| The opportunistic human pathogen, Pseudomonas aeruginosa, can utilize arginine as a sole source of carbon, energy and nitrogen. Under aerobic conditions, L-arginine is converted to L-glutamate via the arginine succinyltransferase (AST) pathway (Haas et al., 1990). Glutamate serves as a source of nitrogen atoms or can be degraded by a catabolic NAD +-dependent glutamate dehydrogenase (NAD-GDH) to alpha-ketoglutarate, which is utilized as a source of carbon and energy by means of the tricarboxylic acid (TCA) cycle.; Recent work (Lu and Abdelal, 2001) has shown that the P. aeruginosa catabolic NAD-GDH is induced by arginine through the mediation of an arginine regulatory protein, ArgR, which is a member of the AraC/XylS family of transcriptional regulators (Park et al., 1997). This study investigates the possible role of ArgR in regulating expression of genes encoding other enzymes of glutamate metabolism, namely the anabolic NADP+-dependent glutamate dehydrogenase (NADP-GDH; gdhA) and glutamate synthase (GOGAT; gltBD).; Measurements of the specific activities of NADP-GDH and GOGAT in cell-free extracts of P. aeruginosa PAO1 under different growth conditions showed that exogenous arginine represses the synthesis of both enzymes. The abolition of repression by arginine in an argR derivative of strain PAO1 established the necessity of ArgR in mediating this effect. Furthermore, low-copy-number gdhA::lacZ and gltB::lacZ translational fusions corroborated the role of ArgR as repressor of the gdhA gene and gltBD operon.; Gel retardation and DNase I footprinting assays demonstrated that purified ArgR (Park et al., 1997) binds and protects DNA sequences in the control regions of gdhA and gltBD that are similar to the consensus sequence of the ArgR binding site. Primer extension experiments showed that the gltBD operon and the gdhA gene are transcribed from single promoters.; Glutamate represses the synthesis of the anabolic NADP-GDH and GOGAT, but this repression was retained in an argR mutant, thus precluding a role for ArgR. The level of NADP-GDH, but not GOGAT, was found to correlate with the effectiveness of other nitrogen sources to support cell growth. Finally, nucleotide analysis of P. fluorescens, P. putida and P. stutzeri genomes suggests that ArgR may regulate operons encoding enzymes of glutamate metabolism in other pseudomonads. |
