The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions. ArgR also induces the operon that encodes a catabolic NAD ؉ -dependent glutamate dehydrogenase (GDH), which converts L-glutamate, the product of the AST pathway, in ␣-ketoglutarate. The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism. Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant. The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments. Measurements of ␤-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression. Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes. DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site. In silica analysis of genomic information for P. fluorescens, P. putida, and P. stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P. aeruginosa likely apply to other pseudomonads.The arginine succinyltransferase (AST) pathway (Fig. 1) is the major route for arginine catabolism under aerobic conditions in Pseudomonas aeruginosa, This pathway converts Larginine into L-glutamate with the concomitant release of three nitrogen moieties (11,13,14). Utilization of arginine as a carbon source entails deamination of glutamate to ␣-ketoglutarate, which is then channeled into the tricarboxylic acid (TCA) cycle. We have recently reported (18) the cloning and characterization of gdhB, which encodes a novel NAD ϩ -dependent glutamate dehydrogenase (NAD-GDH; GdhB). The expression of gdhB was shown to be inducible by exogenous arginine, and this induction was mediated by ArgR, the arginine regulatory protein. The activity of GdhB, a tetramer of equal 180-kDa subunits, was also found to be subject to allosteric activation by arginine. The induction of gdhB expression and the activation by arginine of the encoded enzyme clearly serve as mechanisms that coordinate aerobic utilization of arginine as a carbon source with glutamate utilization via the TCA cycle.The ArgR protein of P. aeruginosa does not exhibit any sequence homology to the arginine regulatory proteins from enteric bacteria (17,19) or Bacillus subtilis (5). Rather, ArgR of P. aeruginosa is a member of the AraC/XylS family of transcriptional regulators (27) and functions like other members of th...