • g protein-1). Because of the very high rates of 14C-methylammonium uptake and the small intracellular volume, this represents an underestimate of the true initial rates of transport. 5 The pH dependence of 14C-methylammonium entry indicated that CH3NH3 + is the permeant species. At micromolar levels, external NH4 + is removed from the assay medium even more rapidly than 14C-methylammonium, so the study of NH4 + inhibition kinetics requires special procedures. If very dilute cell suspensions were used to avoid depletion of external NH4 + during the 14C-methylammonium transport assay, a Ki value of 0.5 μM for competitive inhibition by NH4 + could be estimated. 5 Thus, the apparent affinity of this E. coli system is more than 70 times higher for NH4 + than for CH3NH3 +. The high affinity for NH4 + suggests that this transport system may enable cells to utilize very low concentrations of external ammonium efficiently. Glutamine synthetase, the primary ammonium-utilizing enzyme under nitrogen-limiting growth conditions for E. coli, has a relatively low affinity for NH4 + (Km = 1.8 mM). 6 However, the cells grow well on agar containing less than 20 μM NH4 +. Moreover, mutants with a defect in the high-affinity ammonium/14C-methylammonium transport system are unable to grow on limiting NH4 +. 7 At much higher (>10 mM) concentrations of ammonium, the high-affinity transport system is dispensable and the mutants grow normally on M9 minimal medium with NH4 + as the sole source of nitrogen. Under these conditions, NH4 + may enter E. coli via the Kdp system for K+ transport 8 (see Chapter IIIC). Since E. coli strains with multiple defects in K+ transport (kdp trkA trkD) can utilize 16 mM NH4 + as a nitrogen source, 9 unmediated diffusion of NH3 also may be sufficient to support growth.