Characterization of a Pseudomonas putida ABC transporter (AatJMQP) required for acidic amino acid uptake: biochemical properties and regulation by the Aau two-component system.
We describe an ATP-binding cassette (ABC) transporter in Pseudomonas putida KT2440 that mediates the uptake of glutamate and aspartate. The system (AatJMQP, for acidic amino acid transport) is encoded by an operon involving genes PP1071-PP1068. A deletion mutant with inactivated solute-binding protein (KTaatJ) failed to grow on Glu and Gln as sole sources of carbon and nitrogen, while a mutant lacking a functional nucleotide-binding domain (KTaatP) was able to adapt to growth on Glu after an extended lag phase. Uptake of Glu and Asp by either mutant was greatly impaired at both low and high amino acid concentrations. The purified solute-binding protein AatJ exhibited high affinity towards Glu and Asp (K(d)=0.4 and 1.3 muM, respectively), while Gln and Asn as well as dicarboxylates (succinate and fumarate) were bound with much lower affinity. We further show that the expression of AatJMQP is controlled by the sigma(54)-dependent two-component system AauRS. Binding of the response regulator AauR to the aat promoter was examined by gel mobility shift assays and DNase I footprinting. By in silico screening, the AauR-binding motif (the inverted repeat TTCGGNNNNCCGAA) was detected in further P. putida KT2440 genes with established or putative functions in acidic amino acid utilization, and also occurred in other pseudomonads. The products of these AauR-responsive genes include the H(+)/Glu symporter GltP, a periplasmic glutaminase/asparaginase, AnsB, and phosphoenolpyruvate synthase (PpsA), a key enzyme of gluconeogenesis in Gram-negative bacteria. Based on these findings, we propose that AauR is a central regulator of acidic amino acid uptake and metabolism in pseudomonads.
Singh
Birendra
B
Philipps University, Institute of Physiological Chemistry, D-35043 Marburg, Germany.
Röhm
Klaus-Heinrich
KH
Philipps University, Institute of Physiological Chemistry, D-35043 Marburg, Germany.
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D023061:Gene Order; D018698:Glutamic Acid / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009876:Operon; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016958:Pseudomonas putida / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid