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 Km of 1,400 microM (without globomycin, the Km was 1.5 microM) 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.