Molecular characterization and regulation of operons for asparagine and aspartate uptake and utilization in...

Pseudomonas aeruginosa can utilize proteogenic amino acids as the sole source of carbon and nitrogen. In particular, utilization of l-Asp and l-Asn is insensitive to carbon catabolite repression as strong growth remains in the mutants devoid of the essential CbrAB activators of most catabolic genes. Transcriptome analysis was conducted to identify genes for the catabolism, uptake and regulation of these two amino acids. Gene inactivation and growth phenotype analysis established two asparaginases AsnA and AsnB for the degradation of l-Asn to l-Asp, whereas only AnsB is required for the deamidation of d-Asn to d-Asp. While d-Asp is a dead-end product, conversion of l-Asp to fumarate is catalysed by an aspartase AspA as further evidenced by enzyme kinetics. The results of measuring promoter-lacZ expression in vivo and mobility shift assays in vitro demonstrated that asnR and aspR encode two transcriptional regulators in response to l-Asn and l-Asp, respectively, for the induction of the ansPA operon and the aspA gene. Exogenous l-Glu also caused induction of the aspA gene, most likely due to its conversion to l-Asp by the aspartate transaminase AspC. Expression of several transporters were found inducible by l-Asn and/or l-Asp, including AatJQMP for acid amino acids, DctA and DctPQM for C4-dicarboxylates, and PA5530 for C5-dicarboxylates. In summary, a complete pathway and regulation for l-Asn and l-Asp catabolism was established in this study. Cross induction of three transport systems for dicarboxylic acids may provide a physiological explanation for the insensitivity of l-Asn and l-Asp utilization to carbon catabolite repression.