Identification and characterization of the PhhR regulon in Pseudomonas putida

Abstract

SummaryPseudomonas putida is a soil microorganism that utilizes aromatic amino acids present in root exudates as a nitrogen source. We have previously shown that the PhhR transcriptional regulator induces phhAB genes encoding a phenylalanine hydroxylase. In this study we show, using microarray assays and promoter fusions, that PhhR is a global regulator responsible for the activation of genes essential for phenylalanine degradation, phenylalanine homeostasis and other genes of unknown function. Recently, it has been shown that phenylalanine catabolism occurs through more than one pathway. One of these possible pathways involves the metabolism of phenylalanine via tyrosine, p-hydroxyphenylpyruvate, and homogentisate. We identified two genes within this pathway that encode an acyl-CoA transferase involved in the metabolism of acetoacetate. All genes in this pathway were induced in response to phenylalanine in a PhhR-proficient background. The second potential degradative pathway involves the degradation of phenylalanine to produce phenylpyruvate, which seems to be degraded via phenylacetyl-CoA. A number of mutants in the paa genes encoding phenylacetyl-CoA degradation enzymes fail to grow on phenylpyruvate or phenylacetate, further supporting the existence of this second pathway. We found that the PhhR regulon also includes genes involved in the biosynthesis of aromatic amino acids that are repressed in the presence of phenylalanine, suggesting the possibility of feedback at the transcriptional level. In addition, we found that PhhR modulates the level of expression of the broad-substrate-specificity MexEF/OprN efflux pump. Expression from this pump is under the control of mexT gene product because phenylalanine-dependent transcription from the mexE promoter does not occur in a mexT mutant background. These results place PhhR as an important regulator in the control of bacterial responses to aromatic amino acids.

Characterization of thePseudomonas aeruginosaTranscriptional Response to Phenylalanine and Tyrosine

“…Putative PhhR binding sites were also identified upstream of the hpd (Ϫ138 bp) (22) and dhcA (Ϫ40 bp) transcriptional start sites ( Fig. 2B and C); however, unlike the case in P. putida (11), a DNA sequence similar to the consensus PhhR and TyrR binding sites was not identified in the hmgA promoter (Fig. 2D).…”

“…In Pseudomonas putida, the transcriptional regulator PhhR is required to induce genes encoding enzymes critical for catabolism of these amino acids (12,13). Beyond regulation of genes involved in aromatic amino acid catabolism, PhhR regulates several other classes of genes in P. putida and has thus been described as a global transcriptional regulator (11). P. aeruginosa possesses a PhhR homolog (PA0873 in strain PAO1 and PA14_52980 in strain PA14) with 88% identity to P. putida PhhR that has been proposed to be critical for induction of phenylalanine catabolic genes (27).…”

A Putative ABC Transporter, HatABCDE, Is among Molecular Determinants of Pyomelanin Production inPseudomonas aeruginosa

“…Constituting a branch of the chorismic acid pathway, the conversion of chorismate into HGA (via phenylalanine and tyrosine) is dependent on PhhA/B, PhhC, Hpd, and the regulatory protein PhhR (Fig. 1A) (25). Thus, a transposon insertion in any of the genes encoding any of these proteins would be expected to generate an identifiable phenotype; we hit all of these genes in our screen.…”

Global Regulation of Food Supply by P seudomonas p utida DOT-T1E

“…This multifaceted regulation network (using global regulators and inducer exclusion) serves as an autoregulatory mechanism to maintain sugar utilization at a level harmonized to the bacteria's metabolic capacities rather than to simply establish preferential utilization of certain carbon sources. Consequently, metabolic balance, rather than the term catabolite repression, perhaps better describes the physiological role of this regulatory network (6,18). In pseudomonads, however, catabolite repression does not involve cAMP; indeed, in P. putida and Pseudomonas aeruginosa, cAMP levels remain relatively constant, regardless of the growth conditions (41,48).…”