Corynebacterium glutamicum has two different Amt-type proteins. While AmtB has a low substrate affinity and is not saturable up to 3 mM methylammonium, AmtA has a high substrate affinity and mediates saturable, membrane potential-dependent transport, resulting in a high steady-state accumulation of methylammonium, even in the absence of metabolic trapping.Ammonium uptake systems have been described in many bacteria, although its uncharged form, ammonia (NH 3 ), is supposed to be highly membrane permeable. Until 1998, exclusively energy-dependent, membrane potential-driven ammonium transport was proposed, when Soupene and coworkers argued that AmtB proteins in enteric bacteria increase the rate of equilibration of uncharged ammonia across the cytoplasmic membrane rather than actively transporting and accumulating ammonium (19). According to this concept, transport is driven by metabolic trapping, i.e., intracellular assimilation of ammonium by glutamine synthetase (GS). This model was supported by crystallographic data for AmtB from Escherichia coli and Archaeoglobus fulgidus (2, 7, 21), which indicated the presence of a hydrophobic channel responsible for ammonia transport, and by methylammonium uptake measurements (5).In Corynebacterium glutamicum, two different ammonium transporters are present. AmtA exhibits a relatively high affinity of 44 Ϯ 7 M for methylammonium, an ammonium analogue commonly used for transport measurements (5,12,18,19), and a maximal velocity of 25 Ϯ 5 nmol mg (dry weight) Ϫ1 min Ϫ1 . Uptake is dependent on the membrane potential and can be abolished by the addition of protonophores, like carbonyl cyanide m-chlorophenylhydrazone (CCCP) (18). In the presence of 50 M methylammonium, 10 M of ammonium is sufficient for a half-maximal inhibition of methylammonium uptake, indicating a high affinity of AmtA for this solute (12). In contrast to AmtA, no methylammonium uptake activity was detected for AmtB until now (12).The aim of this study was to investigate the contributions of AmtA and AmtB to methylammonium/ammonium transport and to address the question of the transport mechanism(s) and energetic coupling.Construction of mutant strains. To investigate whether metabolic trapping by GS is a major driving force for methylammonium/ammonium uptake via Amt proteins, glnA2, coding for a GS without known function (15), and gdh, coding for glutamate dehydrogenase (GDH)-in addition to glnA, encoding the only active GS in C. glutamicum-were deleted as described previously (17). The triple-deletion strain DA-2 (⌬glnA ⌬glnA2 ⌬gdh) was generated based on strain TM⌬gdh⌬glnA (13) using plasmid pK18⌬glnA2 (15), which carries an internal glnA2 deletion. Plasmid pK⌬amtB, carrying an internal amtB deletion (14), was used to generate a glnA glnA2 gdh amtB quadruple-mutant strain, MeK-1, based on strain DA-2. The resulting strains were tested with respect to ammonium assimilation and nitrogen control (data not shown). Transcription and Western blot analyses were carried out using probes and antisera directed agai...
