Catabolism of Phenylalanine by Pseudomonas putida: The NtrC-family PhhR Regulator Binds to Two Sites Upstream from the phhA Gene and Stimulates Transcription with σ70

Pseudomonas putida uses l-phenylalanine as the sole nitrogen source for growth by converting l-phenylalanine to l-tyrosine, which acts as a donor of the amino group. This metabolic step requires the products of the phhA and phhB genes, which form an operon. Expression of the phhA promoter is mediated by the phhR gene product in the presence of l-phenylalanine or l-tyrosine. The PhhR protein belongs to the NtrC family of enhancers. In contrast with most members of this family of regulators, transcription from the promoter of the phhAB operon (PphhA) is mediated by RNA polymerase with σ70 rather than with σ54. The PhhR regulator binds two similar but non-identical upstream PhhR motifs (5′-TGTAAAATTATCGTTACG-3′ and 5′-ACAAAAACTGTGTTTCCG-3′) that are located 39 and 97 nucleotides upstream of the proposed −35 hexamer for RNA polymerase, respectively. These motifs are called PhhR proximal and PhhR distal binding motifs because of their position with respect to the RNA polymerase binding site. Affinity of PhhR for its target sequences was determined by isothermal titration calorimetry and was found to be around 30 nM for the proximal site and 2 μM for the distal site, and the binding stoichiometry is of a dimer per binding site. Both target sequences are sine qua non requirements for transcription, since inactivation of either of them resulted in no transcription from the phhA promoter. An IHF binding site overlaps the proximal PhhR proximal motif, which is recognized by IHF with a KD of around 1.2 μM. IHF may consequently compete with PhhR for binding and indeed inhibits PhhR-dependent phhAB operon expression.