To assess the mechanism and function of the glutamate uptake system of gram-positive Corynebacterium glutamicum, a mutant deficient in glutamate uptake was isolated and was then used to isolate a DNA fragment restoring this deficiency. In a low-copy-number vector, this fragment resulted in an increased glutamate uptake rate of 4.9 nmol/min/mg (wild type, 1.5 nmol/min/mg). In addition, carbon source-dependent regulation of the glutamate uptake system was determined with the fragment, showing that the entire structures required for expression and control reside on the fragment isolated. Sequencing of 3,977 bp revealed the presence of a four-gene cluster (gluABCD) with deduced polypeptide sequences characteristic of a nucleotide-binding protein (GluA), a periplasmic binding protein (GluB), and integral membrane proteins (GluC and GluD), identifying the glutamate transporter as a binding protein-dependent system (ABC transporter). This identification was confirmed by the kinetic characteristics obtained for cells grown in the presence of globomycin, which exhibited an increased K m of 1,400 M (without globomycin, the K m was 1.5 M) but a nearly unaltered maximum velocity. By applying gene-directed mutagenesis, a strain with the entire cluster deleted was constructed. With this mutant, the glutamate uptake rate was reduced from 1.4 to less than 0.1 nmol/min/mg, which is proof that this system is the only relevant one for glutamate uptake. With this strain, the glutamate excretion rate was unaffected (18 nmol/min/mg), showing that no component of gluABCD is involved in export but rather that a specific machinery functions for the latter purpose.Gram-positive Corynebacterium glutamicum is in use for the large-scale production of amino acids, in particular for glutamate. However, compared with what is known about many other bacteria, molecular details on the amino acid transport systems of this bacterium are severely restricted. So far, only the structure of the lysine uptake carrier, lysI, is known (33). Functional studies with intact cells on the kinetics and energetics of several amino acid carriers of C. glutamicum, including that of the glutamate uptake system, have been carried out (for a review, see reference 20). This uptake system (i) exhibits high affinity, (ii) results in extremely high internal glutamate accumulation, (iii) is irreversible, and (iv) has activity directly correlated to the cytosolic ATP content of the cell (21). These results were interpreted in terms of a primary, binding proteindependent, ATP-driven uptake system for glutamate. Interestingly, the total catalytic activity of the system is induced when cells are grown on glutamate but it is subject to catabolite repression by glucose (22).Binding protein-dependent systems belong to the large family of ABC transporters (for a review, see reference 11). These multicomponent systems typically consist of two membraneinserted subunits (domains), one component inside the cytoplasm which carries the ATP-binding site, and finally the binding protein ...