Functional and biochemical analysis of acidic amino acid transport and utilization by Pseudomonas putida KT2440

The Gram-negative soil bacterium Pseudomonad putida is able to utilize an unusually large number of organic compounds as sources of energy and biomass, among them the acidic amino acids glutamate (Glu) and aspartate (Asp) and their amides glutamine (Gln) and asparagine (Asn). During growth of P. putida on any of these amino acids, the expression of a defined set of genes is induced that allow for their utilization. As reported previously, the Aau two-component system is involved in this adaptation process. In the present study we analyzed the functional role of the AauR-AauS system in the uptake and metabolism of acidic amino acid by P. putida KT2440. Aau-negative mutants were defective in growth Glu and Asp as sole sources of carbon and nitrogen as well as in Glu and Asp uptake. In addition, the AauR mutant failed to express periplasmic glutaminase/ asparaginase (PGA), an enzyme required for the conversion of Gln and Asn to the respective acidic amino acids. Other enzymes involved in the assimilation of Glu and Asp also showed marked changes in activity. AauR deletion mutants grown on Asn and Asp started to accumulate Glu in the late log phase to levels many times higher than in the wild type and also excreted Glu if glucose was available. The excretion of glutamate in these conditions is probably mediated by Bra (a bi-directional ABC transporter), as reported for TCA cycle mutants of other rhizobacteria. The glutamate accumulation by aau mutants may be due to high aspartase activities which feed the carbon skeletons of Asp and Asn into the TCA cycle where glutamate dehydrogenase converts it to glutamate. Immediately upstream of the aau locus of P. putida KT2440, an operon involving genes PP1068-PP1071 encodes an ABC transporter which we named Aat (for acidic amino acid transporter). Aat is also upregulated when P. putida is grown on acidic amino acids or their amides. AatPMQJ is a polar amino acid transporter belonging to the solute-binding protein family (SBP_Bac_3). The Aat system consists of four subunits, where AatP is the nucleotide binding protein, AatM and Aat Q are membrane spanning permeases and AatJ is the periplasmic solute-binding protein. The expressed and purified solute binding protein (AatJ) showed high binding affinity towards Glu and Asp (Kd = 0.4 μM and 1.3 μM respectively), while Gln and Asn as well as other dicarboxylates were bound with much lower affinity. A modeled structure of AatJ (using the glutamine- binding protein GlnH of E. coli as template) suggested a novel arrangement of active site residues which include several arginine residues. The modeled structure was validated by site directed mutagenesis of several AatJ residues predicted to be in contact with the bound ligand. A data base search suggested that AatJ and at least 15 further proteins constitute a new subfamily of periplasmic acidic amino acid receptors. The role of the AauRS two-component system in the transcriptional regulation of aat and the PGA-encoding ansB gene was analyzed by gel-shift assays. The purified recombinant protein AauR was shown to bind to the promoter regions of both aat and ansB. A DNase I footprinting analysis revealed that the AauR binding motif consists of a well-conserved inverted repeat of six nucleotides (GTTCGGNNNNCCGAAC). By in-silico analysis of the P. putida KT2440 genome, several other genes were detected that contain an AauR interaction motif in their promoters. These genes encode a H+/Glu symporter (GltP), phosphoenolpyruvate synthase (PpsA), a branched-chain amino acid transporter (Bra) and a disulphide exchange protein (DsbC). Based on these data, we give an outline of acidic amino acid uptake and metabolism in P. putida and its regulation by the AauRS two-component system